Site-specific antibody conjugates and antibody-drug conjugates as specific examples thereof

Abstract

The present application relates to a site-specific antibody conjugate and an antibody-drug conjugate as a specific example thereof, a technique that enables a specific number of chemical functional groups or load moieties to be labeled at a specific site of an antibody. The present application can provide an antibody product having a high degree of homogeneity, the product comprising an antibody or a fragment thereof, the antibody or fragment thereof comprises one or more amino acid sequences selected from the group consisting of Formula 8-1, Formula 8-2 and Formula 8-3: [Formula 8-1] G-P-S-V-F-L-F-P-P-(K) '-P-K-D-T-L-M-I [Formula 8-2] G-P-S-V-F-L-F-P-P-K-K'-D-T-L-M-I [Formula 8-3] G-P-S-V-F-L-F-P-P-(K) '-D-T-L-M-I.

Description

[0001] This application is a divisional application of a Chinese patent application filed on March 9, 2020, with application number 202080019814.3, and invention name “Site-specific antibody conjugation and antibody-drug conjugate as a specific example thereof”. Technical Field

[0002] The present invention relates to a technique for selectively labeling specific amino acid residues of an antibody with a substance (a low molecular weight compound, a synthetic polymer, a biopolymer (i.e., a peptide, a carbohydrate, a protein, etc.)) or attaching a molecule to be delivered to a specific target cell or tissue (hereinafter referred to as "cargo") to an antibody. In addition, the present invention relates to a technique for labeling specific sites of an antibody with a desired amount of a substance or attaching a desired amount of a cargo to specific amino acid residues of an antibody. In addition, the present invention encompasses an antibody complex prepared by this method or a method using an antibody fragment complex. Background Art

[0003] Antibodies are biomolecules that have the function of recognizing specific molecules and have been used in various industrial applications. For example, antibodies can be used to detect or screen specific substances and examine the pathways through which specific substances move in the body or cells. In addition, antibodies can be used for therapeutic purposes by inducing an immune response against specific substances.

[0004] In order to expand the function of such antibodies, attempts have been made to improve the ability of antibodies. Typically, attempts are made to label antibodies with foreign substances to supplement or expand the function of antibodies. Typically, when antibodies are labeled with fluorescent substances, the antibodies can be used for fluorescence determination, or when antibodies are labeled with reagents for treating specific diseases, the antibodies can be used to maximize the therapeutic effect of the antibodies. These attempts and techniques are generally referred to as "antibody labeling". The present invention relates to new methods for labeling antibodies.

[0005] Initially, antibody labeling was achieved by randomly attaching foreign substances to antibodies. However, this method has many problems. The problem with the antibodies prepared in this way is that they have poor homogeneity. The problem with these antibodies is that their effects are still not uniform, because the number of substances attached to each antibody is different, and the binding sites of the antibodies are also different. This problem is a big obstacle to the development of antibody-drug conjugate (ADC) technology that requires high safety and reproducibility.

[0006] In addition, this method has the problem that the antibody recognition ability may be significantly reduced. The antibody is composed of a Fab domain including an antigen-binding domain that recognizes the antigen and an Fc domain that participates in antibody crystallization. By allowing foreign substances to bind to the antigen-binding domain of the antibody or a site adjacent to the antigen-binding domain, random labeling leads to highly degraded antibody recognition ability.

[0007] Therefore, there is a need for the technology of uniformly labeling antibodies in a site-specific manner in the related art. Although some technologies have been developed, such as genetically regulated or modified antibodies, most of them are invalid in terms of technology and economy. Therefore, the present invention is designed to solve these problems, and therefore for the technology that can specifically connect a specific substance (or "part") to a specific site of an antibody (this technology does not require any additional regulation of the antibody), and also using antibodies to deliver a specific substance (drug or labeled substance) into a cell or tissue. Summary of the invention

[0008] [Technical issues]

[0009] The present invention provides techniques for specifically transferring chemical functional groups to specific sites of antibodies. In one embodiment, the present invention provides techniques for specifically transferring chemical functional groups to lysine 246 in an antibody. In another embodiment, the present invention provides techniques for specifically transferring chemical functional groups to lysine 248 in an antibody. In yet another specific embodiment, the present invention provides techniques for specifically transferring chemical functional groups to lysine 246 and lysine 248 in an antibody.

[0010] The present invention provides technology capable of attaching a desired number of chemical functional groups to antibodies. In a specific embodiment, the present invention provides antibodies or fragments thereof having two specific parts that bind thereto. In another specific embodiment, the present invention provides antibodies or fragments thereof having four specific parts that bind thereto.

[0011] The present invention provides techniques for specifically linking a payload moiety to a specific site of an antibody. In one embodiment, the present invention provides techniques for specifically linking a payload moiety to lysine 246 in an antibody. In another specific embodiment, the present invention provides techniques for specifically linking a payload moiety to lysine 248 in an antibody. In yet another specific embodiment, the present invention provides techniques for specifically linking a payload moiety to lysine 246 and lysine 248 in an antibody.

[0012] The present invention provides a technique for attaching a desired number of payload moieties to an antibody. In one embodiment, the present invention provides an antibody or fragment thereof having two payload moieties bound thereto. In another specific embodiment, the present invention provides an antibody or fragment thereof having four payload moieties bound thereto.

[0013] The present invention provides methods of using the above antibodies or antibody fragments. In one embodiment, the present invention provides methods of using antibody-drug complexes to treat specific diseases.

[0014] [Technical solution]

[0015] The present application provides a compound of formula 2:

[0016] [Formula 2]

[0017]

[0018] Wherein, H1 is the first click chemistry functional group, and D1 is selected from a covalent bond, C 1～4 Alkylene, C 2～4 Alkenylene, C 2～4 Alkynylidene and C 3～8 Cycloalkylene, X1 is S, D2 is selected from C 1～7 Alkylene, C 2～7 Alkenylene, C 2～7 Alkynylidene and C 3～8 Cycloalkylene, X2 is O, and R2' is N-succinimide, p-nitrophenyl or pentafluorophenyl.

[0019] The present application also provides compounds wherein H1 is selected from terminal alkynes, azides, strained alkynes, dienes, dienophiles, alkenes, thiols and tetrazines. In addition, the present application provides compounds wherein H1 is selected from norbornene, tetrazines, azides and dibenzocyclooctyne-amines.

[0020] In addition, the present application provides a compound wherein R2' is N-succinimide.

[0021] In addition, the present application provides a compound wherein Formula 2 is Formula 2-1, Formula 2-2 or Formula 2-3:

[0022] [Formula 2-1]

[0023]

[0024] [Formula 2-2]

[0025]

[0026] [Formula 2-3]

[0027]

[0028] The present application provides a peptide having a structure of Formula 4-2:

[0029] [Formula 4-2]

[0030] (Xaa) 1-3 -C-(Xaa)2-H-Xa1-G-Xa2-LV-Xa3-C-(Xaa) 1-3

[0031] wherein each Xaa is independently any amino acid residue other than a cysteine ​​residue, C is a cysteine ​​residue, H is a histidine residue, G is a glycine residue, Xa2 is a glutamic acid residue or an asparagine residue, L is a leucine residue, V is a valine residue, Xa3 is selected from a tryptophan residue, a naphthylalanine residue and a phenylalanine residue, and Xa1 is Where D3 is a covalent bond or C 1～3 alkylene, and X3 is NH2, wherein the peptide consists of 13 to 17 amino acid residues, wherein the peptide exhibits an activity of binding to human immunoglobulin G (IgG), and wherein a cysteine ​​residue 2 to 4 amino acids away from the N-terminus is optionally linked to a cysteine ​​residue 2 to 4 amino acids away from the C-terminus.

[0032] The present application also provides a peptide wherein D3 is a covalent bond, a methylene group or an ethylene group.

[0033] In addition, the present application provides a peptide wherein Formula 4-2 is Formula 4-6:

[0034] [Formula 4-6]

[0035] DCAWH-Xa1-GELVWCT

[0036] Among them, D is an aspartic acid residue, A is an alanine residue, E is a glutamic acid residue, W is a tryptophan residue, and T is a threonine residue.

[0037] The present application provides a peptide-compound conjugate having a structure of Formula 6-2:

[0038] [Formula 6-2]

[0039] (Xaa) 1-3 -C-(Xaa)2-H-(Xa1)′-G-Xa2-LV-Xa3-C-(Xaa) 1-3

[0040] wherein each Xaa is independently any amino acid residue other than a cysteine ​​residue, C is a cysteine ​​residue, H is a histidine residue, G is a glycine residue, Xa2 is a glutamic acid residue or an asparagine residue, L is a leucine residue, V is a valine residue, and Xa3 is selected from a tryptophan residue, a naphthylalanine residue and a phenylalanine residue, and

[0041] (Xa1)′ is Wherein H1 is the first click chemistry functional group, D1 is selected from covalent bonds, C 1～4 Alkylene, C 2～4 Alkenylene, C 2～4 Alkynylidene and C 3～8 Cycloalkylene, X1 is S, D2 is selected from C1～7 Alkylene, C 2～7 Alkenylene, C 2～7 Alkynylidene and C 3～8 Cycloalkylene, D3 is a covalent bond or C 1～3 The invention relates to a peptide comprising: a peptide which is composed of 13 to 17 amino acid residues, wherein the peptide exhibits an activity of binding to human immunoglobulin G (IgG), and wherein a cysteine ​​residue which is 2 to 4 amino acids away from the N-terminus and a cysteine ​​residue which is 2 to 4 amino acids away from the C-terminus are optionally linked.

[0042] The present application also provides a peptide-compound conjugate, wherein the distance from the beta carbon of (Xa1)' to the first carbonyl carbon of (Xa1)' is less than about

[0043] In addition, the present application provides a peptide-compound conjugate, wherein D2 is C y Alkylene, C y Alkenylene or C y Alkynylidene, D3 is C x Alkylene, wherein y is an integer greater than or equal to 1, and 1≤x+y≤5.

[0044] In addition, the present application provides a peptide-compound conjugate, wherein the distance from the beta carbon of (Xa1)' to the first carbonyl carbon of (Xa1)' is greater than about

[0045] In addition, the present application provides a peptide-compound conjugate, wherein D2 is C y Alkylene, C y Alkenylene or C y Alkyne, and D3 is C x Alkylene, wherein y is an integer greater than or equal to 1, and 9≤x+y≤12. In addition, the present application provides a peptide-compound conjugate, wherein D2 is C y Alkenylene or C y Alkyne group.

[0046] In addition, the present application provides a peptide-compound conjugate, wherein the distance from the β carbon of (Xa1)' to the first carbonyl carbon of (Xa1)' is about to about

[0047] In addition, the present application provides a peptide-compound conjugate, wherein D2 is C y Alkylene, C y Alkenylene or C y Alkyne, and D3 is C x Alkylene, wherein y is an integer greater than or equal to 1, and 6≤x+y≤8.

[0048] In addition, the present application provides a peptide-compound conjugate wherein Formula 6-2 is Formula 6-3:

[0049] [Formula 6-3]

[0050] DCAWH-(Xa1)′-GELVWCT

[0051] Among them, D is an aspartic acid residue, A is an alanine residue, E is a glutamic acid residue, W is a tryptophan residue, and T is a threonine residue.

[0052] In addition, the present application provides a peptide-compound conjugate, wherein D1 is a covalent bond and D2 is a methylene group.

[0053] The present application provides a method for preparing a reagent for transferring a first click chemistry functional group to an antibody, the method comprising reacting a compound of Formula 2 with a peptide having a structure of Formula 4-2:

[0054] [Formula 2]

[0055]

[0056] Wherein, H1 is the first click chemistry functional group, and D1 is selected from a covalent bond, C 1～4 Alkylene, C 2～4 Alkenylene, C 2～4 Alkynylidene and C 3～8 Cycloalkylene, X1 is S, D2 is selected from C 1～7 Alkylene, C 2～7 Alkenylene, C 2～7 Alkynylidene and C 3～8 Cycloalkylene, X2 is O, R2' is N-succinimide, p-nitrophenyl or pentafluorophenyl;

[0057] [Formula 4-2]

[0058] (Xaa) 1-3 -C-(Xaa)2-H-Xa1-G-Xa2-LV-Xa3-C-(Xaa) 1-3

[0059] wherein each Xaa is independently any amino acid residue other than a cysteine ​​residue, C is a cysteine ​​residue, H is a histidine residue, G is a glycine residue, Xa2 is a glutamic acid residue or an asparagine residue, L is a leucine residue, V is a valine residue, and Xa3 is selected from a tryptophan residue, a naphthylalanine residue and a phenylalanine residue, and

[0060] Xa1 is Where D3 is a covalent bond or C 1～3alkylene, and X3 is NH2, wherein the peptide consists of 13 to 17 amino acid residues, wherein the peptide exhibits activity of binding to human immunoglobulin G (IgG), and wherein a cysteine ​​residue 2 to 4 amino acids away from the N-terminus is optionally linked to a cysteine ​​residue 2 to 4 amino acids away from the C-terminus.

[0061] The present application also provides a method for preparing a reagent for transferring a first click chemistry functional group to an antibody, wherein D2 is C y Alkylene, C y Alkenylene or C y Alkyne, and D3 is C x An alkylene group, wherein y is an integer greater than or equal to 1, and 1≤x+y≤5, characterized in that the reagent for transferring the first click chemistry functional group to the antibody prepared by the method can react with the antibody to specifically deliver the first click chemistry functional group to the lysine 248 residue of the Fc domain of the antibody.

[0062] In addition, the present application provides a method for preparing a reagent for transferring a first click chemistry functional group to an antibody, wherein D2 is C y Alkylene, C y Alkenylene or C y Alkyne, and D3 is C x An alkylene group, wherein y is an integer greater than or equal to 1, and 1≤x+y≤5, characterized in that the reagent for transferring the first click chemistry functional group to the antibody prepared by the method can react with the antibody to specifically deliver the first click chemistry functional group to the lysine 248 residue of the Fc domain of the antibody.

[0063] In addition, the present application provides a method for preparing a reagent for transferring a first click chemistry functional group to an antibody, wherein D2 is C y Alkylene, C y Alkenylene or C y Alkyne, and D3 is C x An alkylene group, wherein y is an integer greater than or equal to 1, and 9≤x+y≤12, characterized in that the reagent for transferring the first click chemistry functional group to the antibody prepared by the method can react with the antibody to specifically deliver the first click chemistry functional group to the lysine 246 residue of the Fc domain of the antibody.

[0064] In addition, the present application provides a method for preparing a reagent for transferring a first click chemistry functional group to an antibody, wherein D2 is C y Alkylene, C y Alkenylene or C y Alkyne, and D3 is C xAn alkylene group, wherein y is an integer greater than or equal to 1, and 6≤x+y≤8, characterized in that the reagent for transferring the first click chemistry functional group to the antibody prepared by the method can react with the antibody to selectively deliver the first click chemistry functional group to the lysine 246 residue or the lysine 248 residue of the Fc domain of the antibody.

[0065] The present application provides a kit for preparing a first click chemistry functional group transferred to an antibody, the kit comprising a compound of Formula 2 and a peptide having a structure of Formula 4-2:

[0066] [Formula 2]

[0067]

[0068] Wherein, H1 is the first click chemistry functional group, and D1 is selected from a covalent bond, C 1～4 Alkylene, C 2～4 Alkenylene, C 2～4 Alkynylidene and C 3～8 Cycloalkylene, X1 is S, D2 is selected from C 1～7 Alkylene, C 2～7 Alkenylene, C 2～7 Alkynylidene and C 3～8 Cycloalkylene, X2 is O, R2' is N-succinimide, p-nitrophenyl or pentafluorophenyl;

[0069] [Formula 4-2]

[0070] (Xaa) 1-3 -C-(Xaa)2-H-Xa1-G-Xa2-LV-Xa3-C-(Xaa) 1-3

[0071] wherein each Xaa is independently any amino acid residue other than a cysteine ​​residue, C is a cysteine ​​residue, H is a histidine residue, G is a glycine residue, Xa2 is a glutamic acid residue or an asparagine residue, L is a leucine residue, V is a valine residue, and Xa3 is selected from a tryptophan residue, a naphthylalanine residue and a phenylalanine residue, and

[0072] Xa1 is Where D3 is a covalent bond or C 1～3 alkylene, and X3 is NH2, wherein the peptide consists of 13 to 17 amino acid residues, wherein the peptide exhibits activity of binding to human immunoglobulin G (IgG), and wherein a cysteine ​​residue 2 to 4 amino acids away from the N-terminus is optionally linked to a cysteine ​​residue 2 to 4 amino acids away from the C-terminus.

[0073] The present application provides an antibody or a fragment thereof, wherein the antibody or the fragment thereof comprises one or more amino acid sequences selected from Formula 8-1, Formula 8-2 and Formula 8-3:

[0074] [Formula 8-1]

[0075] G—P—S—V—F—L—F—P—P—(K)′-P—K—D—T—L—M—I

[0076] [Formula 8-2]

[0077] G—P—S—V—F—L—F—P—P—K—P—(K)′—D—T—L—M—I

[0078] [Formula 8-3]

[0079] G—P—S—V—F—L—F—P—P—(K)′—P—(K)′—D—T—L—M—I

[0080] wherein G is a glycine residue, P is a proline residue, S is a serine residue, V is a valine residue, F is a phenylalanine residue, L is a leucine residue, K is a lysine residue, D is an aspartic acid residue, T is a threonine residue, M is a methionine residue, and I is an isoleucine residue, and

[0081] (K)′ is Wherein H1 is the first click chemistry functional group, D1 is selected from covalent bonds, C 1～4 Alkylene, C 2～4 Alkenylene, C 2～4 Alkynylidene and C 3～8 Cycloalkylene.

[0082] The present application also provides an antibody or a fragment thereof wherein D1 is a covalent bond.

[0083] In addition, the present application provides an antibody or fragment thereof comprising the amino acid sequence of Formula 8-1 but not comprising the amino acid sequences of Formula 8-2 and Formula 8-3. In addition, the present application provides an antibody or fragment thereof comprising the amino acid sequence of Formula 8-1 in both Fc domains.

[0084] In addition, the present application provides an antibody or fragment thereof comprising the amino acid sequence of Formula 8-2 but not the amino acid sequences of Formula 8-1 and Formula 8-3. In addition, the present application provides an antibody or fragment thereof comprising the amino acid sequence of Formula 8-2 in both Fc domains.

[0085] In addition, the present application provides an antibody or fragment thereof comprising the amino acid sequence of Formula 8-3 but not the amino acid sequences of Formula 8-1 and Formula 8-2. In addition, the present application provides an antibody or fragment thereof comprising the amino acid sequence of Formula 8-3 in both Fc domains.

[0086] The present application provides a method for preparing an antibody or a fragment thereof comprising a first click chemistry functional group, the method comprising reacting a peptide-compound conjugate having a structure of Formula 6-2 with an antibody or a fragment thereof:

[0087] [Formula 6-2]

[0088] (Xaa) 1-3 -C-(Xaa)2-H-(Xa1)′-G-Xa2-LV-Xa3-C-(Xaa) 1-3

[0089] wherein each Xaa is independently any amino acid residue other than a cysteine ​​residue, C is a cysteine ​​residue, H is a histidine residue, G is a glycine residue, Xa2 is a glutamic acid residue or an asparagine residue, L is a leucine residue, V is a valine residue, and Xa3 is selected from a tryptophan residue, a naphthylalanine residue and a phenylalanine residue, and

[0090] (Xa1)′ is Wherein H1 is the first click chemistry functional group, D1 is selected from covalent bonds, C 1～4 Alkylene, C 2～4 Alkenylene, C 2～4 Alkynylidene and C 3～8 Cycloalkylene, X1 is S, D2 is selected from C 1～7 Alkylene, C 2～7 Alkenylene, C 2～7 Alkynylidene and C 3～8 Cycloalkylene, D3 is a covalent bond or C 1～3 The invention relates to a peptide comprising: a peptide which is composed of 13 to 17 amino acid residues, wherein the peptide exhibits an activity of binding to human immunoglobulin G (IgG), and wherein a cysteine ​​residue which is 2 to 4 amino acids away from the N-terminus is optionally linked to a cysteine ​​residue which is 2 to 4 amino acids away from the C-terminus.

[0091] The present application provides a kit for preparing an antibody or a fragment thereof comprising a first click chemistry functional group, the kit comprising a peptide-compound conjugate having a structure of Formula 6-2 and an antibody or a fragment thereof:

[0092] [Formula 6-2]

[0093] (Xaa) 1-3 -C-(Xaa)2-H-(Xa1)′-G-Xa2-LV-Xa3-C-(Xaa) 1-3

[0094] wherein each Xaa is independently any amino acid residue other than a cysteine ​​residue, C is a cysteine ​​residue, H is a histidine residue, G is a glycine residue, Xa2 is a glutamic acid residue or an asparagine residue, L is a leucine residue, V is a valine residue, and W is a tryptophan residue, and

[0095] (Xa1)′ is Wherein H1 is the first click chemistry functional group, D1 is selected from covalent bonds, C 1～4 Alkylene, C 2～4 Alkenylene, C 2～4 Alkynylidene and C 3～8 Cycloalkylene, X1 is S, D2 is selected from C 1～7 Alkylene, C 2～7 Alkenylene, C 2～7 Alkynylidene and C 3～8 Cycloalkylene, D3 is a covalent bond or C 1～3 The invention relates to a peptide comprising: a peptide which is composed of 13 to 17 amino acid residues, wherein the peptide exhibits an activity of binding to human immunoglobulin G (IgG), and wherein a cysteine ​​residue which is 2 to 4 amino acids away from the N-terminus is optionally linked to a cysteine ​​residue which is 2 to 4 amino acids away from the C-terminus.

[0096] The present application provides a compound having a structure of Formula 9:

[0097] [Formula 9]

[0098] C m ——————H2

[0099] Among them, C m is the payload part, and H2 is the second click chemistry functional group.

[0100] The present application provides a method for preparing an antibody-drug conjugate, the method comprising reacting an antibody or a fragment thereof comprising one or more amino acid sequences selected from Formula 8-1, Formula 8-2 and Formula 8-3 with a compound having a structure of Formula 9:

[0101] [Formula 8-1]

[0102] G—P—S—V—F—L—F—P—P—(K)′—P—K—D—T—L—M—I

[0103] [Formula 8-2]

[0104] G—P—S—V—F—L—FP—P—K—P—(K)′—D—T—L—M—I

[0105] [Formula 8-3]

[0106] G—P—S—V—F—L—F—P—P—(K)′—P—(K)′—D—T—L—M—I

[0107] wherein G is a glycine residue, P is a proline residue, S is a serine residue, V is a valine residue, F is a phenylalanine residue, L is a leucine residue, K is a lysine residue, D is an aspartic acid residue, T is a threonine residue, M is a methionine residue, and I is an isoleucine residue, and

[0108] (K)′ is Wherein H1 is the first click chemistry functional group, D1 is selected from covalent bonds, C 1～4 Alkylene, C 2～4 Alkenylene, C 2～4 Alkynylidene and C 3～8 Cycloalkylene;

[0109] [Formula 9]

[0110] C m —————H2

[0111] Among them, C m is a load moiety, and H2 is a second click chemistry functional group complementary to the first click chemistry functional group.

[0112] The present application provides a kit for preparing an antibody-drug conjugate, the kit comprising an antibody or a fragment thereof and a compound having a structure of Formula 9, wherein the antibody or a fragment thereof comprises one or more amino acid sequences selected from Formula 8-1, Formula 8-2 and Formula 8-3:

[0113] [Formula 8-1]

[0114] G—P—S—V—F—L—F—P—P—(K)′—P—K—D—T—L—M—I

[0115] [Formula 8-2]

[0116] G—P—S—V—F—L—F—P—P—K—P—(K)′-D—T—L—M—I

[0117] [Formula 8-3]

[0118] G—P—S—V—F—L—F—P—P—(K)′-P-(K)′—D—T—L—M—I

[0119] wherein G is a glycine residue, P is a proline residue, S is a serine residue, V is a valine residue, F is a phenylalanine residue, L is a leucine residue, K is a lysine residue, D is an aspartic acid residue, T is a threonine residue, M is a methionine residue, and I is an isoleucine residue, and

[0120] (K)′ is Wherein H1 is the first click chemistry functional group, D1 is selected from covalent bonds, C 1～4 Alkylene, C 2～4 Alkenylene, C 2～4 Alkynylidene and C 3～8 Cycloalkylene;

[0121] [Formula 9]

[0122] C m ——————H2

[0123] Among them, C m is a load moiety, and H2 is a second click chemistry functional group complementary to the first click chemistry functional group.

[0124] The present application provides an antibody or a fragment thereof, wherein the antibody or the fragment thereof comprises one or more amino acid sequences selected from Formula 10-1, Formula 10-2 and Formula 10-3:

[0125] [Formula 10-1]

[0126] G—P—S—V—F—L—F—P—P—(K)″-P—K—D—T—L—M—I

[0127] [Formula 10-2]

[0128] G—P—S—V—F—L—F—P—P—K—P—(K)″—D—T—L—M—I

[0129] [Formula 10-3]

[0130] G—P—S—V—F—L—F—P—P—(K)″—P—(K)″—D—T—L—M—I

[0131] wherein G is a glycine residue, P is a proline residue, S is a serine residue, V is a valine residue, F is a phenylalanine residue, L is a leucine residue, K is a lysine residue, D is an aspartic acid residue, T is a threonine residue, M is a methionine residue, and I is an isoleucine residue, and

[0132] (K)” Among them C m is the load part, B is selected from

[0133] and Where A1 and A2 are connected to the load section or D1 and they are not both connected to the same section, R x Selected from H, halogen and C 1～3 Alkyl, D1 is selected from a covalent bond, C1～4 Alkylene, C 2～4 Alkenylene, C 2～4 Alkynylidene and C 3～8 Cycloalkylene.

[0134] The present application also provides an antibody or fragment thereof comprising the amino acid sequence of Formula 10-1 but not comprising the amino acid sequences of Formula 10-2 and Formula 10-3. In addition, the present application provides an antibody or fragment thereof comprising the amino acid sequence of Formula 10-1 in both Fc domains.

[0135] In addition, the present application provides an antibody or fragment thereof comprising the amino acid sequence of Formula 10-2 but not the amino acid sequences of Formula 10-1 and Formula 10-3. In addition, the present application provides an antibody or fragment thereof comprising the amino acid sequence of Formula 10-2 in both Fc domains.

[0136] In addition, the present application provides an antibody or fragment thereof comprising the amino acid sequence of Formula 10-3 but not the amino acid sequences of Formula 10-1 and Formula 10-2. In addition, the present application provides an antibody or fragment thereof comprising the amino acid sequence of Formula 10-3 in both Fc domains.

[0137] In addition, the present application provides an antibody or fragment thereof in which the payload portion comprises a drug portion. In addition, the present application provides an antibody or fragment thereof in which the payload portion comprises two or more drug portions. Or further, the present application provides an antibody or fragment thereof in which the drug portion is an anticancer agent. In addition, the present application provides an antibody or a fragment thereof, wherein the anticancer agent is at least one selected from the following: DM1, DM3, DM4, abrin, ricin A, Pseudomonas exotoxin, cholera toxin, diphtheria toxin, tumor necrosis factor, α-interferon, β-interferon, nerve growth factor, platelet-derived growth factor, tissue plasminogen activator, cytokine, apoptotic agent, anti-angiogenic agent, lymphokine, taxane, DNA-alkylating agent, anthracycline, tubulysin analog, duocarmycin analog, auristatin E, auristatin F, maytansinoid, cytotoxic agent containing a reactive polyethylene glycol portion, taxon, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenotoposide, vincristine, vinblastine, t. colchicine (t. ), doxorubicin, daunorubicin, dihydroxyanthraquinone, mitoxantrone, mithramycin, actinomycin D, 1-dihydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin, methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine, nitrogen mustard, thiotepa, chlorambucil, melphalan, carmustine, lomustine, cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, cisplatin, dactinomycin, bleomycin, anthramycin, calicheamicin (ca licheamicin), abiraterone, bendamustine, bortezomib, carboplatin, cabazitaxel, dasatinib, docetaxel, epirubicin, erlotinib, everolimus, gemcitabine, gefitinib, idarubicin, imatinib, hydroxyurea, lapatinib, leuprolide, melphalan, nedaplatin, nilotinib, oxaliplatin, pazopanib, pemetrexed, picoplatin, romidepsin, satraplatin, sorafenib, vemurafenib, sunitinib, teniposide, triplatin, and vinorelbine.

[0138] The present application provides a pharmaceutical composition for treating cancer, wherein the pharmaceutical composition comprises the above-mentioned antibody-drug conjugate.

[0139] The present application also provides a pharmaceutical composition for treating cancer, wherein the cancer is selected from bladder cancer, bone cancer, brain tumor, breast cancer, heart cancer, cervical cancer, colorectal cancer, rectal cancer, esophageal cancer, fibrosarcoma, gastric cancer, gastrointestinal cancer, head and neck cancer, Kaposi's sarcoma, kidney cancer, leukemia, liver cancer, lung cancer, lymphoma, melanoma, myeloma, ovarian cancer, pancreatic cancer, penile cancer, prostate cancer, testicular germ cell cancer, thymoma and thymic carcinoma.

[0140] In addition, the present application provides a pharmaceutical composition for treating cancer, wherein the cancer is breast cancer.

[0141] The present application provides a method for treating cancer, which comprises administering to a subject a pharmaceutical composition comprising the above-mentioned antibody-drug conjugate.

[0142] The present application also provides a method for treating cancer, wherein the cancer is selected from bladder cancer, bone cancer, brain tumor, breast cancer, heart cancer, cervical cancer, colorectal cancer, rectal cancer, esophageal cancer, fibrosarcoma, gastric cancer, gastrointestinal cancer, head and neck cancer, Kaposi's sarcoma, kidney cancer, leukemia, liver cancer, lung cancer, lymphoma, melanoma, myeloma, ovarian cancer, pancreatic cancer, penile cancer, prostate cancer, testicular germ cell cancer, thymoma and thymic carcinoma.

[0143] In addition, the present application provides a method for treating cancer, wherein the cancer is breast cancer.

[0144] [Beneficial Effects]

[0145] The antibody product according to the present invention may have a specific number of chemical functional groups or load moieties labeled at a specific site thereof. Therefore, the present invention may provide an antibody product with high homogeneity. The present invention may also provide an antibody product whose antibody function is not degraded. That is, the present invention may provide an antibody product whose antibody binding affinity and half-life are not reduced. As the first technology that can site-specifically label antibodies without any complex process, the present invention is of great significance. BRIEF DESCRIPTION OF THE DRAWINGS

[0146] Figure 1 A partial sequence of the Fc domain is shown, along with the numbers in the sequence numbered according to the EU numbering system.

[0147] Figure 2 The positions of lysine residues in the Fc domain including lysine 246 and lysine 248 are shown.

[0148] Figure 3 and Figure 4 The topology between SSFI and Fc domains is shown.

[0149] Figure 5 Lysine 246 and lysine 248 in the Fc domain and Xal of SSFI are shown.

[0150] Figure 6 The distance between the amine group of lysine 246 and the beta carbon of Xa1 of SSFI is shown.

[0151] Figure 7 The distance between the amine group of lysine 248 and the beta carbon of Xa1 of SSFI is shown.

[0152] Figure 8 The structure of R1'-L2-SSFI and the distance between the first carbonyl carbon of (Xa1)' and the β carbon (L c ).

[0153] Fig. 9 The topology between R1′-L2-SSFI and the Fc domain is shown so that the side chain of (Xa1)′ has a direction parallel to the x-axis in the figure (dashed arrow).

[0154] Fig.10 Conditions for allowing the first carbonyl carbon to react well with lysine 248 are shown.

[0155] Fig.11 Conditions for allowing the first carbonyl carbon to react well with lysine 246 are shown.

[0156] Fig.12Conditions for allowing the first carbonyl carbon to react selectively with either lysine 246 or lysine 248 are shown.

[0157] Fig.13 The FcRn binding site and lysine 246 and lysine 248 in the Fc domain are shown.

[0158] Fig.14 Analysis of the binding structure between SSFI and the Fc domain compared to the binding site between Fc and FcRn is shown.

[0159] Fig.15 A method for synthesizing Compound I is shown.

[0160] Fig.16 The results of confirming the structure of Compound 1 by mass spectrometry are shown.

[0161] Fig.17 A method for synthesizing compound II is shown.

[0162] Fig.18 The results of confirming the structure of Compound II by mass spectrometry are shown.

[0163] Fig.19 A method for synthesizing compound III is shown.

[0164] Fig. 20 The results of confirming the structure of Compound III by mass spectrometry are shown.

[0165] Fig.21 and Fig. 22 A method for synthesizing compound IV is shown.

[0166] Fig.23 The results of confirming the structure of Compound IV by mass spectrometry are shown.

[0167] Fig.24 The results of confirming the structure of SSFI(6Lys) by mass spectrometry are shown.

[0168] Fig.25 The results of confirming the structure of SSFI(6Orn) by mass spectrometry are shown.

[0169] Fig.26 The results of confirming the structure of SSFI(6Dab) by mass spectrometry are shown.

[0170] Fig. 27 The results of confirming the structure of SSFI(6Dap) by mass spectrometry are shown.

[0171] Fig.28 The results of confirming the structure of DD2 by mass spectrometry are shown.

[0172] Fig.29 The results of confirming the structure of DD3 by mass spectrometry are shown.

[0173] Fig.30 The results of confirming the structure of DD4 by mass spectrometry are shown.

[0174] Fig.31 The results of confirming the structure of DD5 by mass spectrometry are shown.

[0175] Fig.32 The results of confirming the structure of DD6 by mass spectrometry are shown.

[0176] Fig.33 The results of confirming the structure of compound I-SSFI(6Lys) by mass spectrometry are shown.

[0177] Fig.34 The results of confirming the structure of compound II-SSFI(6Lys) by mass spectrometry are shown.

[0178] Fig.35 The results of confirming the structure of compound III-SSFI(6Lys) by mass spectrometry are shown.

[0179] Fig.36 The results of confirming the structure of compound III-SSFI(6Orn) by mass spectrometry are shown.

[0180] Fig.37 The results of confirming the structure of compound III-SSFI(6Dab) by mass spectrometry are shown.

[0181] Fig.38 The results of confirming the structure of compound III-SSFI(6Dap) by mass spectrometry are shown.

[0182] Fig.39 The results of confirming the structure of compound IV-SSFI(6Dap) by mass spectrometry are shown.

[0183] Fig.40 The results of observing the binding reaction using trastuzumab and compound I-SSFI(6Lys) by HIC-HPLC are shown.

[0184] Fig.41 The results of observing the binding reaction using trastuzumab and compound II-SSFI(6Lys) by HIC-HPLC are shown.

[0185] Fig.42 The reaction of the antibodies with Compound III-SSFI (6Dap, Dab, Orn or Lys) is shown.

[0186] Fig.43 The structure of Ab(246 / 248)-norbornene as the final product is shown.

[0187] Fig.44 The results of observing the binding reaction using trastuzumab and compound III-SSFI (6Dap) by HIC-HPLC are shown.

[0188] Fig.45 The results of observing the binding reaction using trastuzumab and compound III-SSFI (6Dab) by HIC-HPLC are shown.

[0189] Fig.46 The results of observing the binding reaction using trastuzumab and compound III-SSFI (6Orn) by HIC-HPLC are shown.

[0190] Fig.47 The results of observing the binding reaction using trastuzumab and compound III-SSFI(6Lys) by HIC-HPLC are shown.

[0191] Fig.48 The results of observing the binding reaction using trastuzumab and compound IV-SSFI (6Dap) by HIC-HPLC are shown.

[0192] Fig.49 and Fig.50 The increase in molecular weight profile resulting from antibody-norbornene binding is shown.

[0193] Figure 51 to Figure 54 MS / MS chromatographic results of trastuzumab and antibody-norbornene complex are shown.

[0194] Fig.55 The results of sequence matching by means of MS / MS spectra are shown.

[0195] Fig.56 Shown is a mass spectrum of trastuzumab measured to confirm the structure of trastuzumab-azide.

[0196] Fig.57 Shown is a mass spectrum of the trastuzumab-azide complex measured to confirm the trastuzumab-azide structure.

[0197] Fig.58 The results of confirming the structure of tetrazine-DM1 by mass spectrometry are shown.

[0198] Fig.59 The structure of the trastuzumab-DM1 conjugate based on Compound III-SSFI(6Dap) is shown.

[0199] Fig.60 The results of observing the formation reaction of the trastuzumab-DM1 conjugate by HIC-HPLC are shown.

[0200] Fig.61 and Fig.62 The increase in molecular weight profile caused by norbornene-tetrazine-DM1 binding is shown.

[0201] Fig.63 Shown are the structures of three designs of payloads conjugated to norbornene-tagged antibodies.

[0202] Fig.64 Shown are the results of analyzing the antigen binding affinity of the antibody-drug conjugates.

[0203] Figure 65 to Figure 67 Shown are the results of analyzing the serum stability of antibody-drug conjugates.

[0204] Figures 68 to 70 Shown are the results of evaluating the efficacy of antibody-drug conjugates at the cellular level.

[0205] Fig.71 and Fig.72 The results of evaluating the efficacy of antibody-drug conjugates at the animal level are shown.

[0206] Fig.73 Shown are the results of pharmacokinetic testing for antibody-drug conjugates. DETAILED DESCRIPTION

[0207] definition

[0208] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those commonly understood by those of ordinary skill in the art to which the present invention belongs. The following references are provided to provide many common definitions of the terms used in the present invention to those skilled in the relevant art: Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd edition, 1994); The Cambridge Dictionary of Science and Technology (Walker, 1988); The Glossary of Genetics, 5th edition, R. Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, The Harper Collins Dictionary of Biology (1991). Unless otherwise expressly indicated, the following terms used in the present invention shall have the following meanings for their description.

[0209] In some embodiments, chemical structures are disclosed together with the corresponding chemical names. When the terms contradict or conflict with each other, the chemical structure takes precedence over the chemical name to understand the meaning of the compound through the chemical structure.

[0210] The term "hetero" used in the present invention refers to a compound or a group of compounds containing at least one heteroatom. The term "heteroatom" refers to an atom other than a carbon atom or a hydrogen atom, and includes, for example, B, Si, N, P, O, S and Se. Preferably, wherein the heteroatom includes a multivalent element such as N, O and S, or a monovalent element such as F, Cl, Br and I, but the present invention is not limited thereto.

[0211] The term "lower" used in the present invention is used to modify hydrocarbons, such as alkylene, and thus means that the corresponding hydrocarbons have 6 or fewer carbon atoms. 1-6 A straight or branched chain alkyl group is referred to by another name, such as a "lower alkyl" group.

[0212] The term "oxy group" used in the present invention refers to a secondary free radical of an oxygen atom (-O-).

[0213] The term "alkyl" or "alkane" refers to a fully saturated straight or branched chain non-aromatic hydrocarbon. Unless otherwise defined, a straight or branched chain alkyl group typically has 1 to about 20 carbon atoms, preferably 1 to about 10 carbon atoms. Straight and branched chain alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, pentyl, hexyl, amyl and octyl.

[0214] The term "alkenyl" or "alkene" refers to a straight or branched non-aromatic hydrocarbon containing at least one double bond. Unless otherwise defined, a straight or branched alkenyl group typically has 1 to about 20 carbon atoms, preferably 1 to about 10 carbon atoms.

[0215] The term "alkynyl" or "alkyne" refers to a straight or branched non-aromatic hydrocarbon containing at least one triple bond. Unless otherwise defined, a straight or branched alkynyl group typically has 1 to about 20 carbon atoms, preferably 1 to about 10 carbon atoms.

[0216] The term "cycloalkane" or "cycloalkyl" refers to a fully saturated cyclic hydrocarbon. "Cycloalkyl" includes monocyclic and polycyclic rings. Unless otherwise defined, monocyclic cycloalkyl groups typically have 3 to about 10 carbon atoms, more typically 3 to 8 carbon atoms. The rings other than the first ring of a polycyclic cycloalkyl can be selected from saturated rings, unsaturated rings, and aromatic rings. The cycloalkyl includes bicyclic molecules containing 1, 2, or 3 or more atoms shared between the two rings. The term "fused cycloalkyl" refers to a polycyclic cycloalkyl in which one ring shares two adjacent atoms with another ring. The rings other than the first ring of a fused polycyclic cycloalkyl can be selected from saturated rings, unsaturated rings, and aromatic rings.

[0217] The term "cycloalkyne" or "cycloalkynyl" refers to a cyclic hydrocarbon containing at least one triple bond, also known as a "strained alkyne". The "cycloalkynyl" includes monocyclic and polycyclic rings. Unless otherwise defined, monocyclic cycloalkynyl groups typically have 3 to about 10 carbon atoms, more typically 3 to 8 carbon atoms. The rings other than the first ring of the polycyclic cycloalkynyl group can be selected from saturated rings, unsaturated rings and aromatic rings. The cycloalkynyl group is a bicyclic molecule containing 1, 2 or 3 or more atoms shared between the two rings. The term "fused cycloalkynyl" refers to a polycyclic cycloalkynyl group in which one ring shares two adjacent atoms with another ring. The rings other than the first ring of the fused polycyclic cycloalkynyl group can be selected from saturated rings, unsaturated rings and aromatic rings.

[0218] The term "alkylene" used as a molecule itself or as part of another molecule refers to a divalent free radical derived from an alkane. For example, the group includes -CH2CH2- and -CH2CH2CH2CH2-, both containing 10 or less carbon atoms, but the present invention is not limited thereto. The term "lower alkylene" refers to a short alkylene group generally having 6 or less carbon atoms. Unless otherwise indicated, the term "alkylene" is intended to encompass the groups represented by "heteroalkylene" in the present invention.

[0219] The term "heteroalkylene" refers to a divalent free radical derived from a heteroalkyl group, and includes, for example, -CH2-CH2-S-CH2-CH2- and -CH2-S-CH2-CH2-NH-CH2-, but the present invention is not limited thereto. A heteroalkylene group may contain the same or different heteroatoms (including alkyleneoxy, alkylenedioxy, alkyleneamino, alkylenediamino, aminooxyalkylene, etc., but the present invention is not limited thereto) at each or all ends of its chain. In addition, the indication of the connection at both ends of the chain is independent of the arrangement of the groups of the formula. For example, the formula -C(O)2R'- refers to -C(O)2R'- and -R'(O)2C-.

[0220] The term "alkenylene" as a molecule itself or as part of another molecule refers to a divalent free radical derived from an alkene. For example, the group includes -CH=CH-, -CH2CH=CHCH2- and -CH=CH-CH=CH-, all of which have 10 or fewer carbon atoms, but the present invention is not limited thereto. Unless otherwise specified, the term "alkenylene" is intended to encompass heteroalkenylene in the present invention.

[0221] The term "alkynylene" as a molecule itself or as part of another molecule refers to a divalent free radical derived from an alkyne. For example, the group includes -C≡C-, -CH2C≡CCH2- and -C≡CC≡C-, all of which have 10 or fewer carbon atoms, but the present invention is not limited thereto. Unless otherwise indicated, the term "alkynylene" is intended to encompass heteroalkynylene groups in the present invention.

[0222] The term "cycloalkylene" as used by itself or as part of another molecule refers to a divalent radical derived from a cycloalkane. Unless otherwise specified, "cycloalkylene" is intended to encompass heterocycloalkylene groups within the present invention.

[0223] The term "alkylene" used in this specification, examples and claims is intended to encompass "unsubstituted alkylene" and "substituted alkylene". Wherein the latter refers to an alkylene group having a substituent replacing a hydrogen atom on one or more carbon atoms of a hydrocarbon. Unless otherwise expressly stated, the substituent may include, for example, halogen, hydroxyl, carbonyl (e.g., carboxyl, alkoxycarbonyl, formyl or acyl), thiocarbonyl (e.g., thioester, thioacetate or thioformate), alkoxy, phosphoryl, phosphoric acid group, phosphonate group, phosphinate group, amino group, amide group, amidine group, imine group, cyano group, nitro group, azido group, sulfhydryl group, alkylthio group, sulfuric acid group, sulfonic acid group, sulfamoyl group, sulfonamido group, sulfonyl group, heterocyclyl group, aralkyl group or aromatic or heteroaromatic group. When suitably substituted, it will be appreciated by those skilled in the art that the substituted residue on the hydrocarbon chain itself may be substituted. For example, the substituent of the substituted alkylidene group can include substituted and unsubstituted amino, azido, imino, amide, phosphoryl (including phosphonate / ester and phosphinate / ester), sulfonyl (including sulfate / ester, sulfonamido, sulfamoyl and sulfonate / ester) and silyl, and can also include ether, alkylthio, carbonyl (including ketone, aldehyde, carboxylate and ester),-CF ,-CN and its equivalent. Exemplary substituted alkyl is as described below.Cycloalkylene can further be replaced by alkyl, alkenyl, alkoxy, alkylthio, aminoalkyl, carbonyl substituted alkyl,-CF ,-CN and its equivalent.This content is also applicable to alkenylene and alkynylene.

[0224] When used with residues such as alkylene, alkenylene or alkynylene, the term "C x-y " is intended to encompass, for example, residues containing from x to y carbon atoms in their chain. For example, the term "C x-y "C0 alkylene" refers to a substituted or unsubstituted, linear or branched alkylene containing x to y carbon atoms in its chain. For example, it is intended to exemplarily include haloalkylene groups such as difluoromethylene, 2,2,2-trifluoroethylene, and the like. C0 alkylene refers to a covalent bond. The term "C0 2-y "Alkenylene" and "C 2-y "Alkyne" refers to a substituted or unsubstituted unsaturated aliphatic residue, to which the definitions of length and possible substitution as described in the definition of alkylene apply. However, this means that each contains at least one double or triple bond.

[0225] The term "click chemistry reaction" is used as a chemical concept introduced by K. Barry Sharpless of Scripps Research Institute to explain chemical reactions and complementary chemical functional groups designed to enable two molecules to quickly and stably form a covalent bond. The click chemistry reaction does not refer to a certain reaction, but to the concept of such a fast and stable reaction. In any embodiment, the click chemistry reaction must be modular, wide in scope, high in yield, produce only negligible by-products, stereospecific, physiologically stable, driven by a thermodynamic driving force (e.g., greater than 84 kJ / mol), and / or have a high atom economy. Some reactions are known to meet the following requirements:

[0226] (1) Huisgen 1,3-dipolar cycloaddition (e.g., including Cu(I)-catalyzed cycloaddition reactions, often referred to as "click reactions"; see Tornoe et al., Journal of Organic Chemistry (2002) 67:3057-3064): Copper or ruthenium is usually used as a catalyst;

[0227] [Schematic diagram of the Huisgen 1,3-dipolar cycloaddition]

[0228]

[0229] (2) Diels-Alder reactions, such as cycloadditions (e.g., strain-promoted cycloadditions (SPAAC)) including normal electron-demanding Diels-Alder reactions and anti-electron-demanding Diels-Alder reactions, but the present invention is not limited thereto);

[0230] [Schematic diagram of Diels-Alder reaction]

[0231]

[0232] [Example of Diels-Alder reaction; TCO and tetrazine]

[0233]

[0234] [Schematic diagram of strain-promoted cycloaddition]

[0235]

[0236] [Example of strain-promoted cycloaddition; azide and DBCO]

[0237]

[0238] (3) Nucleophilic addition to small strained rings such as epoxides and aziridines; (4) Nucleophilic addition to activated carbonyl groups;

[0239] (5) Addition to carbon-carbon double or triple bonds.

[0240] [Addition of thiols and olefins]

[0241]

[0242] The term "click chemistry functional group" used in the present invention refers to a functional group involved in a click chemistry reaction. For example, strained alkynes (e.g., cyclooctyne) correspond to click chemistry functional groups. In general, the click chemistry reaction requires at least two molecules, each of which contains click chemistry functional groups that complement each other. In this way, paired click chemistry functional groups that are reactive to each other are generally referred to as "partner click chemistry functional groups" in the present invention. For example, in the strain-promoted cycloaddition of cyclooctyne and azide, azide is a partner click chemistry functional group of cyclooctyne and other alkynes. Exemplary click chemistry functional groups used in the present invention include terminal alkynes, azides, strained alkynes, dienes, dienophiles, trans-cyclooctene, alkenes, thiols and tetrazines, but the present invention is not limited thereto. Other click chemistry functional groups are known to those skilled in the art.

[0243] The term "leaving group" used in the present invention is the same as the concept known to those skilled in the art (Advanced Organic Chemistry: reactions, mechanisms and structure, 4th edition, Jerry March, John Wiley and Sons, 1992, pp. 351-357), and refers to a chemical functional group connected to any reactant, which moves when the reactant is subjected to a substitution reaction (displacement reaction), such as a nucleophilic substitution reaction. A good leaving group refers to a leaving group that is easily moved during a nucleophilic substitution reaction. Exemplary good leaving groups include halogens (F, Cl, Br and I), toluenesulfonates, mesylates, trifluoromethanesulfonates, acetates, trifluoromethyl acetates, camphorsulfonates, 2-sulfylidenebenzo[d]thiazole-3(2H)-yl, N-hydrosuccinimide, N-aryl oxides, and aryl oxides substituted with one or more electron withdrawing groups (EWG), but the present invention is not limited thereto. For example, aryl oxides substituted with one or more electron withdrawing groups include 2-nitrobenzene oxide, 4-nitrobenzene oxide, 2,4-dinitrobenzene oxide, pentafluorobenzene oxide, 2-chloro-4-nitrobenzene oxide, 2,4-dichlorobenzene oxide, and 2,4,6-chlorobenzene oxide, and the electron withdrawing groups include, for example, halogen (F, Cl, Br or I), -NO2, -CN, -C(O)(C 1-6 alkyl), -C(O)(aryl), -C(O)O(C 1-6alkyl), -C(O)O(aryl), etc.

[0244] The term "interactome" used in the present invention refers to proteins or peptides that participate in protein-protein interactions when there is a protein-protein interaction (PPI) between proteins or between peptides. For example, a chaperone protein and its passenger protein are a mutual interactome. "Protein-protein interaction" refers to two or more protein or peptide molecules interacting to physically contact each other with a high degree of specificity. In this case, causal interactions include electromagnetic forces, hydrogen bonds, and hydrophobic interactions, but the present invention is not limited thereto.

[0245] The term "antibody interaction group" used in the present invention refers to the interaction group of antibodies including immunoglobulins. Exemplary antibody interaction groups are listed in Table 1. Among them, it is known that peptides (such as Fc-III, etc.) have binding activity to the Fc domain of immunoglobulins. In this case, these peptides are also referred to as "Fc interaction groups".

[0246]

[0247]

[0248]

[0249] According to the present invention, the term "antibody" refers to an immunoglobulin molecule or a fragment thereof. Immunoglobulins are generally well known and have the ability to specifically bind to a specific antigen. However, because the antibody according to the present invention is a concept that also encompasses its fragments, the antibody does not have to show the ability to bind to a specific antigen as in the case of an Fc fragment. In addition to naturally occurring immunoglobulins, the antibody is also intended to encompass all recombinant proteins, fusion proteins, chimeric proteins, human immunoglobulins, non-human animal immunoglobulins, etc., having the same or similar structure.

[0250] According to the present invention, the term "conjugate" refers to heterogeneous molecules made when conjugate partners come together to form covalent bonds. The covalent bonds can preferably be formed by click chemistry reactions. The term "conjugate partners" refers to each molecule intended to form a conjugate. In this case, for convenience, when a person who wishes to conjugate intends to connect a certain molecule to any other molecule, the specific molecule can generally be referred to as a "target molecule" or "target protein", and any other molecule can generally be referred to as a "load molecule" or "load moiety".

[0251] According to the present invention, the term "carrier moiety" refers to a part of the molecule constituting the conjugate, i.e., a molecule that acts to enhance the serum stability of the molecule connected thereto or to extend the half-life of the molecule. Molecules that can be used as carrier moieties are well known in the relevant art. Representative examples of carrier moieties include albumin, gelatin, elastin (including tropoelastin), polypeptides derived from elastin (α-elastin and elastin-like polypeptides (ELP)), gliadin, legumin, zein, soy protein (e.g., soy protein isolate (SPI)), lactoprotein, whey protein, bilirubin, etc., but the present invention is not limited thereto.

[0252] According to the present invention, the term "fluorescent moiety" is intended to encompass dyes or dye reagents for fluorescence. Molecules that can be used as dyes and dye reagents are well known in the relevant art. Representative examples of fluorescent moieties are listed in Table 2, but the present invention is not limited thereto (Immunotech-Coulter Corp. catalog, "Cytometry Monoclonal Reagent Guide", 8 / 95, p.3).

[0253] List of fluorescent parts

[0254]

[0255] According to the present invention, the term "drug moiety" refers to a molecule that has a therapeutic effect on any disease. The drug moiety according to the present invention includes drug moieties known to those of ordinary skill in the art that are effective for any disease. Generally, drug moieties with anticancer effects include DM1, DM3, DM4, abrin, ricin A, Pseudomonas exotoxin, cholera toxin, diphtheria toxin, tumor necrosis factor, α-interferon, β-interferon, nerve growth factor, platelet-derived growth factor, tissue plasminogen activator, cytokine, apoptosis inducer, anti-angiogenic agent, lymphokine, taxane, DNA-alkylating agent, anthracycline, Tubulysin analogs, duocarmycin analogs, auristatin E, auristatin F. maytansinoids, cytotoxic agents containing reactive polyethylene glycol residues, taxon, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenotoposide, vincristine, vinblastine, t. colchicine, doxorubicin, daunorubicin, dihydroxyanthraquinone, mitoxantrone, mithramycin, actinomycin D, 1-dihydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin, methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine, nitrogen mustard, thiotepa, chlorambucil, melphalan, carmustine , lomustine, cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, cisplatin, dactinomycin, bleomycin, anthramycin, calicheamicin, abiraterone, bendamustine, bortezomib, carboplatin, cabazitaxel, dasatinib, docetaxel, epirubicin, erlotinib, everolimus, gemcitabine, gefitinib, idarubicin, imatinib, hydroxyurea, lapatinib, leuprolide, melphalan, nedaplatin, nilotinib, oxaliplatin, pazopanib, pemetrexed, picoplatin, romidepsin, satraplatin, sorafenib, vemurafenib, sunitinib, teniposide, triplatin and vinorelbine, but the present invention is not limited thereto.

[0256] According to the present invention, the term "radioactive moiety" refers to a moiety that includes a radioactive isotope. Radioisotope labeling can be used for diagnostic imaging and radiation therapy. Representative radioactive moieties include 18 F. 11 C. 125 I. 123 I. 124 I. 131 I and 99m Tc, but the present invention is not limited thereto.

[0257] The term "pharmaceutically acceptable carrier" can be used in the sense of including an excipient, a diluent or an adjuvant. The carrier can be selected from, for example, the group consisting of lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum arabic, alginate / ester, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, polyvinylpyrrolidone, water, saline, buffers such as PBS, methyl hydroxybenzoate, propyl hydroxybenzoate, talcum, magnesium stearate and mineral oil. The carrier can include a filler, an anti-agglomerating agent, a lubricant, a wetting agent, a flavoring agent, an emulsifier, a preservative or a combination thereof.

[0258] The term "pharmaceutically acceptable salts" refers to salts in which the biological effectiveness and properties of the proteomes and compounds according to the invention are retained and which are not biologically or otherwise undesirable. In many cases, the proteomes and compounds of the invention can form acidic and / or basic salts in the presence of charged groups, such as charged amino and / or carboxyl groups, etc. Pharmaceutically acceptable acid addition salts can be prepared from inorganic and organic acids, while pharmaceutically acceptable base addition salts can be prepared from inorganic and organic bases.

[0259] The term "treatment" refers to an approach to obtaining a beneficial or desired clinical outcome. For purposes of the present invention, non-limiting examples of beneficial or desired clinical outcomes include alleviation of symptoms, reduction in the extent of the disease, stabilization of the disease state (i.e., not worsening), delay in progression or reduction in the rate of progression of the disease, improvement (partial or complete) or temporary remission and alleviation of the disease condition, and whether detectable. The treatment refers to all therapeutic treatments as well as prophylactic or preventative methods. The treatment includes treatment required for disorders to be prevented as well as disorders that have already occurred. "Remission" of a disease means that a range of disease conditions and / or undesirable clinical signs are reduced and / or the time course of disease progression is delayed or prolonged compared to when the disease is not being treated.

[0260] A "therapeutically effective amount" (or "effective amount") refers to an amount of an active ingredient, such as an agent according to the present invention, that is sufficient to achieve treatment when administered to a subject or patient. Thus, one skilled in the relevant art can readily determine what constitutes a therapeutically effective amount of a composition according to the present invention. In the context of vision treatment, a "therapeutically effective amount" is an amount that causes an objectively measured change in one or more parameters associated with the treatment of an ocular disease or condition, including an increase or decrease in the expression of one or more genes associated with an ocular disease or condition, induction of apoptosis or other cell death pathways, clinical improvement of symptoms, reduction in abnormal neovascularization or inflammation, and the like. Of course, the therapeutically effective amount may vary depending on the condition to be treated and the particular subject, the weight and age of the subject, the severity of the disease condition, the particular compound to be selected, the subsequent dosing regimen, dosing time adjustments, dosing mode, and the like, all of which can be readily determined by one skilled in the relevant art. In the context of combination therapy, it should be understood that what constitutes a therapeutically effective amount of an active ingredient may be different from what constitutes a therapeutically effective amount of an active ingredient administered for monotherapy (i.e., a treatment regimen using one chemical entity as the active ingredient).

[0261] "Subject" or "patient" refers to an animal in need of treatment that can be achieved by the molecules of the present invention. Animals to be treated according to the present invention include vertebrates. Particularly preferred examples of animals include mammals, e.g., cattle, dogs, horses, cats, sheep, pigs, and primates (including humans and non-human primates).

[0262] The terms "about" or "approximately" refer to an amount, level, value, amount, frequency, percentage, dimension, size, component, weight or length that varies by 30%, 25%, 20%, 25%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1% relative to a reference amount, level, value, amount, frequency, percentage, dimension, size, component, weight or length.

[0263] 1. Antibodies

[0264] It should be understood that the description of the present invention is intended to provide descriptions of the structures, scientific systems and physiological effects of the provided antibodies to help understand the present invention, and antibodies falling within the scope of the rights of the present invention are not intended to limit the description of the present invention.

[0265] Antibodies consist of two heavy chains and two light chains as known in the art. When antibodies are simply divided in terms of function, they are divided into fragments (Fab) containing the antigen-binding variable regions of the light chains and fragments of the crystallizable regions that are part of the heavy chains. Fab includes a paratope that binds to an antigen and refers to a region that allows an antibody to have specific binding activity to an antigen as known in the art. Since the Fc domain is a ligand for the Fc receptor (FcR) in cells, it plays an important role in inducing an immune response. The Fc domain also plays an important role in extending the half-life of antibodies by binding to the neonatal Fc receptor, allowing the antibody to be repeatedly internalized into cells.

[0266] From these facts, some desirable directionality of labeled antibodies can be inferred: (1), first, it is desirable to label the antibody at a position spaced apart from the paratope of the antibody. When labeling is performed at the paratope or at a position adjacent to the paratope, the binding affinity of the antibody to the antigen may be significantly reduced. (2) Secondly, it is desirable to label the antibody at a position spaced apart from the FcR recognition site including FcRn. When labeling is performed at the recognition site of the receptor or at a position adjacent to the recognition site, the immune response inducing function of the antibody may be reduced, or the half-life of the antibody may be shortened. Information on Fc domain binding activity motifs can be found in DeLano, WL (2000): Convergent Solutions to Binding at a Protein-Protein Interface; Science, 287 (5456), 1279-1283 and W. Lance Martin et al., (2001), Molecular Cell, Vol. 7, 867-877, April 2001.

[0267] In the present invention, when referring to the amino acid sequence of the Fc domain of an antibody, unless otherwise specified, the numbers in the sequence are numbered according to the EU numbering system. After studying the sequence of IgG, the EU numbering system has been widely used as a sequencing system for the Fc domain, such as Edelman GM et al., The covalent structure of an entire gammaGimmunoglobulin molecule; Proc. Natl. Acad. Sci. USA, May 1969; 63 (1): 78-85.

[0268] 1.1. Search for the desired marker site

[0269] The labeling site of an antibody can be designed in view of the criteria for a labeling site, namely, (1) a site that is spaced apart from the paratope; and (2) a site that is spaced apart from the FcR recognition site, including FcRn. Examples of amino acids used in bioconjugation reactions generally include lysine, cysteine, and tyrosine. Lysine 246 (Lys 246 ) and lysine 248 (Lys 248 ) are residues that meet all requirements and are therefore desirable labeling sites. 246 and Lys 248 The sequence of the Fc domain of residues is GPSVFLFPPKPKDTLMI, and Figure 1 The sequence and numbering in the sequence numbered according to the EU numbering system are shown in Figure 1 , SEQ ID NO:1).

[0270] In a specific embodiment, the antibody according to the present invention may include the sequence of SEQ ID NO: 1 or a derivative thereof. The antibody according to the present invention may also include a derivative of SEQ ID NO: 1 in which lysine 246 is substituted. In addition, the antibody according to the present invention may include a derivative of SEQ ID NO: 1 in which lysine 248 is substituted. In addition, the antibody according to the present invention may include a derivative of SEQ ID NO: 1 in which lysine 246 and lysine 248 are substituted.

[0271] The sequence of SEQ ID NO:1 or its derivatives includes sequences that are mutated within an acceptable range. In a specific embodiment, the mutated sequence may have a homology greater than or equal to about 90%, about 85%, about 80%, about 75% or about 70% relative to SEQ ID NO:1 or its derivatives. In the specific embodiments specified below, it should be understood that the derivatives of SEQ ID NO:1 represented by Formula 7-1 to Formula 7-3, Formula 8-1 to Formula 8-3 and Formula 10-1 to Formula 10-3 also include sequences that are mutated within an acceptable range.

[0272] Figure 2 The positions of lysine residues in the Fc domain including lysine 246 and lysine 248 are shown.

[0273] 2. Connector (R1'-L1)

[0274] The present invention discloses novel compounds that can be used to label antibodies. For convenience, such compounds are referred to herein as linkers and are represented by the symbol "R1'-L1".

[0275] The present invention provides a compound having a structure of the following formula 1:

[0276] [Formula 1]

[0277]

[0278] Wherein, R1' is the first chemical functional group,

[0279] D1 is any alkylene, alkenylene or alkynylene group,

[0280] X1 is an element with a higher electronegativity than carbon.

[0281] D2 is any alkylene, alkenylene or alkynylene group,

[0282] X2 is an element more electronegative than carbon, and

[0283] R2' is a second chemical functional group.

[0284] In Formula 1, the carbonyl group connected to D1 is referred to as a first carbonyl group. In addition, the carbonyl group connected to D2 is referred to as a second carbonyl group.

[0285] In a specific embodiment, R1' may include a click chemistry functional group. The click chemistry functional group may also include one or more selected from alkynes, azides, strained alkynes, dienes, dienophiles, alkenes, thiols and tetrazines. In addition, the click chemistry functional group may be selected from azides or strained alkynes. In addition, the click chemistry functional group may be selected from azides or dibenzocyclooctyne-amine. In addition, the click chemistry functional group may be selected from dienes or dienophiles. In addition, the click chemistry functional group may be selected from tetrazines or norbornenes. Alternatively, the click chemistry functional group may be selected from tetrazines or trans-cyclooctene. In addition, R1' may include two or more click chemistry functional groups.

[0286] In other embodiments, R1' may include a carrier moiety, a fluorescent moiety, a drug moiety or a radioactive moiety. R1' may also include a drug moiety. In addition, R1' may include a VC linker. In other embodiments, R1' may include an antibody or an analog thereof, wherein the antibody or an analog thereof includes a paratope.

[0287] In a specific embodiment, D1 may include a covalent bond, a C 1-4 Alkylene, C 2-4 Alkenylene, C 2-4 Alkynylidene and C 3-8Any one of cycloalkylene. D1 can also be -CH2OCH2-. In addition, D1 can be a covalent bond. When D1 is a covalent bond, R1' and the carbon of the first carbonyl group are directly connected to each other. Hereinafter, when describing the structure of a compound, all alkylene, alkenylene, alkynylene and cycloalkylene are intended to include heteroalkylene, heteroalkenylene, heteroalkynylene and heterocycloalkylene, respectively, and their connotations are also specified in the "Definition" section.

[0288] In a specific embodiment, X1 may be NR1, S or O, wherein R1 may be H, halogen or substituted or unsubstituted C 1-3 X1 may also be S. X1 may attract electrons from the carbon of the first carbonyl group to activate the first carbonyl group.

[0289] In a specific embodiment, D2 may include a 1-7 Alkylene, C 2-7 Alkenylene, C 2-7 Alkynylidene and C 3-8 D2 can also be C 1-2 Alkylene. In addition, D2 may be a methylene group.

[0290] In a specific embodiment, X2 may be NR1, S or O, wherein R1 may be H, halogen or substituted or unsubstituted C 1-3 X2 can also be O. X2 can attract electrons from the carbon of the second carbonyl group to activate the second carbonyl group.

[0291] In a specific embodiment, R2' can be halogen, N-succinimide, p-nitrophenyl or pentafluorophenyl.

[0292] In a specific embodiment, R2' and X2 together can form a leaving group. For example, X2 can be O, and R2' can be N-succinimide, p-nitrophenyl or pentafluorophenyl. R2' can also be N-succinimide. When a good leaving group is attached to the second carbonyl group, the reactivity of the second carbonyl group can be enhanced. For example, it is known that N-hydroxysuccinimide esters (NHS esters) show very high reactivity.

[0293] The present invention provides a compound having a structure of the following formula 1-2:

[0294] [Formula 1-2]

[0295]

[0296] Wherein, R1' is the first chemical functional group,

[0297] D1 is any alkylene, alkenylene or alkynylene group, and

[0298] R2' is a second chemical functional group.

[0299] In a specific embodiment, R1' may include a click chemistry functional group. The click chemistry functional group may also include one or more selected from alkynes, azides, strained alkynes, dienes, dienophiles, alkenes, thiols and tetrazines. In addition, the click chemistry functional group may be selected from azides or strained alkynes. In addition, the click chemistry functional group may be selected from azides or dibenzocyclooctyne-amine. In addition, the click chemistry functional group may be selected from dienes or dienophiles. In addition, the click chemistry functional group may be selected from tetrazines or norbornenes. Alternatively, the click chemistry functional group may be selected from tetrazines or trans-cyclooctene. In addition, R1' may include two or more click chemistry functional groups.

[0300] In other embodiments, R1' may include a carrier moiety, a fluorescent moiety, a drug moiety or a radioactive moiety. R1' may also include a drug moiety. In addition, R1' may include a VC linker. In other embodiments, R1' may include an antibody or an analog thereof, wherein the antibody or an analog thereof includes a paratope.

[0301] In a specific embodiment, D1 may include a 1-7 Alkylene, C 2-7 Alkenylene, C 2-7 Alkynylidene and C 3-8 Any one of cycloalkylene. D1 can also be C 1-2 In addition, D1 may be a methylene group.

[0302] In a specific embodiment, R2' and O together can form a leaving group. In this case, R2' can be N-succinimide, p-nitrophenyl or pentafluorophenyl. In addition, R2' can be N-succinimide.

[0303] In a specific embodiment, the compound represented by Formula 1-2 may have a structure of the following Formula 1-3:

[0304] [Formula 1-3]

[0305]

[0306] 2.1. Linkers including the first click chemistry functional group (H1-L1)

[0307] The present invention provides a compound having a structure of the following formula 2:

[0308] [Formula 2]

[0309]

[0310] Among them, H1 is the first click chemistry functional group,

[0311] D1 is any alkylene, alkenylene or alkynylene group,

[0312] X1 is an element with a higher electronegativity than carbon.

[0313] D2 is any alkylene, alkenylene or alkynylene group,

[0314] X2 is an element more electronegative than carbon, and

[0315] R2' is a second chemical functional group. A linker having a structure of Formula 2 is referred to herein as a "linker comprising a first click chemistry functional group", which is indicated by the symbol "H1-L1".

[0316] In Formula 2, the carbonyl group connected to D1 is referred to as a first carbonyl group. In addition, the carbonyl group connected to D2 is referred to as a second carbonyl group.

[0317] In a specific embodiment, H1 may include any one selected from alkynes, azides, strained alkynes, dienes, dienophiles, olefins, thiols, and tetrazines. In addition, H1 may be an azide or a strained alkyne. In addition, H1 may be an azide or a dibenzocyclooctyne-amine. In addition, H1 may be a diene or a dienophile. In addition, H1 may be a tetrazine or a norbornene. Alternatively, H1 may be a tetrazine or a trans-cyclooctene.

[0318] In a specific embodiment, D1 may include a covalent bond, a C 1-4 Alkylene, C 2-4 Alkenylene, C 2-4 Alkynylidene and C 3-8 Any one of cycloalkylene. In addition, D1 can be -CH2OCH2-. In addition, D1 can be a covalent bond.

[0319] In a specific embodiment, X1 may be NR1, S or O, wherein R1 may be H, halogen or substituted or unsubstituted C 1-3 In addition, X1 may be S.

[0320] In a specific embodiment, D2 may include a 1-7 Alkylene, C 2-7 Alkenylene, C 2-7 Alkynylidene and C 3-8 Any one of cycloalkylene. D2 can also be C 1-2 In addition, D2 may be a methylene group.

[0321] In a specific embodiment, X2 may be NR1, S or O, wherein R1 may be H, halogen or substituted or unsubstituted C 1-3In addition, X2 may be O.

[0322] In a specific embodiment, R2' can be halogen, N-succinimide, p-nitrophenyl or pentafluorophenyl.

[0323] In a specific embodiment, R2' and X2 together can form a leaving group. For example, X2 can be O, and R2' can be N-succinimide, p-nitrophenyl or pentafluorophenyl. R2' can also be N-succinimide.

[0324] In a specific embodiment, the compound represented by Formula 2 may have a structure selected from any one of the following Formulas 2-1 to 2-3:

[0325] [Formula 2-1]

[0326]

[0327] [Formula 2-2]

[0328]

[0329] [Formula 2-3]

[0330]

[0331] 2.2. Due to the difference in reactivity between the first carbonyl group and the second carbonyl group, the position where the substitution reaction occurs can be specifically determined

[0332] When designing linkers of Formula 1 and Formula 2 and sub-instances thereof, the site of substitution reaction can be indicated based on the design of X1, X2, and R2'.

[0333] The role of the linker disclosed in the present invention is to transfer R1' to the target molecule through a substitution reaction between the activated first and / or second carbonyl groups. Such a substitution reaction is schematically shown in Scheme 1 below.

[0334] [Solution 1]

[0335]

[0336] In Scheme 1, the target molecule is designated as "Nu:" for convenience. Nu: acts as a nucleophile because it has an unshared electron pair, causing a nucleophilic acyl substitution reaction with the first carbonyl group and / or the second carbonyl group to form a bond with the linker. In the nucleophilic acyl substitution reaction, the reactivity of the carbonyl group may be determined by the basicity of the leaving group. Thus, it is known that the reactivity of the carbonyl group increases in the order of carboxylate, amide, carboxylic acid, ester, thioester, and acyl phosphate. It is also known that carbonyl groups such as NHS esters and the like have high reactivity because the carbonyl group forms a very stable leaving group.

[0337] In a specific embodiment, X1 and X2 can be elements with higher electronegativity than carbon. X1 and X2 can also be NR1, S or O, wherein R1 can be H, halogen or substituted or unsubstituted C 1-3 Alkylene. The carbonyl group can be activated because X1 and X2 together with the residue to which they are attached can form a leaving group.

[0338] In this case, activation of the carbonyl group may optionally allow Nu to: i) preferentially react with the first carbonyl group or ii) preferentially react with the second carbonyl group. This reaction tendency may be determined based on the difference in reactivity between the first carbonyl group and the second carbonyl group. For example, when the basicity of the leaving group including X1 is lower than the basicity of the leaving group including X2, the first carbonyl group may react first. In another embodiment, when the basicity of the leaving group including X2 is lower than the basicity of the leaving group including X1, the second carbonyl group may react first.

[0339] In a preferred embodiment, the reactivity of the second carbonyl group in the joint according to the present invention is preferably higher than the reactivity of the first carbonyl group. According to the present invention, the selective reaction is achieved by allowing X2-R2' to be connected to the second carbonyl group to form a good leaving group, and the first carbonyl group is designed to show mild reactivity amides, thioesters, esters, etc. (showing moderate reactivity). For example, in the case of a joint of formula 1-3, the second carbonyl group can be an NHS ester, so that it reacts faster than the first carbonyl group (thioester).

[0340] The prior art disclosed in publication number US2018 / 0141976 A1 and WO 2018 / 199337 A1 is similar to the present invention in terms of the formation of reagents for transferring the first chemical functional group to the antibody (see Section 4 below), but is different from the present invention in that the cross-linking agent includes two NHS esters. The two carbonyl groups of the cross-linking agent have the same reactivity, and it is difficult to prepare the desired reagent for the transfer of the chemical functional group in high yield. Due to the high reactivity of NHS esters, the cross-linking agent also has a high probability of reacting with two SSFIs. According to the present invention, these problems have been solved by designing the first carbonyl group as a thioester with mild reactivity, etc.

[0341] 3. Site-specific Antibody Interactions (SSAI)

[0342] According to the present invention, novel peptides for bringing a molecule to be labeled into close contact with a specific site of an antibody are disclosed. For convenience, such peptides are referred to herein as site-specific antibody interactors, indicated by the symbol "SSAI".

[0343] The SSAI provided according to the present invention may have binding activity to a specific site of an antibody.

[0344] In a specific embodiment, the SSAI may have binding activity to the Fab domain of the antibody. In this case, the SSAI may preferably have binding activity to a site spaced apart from the paratope of the antibody.

[0345] In a specific embodiment, the SSAI may have binding activity to the Fc domain of the antibody. In this case, the SSAI may preferably have binding activity to the FcRn binding site of the antibody or to a site spaced apart from antibody residues that affect the FcRn binding site of the antibody.

[0346] 3.1. Site-specific Fc interactome (SSFI)

[0347] In the SSAI, peptides having specific binding activity to the Fc domain are referred to herein as "site-specific Fc interactome", which is represented by the symbol "SSFI".

[0348] In a specific embodiment, the SSFI may include an amino acid sequence represented by the following Formula 3:

[0349] [Formula 3]

[0350] (Xaa)2-H-Xa1-G-Xa2-LV-Xa3 (SEQ ID NO: 3)

[0351] wherein each Xaa is independently any amino acid except cysteine,

[0352] H is histidine, G is glycine, Xa2 is glutamic acid or asparagine, L is leucine, V is valine, Xa3 is selected from tryptophan, naphthylalanine and phenylalanine, and

[0353] Xa1 is Where D3 is a covalent bond or C 1-3 Alkylene, and X3 is NH2, OH or SH. As an analog of the sequence AWHLGELVW (SEQ ID NO: 2), this sequence is a sequence found to have binding activity to the Fc domain in the articles "Dias, RLA et al. (2006), Protein Ligand Design: From Phage Display to Synthetic Protein EpitopeMimetics in Human Antibody Fc-Binding Peptidomimetics; Journal of the American Chemical Society, 128 (8), 2726-2732 and DeLano, WL et al., Convergent solutions to binding at a protein-protein interface; Science 2000, 287, 1279-1283". The Fc binding activity motif according to the present invention has the following characteristics: 1) First, the key residues having Fc binding activity are specified. 2) The motif is also designed to allow nucleophilic substitution reaction with the linker by changing the 4th leucine of SEQ ID NO: 2 to Xa1 having a free electron pair. (X) n It means that it is composed of n Xs, and (X) n-m It means that it is composed of n or more and m or less X. Hereinafter, the carbon bonded to D3 is referred to as "β carbon (β-carbon)".

[0354] The amino acid sequence of Formula 3 may also have a structure represented by the following Formula 3-1:

[0355] [Formula 3-1]

[0356] AWH-Xa1-G-Xa2-LV-Xa3 (SEQ ID NO: 4)

[0357] wherein A is alanine, H is histidine, G is glycine, Xa2 is glutamic acid or asparagine, L is leucine, V is valine, and Xa3 is selected from tryptophan, naphthylalanine and phenylalanine, and

[0358] Xa1 is Where D3 is a covalent bond or C 1-3 Alkylene, and X3 is NH2, OH or SH. In Formula 3 and Formula 3-1, X3 can be NH2. Alternatively, Xa2 can be glutamic acid. Alternatively, Xa3 can be tryptophan.

[0359] When the peptide comprising each amino acid sequence of Formula 3 and its sub-examples is in the form of a cyclic peptide in which internal residues are linked to each other, the peptide is known to have better binding activity. The present invention provides a cyclic peptide comprising the amino acid sequence of Formula 3.

[0360] The present invention provides a cyclic peptide having the structure of the following formula 4-1:

[0361] [Formula 4-1]

[0362] L P-(Xaa)2-H-Xa1-G-Xa2-LV-Xa3- D P (SEQ ID NO: 5)

[0363] Among them, the N-terminal L P and D P forms a D-proline-L-proline template,

[0364] Each Xaa is independently any amino acid except cysteine, H is histidine, G is glycine, Xa2 is glutamic acid or asparagine, L is leucine, V is valine, Xa3 is selected from tryptophan, naphthylalanine and phenylalanine, and

[0365] Xa1 is Where D3 is a covalent bond or C 1-3 Alkylene, X3 is NH2, OH or SH.

[0366] In a specific embodiment, X3 may be NH2. In a specific embodiment, (X)2 may be AW. In a specific embodiment, Xa2 may be glutamic acid. In a specific embodiment, Xa3 may be tryptophan.

[0367] The present invention provides a cyclic peptide having the structure of the following formula 4-2:

[0368] [Formula 4-2]

[0369] (Xaa) 1-3 -C-(Xaa)2-H-Xa1-G-Xa2-LV-Xa3-C-(Xaa) 1-3 (SEQ ID NO: 6)

[0370] wherein each Xaa is independently any amino acid except cysteine,

[0371] C is cysteine, H is histidine, G is glycine, Xa2 is glutamic acid or asparagine, L is leucine, V is valine, Xa3 is selected from tryptophan, naphthylalanine and phenylalanine, and

[0372] Xa1 is Where D3 is a covalent bond or C 1-3 Alkylene, X3 is NH2, OH or SH.

[0373] In a specific embodiment, the peptide may consist of more than 13 and less than 17 amino acid residues.

[0374] In a specific embodiment, the cysteine ​​located 2 to 4 amino acids from the N-terminus and the cysteine ​​located 2 to 4 amino acids from the C-terminus may be optionally linked to each other.

[0375] In a specific embodiment, X3 may be NH2. In a specific embodiment, (X)2 may be AW. In a specific embodiment, Xa2 may be glutamic acid. In a specific embodiment, Xa3 may be tryptophan.

[0376] In a specific embodiment, the N-terminal (X) 1-3 One of the residues and the C-terminus (X) 1-3 In one exemplary embodiment, the peptide of Formula 4-2 may have a structure of the following Formula 4-3:

[0377] [Formula 4-3]

[0378] L PDc-(Xaa)2-H-Xa1-G-Xa2-LV-Xa3-CT- D P (SEQ ID NO: 7)

[0379] Among them, the N-terminal L P and D P can form a D-proline-L-proline template.

[0380] In another exemplary embodiment, the peptide of Formula 4-2 may have the structure of the following Formula 4-4:

[0381] [Formula 4-4]

[0382] CDC-(Xaa)2-H-Xa1-G-Xa2-LV-Xa3-CTC (SEQ ID NO: 8)

[0383] Among them, the N-terminal cysteine ​​and the C-terminal cysteine ​​may be linked to each other.

[0384] The present invention provides a cyclic peptide having the structure of the following formula 4-5:

[0385] [Formula 4-5]

[0386] DC-(Xaa)2-H-Xa1-G-Xa2-LV-Xa3-CT (SEQ ID NO: 9)

[0387] Among them, D is aspartic acid, T is threonine,

[0388] Each Xaa is independently any amino acid except cysteine,

[0389] C is cysteine, H is histidine, G is glycine, Xa2 is glutamic acid or asparagine, L is leucine, V is valine, Xa3 is selected from tryptophan, naphthylalanine and phenylalanine, and

[0390] Xa1 is Where D3 is a covalent bond or C 1-3 alkylene, and X3 is NH2, OH or SH.

[0391] In a specific embodiment, the peptide may consist of more than 13 and less than 17 amino acid residues.

[0392] In a specific embodiment, the cysteine ​​located 2 amino acids from the N-terminus and the cysteine ​​located 2 amino acids from the C-terminus can be optionally connected to each other. In a specific embodiment, X3 can be NH2. In a specific embodiment, (X)2 can be AW. In a specific embodiment, Xa2 can be glutamic acid. In a specific embodiment, Xa3 can be tryptophan.

[0393] In a specific embodiment, Formula 4-5 may be the same as the following Formula 4-6:

[0394] [Formula 4-6] (SEQ ID NO: 10)

[0395] DCAWH-Xa1-GELVWCT (SEQ ID NO: 10)

[0396] Among them, A is alanine and E is glutamic acid.

[0397] The peptides having the structures of Formula 4-1 to Formula 4-6 may have binding activity to an antibody. In addition, the peptides may have binding activity to immunoglobulin G (IgG). In addition, the peptides may have binding activity to the Fc domain of an antibody.

[0398] In a specific embodiment, the N-terminus of the SSFI according to the present invention may be succinylated. In a specific embodiment, the SSFI according to the present invention may be succinylated at its N-terminus (X) 1-3 In other embodiments, the SSFI according to the present invention may have a polar amino acid residue at its C-terminus (X) 1-3 The polar amino acid residues include acidic amino acids and basic amino acids. The polar amino acid residues may also include glutamic acid or aspartic acid.

[0399] 3.2. The site-specific Fc interactome according to the present invention can be arranged with the Fc domain of an antibody having a specific topology.

[0400] Herein, the compound of formula 4-6 is provided as an example to help understand the present invention, but the scope of the present invention is not limited thereto. It should be noted that the following description is also applicable to the compounds of formula 3, formula 3-1 and formula 4-1 to formula 4-6, and for convenience, the compound of formula 4-6 is provided only as an example.

[0401] As described above, the SSFI according to the present invention has binding activity to the Fc domain of the antibody. In this case, due to the interaction between the amino acid residues, the SSFI can be arranged together with the Fc domain having a specific topological structure. Representative interactions between the SSFI sequence according to the present invention and the Fc domain include: (1) a salt bond between SSFI and histidine 433 in the Fc domain, (2) a hydrogen bond between SSFI and asparagine 434, (3) a salt bond between SSFI and glutamic acid 380, (4) a salt bond between SSFI and arginine 255, etc. These interactions and specific topological structures formed thereby can be determined from research results known in the art (see DeLano, WL et al., Convergent solutions to binding at a protein-protein interface; Science 2000, 287, 1279-1283).

[0402] When designing the SSFI according to the present invention, it is important that the SSFI forms a stable topological structure with the Fc domain. This is because the reagent for transferring the first chemical functional group to the antibody according to the present invention and the labeling process using the reagent are designed based on the topological structure between the SSFI and the Fc domain found in the study (see Sections 5.2, 5.3 and 5.4 below). When the interaction between the SSFI and the Fc domain becomes unstable during the design process of the SSFI to interfere with the topological structure between its molecules, this is disadvantageous because the interaction may have a negative impact on the labeling process.

[0403] One embodiment of the design principle will be described with reference to exemplary compounds. SSFI represented by Formula 4-6 has the following structure:

[0404] [Formula 4-6]

[0405] DCAWH-Xal-GELVWCT (SEQ ID NO: 10).

[0406] The results of simulating the topological structure between the SSFI and Fc domains shown in SEQ ID NO: 10 based on data from the literature are shown in Figure 3 and Figure 4 In this case, the histidine residue at position 5 in the SSFI forms a salt bond with glutamic acid 380 in the Fc domain, indicating that the salt bond has a significant effect on the topological structure between the SSFI and Fc domain (see DeLano, WL et al., Convergent solutions to binding at a protein-protein interface; Science 2000, 287, 1279-1283). Figure 4 ). Therefore, it is expected that the histidine residue and its position will not be changed during the design process of SSFI (see "H" immediately following the residue Xa1 in Formula 3, Formula 3-1, and Formula 4-1 to Formula 4-6). In addition, it was confirmed that glutamic acid 8 is a residue showing electronegativity, and thus forms a salt bond with arginine 255 showing electronegativity in the Fc domain, thereby significantly affecting the topological structure between SSFI and the Fc domain (see Figure 4 ). Therefore, the corresponding residue is preferably an acidic amino acid that may correspond to glutamic acid and may be replaced by asparagine (see residue Xa2 in Formula 3, Formula 3-1, and Formula 4-1 to Formula 4-6). When an amino acid residue is substituted by other amino acid residues or by any functional group, this may affect the intermolecular interaction and thus the topological structure between the SSFI and the Fc domain.

[0407] In addition, the glycine at position 7 in the sequence of SEQ ID NO: 10 is a small amino acid required to form the bent structure of the SSFI. Therefore, it is desirable not to change the glycine residue and its position during the design of SSFI (see "G" between residues Xa1 and Xa2 in Formula 3, Formula 3-1, and Formula 4-1 to Formula 4-6).

[0408] Figure 5The topology between the lysine residues of the Fc domain and the SSFI is shown. Based on the topology, it can be seen that the lysine residues of the Fc domain closest to residue Xa1 are lysine 246 and lysine 248 ( Figure 5 ).

[0409] The distance between the amine group of lysine 246 and the β-carbon of Xa1 was measured. The results confirmed that when the bonds constituting the lysine side chain rotate, the minimum distance measured is about (hereinafter referred to as "D 246,min ”), and the maximum distance measured is approximately (hereinafter referred to as "D 248,max ”)( Figure 6 ).

[0410] The distance between the amine group of lysine 248 and the beta carbon of Xa1 was measured. The results confirmed that when the bonds constituting the lysine side chain rotate, the minimum distance measured is about (hereinafter referred to as "D 248,min ”), and the maximum distance measured is approximately (hereinafter referred to as "D 248,max ”)( Figure 7 ).

[0411] As described in Section 5.3 below, distance relationships can be an important consideration in the design of the linker and the SSFI as well as reagents for transferring the first chemical functionality to the antibody.

[0412] 4. Reagent for transferring the first chemical functional group to the antibody; conjugate of R1'-L1 and SSAI (R1'-L2-SSAI)

[0413] According to the present invention, a reagent for transferring a first chemical functional group to an antibody is disclosed. This compound is shown herein by the symbol "R1'-L2-SSAI". According to its structure, the compound is also referred to as a conjugate of R1'-L1 and SSAI (R1'-L2-SSAI conjugate).

[0414] The present invention provides R1'-L2-SSAI having the structure of the following formula 5:

[0415] [Formula 5]

[0416]

[0417] Wherein, R1' is the first chemical functional group,

[0418] D1 is any alkylene, alkenylene or alkynylene group,

[0419] X1 is an element with a higher electronegativity than carbon.

[0420] D2 is any alkylene, alkenylene or alkynylene group,

[0421] D3 is a covalent bond or C 1-3 Alkylene,

[0422] X3 is NH, O or S, and

[0423] SSAI is the site-specific antibody interactome.

[0424] In a specific embodiment, R1' may include a click chemistry functional group. In addition, the click chemistry functional group may include one or more selected from alkynes, azides, strained alkynes, dienes, dienophiles, alkenes, thiols and tetrazines. In addition, the click chemistry functional group may be selected from azides or strained alkynes. In addition, the click chemistry functional group may be selected from azides or dibenzocyclooctyne-amine. In addition, the click chemistry functional group may be selected from dienes or dienophiles. In addition, the click chemistry functional group may be selected from tetrazines or norbornenes. Alternatively, the click chemistry functional group may be selected from tetrazines or trans-cyclooctene. In addition, R1' may include two or more click chemistry functional groups.

[0425] In other embodiments, R1' may include a carrier moiety, a fluorescent moiety, a drug moiety or a radioactive moiety. R1' may also include a drug moiety. In addition, R1' may include a VC linker. In other embodiments, R1' may include an antibody or an analog thereof, wherein the antibody or an analog thereof includes a paratope.

[0426] In a specific embodiment, D1 may include a covalent bond, a C 1-4 Alkylene, C 2-4 Alkenylene, C 2-4 Alkynylidene and C 3-8 Any one of cycloalkylene. D1 can also be -CH2OCH2-. In addition, D1 can be a covalent bond.

[0427] In a specific embodiment, X1 may be NR1, S or O, wherein R1 may be H, halogen or substituted or unsubstituted C 1-3 X1 can also be S.

[0428] In a specific embodiment, D2 may include a 1-7 Alkylene, C 2-7 Alkenylene, C 2-7 Alkynylidene and C 3-8 Any of cycloalkylene. D2 can also be C 1-2 In addition, D2 may be a methylene group.

[0429] In a specific embodiment, X3 may be NH.

[0430] In a specific embodiment, the SSAI may be a peptide sequence that has binding activity to Fab. In other specific embodiments, the SSAI may be a peptide sequence that has binding activity to Fc domain.

[0431] When SSAI in Formula 5 is SSFI, it is shown by the symbol "R1'-L2-SSFI". R1'-L2-SSFI according to the present invention is produced by a nucleophilic substitution reaction of Xa1 of SSFI according to the present invention with the second carbonyl group of R1'-L1 (see Section 4-2 and Scheme 2 below). Therefore, R1'-L2-SSFI according to the present invention includes those in which Xa1 of SSFI in Formula 3, Formula 3-1, and Formula 4-1 to Formula 4-6 is substituted with (Xa1)', but the present invention is not limited to the following exemplary embodiments thereof.

[0432] The present invention provides an R1'-L2-SSFI comprising an amino acid sequence of the following formula 5-1:

[0433] [Formula 5-1]

[0434] (Xaa)2-H-(Xa1)'-G-Xa2-LV-Xa3 (SEQ ID NO:11)

[0435] wherein each Xaa is independently any amino acid except cysteine,

[0436] H is histidine, G is glycine, Xa2 is glutamic acid or asparagine, L is leucine, V is valine, Xa3 is selected from tryptophan, naphthylalanine and phenylalanine, and

[0437] (Xa1)' is

[0438] Wherein, R1' is the first chemical functional group,

[0439] D1 is any alkylene, alkenylene or alkynylene group,

[0440] X1 is an element with a higher electronegativity than carbon.

[0441] D2 is any alkylene, alkenylene or alkynylene group,

[0442] D3 is a covalent bond or C 1-3 Alkylene, and

[0443] X3 is NH, O or S.

[0444] In a specific embodiment, R1' may include a click chemistry functional group. The click chemistry functional group may also include one or more selected from alkynes, azides, strained alkynes, dienes, dienophiles, alkenes, thiols and tetrazines. In addition, the click chemistry functional group may be selected from azides or strained alkynes. In addition, the click chemistry functional group may be selected from azides or dibenzocyclooctyne-amine. In addition, the click chemistry functional group may be selected from dienes or dienophiles. In addition, the click chemistry functional group may be selected from tetrazines or norbornenes. Alternatively, the click chemistry functional group may be selected from tetrazines or trans-cyclooctene. In addition, R1' may include two or more click chemistry functional groups.

[0445] In other embodiments, R1' may include a carrier moiety, a fluorescent moiety, or a drug moiety. R1' may also include a VC linker. In addition, R1' may include a radioactive moiety. R1' may also include a drug moiety. In other embodiments, R1' may include an antibody or an analog thereof, wherein the antibody or an analog thereof includes a paratope.

[0446] In a specific embodiment, D1 may include a covalent bond, a C 1-4 Alkylene, C 2-4 Alkenylene, C 2-4 Alkynylidene and C 3-8 Any one of cycloalkylene. In addition, D1 can be -CH2OCH2-. In addition, D1 can be a covalent bond.

[0447] In a specific embodiment, X1 may be NR1, S or O, wherein R1 may be H, halogen or substituted or unsubstituted C 1-3 Alkylene.

[0448] In a specific embodiment, D2 may include a 1-7 Alkylene, C 2-7 Alkenylene, C 2-7 Alkynylidene and C 3-8 Any of cycloalkylene. D2 can also be C 1-2 In addition, D2 may be a methylene group.

[0449] In a specific embodiment, X3 may be NH.

[0450] In a specific embodiment, (X)2 may be AW. In a specific embodiment, Xa2 may be glutamic acid. In a specific embodiment, Xa3 may be tryptophan.

[0451] The present invention provides R1'-L2-SSFI having the structure of the following formula 5-2:

[0452] [Formula 5-2]

[0453] (Xaa) 1-3 -C-(Xaa)2-H-(Xa1)′-G-Xa2-LV-Xa3-C-(Xaa) 1-3 (SEQ ID NO:12)

[0454] wherein each Xaa is independently any amino acid except cysteine,

[0455] C is cysteine, H is histidine, G is glycine, Xa2 is glutamic acid or asparagine, L is leucine, V is valine, Xa3 is selected from tryptophan, naphthylalanine and phenylalanine, and

[0456] (Xa1)' is

[0457] Wherein, R1' is the first chemical functional group,

[0458] D1 is any alkylene, alkenylene or alkynylene group,

[0459] X1 is an element with a higher electronegativity than carbon.

[0460] D2 is any alkylene, alkenylene or alkynylene group,

[0461] D3 is a covalent bond or C 1-3 Alkylene, and

[0462] X3 is NH, O or S.

[0463] In a specific embodiment, R1' may include a click chemistry functional group. The click chemistry functional group may also include one or more selected from alkynes, azides, strained alkynes, dienes, dienophiles, alkenes, thiols and tetrazines. In addition, the click chemistry functional group may be selected from azides or strained alkynes. In addition, the click chemistry functional group may be selected from azides or dibenzocyclooctyne-amine. In addition, the click chemistry functional group may be selected from dienes or dienophiles. In addition, the click chemistry functional group may be selected from tetrazines or norbornenes. Alternatively, the click chemistry functional group may be selected from tetrazines or trans-cyclooctene. In addition, R1' may include two or more click chemistry functional groups.

[0464] In other embodiments, R1' may include a carrier moiety, a fluorescent moiety, a drug moiety or a radioactive moiety. R1' may also include a drug moiety. In addition, R1' may include a VC linker. In other embodiments, R1' may include an antibody or an analog thereof, wherein the antibody or an analog thereof includes a paratope.

[0465] In a specific embodiment, D1 may include a covalent bond, a C 1-4 Alkylene, C2-4 Alkenylene, C 2-4 Alkynylidene and C 3-8 Any one of cycloalkylene. D1 can also be -CH2OCH2-. In addition, D1 can be a covalent bond.

[0466] In a specific embodiment, X1 may be NR1, S or O, wherein R1 may be H, halogen or substituted or unsubstituted C 1-3 Alkylene.

[0467] In a specific embodiment, D2 may include a 1-7 Alkylene, C 2-7 Alkenylene, C 2-7 Alkynylidene and C 3-8 Any one of cycloalkylene. D2 can also be C 1-2 In addition, D2 may be a methylene group.

[0468] In a specific embodiment, Formula 5-2 may be composed of 13 or more and 17 or less amino acid residues (including (Xa1)').

[0469] In a specific embodiment, the cysteine ​​located 2 to 4 amino acids from the N-terminus and the cysteine ​​located 2 to 4 amino acids from the C-terminus may be optionally linked to each other.

[0470] In a specific embodiment, X3 may be NH. In a specific embodiment, (X)2 may be AW. In a specific embodiment, Xa2 may be glutamic acid. In a specific embodiment, Xa3 may be tryptophan.

[0471] In a specific embodiment, the N-terminal (X) 1-3 One of the residues and the C-terminus (X) 1-3 One of the residues can bind to each other.

[0472] In a specific implementation, Formula 5-2 may be the same as Formula 5-3:

[0473] [Formula 5-3]

[0474] DCAWH-(Xa1)′-GELVWCT (SEQ ID NO:13)

[0475] Wherein, A is alanine and E is glutamic acid.

[0476] R1'-L2-SSAI or R1'-L2-SSFI having the structure of Formula 5 and Formula 5-1 to Formula 5-3 may have binding activity to antibodies. R1'-L2-SSAI or R1'-L2-SSFI may also have binding activity to immunoglobulin G (IgG). In addition, R1'-L2-SSAI or R1'-L2-SSFI may have binding activity to the Fc domain of the antibody.

[0477] 4.1. Reagents for transferring the first click chemistry functional group to an antibody; conjugate of H1-L1 and SSAI (H1-L2-SSAI)

[0478] According to the present invention, a reagent for transferring a first click chemistry functional group to an antibody is disclosed. This compound is shown herein by the symbol "H1-L2-SSAI". According to its structure, the compound is also referred to as a conjugate of H1-L1 and SSAI (H1-L2-SSAI conjugate). In this case, when the SSAI is SSFI, it is shown by the symbol "H1-L2-SSFI".

[0479] H1-L2-SSAI or H1-L2-SSFI according to the present invention include those in which R1' comprises a click chemistry functional group in the section "4. Reagent for transferring a first chemical functional group to an antibody", but the present invention is not limited to the following exemplary embodiments thereof.

[0480] The present invention provides an H1-L2-SSFI comprising the amino acid sequence of the following formula 6-1:

[0481] [Formula 6-1]

[0482] (Xaa)2-H-(Xa1)'-G-Xa2-LV-Xa3 (SEQ ID NO:14)

[0483] wherein each Xaa is independently any amino acid except cysteine,

[0484] H is histidine, G is glycine, Xa2 is glutamic acid or asparagine, L is leucine, V is valine, Xa3 is selected from tryptophan, naphthylalanine and phenylalanine, and

[0485] (Xa1)' is

[0486] Where H1 is the first click chemistry functional group,

[0487] D1 is any alkylene, alkenylene or alkynylene group,

[0488] X1 is an element with a higher electronegativity than carbon.

[0489] D2 is any alkylene, alkenylene or alkynylene group,

[0490] D3 is a covalent bond or C 1-3 Alkylene, and

[0491] X3 is NH, O or S.

[0492] In a specific embodiment, H1 may include any one selected from alkynes, azides, strained alkynes, dienes, dienophiles, olefins, thiols, and tetrazines. In addition, H1 may be an azide or a strained alkyne. In addition, H1 may be an azide or a dibenzocyclooctyne-amine. In addition, H1 may be a diene or a dienophile. In addition, H1 may be a tetrazine or a norbornene. Alternatively, H1 may be a tetrazine or a trans-cyclooctene.

[0493] In a specific embodiment, D1 may include a covalent bond, a C 1-4 Alkylene, C 2-4 Alkenylene, C 2-4 Alkynylidene and C 3-8 Any one of cycloalkylene. D1 can also be -CH2OCH2-. In addition, D1 can be a covalent bond.

[0494] In a specific embodiment, X1 may be NR1, S or O, wherein R1 may be H, halogen or substituted or unsubstituted C 1-3 Alkylene.

[0495] In a specific embodiment, D2 may include a 1-7 Alkylene, C 2-7 Alkenylene, C 2-7 Alkynylidene and C 3-8 In addition, D2 can be C 1-2 In addition, D2 may be a methylene group.

[0496] In a specific embodiment, X3 may be NH.

[0497] In a specific embodiment, (X)2 may be AW. In a specific embodiment, Xa2 may be glutamic acid. In a specific embodiment, Xa3 may be tryptophan.

[0498] The present invention provides H1-L2-SSFI having the structure of the following formula 6-2:

[0499] [Formula 6-2]

[0500] (Xaa) 1-3 -C-(Xaa)2-H-(Xa1)′-G-Xa2-LV-Xa3-C-(Xaa) 1-3 (SEQ ID NO:15)

[0501] wherein each Xaa is independently any amino acid except cysteine,

[0502] C is cysteine, H is histidine, G is glycine, Xa2 is glutamic acid or asparagine, L is leucine, V is valine, Xa3 is selected from tryptophan, naphthylalanine and phenylalanine, and

[0503] (Xa1)' is

[0504] Among them, H1 is the first click chemistry functional group,

[0505] D1 is any alkylene, alkenylene or alkynylene group,

[0506] X1 is an element with a higher electronegativity than carbon.

[0507] D2 is any alkylene, alkenylene or alkynylene group,

[0508] D3 is a covalent bond or C 1-3 Alkylene, and

[0509] X3 is NH, O or S.

[0510] In a specific embodiment, H1 may include any one selected from alkynes, azides, strained alkynes, dienes, dienophiles, olefins, thiols, and tetrazines. In addition, H1 may be an azide or a strained alkyne. In addition, H1 may be an azide or a dibenzocyclooctyne-amine. In addition, H1 may be a diene or a dienophile. In addition, H1 may be a tetrazine or a norbornene. Alternatively, H1 may be a tetrazine or a trans-cyclooctene.

[0511] In a specific embodiment, D1 may include a covalent bond, a C 1-4 Alkylene, C 2-4 Alkenylene, C 2-4 Alkynylidene and C 3-8 Any one of cycloalkylene. In addition, D1 can be -CH2OCH2-. In addition, D1 can be a covalent bond.

[0512] In a specific embodiment, X1 may be NR1, S or O, wherein R1 may be H, halogen or substituted or unsubstituted C 1-3 Alkylene.

[0513] In a specific embodiment, D2 may include a 1-7 Alkylene, C 2-7 Alkenylene, C 2-7 Alkynylidene and C 3-8 In addition, D2 can be C 1-2In addition, D2 may be a methylene group.

[0514] In a specific embodiment, Formula 6-2 may be composed of 13 or more and 17 or less amino acid residues (including (Xa1)').

[0515] In a specific embodiment, the cysteine ​​located 2 to 4 amino acids from the N-terminus and the cysteine ​​located 2 to 4 amino acids from the C-terminus may be optionally linked to each other.

[0516] In a specific embodiment, X3 may be NH. In a specific embodiment, (X)2 may be AW. In a specific embodiment, Xa2 may be glutamic acid. In a specific embodiment, Xa3 may be tryptophan.

[0517] In a specific embodiment, the N-terminal (X) 1-3 One of the residues and the C-terminus (X) 1-3 One of the residues can bind to each other.

[0518] In a specific implementation, Formula 6-2 may be the same as Formula 6-3:

[0519] [Formula 6-3](SEQ ID NO:16)

[0520] DCAWH-(Xa1)′-GELVWCT (SEQ ID NO:16)

[0521] Wherein, A is alanine and E is glutamic acid.

[0522] H1-L2-SSAI or H1-L2-SSFI having the structures of Formula 6-1 to Formula 6-3 may have binding activity to antibodies. H1-L2-SSAI or H1-L2-SSFI may also have binding activity to immunoglobulin G (IgG). In addition, H1-L2-SSAI or H1-L2-SSFI may have binding activity to the Fc domain of the antibody.

[0523] 4.2. Method for preparing a reagent for transferring a first chemical functional group to an antibody

[0524] According to the present invention, methods for preparing R1'-L2-SSAI, R1'-L2-SSFI, H1-L2-SSAI and H1-L2-SSFI (hereinafter generally referred to as "R1'-L2-SSAI") are disclosed. It should be noted that the following description of the preparation method is provided to help understand the present invention.

[0525] For example, a method for preparing a compound of Formula 5 will be described. In a specific embodiment, the compound of Formula 5 can be prepared by the reaction of Scheme 2 below.

[0526] [Solution 2]

[0527]

[0528] The R1'-L2-SSAI according to the present invention can be prepared by reacting a site-specific antibody interaction group (SSAI) with a linker (R1'-L1) according to the present invention. The SSAI according to the present invention is designed to include a nucleophile X3. X3 can attack the activated carbonyl group contained in the linker to cause a nucleophilic substitution reaction. In this case, since X3 attacks the carbon of the second carbonyl group, the R1'-L2-SSAI according to the present invention is prepared.

[0529] As a more specific example, a method for preparing a compound of Formula 6-3 will be described. In a specific embodiment, a compound of Formula 6-3 can be prepared by the reaction of Scheme 3 below.

[0530] [Solution 3]

[0531]

[0532] The SSAI comprising the amino acid sequence of Formula 3 or Formula 3-1 according to the present invention and having each of the structures of Formulas 4-1 to 4-6 includes an Xa1 residue comprising a nucleophile X3. Since X3 attacks the second carbonyl group included in the linker, R1'-L2-SSAI according to the present invention is prepared.

[0533] In a specific embodiment, in the joint according to the present invention, the basicity of the leaving group including X2 can be lower than the basicity of the leaving group including X1. In a specific embodiment, the joint according to the present invention can allow X2-R2' connected to the second carbonyl group to form a good leaving group. X2 can also be O, and R2' can be N-succinimide, p-nitrophenyl or pentafluorophenyl. In this case, the reactivity of the second carbonyl group is higher than the reactivity of the first carbonyl group. Therefore, X3 of SSAI can specifically attack the second carbonyl group of the joint.

[0534] According to the present invention, a method for preparing a reagent for transferring a first chemical functional group to an antibody is disclosed.

[0535] The present invention provides a method for preparing R1'-L2-SSAI, the method comprising: reacting the linker according to the present invention with the site-specific antibody interactome according to the present invention.

[0536] In a specific embodiment, the linker can be any one selected from Formula 1, Formula 2, and Formula 2-1 to Formula 2-3.

[0537] In a specific embodiment, the site-specific antibody interaction group may include or have any one structure selected from Formula 3, Formula 3-1, and Formula 4-1 to Formula 4-6.

[0538] The linker may also have a structure of Formula 2, and the site-specific antibody interaction group may have any one structure selected from Formula 4-2 to Formula 4-6. In addition, the site-specific antibody interaction group may have a structure of Formula 4-6.

[0539] According to the present invention, a method for preparing a reagent for transferring a first click chemistry functional group to an antibody is disclosed.

[0540] The present invention provides a method for preparing H1-L2-SSAI, the method comprising: reacting a linker according to the present invention with a site-specific antibody interactome according to the present invention.

[0541] In a specific embodiment, the linker can be any one selected from Formula 2, Formula 2-1 to Formula 2-3.

[0542] In a specific embodiment, the site-specific antibody interaction group may include or have any one structure selected from Formula 3, Formula 3-1, and Formula 4-1 to Formula 4-6.

[0543] The linker may also have a structure of Formula 2, and the site-specific antibody interaction group may have any one structure selected from Formula 4-2 to Formula 4-6. In addition, the site-specific antibody interaction group may have a structure of Formula 4-6.

[0544] According to the present invention, a kit for preparing a reagent for transferring a first chemical functional group to an antibody is disclosed.

[0545] The present invention provides a kit for preparing a reagent for transferring a first chemical functional group to an antibody, the kit comprising a linker according to the present invention and a site-specific antibody interaction group according to the present invention.

[0546] In a specific embodiment, the linker can be any one selected from Formula 1, Formula 2, and Formula 2-1 to Formula 2-3.

[0547] In a specific embodiment, the site-specific antibody interaction group may include or have any one structure selected from Formula 3, Formula 3-1, and Formula 4-1 to Formula 4-6.

[0548] The linker may also have a structure of Formula 2, and the site-specific antibody interaction group may have any one structure selected from Formula 4-2 to Formula 4-6. In addition, the site-specific antibody interaction group may have a structure of Formula 4-6.

[0549] According to the present invention, a kit for preparing a reagent for transferring a first click chemistry functional group to an antibody is disclosed.

[0550] The present invention provides a kit for preparing a reagent for transferring a first click chemistry functional group to an antibody, the kit comprising a linker containing the first click chemistry functional group according to the present invention and a site-specific antibody interaction group.

[0551] In a specific embodiment, the linker can be any one selected from Formula 2, Formula 2-1 to Formula 2-3.

[0552] In a specific embodiment, the site-specific antibody interaction group may include or have any one structure selected from Formula 3, Formula 3-1, and Formula 4-1 to Formula 4-6.

[0553] The linker may also have a structure of Formula 2, and the site-specific antibody interaction group may have any one structure selected from Formula 4-2 to Formula 4-6. In addition, the site-specific antibody interaction group may have a structure of Formula 4-6.

[0554] 5. Antibodies containing the first chemical functional group (R1'-Ab)

[0555] According to the present invention, an antibody containing a first chemical functional group is disclosed. Such a compound is shown herein by the symbol "R1'-Ab".

[0556] The present invention provides R1'-Ab represented by Formula 7:

[0557] [Formula 7]

[0558]

[0559] Wherein, R1' is the first chemical functional group,

[0560] D1 is any alkylene, alkenylene or alkynylene group,

[0561] X4 is NH, O or S, and

[0562] Ab is an antibody.

[0563] In a specific embodiment, R1' can be a click chemistry functional group. R1' can also include any one selected from alkynes, azides, strained alkynes, dienes, dienophiles, alkenes, thiols and tetrazines. In addition, R1' can be an azide or a strained alkyne. In addition, R1' can be an azide or a dibenzocyclooctyne-amine. In addition, R1' can be a diene or a dienophile. In addition, R1' can be a tetrazine or a norbornene. Alternatively, R1' can be a tetrazine or a trans-cyclooctene.

[0564] In other embodiments, R1' may include a carrier moiety, a fluorescent moiety, a drug moiety or a radioactive moiety. R1' may also include a drug moiety. In addition, R1' may include a VC linker. In other embodiments, R1' may include an antibody or an analog thereof, wherein the antibody or an analog thereof includes a paratope.

[0565] In a specific embodiment, D1 may include a covalent bond, a C 1-4 Alkylene, C 2-4 Alkenylene, C 2-4 Alkynylidene and C 3-8 Any one of cycloalkylene. In addition, D1 can be -CH2OCH2-. In addition, D1 can be a covalent bond.

[0566] In one embodiment, X4 may be NH.

[0567] In a specific embodiment, Ab can be a human antibody. In other specific embodiments, Ab can be a non-human animal antibody. In a specific embodiment, Ab can be an immunoglobulin G (IgG). In a specific embodiment, Ab can be a complete antibody. In other specific embodiments, Ab can be a fragment of an antibody.

[0568] In a specific embodiment, X4 and Ab can be connected through the Fab domain of Ab. In other specific embodiments, X4 and Ab can be connected through the Fc domain of Ab. X4 and Ab can also be connected through lysine 246 or lysine 248 in the Fc domain of Ab. In addition, X4 and Ab can be connected through lysine 246 in the Fc domain of Ab. In addition, X4 and Ab can be connected through lysine 248 in the Fc domain of Ab. Or, X4 and Ab can be connected through lysine 246 and lysine 248 in the Fc domain of Ab. In a specific embodiment, X4 and Ab can be connected only through one of the two Fc domains of Ab. In other specific embodiments, X4 and Ab can be connected through two Fc domains of Ab.

[0569] The present invention provides an antibody or a fragment thereof, wherein the antibody or the fragment thereof comprises an amino acid sequence of the following formula 7-1:

[0570] [Formula 7-1]

[0571] GPSVFLFPP-(K)′-PKDTLMI(SEQ ID NO:17)

[0572] wherein G is glycine, P is proline, S is serine, V is valine, F is phenylalanine, L is leucine, K is lysine, D is aspartic acid, T is threonine, M is methionine, and I is isoleucine, and

[0573] (K)' is

[0574] wherein R1' is the first chemical functional group, and

[0575] D1 is any alkylene, alkenylene or alkynylene. The antibody or fragment thereof has R1′ linked via lysine 246 or a site corresponding to lysine 246 in its Fc domain.

[0576] In a specific embodiment, R1' may include a click chemistry functional group. The click chemistry functional group may also include one or more selected from alkynes, azides, strained alkynes, dienes, dienophiles, alkenes, thiols and tetrazines. In addition, the click chemistry functional group may be selected from azides or strained alkynes. In addition, the click chemistry functional group may be selected from azides or dibenzocyclooctyne-amine. In addition, the click chemistry functional group may be selected from dienes or dienophiles. In addition, the click chemistry functional group may be selected from tetrazines or norbornenes. Alternatively, the click chemistry functional group may be selected from tetrazines or trans-cyclooctene. In addition, R1' may include two or more click chemistry functional groups.

[0577] In other embodiments, R1' may include a carrier moiety, a fluorescent moiety, a drug moiety, or a radioactive moiety. In addition, R1' may include a drug moiety. In addition, R1' may include a VC linker. In other embodiments, R1' may include an antibody or an analog thereof, wherein the antibody or an analog thereof includes a paratope.

[0578] In a specific embodiment, D1 may include a covalent bond, a C 1-4 Alkylene, C 2-4 Alkenylene, C 2-4 Alkynylidene and C 3-8 Any one of cycloalkylene. D1 can also be -CH2OCH2-. In addition, D1 can be a covalent bond.

[0579] In a specific embodiment, the antibody can be a human antibody. In other specific embodiments, the antibody can be a non-human animal antibody. In a specific embodiment, the antibody can be an immunoglobulin G (IgG). In a specific embodiment, the antibody can be a complete antibody. In other specific embodiments, the antibody can be a fragment of an antibody.

[0580] In a specific embodiment, the antibody may comprise the amino acid sequence of Formula 7-1 in only one of its two Fc domains. In other specific embodiments, the antibody may comprise the amino acid sequence of Formula 7-1 in both of its two Fc domains.

[0581] The present invention provides an antibody or a fragment thereof, wherein the antibody or the fragment thereof comprises an amino acid sequence of the following formula 7-2:

[0582] [Formula 7-2]

[0583] GPSVFLFPP-(K)′-PKDTLMI(SEQ ID NO:18)

[0584] wherein G is glycine, P is proline, S is serine, V is valine, F is phenylalanine, L is leucine, K is lysine, D is aspartic acid, T is threonine, M is methionine, and I is isoleucine, and

[0585] (K)' is

[0586] wherein R1' is the first chemical functional group, and

[0587] D1 is any alkylene, alkenylene or alkynylene. The antibody or fragment thereof has R1′ linked via lysine 248 or a site corresponding to lysine 248 in its Fc domain.

[0588] In a specific embodiment, R1' may include a click chemistry functional group. The click chemistry functional group may also include one or more selected from alkynes, azides, strained alkynes, dienes, dienophiles, alkenes, thiols and tetrazines. In addition, the click chemistry functional group may be selected from azides or strained alkynes. In addition, the click chemistry functional group may be selected from azides or dibenzocyclooctyne-amine. In addition, the click chemistry functional group may be selected from dienes or dienophiles. In addition, the click chemistry functional group may be selected from tetrazines or norbornenes. Alternatively, the click chemistry functional group may be selected from tetrazines or trans-cyclooctene. In addition, R1' may include two or more click chemistry functional groups.

[0589] In other embodiments, R1' may include a carrier moiety, a fluorescent moiety, a drug moiety or a radioactive moiety. R1' may also include a drug moiety. In addition, R1' may include a VC linker. In other embodiments, R1' may include an antibody or an analog thereof, wherein the antibody or an analog thereof includes a paratope.

[0590] In a specific embodiment, D1 may include a covalent bond, a C 1-4 Alkylene, C 2-4 Alkenylene, C 2-4 Alkynylidene and C 3-8 Any one of cycloalkylene. In addition, D1 can be -CH2OCH2-. In addition, D1 can be a covalent bond.

[0591] In a specific embodiment, the antibody can be a human antibody. In other specific embodiments, the antibody can be a non-human animal antibody. In a specific embodiment, the antibody can be an immunoglobulin G (IgG). In a specific embodiment, the antibody can be a complete antibody. In other specific embodiments, the antibody can be a fragment of an antibody.

[0592] In a specific embodiment, the antibody may comprise the amino acid sequence of Formula 7-2 in only one of its two Fc domains. In other specific embodiments, the antibody may comprise the amino acid sequence of Formula 7-2 in both of its two Fc domains.

[0593] The present invention provides an antibody or a fragment thereof, wherein the antibody or the fragment thereof comprises an amino acid sequence of the following formula 7-3:

[0594] [Formula 7-3](SEQ ID NO:19)

[0595] G (SEQ ID NO: 19)

[0596] wherein G is glycine, P is proline, S is serine, V is valine, F is phenylalanine, L is leucine, K is lysine, D is aspartic acid, T is threonine, M is methionine, and I is isoleucine, and

[0597] (K)' is

[0598] wherein R1' is the first chemical functional group, and

[0599] D1 is any alkylene, alkenylene or alkynylene. The antibody or fragment thereof has R1′ linked through lysine 246 and lysine 248 or a site corresponding to lysine 246 and lysine 248 in its Fc domain.

[0600] In a specific embodiment, R1' may include a click chemistry functional group. The click chemistry functional group may also include one or more selected from alkynes, azides, strained alkynes, dienes, dienophiles, alkenes, thiols and tetrazines. In addition, the click chemistry functional group may be selected from azides or strained alkynes. In addition, the click chemistry functional group may be selected from azides or dibenzocyclooctyne-amine. In addition, the click chemistry functional group may be selected from dienes or dienophiles. In addition, the click chemistry functional group may be selected from tetrazines or norbornenes. Alternatively, the click chemistry functional group may be selected from tetrazines or trans-cyclooctene. In addition, R1' may include two or more click chemistry functional groups.

[0601] In other embodiments, R1' may include a carrier moiety, a fluorescent moiety, a drug moiety or a radioactive moiety. R1' may also include a drug moiety. In addition, R1' may include a VC linker. In other embodiments, R1' may include an antibody or an analog thereof, wherein the antibody or an analog thereof includes a paratope.

[0602] In a specific embodiment, D1 may include a covalent bond, a C 1-4 Alkylene, C 2-4 Alkenylene, C 2-4 Alkynylidene and C 3-8 Any one of cycloalkylene. D1 can also be -CH2OCH2-. In addition, D1 can be a covalent bond.

[0603] In a specific embodiment, the antibody can be a human antibody. In other specific embodiments, the antibody can be a non-human animal antibody. In a specific embodiment, the antibody can be an immunoglobulin G (IgG). In a specific embodiment, the antibody can be a complete antibody. In other specific embodiments, the antibody can be a fragment of an antibody.

[0604] In a specific embodiment, the antibody may comprise the amino acid sequence of Formula 7-3 in only one of its two Fc domains. In other specific embodiments, the antibody may comprise the amino acid sequence of Formula 7-3 in both of its two Fc domains.

[0605] The present invention provides an antibody or a fragment thereof, wherein the antibody or the fragment thereof comprises one or more amino acid sequences selected from Formula 7-1, Formula 7-2 and Formula 7-3. In this case, the contents of the sequences of Formula 7-1 to Formula 7-3 are as described above.

[0606] In a specific embodiment, D1 can be a covalent bond.

[0607] In a specific embodiment, the antibody or its fragment may include only the amino acid sequence of Formula 7-1, and may not include the amino acid sequences of Formula 7-2 and Formula 7-3. The antibody or its fragment may also include the amino acid sequence of Formula 7-1 only in one of its two Fc domains. In addition, the antibody may include the amino acid sequence of Formula 7-1 in both of its two Fc domains.

[0608] In other embodiments, the antibody or fragment thereof may include only the amino acid sequence of Formula 7-2, but may not include the amino acid sequences of Formula 7-1 and Formula 7-3. The antibody or fragment thereof may also include the amino acid sequence of Formula 7-2 only in one of its two Fc domains. In addition, the antibody may include the amino acid sequence of Formula 7-2 in both of its two Fc domains.

[0609] In a specific embodiment, the antibody or its fragment may include only the amino acid sequence of Formula 7-3, and may not include the amino acid sequences of Formula 7-1 and Formula 7-2. The antibody or its fragment may also include the amino acid sequence of Formula 7-3 only in one of its two Fc domains. In addition, the antibody may include the amino acid sequence of Formula 7-3 in both of its two Fc domains.

[0610] 5.1. Antibodies with first click chemistry functional groups

[0611] According to the present invention, an antibody containing a first click chemistry functional group is disclosed. Such a compound is shown herein by the symbol "H1-Ab".

[0612] The present invention provides H1-Ab represented by Formula 8:

[0613] [Formula 8]

[0614]

[0615] Among them, H1 is the first click chemistry functional group,

[0616] D1 is any alkylene, alkenylene or alkynylene group,

[0617] X4 is NH, O or S, and

[0618] Ab is an antibody.

[0619] In a specific embodiment, H1 may include any one selected from alkynes, azides, strained alkynes, dienes, dienophiles, olefins, thiols, and tetrazines. In addition, H1 may be an azide or a strained alkyne. In addition, H1 may be an azide or a dibenzocyclooctyne-amine. In addition, H1 may be a diene or a dienophile. In addition, H1 may be a tetrazine or a norbornene. Alternatively, H1 may be a tetrazine or a trans-cyclooctene.

[0620] In a specific embodiment, D1 may include a covalent bond, a C 1-4 Alkylene, C 2-4 Alkenylene, C 2-4 Alkynylidene and C 3-8 Any one of cycloalkylene. D1 can also be -CH2OCH2-. In addition, D1 can be a covalent bond.

[0621] In a specific embodiment, X4 may be NH.

[0622] In a specific embodiment, Ab can be a human antibody. In other specific embodiments, Ab can be a non-human animal antibody. In a specific embodiment, Ab can be an immunoglobulin G (IgG). In a specific embodiment, Ab can be a complete antibody. In other specific embodiments, Ab can be a fragment of an antibody. In a specific embodiment, Ab can be a wild-type antibody. In other specific embodiments, Ab can be a manipulated antibody.

[0623] In a specific embodiment, X4 and Ab can be connected through the Fab domain of Ab. In other specific embodiments, X4 and Ab can be connected through the Fc domain of Ab. X4 and Ab can also be connected through lysine 246 or lysine 248 in the Fc domain of Ab. In addition, X4 and Ab can be connected through lysine 246 in the Fc domain of Ab. In addition, X4 and Ab can be connected through lysine 248 in the Fc domain of Ab. Or, X4 and Ab can be connected through lysine 246 and lysine 248 in the Fc domain of Ab. In a specific embodiment, X4 and Ab can be connected only through one of the two Fc domains of Ab. In other specific embodiments, X4 and Ab can be connected through the two Fc domains of Ab.

[0624] The present invention provides an antibody or a fragment thereof, wherein the antibody or the fragment thereof comprises an amino acid sequence of the following formula 8-1:

[0625] [Formula 8-1]

[0626] GPSVFLFPP-(K)′-PKDTLMI(SEQ ID NO:20)

[0627] wherein G is glycine, P is proline, S is serine, V is valine, F is phenylalanine, L is leucine, K is lysine, D is aspartic acid, T is threonine, M is methionine, and I is isoleucine, and

[0628] (K)' is

[0629] Wherein, H1 is the first click chemistry functional group, and

[0630] D1 is any alkylene, alkenylene or alkynylene. The antibody or fragment thereof has H1 linked via lysine 246 or a site corresponding to lysine 246 in its Fc domain.

[0631] In a specific embodiment, H1 may include any one selected from alkynes, azides, strained alkynes, dienes, dienophiles, olefins, thiols, and tetrazines. In addition, H1 may be an azide or a strained alkyne. In addition, H1 may be an azide or a dibenzocyclooctyne-amine. In addition, H1 may be a diene or a dienophile. In addition, H1 may be a tetrazine or a norbornene. Alternatively, H1 may be a tetrazine or a trans-cyclooctene.

[0632] In a specific embodiment, D1 may include a covalent bond, a C 1-4 Alkylene, C 2-4 Alkenylene, C 2-4 Alkynylidene and C 3-8 Any one of cycloalkylene. D1 can also be -CH2OCH2-. In addition, D1 can be a covalent bond.

[0633] In specific embodiments, the antibody may be a human antibody. In other specific embodiments, the antibody may be a non-human animal antibody. In specific embodiments, the antibody may be an immunoglobulin G (IgG). In specific embodiments, the antibody may be a complete antibody. In other specific embodiments, the antibody may be a fragment of an antibody. In specific embodiments, the antibody may be a wild-type antibody. In other specific embodiments, the antibody may be a manipulated antibody.

[0634] In a specific embodiment, the antibody may comprise the amino acid sequence of Formula 8-1 in only one of its two Fc domains. In other specific embodiments, the antibody may comprise the amino acid sequence of Formula 8-1 in both of its two Fc domains.

[0635] The present invention provides an antibody or a fragment thereof, wherein the antibody or the fragment thereof comprises an amino acid sequence of the following formula 8-2:

[0636] [Formula 8-2]

[0637] GPSVFLFPPKP-(K)′-DTLMI(SEQ ID NO:21)

[0638] wherein G is glycine, P is proline, S is serine, V is valine, F is phenylalanine, L is leucine, K is lysine, D is aspartic acid, T is threonine, M is methionine, and I is isoleucine, and

[0639]

[0640] Wherein, H1 is the first click chemistry functional group, and

[0641] D1 is any alkylene, alkenylene or alkynylene. The antibody or fragment thereof has H1 linked via lysine 248 or a site corresponding to lysine 248 in its Fc domain.

[0642] In a specific embodiment, H1 may include any one selected from alkynes, azides, strained alkynes, dienes, dienophiles, olefins, thiols, and tetrazines. In addition, H1 may be an azide or a strained alkyne. In addition, H1 may be an azide or a dibenzocyclooctyne-amine. In addition, H1 may be a diene or a dienophile. In addition, H1 may be a tetrazine or a norbornene. Alternatively, H1 may be a tetrazine or a trans-cyclooctene.

[0643] In a specific embodiment, D1 may include a covalent bond, a C 1-4 Alkylene, C 2-4 Alkenylene, C 2-4 Alkynylidene and C 3-8 Any one of cycloalkylene. D1 can also be -CH2OCH2-. In addition, D1 can be a covalent bond.

[0644] In specific embodiments, the antibody may be a human antibody. In other specific embodiments, the antibody may be a non-human animal antibody. In specific embodiments, the antibody may be an immunoglobulin G (IgG). In specific embodiments, the antibody may be a complete antibody. In other specific embodiments, the antibody may be a fragment of an antibody. In specific embodiments, the antibody may be a wild-type antibody. In other specific embodiments, the antibody may be a manipulated antibody.

[0645] In a specific embodiment, the antibody may comprise the amino acid sequence of Formula 8-1 in only one of its two Fc domains. In other specific embodiments, the antibody may comprise the amino acid sequence of Formula 8-2 in both of its two Fc domains.

[0646] The present invention provides an antibody or a fragment thereof, wherein the antibody or the fragment thereof comprises an amino acid sequence of the following formula 8-3:

[0647] [Formula 8-3](SEQ ID NO:22)

[0648] GPSVFLFPP-(K)′-P-(K)′-DTLMI(SEQ ID NO:22)

[0649] wherein G is glycine, P is proline, S is serine, V is valine, F is phenylalanine, L is leucine, K is lysine, D is aspartic acid, T is threonine, M is methionine, and I is isoleucine, and

[0650] (K)′ is

[0651] Wherein, H1 is the first click chemistry functional group, and

[0652] D1 is any alkylene, alkenylene or alkynylene. The antibody or fragment thereof has H1 linked via lysine 246 and lysine 248 or sites corresponding to lysine 246 and lysine 248 in its Fc domain.

[0653] In a specific embodiment, H1 may include any one selected from alkynes, azides, strained alkynes, dienes, dienophiles, olefins, thiols, and tetrazines. In addition, H1 may be an azide or a strained alkyne. In addition, H1 may be an azide or a dibenzocyclooctyne-amine. In addition, H1 may be a diene or a dienophile. In addition, H1 may be a tetrazine or a norbornene. Alternatively, H1 may be a tetrazine or a trans-cyclooctene.

[0654] In a specific embodiment, D1 may include a covalent bond, a C 1-4 Alkylene, C 2-4 Alkenylene, C 2-4 Alkynylidene and C 3-8 Any one of cycloalkylene. D1 can also be -CH2OCH2-. In addition, D1 can be a covalent bond.

[0655] In specific embodiments, the antibody may be a human antibody. In other specific embodiments, the antibody may be a non-human animal antibody. In specific embodiments, the antibody may be an immunoglobulin G (IgG). In specific embodiments, the antibody may be a complete antibody. In other specific embodiments, the antibody may be a fragment of an antibody. In specific embodiments, the antibody may be a wild-type antibody. In other specific embodiments, the antibody may be a manipulated antibody.

[0656] In a specific embodiment, the antibody may comprise the amino acid sequence of Formula 8-3 in only one of its two Fc domains. In other specific embodiments, the antibody may comprise the amino acid sequence of Formula 8-3 in both of its two Fc domains.

[0657] The present invention provides an antibody or a fragment thereof, wherein the antibody or the fragment thereof comprises one or more amino acid sequences selected from Formula 8-1, Formula 8-2 and Formula 8-3. In this case, the contents of the sequences of Formula 8-1 to Formula 8-3 are as described above.

[0658] In a specific embodiment, D1 can be a covalent bond.

[0659] In a specific embodiment, the antibody or its fragment may include only the amino acid sequence of formula 8-1 and may not include the amino acid sequences of formula 8-2 and formula 8-3. The antibody or its fragment may also include the amino acid sequence of formula 8-1 only in one of its two Fc domains. In addition, the antibody may include the amino acid sequence of formula 8-1 in both of its two Fc domains.

[0660] In other embodiments, the antibody or its fragment may include only the amino acid sequence of formula 8-2, but may not include the amino acid sequences of formula 8-1 and formula 8-3. The antibody or its fragment may also include the amino acid sequence of formula 8-2 only in one of its two Fc domains. In addition, the antibody may include the amino acid sequence of formula 8-2 in both of its two Fc domains.

[0661] In a specific embodiment, the antibody or its fragment may include only the amino acid sequence of formula 8-3, and may not include the amino acid sequences of formula 8-1 and formula 8-2. The antibody or its fragment may also include the amino acid sequence of formula 8-3 only in one of its two Fc domains. In addition, the antibody may include the amino acid sequence of formula 8-3 in both of its two Fc domains.

[0662] In specific embodiments, the antibody may be a human antibody. In other specific embodiments, the antibody may be a non-human animal antibody. In specific embodiments, the antibody may be an immunoglobulin G (IgG). In specific embodiments, the antibody may be a complete antibody. In other specific embodiments, the antibody may be a fragment of an antibody. In specific embodiments, the antibody may be a wild-type antibody. In other specific embodiments, the antibody may be a manipulated antibody.

[0663] 5.2. Methods for preparing antibodies containing a first chemical functional group

[0664] According to the present invention, methods for preparing R1'-Ab and H1-Ab (hereinafter generally referred to as "R1'-Ab") are disclosed. It should be noted that the following description of the preparation method is provided to help understand the present invention.

[0665] For example, a method for preparing a compound of Formula 7 will be described. In a specific embodiment, the compound of Formula 7 can be prepared by the reaction of Scheme 4 below.

[0666] [Solution 4]

[0667]

[0668] Since the first carbonyl group of the joint according to the present invention has mild reactivity, when the first carbonyl group of the joint has a close positional relationship with the amine group of the antibody, cross-linking can be achieved. First, the R1'-L2-SSAI represented by Formula 5 is in close contact with the specific part of the antibody to produce an environment where a reaction may occur. Because the R1'-L2-SSAI according to the present invention has an activated carbonyl group corresponding to the first carbonyl group of the joint (i.e., a carbonyl group connected to X1), the R1'-L2-SSAI of the present invention can trigger a nucleophilic substitution reaction. In this case, since the atom (X4) with a free electron pair present in the antibody acts as a nucleophile to attack the carbonyl group, a compound of Formula 7 can be prepared. In this case, by the design of the joint, the site-specific antibody interaction group (SSAI) leaves when it is contained in the leaving group, and the SSAI is removed from the final product. These characteristics have a positive impact on the physical properties of the antibody product, as shown in Section 5.8 below.

[0669] In a specific embodiment, X4 may be NH2. X4 may also be the NH2 of a lysine residue. In other specific embodiments, X4 may be SH. X4 may also be the SH of a cysteine ​​residue. In other specific embodiments, X4 may be OH.

[0670] As a more specific example, a method for preparing an antibody or a fragment thereof comprising the amino acid sequence of Formula 8-3 will be described. In a specific embodiment, a compound comprising the amino acid sequence of Formula 8-3 can be prepared by the reaction of Scheme 5 below.

[0671] [Solution 5]

[0672]

[0673] The compound with the structure of formula 6-3 (a structure in which two cysteine ​​residues are optionally connected) is directed to the Fc domain of the antibody because the compound has an SSFI sequence (see Section 3.2). In this case, the antibody including the amino acid sequence of SEQ ID NO:1 includes a lysine residue in its Fc domain, and such a lysine residue can act as a nucleophile. An exemplary embodiment shows the case where lysine 246 in the Fc domain or a residue corresponding to lysine 246 (hereinafter referred to as "lysine 246") acts as a nucleophile. In this case, the amine group of lysine 246 attacks the first carbonyl group of formula 6-3 to produce a compound of formula 8-1. In this way, H1 or R1 can be transferred to a lysine residue in the Fc domain of the antibody. In this case, which lysine residue the chemical functional group is transferred to may depend on the design of the linker, SSAI and R1'-L2-SSAI, as described in Sections 5.3 and 5.4 below.

[0674] According to the present invention, a method for preparing an antibody comprising a first chemical functional group is disclosed.

[0675] The present invention provides a method for preparing R1'-Ab, the method comprising: reacting a reagent for transferring a first chemical functional group to the antibody according to the present invention with the antibody or a fragment thereof.

[0676] In a specific embodiment, the reagent for transferring the first chemical functional group to the antibody can be any one selected from Formula 5, Formula 5-1 to Formula 5-3, and Formula 6-1 to Formula 6-3.

[0677] In specific embodiments, the antibody or fragment thereof may be a human antibody. In other specific embodiments, the antibody or fragment thereof may be a non-human animal antibody. In specific embodiments, the antibody or fragment thereof may be an immunoglobulin G (IgG). In specific embodiments, the antibody or fragment thereof may be a complete antibody. In other specific embodiments, the antibody or fragment thereof may be a fragment of an antibody. In specific embodiments, the antibody or fragment thereof may be a wild-type antibody. In other specific embodiments, the antibody or fragment thereof may be a manipulated antibody.

[0678] In a specific embodiment, the invention provides a method for preparing an antibody having a first chemical functional group transferred to a specific lysine residue of its Fc domain. In this case, those aspects described in Section 5.4 below can be used as reagents for transferring the first chemical functional group to the antibody.

[0679] According to the present invention, a method for preparing an antibody containing a first click chemistry functional group is disclosed.

[0680] The present invention provides a method for preparing H1-Ab, comprising: reacting a reagent for transferring a first click chemistry functional group to the antibody according to the present invention with the antibody or a fragment thereof.

[0681] In a specific embodiment, the reagent for transferring the first click chemistry functional group to the antibody can be any one selected from Formula 6-1 to Formula 6-3. The reagent for transferring the first click chemistry functional group to the antibody can also have the structure of Formula 6-3.

[0682] In specific embodiments, the antibody or fragment thereof may be a human antibody. In other specific embodiments, the antibody or fragment thereof may be a non-human animal antibody. In specific embodiments, the antibody or fragment thereof may be an immunoglobulin G (IgG). In specific embodiments, the antibody or fragment thereof may be a complete antibody. In other specific embodiments, the antibody or fragment thereof may be a fragment of an antibody. In specific embodiments, the antibody or fragment thereof may be a wild-type antibody. In other specific embodiments, the antibody or fragment thereof may be a manipulated antibody.

[0683] In a specific embodiment, the present invention provides a method for preparing an antibody having a first click chemistry functional group transferred to a specific lysine residue of its Fc domain. In this case, the aspects described in Section 5.4 below can be used as reagents for transferring the first click chemistry functional group to the antibody.

[0684] According to the present invention, a kit for preparing an antibody or a fragment thereof containing a first chemical functional group is disclosed.

[0685] The present invention provides a kit for preparing an antibody or a fragment thereof containing a first chemical functional group, the kit comprising a reagent for transferring the first chemical functional group to the antibody according to the present invention, and the antibody or a fragment thereof.

[0686] In a specific embodiment, the reagent for transferring the first chemical functional group to the antibody can be any one selected from Formula 5, Formula 5-1 to Formula 5-3, and Formula 6-1 to Formula 6-3.

[0687] In specific embodiments, the antibody or fragment thereof may be a human antibody. In other specific embodiments, the antibody or fragment thereof may be a non-human animal antibody. In specific embodiments, the antibody or fragment thereof may be an immunoglobulin G (IgG). In specific embodiments, the antibody or fragment thereof may be a complete antibody. In other specific embodiments, the antibody or fragment thereof may be a fragment of an antibody. In specific embodiments, the antibody or fragment thereof may be a wild-type antibody. In other specific embodiments, the antibody or fragment thereof may be a manipulated antibody.

[0688] In addition, the present invention provides a kit for preparing an antibody or a fragment thereof containing a first chemical functional group, the kit comprising:

[0689] A linker (R1'-L1) according to the invention;

[0690] The site-specific antibody interactome according to the invention; and

[0691] Antibodies or fragments thereof.

[0692] In a specific embodiment, the linker can be any one selected from Formula 1, Formula 2, and Formula 2-1 to Formula 2-3.

[0693] In a specific embodiment, the site-specific antibody interaction group can be any one selected from Formula 3, Formula 3-1, and Formula 4-1 to Formula 4-6.

[0694] In specific embodiments, the antibody or fragment thereof may be a human antibody. In other specific embodiments, the antibody or fragment thereof may be a non-human animal antibody. In specific embodiments, the antibody or fragment thereof may be an immunoglobulin G (IgG). In specific embodiments, the antibody or fragment thereof may be a complete antibody. In other specific embodiments, the antibody or fragment thereof may be a fragment of an antibody. In specific embodiments, the antibody or fragment thereof may be a wild-type antibody. In other specific embodiments, the antibody or fragment thereof may be a manipulated antibody.

[0695] The present invention provides a kit for preparing an antibody having a first chemical functional group transferred to a specific lysine residue of its Fc domain. In this case, the aspects described in Section 5.4 below can be used as reagents for transferring the first chemical functional group to antibodies, linkers, and site-specific antibody interactomes.

[0696] According to the present invention, a kit for preparing an antibody or a fragment thereof containing a first click chemistry functional group is disclosed.

[0697] The present invention provides a kit for preparing an antibody or a fragment thereof containing a first click chemistry functional group, the kit comprising a reagent for transferring the first click chemistry functional group to the antibody according to the present invention, and the antibody or a fragment thereof.

[0698] In a specific embodiment, the reagent for transferring the first click chemistry functional group to the antibody can be any one selected from Formula 6-1 to Formula 6-3. The reagent for transferring the first click chemistry functional group to the antibody can also have the structure of Formula 6-3.

[0699] In specific embodiments, the antibody or fragment thereof may be a human antibody. In other specific embodiments, the antibody or fragment thereof may be a non-human animal antibody. In specific embodiments, the antibody or fragment thereof may be an immunoglobulin G (IgG). In specific embodiments, the antibody or fragment thereof may be a complete antibody. In other specific embodiments, the antibody or fragment thereof may be a fragment of an antibody. In specific embodiments, the antibody or fragment thereof may be a wild-type antibody. In other specific embodiments, the antibody or fragment thereof may be a manipulated antibody.

[0700] The present invention also provides a kit for preparing an antibody or a fragment thereof containing a first click chemistry functional group, the kit comprising:

[0701] A joint (H1-L1) according to the invention;

[0702] The site-specific antibody interactome according to the invention; and

[0703] Antibodies or fragments thereof.

[0704] In a specific embodiment, the linker can be any one selected from Formula 2, Formula 2-1 to Formula 2-3.

[0705] In a specific embodiment, the site-specific antibody interaction group can be any one selected from Formula 3, Formula 3-1, and Formula 4-1 to Formula 4-6.

[0706] In specific embodiments, the antibody or fragment thereof may be a human antibody. In other specific embodiments, the antibody or fragment thereof may be a non-human animal antibody. In specific embodiments, the antibody or fragment thereof may be an immunoglobulin G (IgG). In specific embodiments, the antibody or fragment thereof may be a complete antibody. In other specific embodiments, the antibody or fragment thereof may be a fragment of an antibody. In specific embodiments, the antibody or fragment thereof may be a wild-type antibody. In other specific embodiments, the antibody or fragment thereof may be a manipulated antibody.

[0707] The present invention provides a kit for preparing an antibody having a first chemical functional group transferred to a specific lysine residue of its Fc domain. In this case, the aspects described in Section 5.4 below can be used as reagents for transferring the first chemical functional group to antibodies, linkers, and site-specific antibody interactomes.

[0708] 5.3. The role of (Xa1)' and the design principle of its position on R1'-L2-SSFI

[0709] Herein, the compound of formula 6-3 is provided as an example to help understand the present invention, but the scope of the present invention is not limited thereto. It should be noted that the following description is also applicable to the compounds of formula 5, formula 5-1 to formula 5-3 and formula 6-1 to formula 6-3, and for convenience, the compound of formula 6-3 is provided only as an example of the compound.

[0710] As discussed in Section 5.2, the role of (Xa1)' of R1'-L2-SSFI is to transfer R1' to the antibody via a nucleophilic substitution reaction. According to the present invention, it is believed that the conditions that promote the nucleophilic substitution reaction meet the following requirements: (1) (Xa1)' is adjacent to a lysine residue of the Fc domain, and (2) the side chain to which R1' binds is directed toward the lysine residue. It is expected that the yield and uniformity of the process will decrease as the position of (Xa1)' and the direction of the side chain become further away from the lysine residue of the Fc domain. (3) It will also be preferred that the substitution position of (Xa1)' does not have much effect on the interaction between SSFI and the Fc domain (see Section 3.2).

[0711] like Figure 3 and Figure 5 Indirectly, based on the following formula 6-3, it was confirmed that the positions in SSFI that satisfy the requirement (1) are position 5, position 6, position 7, and position 8 regarding lysine 246 and lysine 248 in the Fc domain.

[0712] [Formula 6-3]

[0713] DCAWH-(Xa1)′-GELVWCT

[0714] In this case, since the histidine at the corresponding position 5 forms a salt bond with glutamic acid 380 in the Fc domain, the replacement of histidine may affect the interaction between the SSFI and the Fc domain. Therefore, this does not meet the requirement of (3). Since the direction of this side chain is not close to lysine 246 and lysine 248, this does not meet the requirement of (2). Since the glycine at the corresponding position 7 contributes to the formation of the curved structure of the SSFI, it is not desirable to replace the glycine residue with a large (Xa1)' residue. Since the glutamic acid at the corresponding position 8 forms a salt bond with arginine 255 in the Fc domain, the replacement of glutamic acid may affect the interaction between the SSFI and the Fc domain. Therefore, this does not meet the requirement of (3) (see Section 3.2). It is judged that position 6 is the most suitable position for (Xa1)' because position 6 meets all the requirements of (1), (2) and (3). Therefore, based on these facts, R1'-L2-SSFI according to the present invention is completed.

[0715] 5.4. The position of R1' transferred to the antibody may vary depending on the length of D2 in R1'-L1 and D3 in SSFI.

[0716] This article aims to explain the preferred design principle of R1'-L2-SSFI according to the present invention. According to the design of R1'-L2-SSFI, R1' can be specifically transferred to a specific lysine residue of the Fc domain. In addition, this article aims to explain the design principle of R1'-L1 and SSFI to prepare the preferred R1'-L2-SSFI.

[0717] A person skilled in the art who has read the above section 5.2 can recognize that when the first carbonyl carbon of (Xa1)' of R1'-L2-SSFI is positioned adjacent to the amine group of lysine of the Fc domain, a nucleophilic substitution reaction may occur. Based on the description in the above section 3.2, a person skilled in the art can also recognize that R1'-L2-SSFI is arranged together with an Fc domain having a specific topological structure, in which the amine groups of lysine 246 and lysine 248 in the Fc domain are spaced a certain distance from the β carbon of (Xa1)' ( Figure 5 By using this combination to design R1'-L2-SSFI, it is expected that when the distance between the β carbon of (1)(Xa1)' and the first carbonyl carbon (hereinafter generally referred to as "L c When the distances between the β carbon of (2)(Xa1)' and the amine groups of lysine 246 and lysine 248 in the Fc domain of a specific topology are the same or similar, it can specifically label the desired lysine residues.

[0718] According to the structure of R1'-L2-SSFI of the present invention and the L c exist Figure 8 As shown in Figure 8 As shown, D3, X3, carbon atom, D2 and X1 are located between the β carbon of (Xa1)' and the first carbonyl carbon. Among them, D3 and X3 are related to the design of SSFI, and D2 and X1 are related to the design of L1-R1'.

[0719] For convenience, the present invention is embodied with the following assumptions: D3 is C x Alkylene, X3 is N, D2 is C y Alkylene, C y Alkenylene or C y alkynylene, and X1 is S, wherein y is an integer greater than or equal to 1. In this case, it is assumed that the alkylene, alkenylene and alkynylene groups have the same length. Figure 8 The structure of the linker shown was modeled using Discovery Studio software to calculate L c As a result, the L value determined relative to the x+y valuec The values ​​are listed in Table 3. During the calculations, the chain was modeled when it had the longest length.

[0720] L relative to x+y value c value

[0721]

[0722] In the following, based on Fig. 9 A drawing showing the topological structure between the R1′-L2-SSFI and the lysine residues of the Fc domain is provided. Fig. 9 The topological structure between R1′-L2-SSFI and the Fc domain is shown, so that the direction of the side chain of (Xa1)′ (dashed arrow) is parallel to the x-axis in the figure. Fig. 9 As shown in the figure, it can be seen that the side chain of (Xa1)′ points to the amine groups of lysine 246 and lysine 248 (see Section 5.3). It can also be seen from the figure that depending on L c The length of (Xa1)′ may react specifically with lysine 246 or lysine 248. Figure 10-12 is along the exemplary L c The length is observed in the direction parallel to the y-axis in the figure (thick solid arrow) Fig. 9 Illustration of a chart.

[0723] The distance (D) between the β carbon of (Xa1)′ and the amine groups of lysine 246 and lysine 248 in the Fc domain 246,min , D 246,max , D 248,min and D 248,max ) is described in Section 3.2. The beta carbon of (Xa1)' is closer to lysine 248 in the Fc domain than the lysine residue at position 246. Therefore, it is expected that when L c Value shorter than D 246，min ( Fig.10 ) when the first carbonyl carbon reacts well with lysine 248. c Value longer than D 248,max ( Fig.11 ) when the first carbonyl carbon reacts well with lysine 246, and when L c Value longer than or equal to D 246,min and shorter than or equal to D 248,max ( Fig.12 ) when the first carbonyl carbon will react selectively with lysine 246 and lysine 248.

[0724] According to the present invention, R1′-L2-SSFI (see Fig.11 ).

[0725] The present invention provides a reagent for transferring a first chemical functional group to an antibody, characterized in that the distance (L) between the beta carbon of (Xa1)' and the first carbonyl carbon is c ) is shorter than D 246,min (about ). In this case, L c Can have about about about about about about or about The value of .

[0726] In a specific embodiment, when the R1'-L2-SSFI has any one structure selected from Formula 5, Formula 5-1 to Formula 5-3 and Formula 6-1 to Formula 6-3, D3 is C x Alkylene, X3 is N, D2 is C y Alkylene, C y Alkenylene or C y Alkyne group, X1 is S, wherein y may be an integer greater than or equal to 1, and the sum of x and y may be 1≤x+y≤5. The sum of x and y may also be 1, 2, 3, 4 or 5. For example, x=0, and y may be 1≤y≤5. In another exemplary embodiment, x=1, and y may be 1≤y≤4. In yet another exemplary embodiment, x=2, and y may be 1≤y≤3. In yet another exemplary embodiment, x=3, and y may be 1≤y≤2. The corresponding numerical range is determined based on the values ​​listed in Table 3.

[0727] According to the present invention, R1'-L2-SSFI for specifically transferring a first chemical functional group to lysine 246 in the Fc domain of an antibody is disclosed.

[0728] According to one aspect of the present invention, the present invention provides a reagent for transferring a first chemical functional group to an antibody, characterized in that the distance (L) between the beta carbon of (Xa1)' and the first carbonyl carbon is c ) is longer than D 248,max (about ). For example, L c Can have about about about about about or about The value of .

[0729] In a specific embodiment, when R1'-L2-SSFI has any one structure selected from Formula 5, Formula 5-1 to Formula 5-3 and Formula 6-1 to Formula 6-3, D3 is C x Alkylene, X3 is N, D2 is C y Alkylene, C y Alkenylene or C y Alkyne group, X1 is S, wherein y may be an integer greater than or equal to 1, and the sum of x and y may be greater than or equal to 9. The sum of x and y may also be 9, 10, 11 or 12. For example, x=0, and y may be 9≤y≤12. In another exemplary embodiment, x=1, and y may be 8≤y≤11. In yet another exemplary embodiment, x=2, and y may be 7≤y≤10. In yet another exemplary embodiment, x=3, and y may be 6≤y≤9. Alternatively, D2 may be an alkynyl group. When D2 is an alkynyl group, the side chain of (Xa1)' may become stereorigid, thereby preventing bending of the side chain.

[0730] According to the present invention, R1′-L2-SSFI for selectively transferring a first chemical functional group to lysine 246 or lysine 248 in the Fc domain of an antibody is disclosed.

[0731] According to one aspect of the present invention, the present invention provides a reagent for transferring a first chemical functional group to an antibody, characterized in that the distance (L) between the beta carbon of (Xa1)' and the first carbonyl carbon is c ) has a length greater than or equal to D 246,min (about ) and shorter than or equal to D 248,max (about ) value. In this case, L c Can have about about about about about about about or about The value of .

[0732] In a specific embodiment, when R1'-L2-SSFI has any one structure selected from Formula 5, Formula 5-1 to Formula 5-3 and Formula 6-1 to Formula 6-3, D3 is C x Alkylene, X3 is N, D2 is C y Alkylene, C y Alkenylene or C yAlkyne group, X1 is S, wherein y may be an integer greater than or equal to 1, and the sum of x and y may be 6≤x+y≤8. In this case, the sum of x and y may be 6, 7, or 8. For example, x=0, and y may be 6≤y≤8. In another exemplary embodiment, x=1, and y may be 5≤y≤7. In another exemplary embodiment, x=2, and y may be 4≤y≤6. In another exemplary embodiment, x=3, and y may be 3≤y≤5.

[0733] According to the present invention, a method for preparing R1'-L2-SSFI for specifically transferring a first chemical functional group to lysine 248 in the Fc domain of an antibody is disclosed.

[0734] As an example of the method for preparing a reagent for transferring a first chemical functional group to an antibody as described in Section 4.2, the present invention provides a method for preparing R1'-L2-SSAI, characterized in that it comprises: reacting a linker according to the present invention with a site-specific antibody interactor according to the present invention,

[0735] Wherein, D2 in the joint is C y Alkylene, C y Alkenylene or C y Alkyne groups, and

[0736] D3 in the site-specific antibody interactome is C x Alkylene,

[0737] Wherein, y is an integer greater than or equal to 1, and the sum of x and y is 1≤x+y≤5. The R1'-L2-SSAI prepared by this method can also react with an antibody to specifically transfer the first chemical functional group to lysine 248 of the Fc domain of the antibody.

[0738] According to the present invention, a kit for preparing R1′-L2-SSFI is disclosed, which is used to specifically transfer a first chemical functional group to lysine 248 in the Fc domain of an antibody.

[0739] As an example of the method for preparing a reagent for transferring a first chemical functional group to an antibody as described in Section 4.2, the present invention provides a kit for preparing R1'-L2-SSAI, characterized in that it comprises:

[0740] A joint according to the invention; and

[0741] According to the site-specific antibody interactome of the present invention,

[0742] Wherein, D2 in the joint is C y Alkylene, Cy Alkenylene or C y an alkynylene group, and

[0743] D3 in the site-specific antibody interactome is C x Alkylene,

[0744] Here, y is an integer greater than or equal to 1, and the sum of x and y is 1≤x+y≤5.

[0745] According to the present invention, a method for preparing R1'-L2-SSFI is disclosed, which is used to specifically transfer a first chemical functional group to lysine 246 in the Fc domain of an antibody.

[0746] As an example of the method for preparing a reagent for transferring a first chemical functional group to an antibody as described in Section 4.2, the present invention provides a method for preparing R1'-L2-SSAI, characterized in that it comprises:

[0747] reacting a linker according to the invention with a site-specific antibody interactome according to the invention,

[0748] Wherein, D2 in the joint is C y Alkylene, C y Alkenylene or C y an alkynylene group, and

[0749] D3 in the site-specific antibody interactome is C x Alkylene,

[0750] Wherein, y is an integer greater than or equal to 1, and the sum of x and y is 9≤x+y≤12. The R1'-L2-SSAI prepared by this method can also react with an antibody, thereby specifically transferring the first chemical functional group to lysine 246 in the Fc domain of the antibody.

[0751] According to the present invention, a kit for preparing R1'-L2-SSFI is disclosed, wherein the kit is used to specifically transfer a first chemical functional group to lysine 246 in the Fc domain of an antibody.

[0752] As an example of the method for preparing a reagent for transferring a first chemical functional group to an antibody as described in Section 4.2, the present invention provides a kit for preparing R1'-L2-SSAI, characterized in that it comprises:

[0753] A joint according to the invention; and

[0754] According to the site-specific antibody interactome of the present invention,

[0755] Wherein, D2 in the joint is Cy Alkylene, C y Alkenylene or C y an alkynylene group, and

[0756] D3 in the site-specific antibody interactome is C x Alkylene,

[0757] Here, y is an integer greater than or equal to 1, and the sum of x and y is 9≤x+y≤12.

[0758] According to the present invention, a method for preparing R1'-L2-SSFI for selectively transferring a first chemical functional group to lysine 246 or lysine 248 in the Fc domain of an antibody is disclosed.

[0759] As an example of the method for preparing a reagent for transferring a first chemical functional group to an antibody as described in Section 4.2, the present invention provides a method for preparing R1'-L2-SSAI, characterized in that it comprises:

[0760] reacting a linker according to the invention with a site-specific antibody interactome according to the invention,

[0761] Wherein, D2 in the joint is C y Alkylene, C y Alkenylene or C y Alkyne groups, and

[0762] D3 in the site-specific antibody interactome is C x Alkylene,

[0763] wherein y is an integer greater than or equal to 1, and the sum of x and y is 6≤x+y≤8. The R1'-L2-SSAI prepared by this method can react with an antibody to selectively transfer the first chemical functional group to lysine 246 or lysine 248 in the Fc domain of the antibody.

[0764] According to the present invention, a kit for preparing R1′-L2-SSFI is disclosed, wherein the kit is used for selectively transferring a first chemical functional group to lysine 246 or lysine 248 in the Fc domain of an antibody.

[0765] As an example of the method for preparing a reagent for transferring a first chemical functional group to an antibody as described in Section 4.2, the present invention provides a kit for preparing R1'-L2-SSAI, characterized in that it comprises:

[0766] A joint according to the invention; and

[0767] According to the site-specific antibody interactome of the present invention,

[0768] Wherein, D2 in the joint is C y Alkylene, C y Alkenylene or C y an alkynylene group, and

[0769] D3 in the site-specific antibody interactome is C x Alkylene,

[0770] Wherein, y is an integer greater than or equal to 1, and the sum of x and y is 6≤x+y≤8.

[0771] According to the present invention, a method of preparing an antibody having a first chemical functional group specifically transferred to lysine 248 in its Fc domain is disclosed.

[0772] As an example of the method for preparing an antibody containing a first chemical functional group as described in Section 5.2, the present invention provides a method for preparing R1'-Ab, characterized in that it comprises:

[0773] The reagent for transferring the first chemical functional group to the antibody according to the present invention reacts with the antibody or a fragment thereof,

[0774] Wherein, in the reagent for transferring the first chemical functional group to the antibody, the distance (L) between the β carbon of (Xa1)' and the first carbonyl carbon c ) is shorter than about

[0775] In a specific embodiment, in the reagent for transferring the first chemical functional group to the antibody, D3 is C x Alkylene, and D2 is C y Alkylene, C y Alkenylene or C y Alkynylidene,

[0776] Here, y may be an integer greater than or equal to 1, and the sum of x and y may be 1≤x+y≤5.

[0777] According to the present invention, a kit for preparing an antibody having a first chemical functional group specifically transferred to lysine 248 in its Fc domain is disclosed.

[0778] As an example of the method for preparing an antibody containing a first chemical functional group as described in Section 5.2, the present invention provides a kit for preparing R1'-Ab, characterized in that it comprises:

[0779] A reagent for transferring a first chemical functional group to the antibody according to the present invention; and

[0780] an antibody or a fragment thereof,

[0781] Wherein, in the reagent for transferring the first chemical functional group to the antibody, the distance (L) between the β carbon of (Xa1)' and the first carbonyl carbon c ) is shorter than about

[0782] In a specific embodiment, in the reagent for transferring the first chemical functional group to the antibody, D3 is C x Alkylene, and D2 is C y Alkylene, C y Alkenylene or C y Alkynylidene,

[0783] Here, y may be an integer greater than or equal to 1, and the sum of x and y may be 1≤x+y≤5.

[0784] Alternatively, as an example of the method for preparing an antibody containing a first chemical functional group as described in Section 5.2, the present invention provides a kit for preparing R1'-Ab, characterized in that it comprises:

[0785] A joint according to the invention;

[0786] The site-specific antibody interactome according to the invention; and

[0787] an antibody or a fragment thereof,

[0788] Wherein, D2 in the joint is C y Alkylene, C y Alkenylene or C y an alkynylene group, and

[0789] D3 in the site-specific antibody interactome is C x Alkylene,

[0790] Here, y is an integer greater than or equal to 1, and the sum of x and y is 1≤x+y≤5.

[0791] According to the present invention, a method of preparing an antibody having a first chemical functional group specifically transferred to lysine 246 in its Fc domain is disclosed.

[0792] As an example of the method for preparing an antibody containing a first chemical functional group as described in Section 5.2, the present invention provides a method for preparing R1'-Ab, characterized in that it comprises:

[0793] allowing the antibody or fragment thereof to react with a reagent for transferring a first chemical functional group to the antibody according to the present invention,

[0794] Wherein, in the reagent for transferring the first chemical functional group to the antibody, the distance (L) between the β carbon of (Xa1)' and the first carbonyl carbon c) is longer than about

[0795] In a specific embodiment, in the reagent for transferring the first chemical functional group to the antibody, D3 is C x Alkylene, and D2 is C y Alkylene, C y Alkenylene or C y Alkynylidene,

[0796] Here, y may be an integer greater than or equal to 1, and the sum of x and y may be 9≤x+y≤12.

[0797] According to the present invention, a kit for preparing an antibody having a first chemical functional group specifically transferred to lysine 246 in its Fc domain is disclosed.

[0798] As an example of the method for preparing an antibody containing a first chemical functional group as described in Section 5.2, the present invention provides a kit for preparing R1'-Ab, characterized in that it comprises:

[0799] A reagent for transferring a first chemical functional group to an antibody of the invention; and

[0800] an antibody or a fragment thereof,

[0801] Wherein, in the reagent for transferring the first chemical functional group to the antibody, the distance (L) between the β carbon of (Xa1)' and the first carbonyl carbon c ) is longer than about

[0802] In a specific embodiment, in the reagent for transferring the first chemical functional group to the antibody, D3 is C x Alkylene, and D2 is C y Alkylene, C y Alkenylene or C y Alkynylidene,

[0803] Here, y may be an integer greater than or equal to 1, and the sum of x and y may be 9≤x+y≤12.

[0804] Alternatively, as an example of the method for preparing an antibody containing a first chemical functional group as described in Section 5.2, the present invention provides a kit for preparing R1'-Ab, characterized in that it comprises:

[0805] A joint according to the invention;

[0806] The site-specific antibody interactome according to the invention; and

[0807] an antibody or a fragment thereof,

[0808] Wherein, D2 in the joint is C y Alkylene, C y Alkenylene or C y an alkynylene group, and

[0809] D3 in the site-specific antibody interaction group is C x Alkylene,

[0810] Here, y is an integer greater than or equal to 1, and the sum of x and y is 9≤x+y≤12.

[0811] According to the present invention, a method for preparing an antibody having a first chemical functional group selectively transferred to lysine 246 or lysine 248 in its Fc domain is disclosed.

[0812] As an example of the method for preparing an antibody containing a first chemical functional group as described in Section 5.2, the present invention provides a method for preparing R1'-Ab, characterized in that it comprises:

[0813] The reagent for transferring the first chemical functional group to the antibody according to the present invention reacts with the antibody or a fragment thereof,

[0814] Wherein, in the reagent for transferring the first chemical functional group to the antibody, the distance (L) between the β carbon of (Xa1)' and the first carbonyl carbon c ) is longer than or equal to approximately and shorter than or equal to approximately

[0815] In a specific embodiment, in the reagent for transferring the first chemical functional group to the antibody, D3 is C x Alkylene, and D2 is C y Alkylene, C y Alkenylene or C y Alkynylidene,

[0816] Here, y may be an integer greater than or equal to 1, and the sum of x and y may be 6≤x+y≤8.

[0817] According to the present invention, a kit for preparing an antibody having a first chemical functional group selectively transferred to lysine 246 or lysine 248 in its Fc domain is disclosed.

[0818] As an example of the method for preparing an antibody containing a first chemical functional group as described in Section 5.2, the present invention provides a kit for preparing R1'-Ab, characterized in that it comprises:

[0819] A reagent for transferring a first chemical functional group to the antibody according to the present invention; and

[0820] an antibody or a fragment thereof,

[0821] Wherein, in the reagent for transferring the first chemical functional group to the antibody, the distance (L) between the β carbon of (Xa1)' and the first carbonyl carbon c ) is longer than or equal to approximately and shorter than or equal to approximately

[0822] In a specific embodiment, in the reagent for transferring the first chemical functional group to the antibody, D3 is C x Alkylene, and D2 is C y Alkylene, C y Alkenylene or C y Alkynylidene,

[0823] Here, y may be an integer greater than or equal to 1, and the sum of x and y may be 6≤x+y≤8.

[0824] Alternatively, as an example of the method for preparing an antibody containing a first chemical functional group as described in Section 5.2, the present invention provides a kit for preparing R1'-Ab, characterized in that it comprises:

[0825] A joint according to the invention;

[0826] The site-specific antibody interactome according to the invention; and

[0827] an antibody or a fragment thereof,

[0828] Wherein, D2 in the joint is C y Alkylene, C y Alkenylene or C y an alkynylene group, and

[0829] D3 in the site-specific antibody interaction group is C x Alkylene,

[0830] Wherein, y is an integer greater than or equal to 1, and the sum of x and y is 6≤x+y≤8.

[0831] According to the present invention, a method of making an antibody having a first chemical functional group transferred to both lysines 246 and 248 in its Fc domain is disclosed.

[0832] The present invention provides a method for preparing R1'-Ab, the method comprising:

[0833] a first reagent for transferring a first chemical functional group to the antibody reacts with the antibody or a fragment thereof; and

[0834] A second reagent for transferring a first chemical functional group to the antibody reacts with the antibody or a fragment thereof.

[0835] In a specific embodiment, the first reagent for transferring the first chemical functional group to the antibody can be R1'-L2-SSFI, which is used to specifically transfer the first chemical functional group to lysine 248 in the Fc domain as described above, and the second reagent for transferring the first chemical functional group to the antibody can be R1'-L2-SSFI, which is used to specifically transfer the first chemical functional group to lysine 246 in the Fc domain as described above.

[0836] In a specific embodiment, the first reagent for transferring the first chemical functional group to the antibody can be R1'-L2-SSFI, which is used to specifically transfer the first chemical functional group to lysine 246 in the Fc domain as described above, and the second reagent for transferring the first chemical functional group to the antibody can be R1'-L2-SSFI, which is used to specifically transfer the first chemical functional group to lysine 248 in the Fc domain as described above.

[0837] In a specific embodiment, the reaction of the first reagent for transferring the first chemical functional group to the antibody with the antibody or a fragment thereof, and the reaction of the second reagent for transferring the first chemical functional group to the antibody with the antibody or a fragment thereof can be performed sequentially.

[0838] According to the present invention, a kit for preparing an antibody having a first chemical functional group transferred to both lysine 246 and lysine 248 in its Fc domain is disclosed.

[0839] The present invention provides a kit for preparing R1'-Ab, the kit comprising:

[0840] a first reagent for transferring a first chemical functional group to the antibody;

[0841] a second reagent for transferring the first chemical functional group to the antibody; and

[0842] Antibodies or fragments thereof.

[0843] In a specific embodiment, the first reagent for transferring the first chemical functional group to the antibody can be R1'-L2-SSFI, which is used to specifically transfer the first chemical functional group to lysine 248 in the Fc domain as described above, and the second reagent for transferring the first chemical functional group to the antibody can be R1'-L2-SSFI, which is used to specifically transfer the first chemical functional group to lysine 246 in the Fc domain as described above.

[0844] In a specific embodiment, the first reagent for transferring the first chemical functional group to the antibody can be R1'-L2-SSFI, which is used to specifically transfer the first chemical functional group to lysine 246 in the Fc domain as described above, and the second reagent for transferring the first chemical functional group to the antibody can be R1'-L2-SSFI, which is used to specifically transfer the first chemical functional group to lysine 248 in the Fc domain as described above.

[0845] 5.5. By adjusting the reactivity of the first carbonyl group, the first chemical functional group can be transferred more site-specifically.

[0846] As described in Section 5.2 above, an antibody containing a first chemical functional group can be prepared by attacking the first carbonyl group of R1'-L2-SSFI by a nucleophile of the antibody. The first carbonyl group is characterized in that it has a milder reactivity than the second carbonyl group of the linker (see Section 2.2). The reactivity of the first carbonyl group can be adjusted so that a specific amine group of the antibody can react with the first carbonyl group only when the specific amine group is near the first carbonyl group. In this way, the present invention allows the first carbonyl group to not react well with any amine group through the vicinity conditions of the reaction, and makes the position specificity of the reaction high.

[0847] The prior art disclosed in publication number US2018 / 0141976A1 and WO 2018 / 199337A1 aims to selectively regulate lysine 246 or lysine 248 in the Fc domain using analogs of Fc-III. Because the prior art uses a disuccinimide cross-linker, the first carbonyl group and the second carbonyl group have the same high reactivity (see Section 2.2). Therefore, since the first carbonyl group has high reactivity and does not have nearby conditions for reaction, any lysine residue is very likely to be labeled.

[0848] 5.6. The antibody containing the first chemical functional group prepared according to the present invention has high homogeneity and yield.

[0849] The technical problem and technical solution of specifically transferring (1) the "desired number" of first chemical functional groups to (2) "specific sites of antibodies" have been described in detail with reference to Sections 5.3 and 5.4. As can be seen from the following experimental examples, it can be seen that the chemical functional groups of antibodies containing first chemical functional groups prepared by the present technical solution are transferred to specific lysine residues in their Fc domains with high specificity and high yield (see Experimental Example 3.3). Therefore, the present invention is completed based on these facts.

[0850] As shown in the background art, the conjugate product having high uniformity due to antibody labeling has the following advantages: (1) the function of the antibody conjugate is uniformly guaranteed, (2) the antibody conjugate is safe because its effect is predictable, and (3) the decline of antibody function can be avoided because the functional region of the antibody can be avoided while labeling the antibody.

[0851] The prior art disclosed in publication Nos. US2018 / 0141976A1 and WO 2018 / 199337A1 aims to selectively regulate lysine 246 or lysine 248 in the Fc domain using analogs of Fc-III. However, according to the prior art, the uniformity and yield of the antibody may be inevitably low for two reasons. According to the prior art, first, due to the distance (L) between the β carbon of (Xa1)' and the first carbonyl carbon, c ) is about Therefore, it is possible to selectively label lysine at position 246 or 248, but it is not possible to specifically select one of lysine 246 and lysine 248 to be labeled. Secondly, the first carbonyl group of the cross-linking agent according to the prior art has no proximity conditions for reaction due to high reactivity (see Section 5.5). As a result, the first carbonyl group is very likely to react with any lysine residue. Therefore, the prior art has the following disadvantages: the uniformity and yield of the antibody-labeled product are not as good as those of the present invention.

[0852] 5.7. Since the antibody containing the first chemical functional group according to the present invention does not have a blocked FcRn binding site therein, the function of the antibody is not reduced.

[0853] The design principles of the antibody labeling site are: (1) spaced from the paratope; and (2) spaced from the FcR recognition site including FcRn described in Section 1.1 above. Since lysine 246 and lysine 248 are included in the Fc domain, lysine 246 and lysine 248 are spaced from the paratope, but the corresponding site may overlap with the recognition site of FcRn, which may be problematic.

[0854] FcRn is known to have multiple functions, and in particular, plays an important role in extending the half-life of antibodies by participating in IgG recycling. When the antibody is used in vivo, that is, when the antibody is used as a therapeutic agent, contrast agent, etc., the interaction between FcRn and the antibody may not occur smoothly, thereby making the antibody unable to function normally due to the short half-life of the antibody.

[0855] 5.7.1. Lysine 246 and Lysine 248 are spaced from the FcRn binding site of the antibody

[0856] The spacing between a lysine residue in the Fc domain and the FcRn binding site of Fc is an important consideration in selecting a lysine residue in the Fc domain. Fig.13 The binding structure between the Fc domain and FcRn and the positions of the FcRn binding site and lysine 246 and lysine 248 in the Fc domain are shown. Papers and computer modeling were used to show the binding structure between the Fc domain and FcRn (Ying T, Ju TW, Wang Y, Prabakaran P, Dimitrov DS., Interactions of IgG1 CH2 and CH3Domains with FcRn, Front Immunol. 2014; 5: 146; Monnet C, Jorieux S, Urbain R et al., Selection of IgG Variants with Increased FcRnBinding Using Random and Directed Mutagenesis: Impact on EffectorFunctions. Front Immunol. 2015; 6: 39) (see Fig.13 The FcRn binding site disclosed in the same article data and lysine 246 and lysine 248 are shown in the Fc domain (see Fig.13 Referring to these two figures, it can be seen that the side chains of lysine 246 and lysine 248 point in the opposite direction to the FcRn binding site.

[0857] From these facts, it is expected that when selecting an antibody tag site, the antibody tag site will not affect the interaction between FcRn and the Fc domain. In fact, it was confirmed that the half-life of the prepared antibody was not shortened, so that the antibody tag site would not affect the combination of FcRn as intended (see Experimental Example 5).

[0858] 5.8. The method for preparing an antibody containing a first chemical functional group according to the invention is characterized in that SSAI leaves

[0859] As can be seen from Scheme 4 and Scheme 5 of Section 5.2, the process for preparing R1'-Ab according to the present invention is characterized in that SSAI leaves the final product R1'-Ab because SSAI is included in the leaving group of the nucleophilic substitution reaction. Compared with the binding site between Fc and FcRn, the binding structure between SSFI according to the present invention and the Fc domain was analyzed (see Section 3.2). The results confirmed that a portion of the FcRn binding site was covered by SSFI ( Fig.14 In this regard, it can be expected that when the SSFI is not removed during the preparation of R1'-Ab, the SSFI has a negative impact on the physical properties of the antibody (eg, half-life, etc.).

[0860] The prior art disclosed in publication number US2018 / 0141976A1 aims to selectively regulate lysine 246 or lysine 248 in the Fc domain using an analog site of Fc-III. However, the corresponding prior art is different from the present invention in that SSFI is included in the final antibody-labeled product because the SSFI does not leave during the preparation process.

[0861] The prior art disclosed in publication number WO 2018 / 199337A1 aims to selectively regulate lysine 246 or lysine 248 in the Fc domain using an analog of Fc-III, wherein SSFI is not included in the final antibody-labeled product. However, the corresponding prior art further includes cutting "a cleavable linker which is a divalent group" during the preparation process to remove the SSFI, but the advantage of the present invention is that SSFI leaves during the coupling reaction without any additional process. The cutting of the cleavable linker which is a divalent group according to the corresponding invention has the disadvantage that the disulfide bond forming the antibody structure can be cut. Since the present invention adopts a nucleophilic substitution reaction, which can easily occur without any special conditions, this problem can be significantly improved.

[0862] 5.9. Since the antibody comprising a first click chemistry functional group according to the present invention does not comprise a highly bioreactive chemical functional group, no side reactions other than the conjugate formation reaction occur.

[0863] The antibody prepared by the method for preparing R1'-Ab of the present invention has a structure represented by Formula 7:

[0864] [Formula 7]

[0865]

[0866] When R1' is transferred to lysine 246 or lysine 248, X4 can be NH, and D1 can be any alkylene, alkenylene or alkynylene. In this case, the -NH-(CO)- connected to the antibody is an amide bond that is generally stable in vivo. D1 also generally has a structure with low biological reactivity. Therefore, it is expected that the R1'-Ab prepared according to the present invention has high safety because the high biological reactivity structure is not added to molecules other than the labeled molecule (R1').

[0867] In particular, when R1'-Ab is H1-Ab having a click chemistry residue transferred thereto, a highly bioreactive structure is not added to R1'-Ab. Therefore, the yield of the click chemistry reaction can be improved because no secondary reaction occurs in addition to the click chemistry reaction.

[0868] The prior art disclosed in publication number WO 2018 / 199337A1 aims to use analogs of Fc-III and use biorthogonal functional groups to site-selectively regulate lysine 246 or lysine 248 in the Fc domain. However, the difference between the corresponding prior art and the present invention is that in the cleavage process of "cleavable linker as a divalent group", the antibody product according to the corresponding prior art is allowed to have additional chemical functional groups, such as thiol, hydroxyl, carboxylic acid, phosphoric acid, amine, etc. This additional chemical functional group is a bioreactive functional group and may therefore affect the safety of the antibody product.

[0869] 6. Payload (C m -H2)

[0870] According to the present invention, a payload is disclosed. Such a compound is referred to herein by the symbol "C m -H2" is shown.

[0871] The present invention provides C represented by Formula 9 m -H2:

[0872] [Formula 9]

[0873] C m ————H2

[0874] Among them, C m is the load part,

[0875] H2 is the second click chemistry functional group.

[0876] In a specific embodiment, C mIt may include a carrier moiety, a fluorescent moiety, a drug moiety or a radioactive moiety. R1' may also include a drug moiety. In addition, R1' may include a VC linker. In other embodiments, R1' may include an antibody or an analog thereof, wherein the antibody or the analog thereof includes a paratope.

[0877] In a specific embodiment, when C m When a drug moiety is included, the drug moiety can be an anticancer drug. The anticancer drug can also include one or more selected from the following: DM1, DM3, DM4, abrin, ricin A, Pseudomonas exotoxin, cholera toxin, diphtheria toxin, tumor necrosis factor, α-interferon, β-interferon, nerve growth factor, platelet-derived growth factor, tissue plasminogen activator, cytokine, apoptosis inducer, anti-angiogenic agent, lymphokine, taxane, DNA-alkylating agent, anthracycline, Tubulysin analog, duocarmycin analog, auristatin E, auristatin F. maytansinoids, cytotoxic agents containing reactive polyethylene glycol residues, taxon, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenotoposide, vincristine, vinblastine, t. colchicine, doxorubicin, daunorubicin, dihydroxyanthraquinone, mitoxantrone, mithramycin, actinomycin D, 1-dihydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin, methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbamide, nitrogen mustard, thiotepa, chlorambucil, melphalan , carmustine, lomustine, cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, cisplatin, dactinomycin, bleomycin, anthramycin, calicheamicin, abiraterone, bendamustine, bortezomib, carboplatin, cabazitaxel, dasatinib, docetaxel, epirubicin, erlotinib, everolimus, gemcitabine, gefitinib, idarubicin, imatinib, hydroxyurea, lapatinib, leuprolide, melphalan, nedaplatin, nilotinib, oxaliplatin, pazopanib, pemetrexed, picoplatin, romidepsin, satraplatin, sorafenib, vemurafenib, sunitinib, teniposide, triplatin, and vinorelbine.

[0878] In a specific embodiment, C m It may include multiple carrier moieties, fluorescent moieties, drug moieties, or radioactive moieties. m Two or more drug moieties may also be included. m A VC connector may be included.

[0879] In a specific embodiment, H2 may include any one selected from alkynes, azides, strained alkynes, dienes, dienophiles, olefins, thiols, and tetrazines. In addition, H2 may be an azide or a strained alkyne. In addition, H2 may be an azide or a dibenzocyclooctyne-amine. In addition, H2 may be a diene or a dienophile. In addition, H2 may be a tetrazine or a norbornene. Alternatively, H2 may be a tetrazine or a trans-cyclooctene.

[0880] In a specific embodiment, when H2 reacts with the H1-Ab according to the present invention, H2 can be complementary to the first click chemistry functional group (H1).

[0881] 7. Antibody-Payload Conjugates (C m -Ab)

[0882] According to the present invention, conjugates of antibodies and payloads (i.e., antibody-payload conjugates) are disclosed. Such compounds are referred to herein by the symbol "C m -Ab" is shown.

[0883] The present invention provides C represented by Formula 10 m -Ab:

[0884] [Formula 10]

[0885]

[0886] Among them, C m is the load part,

[0887] B is a structure formed by a click chemistry reaction between the first click chemistry functional group and the second click chemistry functional group,

[0888] D1 is any alkylene, alkenylene or alkynylene group,

[0889] X4 is NH, O or S, and

[0890] Ab is an antibody.

[0891] The contents of the payload section apply to the contents disclosed in Section “6. Payload”.

[0892] In a specific embodiment, B may be a structure formed by a click chemistry reaction between any one selected from alkynes, azides, strained alkynes, dienes, dienophiles, alkenes, thiols and tetrazines and their partner "click chemistry" functional groups. B may also be any one selected from the following:

[0893] Where A1 and A2 are both connected to the load section or D1 so that A1 and A2 are not connected to the same section, and Rx Can be selected from H, halogen and C 1-3 In addition, B can be In addition, B can be

[0894] In a specific embodiment, D1 may include a covalent bond, a C 1-4 Alkylene, C 2-4 Alkenylene, C 2-4 Alkynylidene and C 3-8 Any of cycloalkylene. In addition, D1 may be -CH2OCH2-. In addition, D1 may be a covalent bond.

[0895] In a specific embodiment, X4 may be NH.

[0896] In a specific embodiment, Ab can be a human antibody. In other specific embodiments, Ab can be a non-human animal antibody. In a specific embodiment, Ab can be an immunoglobulin G (IgG). In a specific embodiment, Ab can be a complete antibody. In other specific embodiments, Ab can be a fragment of the antibody.

[0897] In a specific embodiment, X4 and Ab can be connected through the Fab domain of Ab. In other specific embodiments, X4 and Ab can be connected through the Fc domain of Ab. In addition, X4 and Ab can be connected through lysine 246 or lysine 248 in the Fc domain of Ab. In addition, X4 and Ab can be connected through lysine 246 in the Fc domain of Ab. In addition, X4 and Ab can be connected through lysine 248 in the Fc domain of Ab. Or, X4 and Ab can be connected through lysine 246 and lysine 248 in the Fc domain of Ab. In a specific embodiment, X4 and Ab can be connected only through one of the two Fc domains of Ab. In other specific embodiments, X4 and Ab can be connected through two Fc domains of Ab.

[0898] The present invention provides an antibody or a fragment thereof comprising the amino acid sequence of the following formula 10-1:

[0899] [Formula 10-1]

[0900] GPSVFLFPP-(K)″-PKDTLMI(SEQ ID NO:23)

[0901] wherein G is glycine, P is proline, S is serine, V is valine, F is phenylalanine, L is leucine, K is lysine, D is aspartic acid, T is threonine, M is methionine, and I is isoleucine, and

[0902] (K)”

[0903] Among them, C m is the load part,

[0904] B is a structure formed by a click chemistry reaction between the first click chemistry functional group and the second click chemistry functional group, and

[0905] D1 is any alkylene, alkenylene or alkynylene. The antibody or fragment thereof has a payload moiety coupled to lysine 246, or a site corresponding to lysine 246, in its Fc domain.

[0906] The contents of the payload section apply to the contents disclosed in Section “6. Payload”.

[0907] In a specific embodiment, B can be a structure formed by a click chemistry reaction between any one selected from alkynes, azides, strained alkynes, dienes, dienophiles, alkenes, thiols and tetrazines and their partner "click chemistry functional groups". B can also be any one selected from the following:

[0908] Where both A1 and A2 are connected to the load part or D1 so that A1 and A2 are not connected to the same part, and R x Can be selected from H, halogen and C 1-3 In addition, B can be In addition, B can be

[0909] In a specific embodiment, D1 may include a covalent bond, a C 1-4 Alkylene, C 2-4 Alkenylene, C 2-4 Alkynylidene and C 3-8 Any one of cycloalkylene. D1 can also be -CH2OCH2-. In addition, D1 can be a covalent bond.

[0910] In a specific embodiment, the antibody can be a human antibody. In other specific embodiments, the antibody can be a non-human animal antibody. In a specific embodiment, the antibody can be an immunoglobulin G (IgG). In a specific embodiment, the antibody can be a complete antibody. In other specific embodiments, the antibody can be a fragment of an antibody.

[0911] In a specific embodiment, the antibody may comprise the amino acid sequence of Formula 10-1 in only one of its two Fc domains. In other specific embodiments, the antibody may comprise the amino acid sequence of Formula 10-1 in both of its two Fc domains.

[0912] The present invention provides an antibody or a fragment thereof comprising the amino acid sequence of the following formula 10-2:

[0913] [Formula 10-2]

[0914] GPSVFLFPPKP-(K)″-DTLMI(SEQ ID NO:24)

[0915] wherein G is glycine, P is proline, S is serine, V is valine, F is phenylalanine, L is leucine, K is lysine, D is aspartic acid, T is threonine, M is methionine, and I is isoleucine, and

[0916] (K)”

[0917] Among them, C m is the load part,

[0918] B is a structure formed by a click chemistry reaction between the first click chemistry functional group and the second click chemistry functional group, and

[0919] D1 is any alkylene, alkenylene or alkynylene. The antibody or fragment thereof has a payload moiety coupled to lysine 246, or a site corresponding to lysine 246, in its Fc domain.

[0920] The contents of the payload section apply to the contents disclosed in Section “6. Payload”.

[0921] In a specific embodiment, B can be a structure formed by a click chemistry reaction between any one selected from alkynes, azides, strained alkynes, dienes, dienophiles, alkenes, thiols and tetrazines and their partner "click chemistry functional groups". B can also be any one selected from the following:

[0922] Where both A1 and A2 are connected to the load part or D1 so that A1 and A2 are not connected to the same part, and R x Can be selected from H, halogen and C 1-3 In addition, B can be In addition, B can be

[0923] In a specific embodiment, D1 may include a covalent bond, a C 1-4 Alkylene, C 2-4 Alkenylene, C 2-4 Alkynylidene and C 3-8Any of cycloalkylene. D1 can also be -CH2OCH2-. In addition, D1 can be a covalent bond.

[0924] In a specific embodiment, the antibody can be a human antibody. In other specific embodiments, the antibody can be a non-human animal antibody. In a specific embodiment, the antibody can be an immunoglobulin G (IgG). In a specific embodiment, the antibody can be a complete antibody. In other specific embodiments, the antibody can be a fragment of an antibody.

[0925] In a specific embodiment, the antibody may comprise the amino acid sequence of Formula 10-2 in only one of its two Fc domains. In other specific embodiments, the antibody may comprise the amino acid sequence of Formula 10-2 in both of its two Fc domains.

[0926] The present invention provides an antibody or a fragment thereof comprising the amino acid sequence of the following formula 10-3:

[0927] [Formula 10-3]

[0928] GPSVFLFPP-(K)″-DTLMI(SEQ ID NO:25)

[0929] wherein G is glycine, P is proline, S is serine, V is valine, F is phenylalanine, L is leucine, K is lysine, D is aspartic acid, T is threonine, M is methionine, and I is isoleucine, and

[0930] (K)” are each independently Among them C m is the load part,

[0931] B is a structure formed by a click chemistry reaction between the first click chemistry functional group and the second click chemistry functional group, and

[0932] D1 is any alkylene, alkenylene or alkynylene. The antibody or fragment thereof has a payload moiety coupled to lysine 246, or a site corresponding to lysine 246, in its Fc domain.

[0933] The contents of the payload section apply to the contents disclosed in Section “6. Payload”.

[0934] In a specific embodiment, B may be a structure formed by a click chemistry reaction between any one selected from alkynes, azides, strained alkynes, dienes, dienophiles, alkenes, thiols and tetrazines and their partner "click chemistry" functional groups. B may also be any one selected from the following:

[0935] Where both A1 and A2 are connected to the load part or D1 so that A1 and A2 are not connected to the same part, and R x Can be selected from H, halogen and C 1-3 In addition, B can be In addition, B can be

[0936] In a specific embodiment, D1 may include a covalent bond, a C 1-4 Alkylene, C 2-4 Alkenylene, C 2-4 Alkynylidene and C 3-8 Any of cycloalkylene. D1 can also be -CH2OCH2-. In addition, D1 can be a covalent bond.

[0937] In a specific embodiment, the antibody can be a human antibody. In other specific embodiments, the antibody can be a non-human animal antibody. In a specific embodiment, the antibody can be an immunoglobulin G (IgG). In a specific embodiment, the antibody can be a complete antibody. In other specific embodiments, the antibody can be a fragment of an antibody.

[0938] In a specific embodiment, the antibody may comprise the amino acid sequence of Formula 10-3 in only one of its two Fc domains. In other specific embodiments, the antibody may comprise the amino acid sequence of Formula 10-3 in both of its two Fc domains.

[0939] The present invention provides an antibody or a fragment thereof comprising one or more amino acid sequences selected from Formula 10-1, Formula 10-2 and Formula 10-3. In this case, the contents of Formula 10-1 to Formula 10-3 are as described above.

[0940] In a specific embodiment, D1 can be a covalent bond.

[0941] In a specific embodiment, the antibody or fragment thereof may include only the amino acid sequence of Formula 10-1 and may not include the amino acid sequences of Formula 10-2 and Formula 10-3. In addition, the antibody or fragment thereof may include the amino acid sequence of Formula 10-1 only in one of its two Fc domains. In addition, the antibody may include the amino acid sequence of Formula 10-1 in both of its two Fc domains.

[0942] In other embodiments, the antibody or fragment thereof may include only the amino acid sequence of Formula 10-2, but may not include the amino acid sequences of Formula 10-1 and Formula 10-3. The antibody or fragment thereof may also include the amino acid sequence of Formula 10-2 in only one of its two Fc domains. In addition, the antibody may include the amino acid sequence of Formula 10-2 in both of its two Fc domains.

[0943] In a specific embodiment, the antibody or fragment thereof may include only the amino acid sequence of Formula 10-3, and may not include the amino acid sequences of Formula 10-1 and Formula 10-2. The antibody or fragment thereof may also include the amino acid sequence of Formula 10-3 only in one of its two Fc domains. In addition, the antibody may include the amino acid sequence of Formula 10-3 in both of its two Fc domains.

[0944] 7.1. Antibody-drug conjugates (ADCs)

[0945] According to the present invention, novel antibody-drug conjugates (ADCs) are disclosed. The ADCs according to the present invention refer to payloads in antibody-payload conjugates that include a drug moiety.

[0946] The present invention provides C m -Ab, wherein the cargo moiety comprises a drug moiety.

[0947] In specific embodiments, the payload moiety may include two or more drug moieties.

[0948] In a specific embodiment, the drug moiety can be an anticancer drug. The anticancer drug can also include one or more selected from the following: DM1, DM3, DM4, abrin, ricin A, Pseudomonas exotoxin, cholera toxin, diphtheria toxin, tumor necrosis factor, α-interferon, β-interferon, nerve growth factor, platelet-derived growth factor, tissue plasminogen activator, cytokine, apoptosis inducer, anti-angiogenic agent, lymphokine, taxane, DNA-alkylating agent, anthracycline, Tubulysin analog, duocarmycin analog, auristatin E, auristatin F. maytansinoids, cytotoxic agents containing reactive polyethylene glycol residues, taxon, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenotoposide, vincristine, vinblastine, t. colchicine, doxorubicin, daunorubicin, dihydroxyanthraquinone, mitoxantrone, mithramycin, actinomycin D, 1-dihydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin, methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbamide, nitrogen mustard, thiotepa, chlorambucil, melphalan , carmustine, lomustine, cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, cisplatin, dactinomycin, bleomycin, anthramycin, calicheamicin, abiraterone, bendamustine, bortezomib, carboplatin, cabazitaxel, dasatinib, docetaxel, epirubicin, erlotinib, everolimus, gemcitabine, gefitinib, idarubicin, imatinib, hydroxyurea, lapatinib, leuprolide, melphalan, nedaplatin, nilotinib, oxaliplatin, pazopanib, pemetrexed, picoplatin, romidepsin, satraplatin, sorafenib, vemurafenib, sunitinib, teniposide, triplatin, and vinorelbine.

[0949] In a specific embodiment, the load-bearing portion may include a VC connector.

[0950] 7.2. Methods for Preparing Antibody-Payload Conjugates

[0951] According to the present invention, methods of preparing antibody-payload conjugates are disclosed.

[0952] The antibody-payload conjugates of the present invention can be prepared by using C m -H2 is prepared by reacting with H1-Ab. In this case, the contents of H1-Ab apply to the contents disclosed in Section 5.1, and the contents disclosed in Section 6 apply to the C m -H2. When the first click chemistry functional group of H1-Ab and C mWhen the second click chemistry functional group of H1-Ab and C m When the second click chemistry functional group of -H2 acts as a partner click chemistry functional group, a click chemistry reaction can occur to prepare the antibody-payload conjugate according to the present invention. The content of the click chemistry reaction is fully described in the "Definition" section above. The click chemistry reaction is a biorthogonal reaction, and the advantage is that the reaction occurs at a very fast reaction speed and is used to form a strong binding structure. Therefore, the click chemistry reaction has the same advantages as the following Sections 7.3 and 7.4.

[0953] The present invention provides a method for preparing an antibody-payload conjugate, characterized in that the method comprises reacting an antibody containing a first click chemistry functional group according to the present invention with a payload according to the present invention, wherein H2 of the payload is a second click chemistry functional group complementary to the first click chemistry functional group.

[0954] In this case, the antibody provided in Section 5.1 applies to the antibody containing the first click chemistry functional group, and the payload provided in Section 6 applies to the payload. Because all antibodies containing the first click chemistry functional group in Lysine 246 and / or Lysine 248 are disclosed in Section 5.1, it is expected that the antibody-payload conjugates in which the payload moiety is coupled to Lysine 246 and / or Lysine 248 are disclosed to a fully reproducible degree (see Formula 10 and Formula 10-1 to Formula 10-3 in Section 7).

[0955] The present invention provides a kit for preparing an antibody-payload conjugate, characterized in that the kit comprises an antibody containing a first click chemistry functional group according to the present invention; and a payload according to the present invention, wherein H2 of the payload is a second click chemistry functional group complementary to the first click chemistry functional group.

[0956] In this case, the antibody provided in Section 5.1 is applied to the antibody containing the first click chemistry functional group, and the payload provided in Section 6 is applied to the payload.

[0957] Alternatively, the present invention provides a kit for preparing an antibody-payload conjugate, the kit comprising:

[0958] Antibody;

[0959] Reagents for transferring a first click chemistry functional group to an antibody according to the invention;

[0960] Payload according to the invention.

[0961] In a specific embodiment, the reagent for transferring the first click chemistry functional group to the antibody may be H1-L2-SSFI, which is used to specifically transfer the first click chemistry functional group to lysine 246 or lysine 248 in the Fc domain of the antibody according to the present invention. In other specific embodiments, the reagent for transferring the first click chemistry functional group to the antibody may be H1-L2-SSFI, which is used to specifically transfer the first click chemistry functional group to lysine 246 in the Fc domain of the antibody according to the present invention. In another specific embodiment, the reagent for transferring the first click chemistry functional group to the antibody may be H1-L2-SSFI, which is used to specifically transfer the first click chemistry functional group to lysine 248 in the Fc domain of the antibody according to the present invention.

[0962] Alternatively, the present invention provides a kit for preparing an antibody-payload conjugate, the kit comprising:

[0963] Antibody;

[0964] A joint according to the invention;

[0965] The site-specific antibody interactome according to the invention; and

[0966] Payload according to the invention.

[0967] In a specific embodiment, D2 in the linker is C y Alkylene, C y Alkenylene or C y Alkyne, and D3 in the site-specific antibody interaction group is C x Alkylene, wherein y may be an integer greater than or equal to 1. The sum of x and y may also be 1≤x+y≤5. In addition, the sum of x and y may be 6≤x+y≤8. Further, the sum of x and y may be 9≤x+y≤12.

[0968] 7.3. Antibody-payload conjugates according to the invention use a biorthogonal reaction to form a stable bond.

[0969] The antibody-payload conjugate according to the present invention is formed by a click chemistry reaction. The click chemistry reaction is a biorthogonal reaction that does not affect biochemical phenomena that occur naturally in the body. The bonds formed by the biorthogonal reaction are also not recognized by cleavage enzymes in the body. Therefore, the advantage of the antibody-payload conjugate according to the present invention is that the antibody and the payload moiety form a very stable bond in vivo.

[0970] 7.4. Antibody-payload conjugates prepared according to the present invention have high uniformity and yield.

[0971] As described above in Section 5.6, the antibodies according to the present invention containing the first click chemistry functional group have high homogeneity and yield. Therefore, the advantage of the antibody-payload conjugate prepared from the antibody according to the present invention is that it also has high homogeneity and yield. m -The homogeneity of the Ab must be high so that the antibody conjugate has uniform properties.

[0972] In the case of antibody conjugates prepared by the prior art disclosed in Publication Nos. US2018 / 0141976A1 and WO 2018 / 199337A1, since the antibody conjugates have poor yield and uniformity, as described in Section 5.6 above, there may be a negative impact on the uniformity of performance.

[0973] 7.5. Because the antibody-payload conjugates according to the present invention do not have a blocked FcRn binding site therein, the function of the antibody is not reduced.

[0974] According to the present invention, C m -Abs have the same advantages as described in Sections 5.7 and 5.8. In particular, the pharmacokinetic (PK) properties of ADCs for in vivo use may be greatly improved because the ADCs have an increased half-life.

[0975] The FcRn binding site of the ADC prepared by the prior art disclosed in Publication No. US20180141976A1 is blocked because the final antibody-labeled product contains SSFI. Therefore, compared with the ADC according to the corresponding prior art, the ADC according to the present invention has excellent PK properties.

[0976] 7.6. The antibody-drug conjugates according to the present invention are stable because the antibody-drug conjugates do not contain highly bioreactive chemical functional groups.

[0977] According to the present invention, C m -Abs have the same advantages as described in Section 5.9. In particular, the pharmacodynamic (PD) properties of ADCs used in vivo may be greatly improved because ADCs enable smooth antibody drug effects.

[0978] In the case of ADC prepared by the prior art disclosed in Publication No. WO 2018199337A1, additional chemical functional groups are included in the final antibody-labeled product, such as thiol, hydroxyl, carboxylic acid, phosphoric acid, amine, etc. Therefore, the ADC according to the present invention has excellent PD characteristics compared with the ADC according to the corresponding prior art.

[0979] 8. Composition

[0980] According to the present invention, a composition comprising an antibody is disclosed. In this case, the antibody may be an antibody comprising a first chemical functional group according to the present invention. Alternatively, the antibody may be an antibody-payload conjugate according to the present invention. The antibody may also be an antibody-drug conjugate according to the present invention.

[0981] Depending on the function of the antibody contained in the composition, the composition according to the present invention can be used for various applications. For example, when the first chemical functional group or the load part includes a radioactive part, the corresponding composition can be used as a radiocontrast agent, etc. Alternatively, when the first chemical functional group or the load part includes a fluorescent part, the corresponding composition can be used as a label used in enzyme-linked immunosorbent assay (ELISA), etc. Alternatively, when the first chemical functional group or the load part includes a drug part, the corresponding composition can be used as a pharmaceutical composition. In this case, the components of the composition commonly used in the related art fall within the scope of the present invention. Moreover, the composition ratio of the corresponding components commonly used in this area also falls within the scope of the present invention.

[0982] The present invention provides a composition comprising an antibody containing a first chemical functional group according to the present invention. In a specific embodiment, the antibody containing the first chemical functional group can be at least one selected from Formula 7, Formula 7-1 to Formula 7-3, Formula 8, and Formula 8-1 to Formula 8-3.

[0983] The present invention also provides a composition, which includes an antibody containing the first click chemistry functional group according to the present invention. In a specific embodiment, the antibody containing the first click chemistry functional group can be at least one selected from Formula 8 and Formula 8-1 to Formula 8-3.

[0984] Furthermore, the present invention provides a composition comprising an antibody-payload conjugate according to the present invention. In this case, the contents described in Section 7 apply to the contents of the antibody-payload conjugate.

[0985] Alternatively, the present invention provides a composition comprising an antibody-drug conjugate according to the present invention. In this case, the contents described in Section 7.1 apply to the contents of the antibody-drug conjugate.

[0986] Pharmaceutical composition

[0987] The following content relates to a composition, which is a pharmaceutical composition for diagnosis, prevention and / or treatment purposes. Only in the context of "pharmaceutical composition" herein, according to the present invention, R1'-Ab, H1-Ab, C m -Ab and ADC are both used interchangeably with the term "antibody or fragment thereof".

[0988] The present invention provides a pharmaceutical composition comprising an antibody or fragment thereof according to the present invention. The pharmaceutical composition can also be a composition for treating cancer. In addition, the cancer can include any one selected from the following: bladder cancer, bone cancer, brain cancer, breast cancer, heart cancer, cervical cancer, colon cancer, rectal cancer, esophageal cancer, fibrosarcoma, gastric cancer, stomach cancer, head and neck cancer, Kaposi's sarcoma, kidney cancer, leukemia, liver cancer, lung cancer, lymphoma, melanoma, myeloma, ovarian cancer, pancreatic cancer, penile cancer, prostate cancer, testicular germ cell cancer, thymoma and thymic cancer. In addition, the cancer can be breast cancer.

[0989] The present invention provides a method of treatment, which comprises administering to a subject a pharmaceutical composition comprising an antibody or fragment thereof according to the present invention. The method of treatment can also be a method for treating cancer. In addition, the cancer can include any one selected from the following: bladder cancer, bone cancer, brain cancer, breast cancer, heart cancer, cervical cancer, colon cancer, rectal cancer, esophageal cancer, fibrosarcoma, gastric cancer, gastric cancer, head and neck cancer, Kaposi's sarcoma, kidney cancer, leukemia, liver cancer, lung cancer, lymphoma, melanoma, myeloma, ovarian cancer, pancreatic cancer, penile cancer, prostate cancer, testicular germ cell cancer, thymoma and thymic cancer. In addition, the cancer can be breast cancer.

[0990] In order to prepare a pharmaceutical composition or aseptic composition comprising an antibody or its fragment, the antibody or its fragment according to the present invention can be mixed with a pharmaceutically acceptable carrier or excipient. The composition can further include one or more other therapeutic agents suitable for treating or preventing cancer, such as breast cancer, colorectal cancer, lung cancer, multiple myeloma, ovarian cancer, liver cancer, gastric cancer, pancreatic cancer, acute myeloid leukemia, chronic myeloid leukemia, osteosarcoma, squamous cell carcinoma, peripheral nerve sheath tumor, schwannoma, head and neck cancer, bladder cancer, esophageal cancer, Barrett's esophagus cancer, glioblastoma, soft tissue clear cell sarcoma, malignant mesothelioma, neurofibromatosis, renal cancer, melanoma, prostate cancer, benign prostatic hypertrophy (BPH), gynecomastia, rhabdomyosarcoma and endometriosis.

[0991] Formulations of therapeutic and diagnostic agents can be prepared by mixing with physiologically acceptable carriers, excipients or stabilizers, for example, in the form of lyophilized powders, syrups, aqueous solutions, lotions or suspensions (see, e.g., Hardman et al., Goodman and Gilman's The Pharmacological Basis of Therapeutics, McGraw-Hill, New York, NY, 2001; Gennaro, Remington: The Science and Practice of Pharmacy, Lippincott, Williams and Wilkins, New York, NY, 2000; Avis et al. (eds.), Pharmaceutical Dosage Forms: Parenteral Medications, Marcel Dekker, NY, 1993; Lieberman et al. (eds.), Pharmaceutical Dosage Forms: Tablets, Marcel Dekker, NY, 1990; Lieberman et al. (eds.), Pharmaceutical Dosage Forms: Disperse Systems, Marcel Dekker, NY, 1994). Dekker, NY, 1990; Weiner and Kotkoskie, Excipient Toxicity and Safety, Marcel Dekker, Inc., New York, NY, 2000).

[0992] In a specific embodiment, the clinical service form (CSF) of the antibody-drug conjugate according to the present invention is a lyophilisate in a vial containing ADC, sodium succinate and polysorbate 20. The lyophilisate can be reconstituted with water for injection, and the solution includes ADC, sodium succinate, sucrose and polysorbate 20, and the pH value is about 5.0. For continuous intravenous administration, the resulting solution is usually further diluted into a carrier solution.

[0993] The choice of treatment dosing regimen depends on a variety of factors, including the serum or tissue replacement rate of the substance, the level of symptoms, the immunogenicity of the substance, and the accessibility of target cells in the biological matrix. In a specific embodiment, the dosing regimen maximizes the amount of the therapeutic agent to be delivered to the patient to meet an acceptable level of side effects. Therefore, the amount of the biological agent to be administered depends in part on the severity of the specific substance and the condition being treated. Guidelines are available for selecting appropriate doses of antibodies, cytokines, and small molecules (see, e.g., Wawrzynczak, Antibody Therapy, Bios Scientific Pub. Ltd, Oxfordshire, UK, 1996; Kresina (ed.), Monoclonal Antibodies, Cytokines and Arthritis, Marcel Dekker, New York, NY, 1991; Bach (ed.), Monoclonal Antibodies and Peptide Therapy in Autoimmune Diseases, Marcel Dekker, New York, NY, 1993; Baert et al., New Engl. J. Med. 348:601-608, 2003; Milgrom et al., New Engl. J. Med. 341:1966-1973, 1999; Slamon et al., New Engl.J.Med.344:783-792, 2001; Beniaminovitz et al., New Engl.J.Med.342:613-619, 2000; Ghosh et al., New Engl.J.Med.348:24-32, 2003; Lipsky et al., New Engl. J. Med. 343:1594-1602, 2000).

[0994] Suitable dosage is for example determined by a clinician using parameters or factors known in the relevant field or suspected to affect treatment, or using parameters or factors expected to affect treatment. Typically, the dosage starts with an amount slightly smaller than the optimal dose, and then increases in small increments until the desired or optimal effect is achieved with respect to any negative side effects. For example, important diagnostic measures include inflammatory symptoms or the level of the inflammatory cytokines produced.

[0995] The actual dosage level of the active ingredient in the pharmaceutical composition according to the present invention can be varied to achieve an effective amount of the active ingredient that achieves the desired therapeutic response in a particular patient, composition and mode of administration without causing any toxicity in the patient. The selected dosage level can be determined depending on various pharmacokinetic factors, including: the activity of a certain composition of the present invention or its ester, salt or amide used, the route of administration, the time of administration, the secretion rate of the specific compound used, the duration of treatment, other drugs, substances and / or compounds used in combination with the specific compound used, age, sex, weight, condition, overall health and previous medical history of the patient to be treated, and other factors known in the medical field.

[0996] Compositions comprising antibodies or fragments thereof according to the present invention can be administered by continuous infusion, or in a single dose, e.g., daily, weekly, one to seven times per week, once every two weeks, once every three weeks, once every four weeks, once every five weeks, once every six weeks, once every seven weeks, or once every eight weeks. The dosage can be administered intravenously, subcutaneously, topically, orally, intranasally, intrarectally, intramuscularly, intracerebrally, or by inhalation. Specific dosing regimens involve maximum doses or dosing frequencies that avoid significant unwanted side effects.

[0997] For the antibody or fragment thereof according to the present invention, the dosage to be administered to a patient may be in the range of 0.0001 mg / kg to 100 mg / kg (patient body weight). The dosage may be in the range of 0.0001 mg / kg to 20 mg / kg, 0.0001 mg / kg to 10 mg / kg, 0.0001 mg / kg to 5 mg / kg, 0.0001 to 2 mg / kg, 0.0001 to 1 mg / kg, 0.0001 mg / kg to 0.75 mg / kg, 0.0001 mg / kg to 0.5 mg / kg, 0.0001 mg / kg to 0.25 mg / kg, 0.0001 mg / kg to 0.15 mg / kg, 0.0001 mg / kg to 0.10 mg / kg, 0.0001 mg / kg to 0.05 mg / kg, 0.01 mg / kg to 0.25 mg / kg, or 0.01 mg / kg to 0.10 mg / kg (patient body weight). The dosage of the antibody or fragment thereof according to the present invention can be calculated by multiplying the patient's body weight (kilograms (kg)) by the dose to be administered (mg / kg).

[0998] Antibodies according to the present invention or fragments thereof may be administered repeatedly, and may be administered at intervals of at least 1 day, 2 days, 3 days, 5 days, 10 days, 15 days, 30 days, 45 days, 2 months, 75 days, 3 months or at least 6 months. In a specific embodiment, antibodies according to the present invention or fragments thereof may be administered repeatedly every three weeks.

[0999] The effective amount to be administered to a particular patient may vary depending on factors such as the condition to be treated, the patient's overall health, the manner, route and dosage of administration, and the severity of side effects (see, e.g., Maynard et al., A Handbook of SOPs for Good Clinical Practice, Interpharm Press, Boca Raton, Fla., 1996; Dent, Good Laboratory and Good Clinical Practice, Urch Publ, London, UK, 2001).

[1000] Administration can be, for example, by topical or cutaneous application, by intravenous, intraperitoneal, intracerebral, intramuscular, intraocular, intraarterial, intramedullary, intralesional injection or infusion, or by sustained release systems or implants (see, e.g., Sidman et al., Biopolymers 22:547-556, 1983; Langer et al., J. Biomed. Mater. Res. 15:167-277, 1981; Langer, Chem. Tech. 12:98-105, 1982; Epstein et al., Proc. Natl. Acad. Sci. USA 82:3688-3692, 1985; Hwang et al., Proc. Natl. Acad. Sci. USA 77:4030-4034, 1980; U.S. Pat. Nos. 6,350,466 and 6,316,024). If necessary, the composition may also include a solubilizer and a local anesthetic (e.g., lidocaine) to relieve pain at the injection site. For example, the composition may also be prepared using an inhaler or a nebulizer, or may be prepared for pulmonary administration using an atomizer. See, for example, U.S. Patent Nos. 6,019,968, 5,985,320, 5,985,309, 5,934,272, 5,874,064, 5,855,913, 5,290,540, and 4,880,078; and PCT Publication Nos. WO 92 / 19244, WO 97 / 32572, WO 97 / 44013, WO 98 / 31346, and WO 99 / 66903, the entire contents of which are incorporated herein by reference.

[1001] The composition of the present invention can also be administered by one or more various methods known in the relevant field. As recognized by those of ordinary skill in the art, the route of administration and / or mode will depend on the desired result. The route of administration selected according to the antibody or its fragment of the present invention includes intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, intraspinal or other parenteral routes of administration, such as by injection or infusion. Parenteral administration generally represents a mode of administration by injection different from enteral and topical administration, and includes intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcutaneous, intraepidermal, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion, but the present invention is not limited thereto. Alternatively, the composition of the present invention can be administered by non-parenteral routes of administration, such as local, intraepidermal or intramucosal routes of administration, and can be administered, for example, intranasally, orally, vaginally, rectally, sublingually, or topically. In an exemplary embodiment, the antibody or its fragment according to the present invention can be administered by infusion. In another exemplary embodiment, the antibody or fragment thereof according to the present invention may be administered subcutaneously.

[1002] When the antibody or fragment thereof according to the present invention is administered using a controlled release system or a delayed release system, a pump can be used to achieve controlled release or delayed release (see Langer, supra; Sefton, CRC Crit. Ref Biomed. Eng. 14:20, 1987; Buchwald et al., Surgery 88:507, 1980; Saudek et al., N. Engl. J. Med. 321:574, 1989). Polymeric materials can be used to achieve such controlled or delayed release in the treatment of the present invention (see, e.g., Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton, Fla., 1974; Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen and Ball (eds.), Wiley, New York, 1984; Ranger and Peppas, J. Macromol. Sci. Rev. Macromol. Chem. 23:61, 1983; see also Levy et al., Science 228:190, 1985; During et al., Ann. Neurol. 25:351, 1989; Howard et al., J. Neurosurg. 7:71, 1989). 1:105, 1989; U.S. Pat. No. 5,679,377; U.S. Pat. No. 5,916,597; U.S. Pat. No. 5,912,015; U.S. Pat. No. 5,989,463; U.S. Pat. No. 5,128,326; PCT Publication No. WO 99 / 15154; and PCT Publication No. WO 99 / 20253). Examples of polymers used in sustained-release formulations include poly(2-hydroxyethyl methacrylate), poly(methyl methacrylate), poly(acrylic acid), poly(ethylene-co-vinyl acetate), poly(methacrylic acid), polyglycolide (PLG), polyanhydride, poly(N-vinyl pyrrolidone), poly(vinyl alcohol), polyacrylamide, poly(ethylene glycol), polylactide (PLA), poly(lactide-co-glycolide) (PLGA), and polyorthoesters, but the present invention is not limited thereto. In an exemplary embodiment, the polymer used for the sustained-release formulation is inactive, has no filterable impurities, is stable during storage, sterilization, and is biodegradable. A controlled-release system or delayed-release system may be located near a preventive or therapeutic target and therefore requires a fraction of a systemic dose (see, e.g., Goodson, in Medical Applications of Controlled Release, supra, Vol. 2, pp. 115-138, 1984).

[1003] Controlled release systems are discussed in the review article "Langer, Science 249: 1527-1533, 1990." Any existing technology known to those of ordinary skill in the art can be used to produce a sustained release formulation comprising one or more antibodies or fragments thereof according to the invention. See, e.g., U.S. Pat. No. 4,526,938, PCT Publication No. WO 91 / 05548, PCT Publication No. WO 96 / 20698, Ning et al., Radiotherapy & Oncology 39:179-189, 1996; Song et al., PDA Journal of Pharmaceutical Science & Technology 50:372-397, 1995; Cleek et al., Pro. Int'l. Symp. Control. Rel. Bioact. Mater, 24:853-854, 1997; and Lam et al., Proc. Int'l. Symp. Control. Rel. Bioact. Mater, 24:759-760, 1997, the entire contents of which are incorporated herein by reference.

[1004] When the antibody or its fragment according to the present invention is topically administered, the antibody or its fragment can be formulated into an ointment, cream, transdermal patch, lotion, gel, shampoo, spray, aerosol, solution or emulsion or other forms known to those of ordinary skill in the art. For example, see Remington's Pharmaceutical Sciences and Introduction to Pharmaceutical Dosage Forms, 19th edition, Mack Pub. Co., Easton, Pa. (1995). Non-sprayable topical dosage forms include carriers or one or more excipients suitable for topical application. In some cases, viscous, semi-solid or solid forms with a higher kinematic viscosity than water are generally used as topical dosage forms. Suitable preparations include solutions, suspensions, emulsions, creams, ointments, powders, liniments, ointments, etc., all of which are sterilized or mixed with adjuvants (e.g., preservatives, stabilizers, wetting agents, buffers or salts) that affect various properties (e.g., if necessary, such as osmotic pressure), but the present invention is not limited thereto. In some cases, other suitable topical dosage forms include sprayable aerosol formulations filled in a mixture of compressed volatile substances (e.g., gaseous propellants, such as freon) or squeeze bottles after the active ingredient is combined with a solid or liquid inactive carrier. If necessary, moisturizers or wetting agents can also be added to the pharmaceutical composition and dosage form. Examples of such additional components are known in the prior art.

[1005] When the composition comprising the antibody or its fragment is administered intranasally, the composition can be formulated into the form of an aerosol, a spray, atomizer or drops. In particular, suitable propellants (e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gases) can be used for the preventive or therapeutic agent of the present invention to conveniently deliver in an aerosol spray form from a pressurized package or atomizer. In the case of a pressurized aerosol, the dosage unit can be determined by providing a valve to deliver a metered amount. Capsules and cartridges (e.g., composed of gelatin) for inhalers or insufflators can be formulated as a powder mixture comprising a compound and a suitable powder base (e.g., lactose or starch).

[1006] Methods for co-administering or treating with a second therapeutic agent (e.g., a cytokine, a steroid, a chemotherapeutic agent, an antibiotic, or a radiotherapeutic agent) are known in the relevant art, see, for example, Hardman et al., (eds.) (2001) Goodman and Gilman's The Pharmacological Basis of Therapeutics, 10th ed., McGraw-Hill, New York, NY,; Poole and Peterson (eds.) (2001) Pharmacotherapeutics for Advanced Practice: A Practical Approach, Lippincott, Williams & Wilkins, Phila, PA.; Chabner and Longo (eds.) (2001) Cancer Chemotherapy and Biotherapy, Lippincott, Williams & Wilkins, Phila., Pa. The effective amount of the therapeutic agent can reduce symptoms by at least 10%, at least 20%, at least about 30%, at least 40%, or at least 50%.

[1007] Additional therapies (e.g., prophylactic or therapeutic agents) that can be administered in combination with an antibody or fragment thereof according to the present invention can be administered with an antibody or fragment thereof according to the present invention at intervals of less than 5 minutes, less than 30 minutes, 1 hour, about 1 hour, about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 7 hours, about 8 hours, about 9 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 12 hours to 18 hours, 18 hours to 24 hours, 24 hours to 36 hours, 36 hours to 48 hours, 48 ​​hours to 52 hours, 52 hours to 60 hours, 60 hours to 72 hours, 72 hours to 84 hours, 84 hours to 96 hours, or 96 hours to 120 hours. Two or more therapeutic agents may be administered during the same visit to the patient.

[1008] In a specific embodiment, the antibody or fragment thereof according to the present invention can be formulated to ensure appropriate in vivo distribution. For example, the blood-brain barrier (BBB) ​​excludes many highly hydrophilic compounds. In order to ensure that the therapeutic compounds of the present invention pass through the blood-brain barrier (if necessary), they can be formulated in liposomes, for example. For methods of preparing liposomes, for example, see U.S. Patent Nos. 4,522,811, 5,374,548 and 5,399,331. The liposome may include one or more parts that are selectively transported to specific cells or organs, thereby enhancing the delivery of targeted drugs (see, for example, Ranade, (1989) J.Clin.Pharmacol.29:685). Exemplary targeting moieties include folic acid or biotin (see, e.g., U.S. Pat. No. 5,416,016 (to Low et al.)), mannosides (see Umezawa et al., (1988) Biochem. Biophys. Res. Commun. 153:1038), antibodies (see Bloeman et al., (1995) FEBS Lett. 357:140; Owais et al., (1995) Antimicrob. Agents Chemother. 39:180), surfactant protein A receptor (see Briscoe et al., (1995) Am. J. Physiol. 1233:134), p120 (see Schreier et al., (1994) J. Biol. Chem. 269:9090), and the like. See also K. Keinanen; ML Laukkanen (1994) FEBS Lett. 346:123; JJ Killion; IJ Fidler (1994) Immunomethods 4:273.

[1009] The present invention provides a scheme for administering a pharmaceutical composition to a subject in need thereof, alone or in combination with other therapies, the pharmaceutical composition including an antibody or fragment thereof according to the present invention. The therapeutic agent (e.g., a prophylactic or therapeutic agent) for combined therapy of the present invention can be administered to a subject simultaneously or sequentially. The therapeutic agent (e.g., a prophylactic or therapeutic agent) for combined therapy of the present invention can also be administered periodically. Cyclic therapy includes administering a first therapeutic agent (e.g., a first prophylactic or therapeutic agent) for a predetermined time period, administering a second therapeutic agent (e.g., a second prophylactic or therapeutic agent) for a predetermined time period, and then repeating the administration step in sequence, i.e., to reduce the occurrence of drug resistance to one (e.g., an agonist) in the therapeutic agent, and / or prevent or reduce the side effects of one (e.g., an agonist) in the therapeutic agent, and / or improve the predetermined cycle of the efficacy of the therapeutic agent.

[1010] The therapeutic agents (eg, prophylactic agents or therapeutic agents) used in the combination therapies of the invention can be administered to a subject simultaneously.

[1011] The term "simultaneously" means that the treatment (e.g., a prophylactic or therapeutic agent) does not need to be administered at exactly the same time without restriction, but is administered to the subject sequentially with the pharmaceutical composition comprising the antibody or fragment thereof according to the present invention, and is administered at a time interval that can be used to provide a higher benefit compared to when the antibody of the present invention is administered at a different time from other therapies. For example, each therapeutic agent can be administered to the subject in any order at the same time or at different time points; but it should be administered at sufficiently short time intervals to provide the desired therapeutic or preventive effect when administered at different times. Each therapeutic agent can be administered to the subject in any appropriate form by any suitable route of administration. In various exemplary embodiments, the therapeutic agent (e.g., a prophylactic or therapeutic agent) can be administered to a subject at intervals of less than 15 minutes, less than 30 minutes, less than 1 hour, about 1 hour, about 1 hour to about 2 hours, about 2 hours to about 3 hours, about 3 hours to about 4 hours, about 4 hours to about 5 hours, about 5 hours to about 6 hours, about 6 hours to about 7 hours, about 7 hours to about 8 hours, about 8 hours to about 9 hours, about 9 hours to about 10 hours, about 10 hours to about 11 hours, about 11 hours to about 12 hours, 24 hours, 48 ​​hours, 72 hours, or one week. In other exemplary embodiments, two or more therapeutic agents (e.g., prophylactic or therapeutic agents) can be administered during the same visit of a patient.

[1012] The prophylactic or therapeutic agent for combined therapy can be administered to the subject in the same pharmaceutical composition. Alternatively, the prophylactic or therapeutic agent for combined therapy can be administered to the subject simultaneously in a separate pharmaceutical composition. The prophylactic or therapeutic agent can be administered to the subject by the same or different routes of administration.

[1013] Experimental Examples

[1014] 1. Example of Preparation of Compounds for Preparing Reagents for Transferring Site-Specific First Click Chemistry Functional Groups to Antibodies

[1015] 1.1. Synthesis and confirmation of the linker (H1-L1) structure

[1016] 1.1.1. Compound I (SO1 linker: NHS & norbornene)

[1017] [Solution 6]

[1018]

[1019] Synthesis of Compound I (Scheme 6, Fig.15 )

[1020] Synthesis of compound 1

[1021] 2 g (10.98 mmol, 1.0 eq.) of 2-(bicyclo[2.2.1]hept-5-en-2-ylmethoxy)acetic acid was dissolved in 50 mL of DCM, and then 0.085 mL (1.098 mmol, 0.1 eq.) of DMF and 1.91 mL (21.96 mmol, 2.0 eq.) of oxalyl chloride were added dropwise while stirring at room temperature. After the reaction solution was stirred for 3 hours, it was concentrated under reduced pressure to obtain 1.97 g of the target compound (yield 90%).

[1022] Synthesis of compound 2

[1023] 1.97g (9.88mmol, 1.0eq.) of compound 1 was dissolved in 20mL of acetonitrile (ACN), and then 0.68mL (9.88mmol, 1.0eq.) of thioglycolic acid and 2.06mL (14.82mmol, 1.5eq.) of triethylamine were added dropwise while stirring at room temperature. The reaction solution was stirred for 18 hours and then concentrated under reduced pressure. Subsequently, water was added to the reaction solution, and the reaction solution was extracted 3 times with ethyl acetate (EA). The organic layer was dried over magnesium sulfate, concentrated under reduced pressure, and then purified by column chromatography (DCM: MeOH = 10: 1) to obtain 2.05g of the target compound (yield: 79%).

[1024] 1 H NMR (500 MHz, CDCl3) δ 1 H NMR (500MHz, CDCl3) δ6.15-6.01 (m, 2H), 4.20 (t, J=5.0Hz, 1H), 3.77-3.71 (m, 2H), 3.68-3.57 (m, 1H), 3.56-3.45 (m, 1H), 2.84 (s, 2H), 1.81-1.69 (m, 2H), 1.30 (ddd, J=19.8, 14.5, 8.6Hz, 4H), 1.15 (ddd, J=16.0, 7.6, 3.3Hz, 1H).

[1025] Synthesis of Compound I

[1026] 2.05 g (7.81 mmol, 1.0 eq.) of compound 2 was dissolved in 50 mL of ACN, and then 2.76 g (14.44 mmol, 1.8 eq.) of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDCi) and 2.21 g (19.25 mmol, 2.46 eq.) of N-hydroxysuccinimide (NHS) were added while stirring at room temperature. The reaction solution was stirred for 18 hours and then concentrated under reduced pressure. Subsequently, water was added to the reaction solution, and the reaction solution was extracted 3 times with EA. The organic layer was recovered, dried with magnesium sulfate, and then concentrated under reduced pressure. Then, the residue was purified on a silica gel column using chromatography (EA: Hex = 2: 1) to obtain 2.7 g of the target compound (yield: 98%).

[1027] 1 H NMR(500MHz, CDCl3)δ 6.15-6.03 (m, 2H), 4.45 (s, 1H), 4.21 (d, J=5.0Hz, 1H), 3.97 (s, 1H), 3.65 (dd, J=12.0, 7.3Hz, 1H), 3.52 (t, J=8.9Hz, 1H ), 2.83 (dd, J=24.4, 11.8Hz, 6H), 1.81-1.67 (m, 1H), 1.30 (dq, J=27.0, 9.5Hz, 4H), 1.16 (ddd, J=15.3.7.4, 3.8Hz, 1H).

[1028] Confirmation of the structure of compound I

[1029] 1 H NMR (500MHz, CDCl3) δ6.15-6.03 (m, 2H), 4.45 (s, 1H), 4.21 (d, J=5.0Hz, 1H), 3.97 (s, 1H), 3.65 (dd, J=12.0.7.3Hz, 1H), 3.52 (t, J =8.9Hz, 1H), 2.83 (dd, J=24.4, 11.8Hz, 6H), 1.81-1.67 (m, 1H), 1.30 (dq, J=27.0, 9.5Hz, 4H), 1.16 (ddd, J=15.3, 7.4, 3.8Hz, 1H).

[1030] LRMS (ESI): m / z 371.2 [M+NH4 + ]

[1031] The structure of compound I was confirmed by mass spectrometry. The results are shown in Fig.16 .

[1032] 1.1.2. Compound II (SO2 linker: NHS & norbornene)

[1033] [Solution 7]

[1034]

[1035] Synthesis of Compound II (Scheme 7, Fig.17 )

[1036] Synthesis of compound 3

[1037] 0.55 g (2.33 mmol, 1.0 eq.) of compound 1 was dissolved in 5 mL of acetonitrile (ACN), and then 0.2 mL (2.33 mmol, 1.0 eq.) of 3-mercaptopropionic acid and 0.49 mL (3.49 mmol, 1.5 eq.) of triethylamine were added dropwise while stirring at room temperature. The reaction solution was stirred for 11 hours and then concentrated under reduced pressure. Subsequently, water was added to the reaction solution, and the reaction solution was extracted 3 times with ethyl acetate (EA). The organic layer was dried over magnesium sulfate, concentrated under reduced pressure, and then purified by silica gel column chromatography (DCM: MeOH = 10: 1) to obtain 0.56 g of the target compound (yield: 89%).

[1038] Synthesis of compound II

[1039] 0.56 g (2.07 mmol, 1.0 eq.) of compound 3 was dissolved in 10 mL of acetonitrile (ACN), and then 0.81 g (4.21 mmol, 2.0 eq.) of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDCi) and 0.65 g (5.61 mmol, 2.7 eq.) of N-hydroxysuccinimide (NHS) were added while stirring at room temperature. The reaction solution was stirred for 12 hours and then concentrated under reduced pressure. Subsequently, water was added to the reaction solution, and the reaction solution was extracted 3 times with EA. The organic layer was dried over magnesium sulfate and concentrated under reduced pressure. Then, the residue was purified by silica gel column chromatography (EA: Hex = 2: 1) to obtain 0.63 g of the target compound (yield: 83%).

[1040] Confirmation of the structure of compound II

[1041] 1 H NMR (500MHz, CDCl3) δ6.16-6.14 (m, 2H), 4.45 (s, 1H), 4.21-4.08 (m, 1H), 3.69-3.58 (m, 1H), 3.51 (dt, J=14.5, 8.9Hz, 1H), 3.22 (t, J=7.1Hz, 2H), 2.96 (t, J=7.1Hz, 2H), 2.79-2.83 (m, 6H), 1.81-1.67 (m, 1H), 1.37-1.21 (m, 4H), 1.13-1.18 (m, 1H).

[1042] LRMS (ESI): m / z 385.1 [M+NH4 + ]

[1043] The structure of compound II was confirmed by mass spectrometry. The results are shown in Fig.18 .

[1044] 1.1.3. Compound III (SO3 linker: NHS & norbornene)

[1045] [Scheme 8]

[1046]

[1047] Synthesis of Compound III (Scheme 8, Fig.19 )

[1048] Synthesis of compound 4

[1049] 0.5 g (3.62 mmol, 1.0 eq.) of exo-5-norbornene carboxylic acid was dissolved in 20 mL of DCM, and then 0.028 mL (0.37 mmol, 0.94 eq.) of DMF and 0.63 mL (7.24 mmol, 2.0 eq.) of oxalyl chloride were added dropwise while stirring at room temperature. After the reaction solution was stirred for 3 hours, it was concentrated under reduced pressure to obtain 0.47 g of the target compound (yield 83%).

[1050] Synthesis of compound 5

[1051] 0.47 g (3.0 mmol, 1.0 eq.) of compound 4 was dissolved in 15 mL of acetonitrile (ACN), and then 0.21 mL (3.0 mmol, 1.0 eq.) of thioglycolic acid and 0.63 mL (4.5 mmol, 1.5 eq.) of triethylamine were added dropwise while stirring at room temperature. The reaction solution was stirred for 18 hours and then concentrated under reduced pressure. Subsequently, water was added to the reaction solution, and the reaction solution was extracted 3 times with ethyl acetate (EA). The organic layer was dried over magnesium sulfate, concentrated under reduced pressure, and then purified by column chromatography (DCM: MeOH = 10: 1) to obtain 0.3 g of the target compound (yield: 47%).

[1052] 1 H NMR (500MHz, CDCl3) δ9.82 (brs, 1H), 6.24-6.07 (m, 2H), 3.76 (s, 2H), 3.12 (s, 1H), 2.97 (s, 1 H), 2.54 (dd, J=9.0, 4.7Hz, 1H), 2.04-1.87 (m, 1H), 1.57 (d, J=8.5Hz, 1H), 1.48-1.35 (m, 2H).

[1053] Synthesis of compound III

[1054] 0.26 g (1.22 mmol, 1.0 eq.) of compound 5 was dissolved in 15 mL of acetonitrile (ACN), and then 0.35 g (1.83 mmol, 1.5 eq.) of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDCi) and 0.28 g (2.44 mmol, 2.0 eq.) of N-hydroxysuccinimide (NHS) were added while stirring at room temperature. The reaction solution was stirred for 3 hours and then concentrated under reduced pressure. Subsequently, water was added to the reaction solution, and the reaction solution was extracted 3 times with EA. The organic layer was recovered, dried over magnesium sulfate, and then concentrated under reduced pressure. Then, the residue was purified on a silica gel column using chromatography (EA: Hex = 2: 1) to obtain 0.32 g of the target compound (yield: 85%).

[1055] Confirmation of the structure of compound III

[1056] 1 H NMR (500MHz, CDCl3) δ6.21 (dd, J=5.5, 3.0Hz, 1H), 6.15 (dd, J=5.5, 3.1Hz, 1H), 4.01 (s, 2H), 3.15 (s, 1H), 2.98 (s, 1H) ), 2.86 (s, 4H), 2.54 (dd, J=9.2, 4.6Hz, 1H), 2.02 (dt, J=11.9, 4.0Hz, 1H), 1.59 (d, J=8.6Hz, 1H), 1.46-1.36 (m, 2H).

[1057] LCMS (ESI): m / z 332.16 [M+Na + ]

[1058] The structure of compound III was confirmed by mass spectrometry. Fig. 20 .

[1059] 1.1.4. Compound IV (SO4 linker: NHS & azide)

[1060] [Solution 9]

[1061]

[1062] Synthesis of Compound IV (Scheme 9, Fig.21 and Fig. 22 )

[1063] Synthesis of compound 6

[1064] 2-(2-chloroethoxy)ethanol (2mL, 18.94mmol) was dissolved in distilled water (12mL), and NaN3 (3.08g, 47.35mmol, 2.5eq.) was added thereto. The resulting mixture was stirred at 80°C for 16 hours. The reaction mixture was cooled to room temperature and poured into 5% NaOH (aq.) (20mL) and then stirred for about 10 minutes. The reaction mixture was extracted 3 times with ether, and the organic layer was dried over magnesium sulfate and filtered. The filtrate was concentrated under reduced pressure to obtain 2.47g of the target compound (yield 99%).

[1065] Synthesis of compound 7

[1066] 2.47 g (18.84 mmol) of compound 6 was dissolved in acetone (50 mL), and then 1 M Jones reagent (75.36 mL, 75.36 mmol, 4 eq.) was slowly added at 0 ° C. The reaction mixture was stirred at 0 ° C for 3 hours, warmed to room temperature, and then stirred for about 10 minutes. The reaction mixture was extracted 3 times with ethyl acetate (EA), and the organic layer was dried over magnesium sulfate and filtered. The filtrate was concentrated under reduced pressure to obtain 2.69 g of the target compound (yield 98%).

[1067] Synthesis of compound 8

[1068] 2.69 g (18.57 mmol, 1.0 eq.) of compound 7 was dissolved in 50 mL of DCM, and then 0.1 mL (1.29 mmol, 0.07 eq.) of DMF and 2.43 mL (27.86 mmol, 1.5 eq.) of oxalyl chloride were added dropwise while stirring at room temperature. After the reaction solution was stirred for 3 hours, it was concentrated under reduced pressure to obtain 2.42 g of the target compound (yield 80%).

[1069] Synthesis of compound 9

[1070] 0.88 g (5.40 mmol, 1.0 eq.) of compound 8 was dissolved in 30 mL of DCM, and then 0.8 g (5.40 mmol, 1.0 eq.) of tert-butyl 2-mercaptoacetate and 1.41 mL (8.10 mmol, 1.5 eq.) of N, N-diisopropylethylamine were added dropwise while stirring at room temperature. The reaction mixture was stirred for 2 hours and then concentrated under reduced pressure. Subsequently, water was added to the reaction solution, and the reaction solution was extracted 3 times with dichloromethane (DCM). The organic layer was dried over magnesium sulfate, concentrated under reduced pressure, and then purified by column chromatography (Hex: EA = 5: 1) to obtain 0.73 g of the target compound (yield: 49%).

[1071] Synthesis of compound 10

[1072] 0.73 g (2.66 mmol, 1 eq.) of compound 9 was dissolved in 10 mL of DCM, and then 10 mL (129.8 mmol, 48 eq.) of trifluoroacetic acid was added dropwise while stirring at room temperature. The reaction solution was stirred for 8 hours and then concentrated under reduced pressure to obtain 0.40 g of the target compound (yield 70%).

[1073] Synthesis of compound IV

[1074] 1.97 g (9.0 mmol, 1.0 eq.) of compound 10 was dissolved in 25 mL of acetonitrile (ACN), and 2.58 g (13.5 mmol, 1.5 eq.) of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDCi) and 2.07 g (18.0 mmol, 2.0 eq.) of N-hydroxysuccinimide (NHS) were added while stirring at room temperature. The reaction solution was stirred for 3 hours and then concentrated under reduced pressure. Subsequently, water was added to the reaction solution, and the reaction solution was extracted 3 times with EA. The organic layer was recovered, dried over magnesium sulfate, and then concentrated under reduced pressure. Then, the residue was purified on a silica gel column using chromatography (EA: Hex = 2: 1) to obtain 1.03 g of the target compound (yield: 36%).

[1075] Confirmation of the structure of compound IV

[1076] 1 H NMR (500MHz, CDCl3) δ4.30 (s, 2H), 4.00 (s, 2H), 3.87-3.75 (m, 2H), 3.55-3.47 (m, 2H), 2.86 (s, 4H).

[1077] LRMS (ESI): m / z 334.0 [M+NH4 + ]

[1078] The structure of compound IV was confirmed by mass spectrometry. The results are shown in Fig.23 .

[1079] 1.2. Synthesis and structure confirmation of site-specific Fc interactome (SSFI)

[1080] 1.2.1. Synthesis and structure confirmation of SSFI (where Xa1 is lysine)

[1081] Synthesis of SSFI(6Lys)

[1082]

[1083] List and order of introduction of Fmoc amino acids used

[1084] Fmoc-L-Thr(tBu)-OH, Fmoc-Cys(Trt)-OH, Fmoc-L-Trp(Boc)-OH, Fmoc-L-Val-OH, Fmoc-L-Leu-OH, Fmoc-L-Glu(OtBu)-OH, Fmoc- Gly-OH, Fmoc-Lys(Boc)-OH, Fmoc-L-His(Trt)-OH, Fmoc-L-Trp(Boc)-OH, Fmoc-Ala-OH, Fmoc-Cys(Trt)-OH, Fmoc-Asp(tBu)-OH.

[1085] Preparation method

[1086] (a) Introduction of amino acids

[1087] The amount of reagents used in the following process is based on 0.25 mmol. 0.5 g (0.48 mmol / g) of transparent amide resin (Peptide International Inc., USA) was placed in a synthesis reactor, and 1 mmol of each Fmoc-amino acid block was weighed to prepare a peptide amino acid sequence from C-terminus to N-terminus in the above order.

[1088] The reaction for activating the Fmoc-amino acid to attach the activated residue to the transparent amide resin was performed sequentially from the C-terminal amino acid.

[1089] Removal of Fmoc was carried out in DMF containing 20% ​​piperidine, and activation and introduction of the residue were carried out by mixing the amino acid prepared corresponding to the sequence with 2 mL of a 0.5 M DMF solution containing HOBt, 2 mL of a 0.5 M DMF solution containing HBTU and 174 μL of DIPEA for 5 minutes, and the resulting mixture was mixed in a reactor containing the resin for 2 hours.

[1090] Confirmation of the introduction reaction was performed using the Kaiser test method. When it was confirmed that the introduction reaction was incomplete, the introduction reaction was repeated again, or a DMF solution containing 20% ​​Ac2O was used for end-capping. In each introduction reaction and Fmoc removal, the resin was thoroughly washed with DMF and DCM and then moved to the next step. This process was repeated until the target peptide sequence was completed.

[1091] (b) Introduction of H-PEG8-OH

[1092] In order to introduce H-PEG8-OH to the N-terminus of the sequence after the introduction of the amino acid was completed, 1 mL of a 0.5 M Fmoc-N-amido-dPEG8-acid-in-DMF solution, 1 mL of a 0.5 M DMF solution containing HBTU, 1 mL of a 0.5 M DMF solution containing HOBt, and 87 μL of DIPEA were mixed for 5 minutes, and the resulting mixture was mixed in a reactor containing the reactive resin for 2 hours.

[1093] The reaction progress was monitored by the Kaiser test. When unreacted amine was found to remain, the reaction time was extended by another 1 to 3 hours, or the reaction solution was discarded and the above reaction process was repeated again. The N-terminal Fmoc protecting group was removed using DMF containing 20% ​​piperidine, and then the resin with the peptide attached was dried and weighed.

[1094] (c) 250 mg of the resin with the peptide attached prepared in step (b) was stirred with 2 mL of a mixed solution of TFA, TIS, water and EDT (94:1.0:2.5:2.5) at room temperature for 120 minutes to cleave the peptide from the resin. The cleaved mixture was filtered and the filtrate was concentrated to about half its volume using nitrogen. Subsequently, diethyl ether was poured into the mixture to precipitate the peptide. The precipitated peptide was washed 3 times with diethyl ether and dried under nitrogen. The dried precipitate was dissolved in water containing 0.1% TFA-30% ACN, stirred for 6 hours, and then concentrated.

[1095] The concentrate was dissolved in a 0.01M ammonium acetate buffer (pH 6.5) solution containing 5%-DMSO-20%-ACN at a concentration of 0.1 mg / mL, and then stirred for 3 days while exposed to air. The progress of the disulfide bond formation reaction was monitored by HPLC. When it was found that the reaction was no longer progressing, the reaction solution was lyophilized to obtain a peptide precipitate.

[1096] (d) Purification

[1097] The peptide precipitate obtained by lyophilization in step (c) was separated under the preparative LC primary purification conditions listed in Table 4 below, further purified under the preparative LC secondary purification conditions listed in Table 5 below, and then lyophilized. The obtained peptide was confirmed to have a purity of 90% or more by analytical HPLC, and the molecular weight of the synthetic peptide was confirmed using an LC / MS mass spectrometer.

[1098] H-PEG8-Asp-Cys*-Ala-Trp-His-Lys-Gly-Glu-Leu-Val-Trp-Cys*-Thr-NH2 (Cys*: disulfide bond site)

[1099] Preparative LC primary purification conditions

[1100]

[1101] Preparative LC secondary purification conditions

[1102]

[1103] Confirmation of the structure of SSFI(6Lys)

[1104] Molecular weight measurements by matrix-assisted laser desorption / ionization (MALDI) mass spectrometry confirmed the synthesis of SSFI(6Lys).

[1105] Measuring equipment: Ultraflextreme (Bruker)

[1106] Measurement matrix: CHCA (α-cyano-4-hydroxycinnamic acid) and DHB (2,5-dihydroxybenzoic acid)

[1107] Calculated molecular weight: 2010.29 g / mol

[1108] Measured molecular weight (M+H) + :2011.89g / mol

[1109] The mass spectrometry results of SSFI (6Lys) were Fig.24 Shown in.

[1110] 1.2.2. Synthesis and structure confirmation of SSFI (where Xa1 is ornithine)

[1111]

[1112] Synthesis of SSFI(6Orn)

[1113] The order and list of Fmoc amino acids used

[1114] Fmoc-L-Thr(tBu)-OH, Fmoc-Cys(Trt)-OH, Fmoc-L-Trp(Boc)-OH, Fmoc-L-Val-OH, Fmoc-L-Leu-OH, F moc-L-Glu(OtBu)-OH, Fmoc-Gly-OH, Fmoc-Orn(Boc)-OH, Fmoc-L-His(Trt)-OH, Fmoc-L-Trp(Boc)-OH Fmoc-Ala-OH, Fmoc-Cys(Trt)-OH, Fmoc-Asp(tBu)-OH.

[1115] Preparation method

[1116] (a) Introduction of amino acids

[1117] The amount of reagents used in the following process is based on 0.25 mmol. 0.5 g (0.48 mmol / g) of transparent amide resin (Peptide International Inc., USA) was placed in a synthesis reactor, and 1 mmol of each Fmoc-amino acid block was weighed to prepare a peptide amino acid sequence from C-terminus to N-terminus in the above order.

[1118] The reaction for activating the Fmoc-amino acid to attach the activated residue to the transparent amide resin was performed sequentially from the C-terminal amino acid.

[1119] Removal of Fmoc was carried out in DMF containing 20% ​​piperidine, and activation and introduction of the residue were carried out by mixing the amino acid prepared corresponding to the sequence with 2 mL of a 0.5 M DMF solution containing HOBt, 2 mL of a 0.5 M DMF solution containing HBTU and 174 μL of DIPEA for 5 minutes, and the resulting mixture was mixed in a reactor containing the resin for 2 hours.

[1120] Confirmation of the introduction reaction was performed using the Kaiser test method. When it was confirmed that the introduction reaction was incomplete, the introduction reaction was repeated again, or a DMF solution containing 20% ​​Ac2O was used for end-capping. In each introduction reaction and Fmoc removal, the resin was thoroughly washed with DMF and DCM and then moved to the next step. This process was repeated until the target peptide sequence was completed.

[1121] (b) Introduction of H-PEG8-OH

[1122] In order to introduce H-PEG8-OH to the N-terminus of the sequence after the introduction of the amino acid was completed, 1 mL of a 0.5 M Fmoc-N-amido-dPEG8-acid-in-DMF solution, 1 mL of a 0.5 M DMF solution containing HBTU, 1 mL of a 0.5 M DMF solution containing HOBt, and 87 μL of DIPEA were mixed for 5 minutes, and the resulting mixture was mixed in a reactor containing the reactive resin for 2 hours.

[1123] The reaction progress was monitored by the Kaiser test. When unreacted amine was found to remain, the reaction time was extended by another 1 to 3 hours, or the reaction solution was discarded and the above reaction process was repeated again. The N-terminal Fmoc protecting group was removed using DMF containing 20% ​​piperidine, and then the resin with the peptide attached was dried and weighed.

[1124] (c) 250 mg of the resin with the peptide attached prepared in step (b) was stirred with 2 mL of a mixed solution of TFA, TIS, water and EDT (94:1.0:2.5:2.5) at room temperature for 120 minutes to cleave the peptide from the resin. The cleaved mixture was filtered and the filtrate was concentrated to about half its volume using nitrogen. Subsequently, diethyl ether was poured into the mixture to precipitate the peptide. The precipitated peptide was washed 3 times with diethyl ether and dried under nitrogen. The dried precipitate was dissolved in water containing 0.1% TFA-30% ACN, stirred for 6 hours, and then concentrated.

[1125] The concentrate was dissolved in a 0.01M ammonium acetate buffer (pH 6.5) solution containing 5%-DMSO-20%-ACN at a concentration of 0.1 mg / mL, and then stirred for 3 days while exposed to air. The progress of the disulfide bond formation reaction was monitored by HPLC. When it was found that the reaction was no longer progressing, the reaction solution was lyophilized to obtain a peptide precipitate.

[1126] (d) Purification

[1127] The peptide precipitate obtained by lyophilization in step (c) was separated under the preparative LC primary purification conditions listed in Table 6 below, further purified under the preparative LC secondary purification conditions listed in Table 7 below, and then lyophilized. The obtained peptide was confirmed to have a purity of 90% or more by analytical HPLC, and the molecular weight of the synthetic peptide was confirmed using an LC / MS mass spectrometer.

[1128] H-PEG8-Asp-Cys*-Ala-Trp-His-Om-Gly-Glu-Leu-Va1-Trp-Cys*-Thr-NH2 (Cys*: disulfide bond site)

[1129] Preparative LC primary purification conditions

[1130]

[1131] Preparative LC secondary purification conditions

[1132]

[1133] Confirmation of the structure of SSFI(6Om)

[1134] Molecular weight measurements by matrix-assisted laser desorption / ionization (MALDI) mass spectrometry confirmed the synthesis of SSFI(6-ornithine).

[1135] Measuring equipment: Ultraflextreme (Bruker)

[1136] Measurement matrix: CHCA (α-cyano-4-hydroxycinnamic acid) and DHB (2,5-dihydroxybenzoic acid)

[1137] Calculated molecular weight: 1996.26 g / mol

[1138] Measured molecular weight (M+2H) 2+ :999.13g / mol

[1139] The mass spectrum results of SSFI (6Om) were Fig.25 Shown in.

[1140] 1.2.3. Synthesis and structure confirmation of SSFI (where Xa1 is diaminobutyric acid (Dab))

[1141]

[1142] Synthesis of SSFI(6Dab)

[1143] The order and list of Fmoc amino acids used

[1144] Fmoc-L-Thr(tBu)-OH, Fmoc-Cys(Trt)-OH, Fmoc-L-Trp(Boc)-OH, Fmoc-L-Val-OH, Fmoc-L-Leu-OH, F moc-L-Glu(OtBu)-OH, Fmoc-Gly-OH, Fmoc-Dab(Boc)-OH, Fmoc-L-His(Trt)-OH, Fmoc-L-Trp(Boc)-OH Fmoc-Ala-OH, Fmoc-Cys(Trt)-OH, Fmoc-Asp(tBu)-OH.

[1145] Preparation method

[1146] (a) Introduction of amino acids

[1147] The amount of reagents used in the following process is based on 0.25 mmol. 0.5 g (0.48 mmol / g) of transparent amide resin (Peptide International Inc., USA) was placed in a synthesis reactor, and 1 mmol of each Fmoc-amino acid block was weighed to prepare a peptide amino acid sequence from C-terminus to N-terminus in the above order.

[1148] The reaction for activating the Fmoc-amino acid to attach the activated residue to the transparent amide resin was performed sequentially from the C-terminal amino acid.

[1149] Removal of Fmoc was carried out in DMF containing 20% ​​piperidine, and activation and introduction of the residue were carried out by mixing the amino acid prepared corresponding to the sequence with 2 mL of a 0.5 M DMF solution containing HOBt, 2 mL of a 0.5 M DMF solution containing HBTU and 174 μL of DIPEA for 5 minutes, and the resulting mixture was mixed in a reactor containing the resin for 2 hours.

[1150] Confirmation of the introduction reaction was performed using the Kaiser test method. When it was confirmed that the introduction reaction was incomplete, the introduction reaction was repeated again, or a DMF solution containing 20% ​​Ac2O was used for end-capping. In each introduction reaction and Fmoc removal, the resin was thoroughly washed with DMF and DCM and then moved to the next step. This process was repeated until the target peptide sequence was completed.

[1151] (b) Introduction of H-PEG8-OH

[1152] In order to introduce H-PEG8-OH to the N-terminus of the sequence after the introduction of the amino acid was completed, 1 mL of a 0.5 M Fmoc-N-amido-dPEG8-acid-in-DMF solution, 1 mL of a 0.5 M DMF solution containing HBTU, 1 mL of a 0.5 M DMF solution containing HOBt, and 87 μL of DIPEA were mixed for 5 minutes, and the resulting mixture was mixed in a reactor containing the reactive resin for 2 hours.

[1153] The reaction progress was monitored by the Kaiser test. When unreacted amine was found to remain, the reaction time was extended by another 1 to 3 hours, or the reaction solution was discarded and the above reaction process was repeated again. The N-terminal Fmoc protecting group was removed using DMF containing 20% ​​piperidine, and then the resin with the peptide attached was dried and weighed.

[1154] (c) 250 mg of the resin with the peptide attached prepared in step (b) was stirred with 2 mL of a mixed solution of TFA, TIS, water and EDT (94:1.0:2.5:2.5) at room temperature for 120 minutes to cleave the peptide from the resin. The cleaved mixture was filtered and the filtrate was concentrated to about half its volume using nitrogen. Subsequently, diethyl ether was poured into the mixture to precipitate the peptide. The precipitated peptide was washed 3 times with diethyl ether and dried under nitrogen. The dried precipitate was dissolved in water containing 0.1% TFA-30% ACN, stirred for 6 hours, and then concentrated.

[1155] The concentrate was dissolved in a 0.01M ammonium acetate buffer (pH 6.5) solution containing 5%-DMSO-20%-ACN at a concentration of 0.1 mg / mL, and then stirred for 3 days while exposed to air. The progress of the disulfide bond formation reaction was monitored by HPLC. When it was found that the reaction was no longer progressing, the reaction solution was lyophilized to obtain a peptide precipitate.

[1156] (d) Purification

[1157] The peptide precipitate obtained by lyophilization in step (c) was separated under the preparative LC primary purification conditions listed in Table 8 below, further purified under the preparative LC secondary purification conditions listed in Table 9 below, and then lyophilized. The obtained peptide was confirmed to have a purity of 90% or more by analytical HPLC, and the molecular weight of the synthetic peptide was confirmed using an LC / MS mass spectrometer.

[1158] H-PEG8-Asp-Cys*-Ala-Trp-His-Dab-Glv-Glu-Leu-Va1-Trp-Cvs*-Thr-NH2 (Cys*: disulfide bond site)

[1159] Preparative LC primary purification conditions

[1160]

[1161] Preparative LC secondary purification conditions

[1162]

[1163] Confirmation of the structure of SSFI(6Dab)

[1164] Molecular weight measurements by matrix-assisted laser desorption / ionization (MALDI) mass spectrometry confirmed the synthesis of SSFI(6Dab).

[1165] Measuring equipment: Ultraflextreme (Bruker)

[1166] Measurement matrix: CHCA (α-cyano-4-hydroxycinnamic acid) and DHB (2,5-dihydroxybenzoic acid)

[1167] Calculated molecular weight: 1982.24 g / mol

[1168] Measured molecular weight (M+2H) 2+ :992.33g / mol

[1169] The mass spectrometry results of SSFI (6Dab) were Fig.26 Shown in.

[1170] 1.2.4. Synthesis and structure confirmation of SSFI (where Xa1 is diaminopropionic acid (Dap))

[1171]

[1172] Synthesis of SSFI(6Dap)

[1173] Sequence: Nor.-PEG8-DCAWHA (β-aminoalanine, Dap)GELVWCT-CONH2

[1174] The order and list of Fmoc amino acids used

[1175] Fmoc-L-Thr(tBu)-OH, Fmoc-Cys(Trt)-OH, Fmoc-L-Trp(Boc)-OH, Fmoc-L-Val-OH, Fmoc-L-Leu-OH, F moc-L-Glu(OtBu)-OH, Fmoc-Gly-OH, Fmoc-Dap(Boc)-OH, Fmoc-L-His(Trt)-OH, Fmoc-L-Trp(Boc)-OH Fmoc-Ala-OH, Fmoc-Cys(Trt)-OH, Fmoc-Asp(tBu)-OH.

[1176] Preparation method

[1177] (a) Introduction of amino acids

[1178] The amount of reagents used in the following process is based on 0.25 mmol. 0.5 g (0.48 mmol / g) of transparent amide resin (Peptide International Inc., USA) was placed in a synthesis reactor, and 1 mmol of each Fmoc-amino acid block was weighed to prepare a peptide amino acid sequence from C-terminus to N-terminus in the above order.

[1179] The reaction for activating the Fmoc-amino acid to attach the activated residue to the transparent amide resin was performed sequentially from the C-terminal amino acid.

[1180] Removal of Fmoc was carried out in DMF containing 20% ​​piperidine, and activation and introduction of the residue were carried out by mixing the amino acid prepared corresponding to the sequence with 2 mL of a 0.5 M DMF solution containing HOBt, 2 mL of a 0.5 M DMF solution containing HBTU and 174 μL of DIPEA for 5 minutes, and the resulting mixture was mixed in a reactor containing the resin for 2 hours.

[1181] Confirmation of the introduction reaction was performed using the Kaiser test method. When it was confirmed that the introduction reaction was incomplete, the introduction reaction was repeated again, or a DMF solution containing 20% ​​Ac2O was used for end-capping. In each introduction reaction and Fmoc removal, the resin was thoroughly washed with DMF and DCM and then moved to the next step. This process was repeated until the target peptide sequence was completed.

[1182] (b) Introduction of H-PEG8-OH

[1183] In order to introduce H-PEG8-OH to the N-terminus of the sequence after the introduction of the amino acid was completed, 1 mL of a 0.5 M Fmoc-N-amido-dPEG8-acid-in-DMF solution, 1 mL of a 0.5 M DMF solution containing HBTU, 1 mL of a 0.5 M DMF solution containing HOBt, and 87 μL of DIPEA were mixed for 5 minutes, and the resulting mixture was mixed in a reactor containing the reactive resin for 2 hours.

[1184] The reaction progress was monitored by the Kaiser test. When unreacted amine was found to remain, the reaction time was extended by another 1 to 3 hours, or the reaction solution was discarded and the above reaction process was repeated again. The N-terminal Fmoc protecting group was removed using DMF containing 20% ​​piperidine, and then the resin with the peptide attached was dried and weighed.

[1185] (c) 250 mg of the resin with the peptide attached prepared in step (b) was stirred with 2 mL of a mixed solution of TFA, TIS, water and EDT (94:1.0:2.5:2.5) at room temperature for 120 minutes to cleave the peptide from the resin. The cleaved mixture was filtered and the filtrate was concentrated to about half its volume using nitrogen. Subsequently, diethyl ether was poured into the mixture to precipitate the peptide. The precipitated peptide was washed 3 times with diethyl ether and dried under nitrogen. The dried precipitate was dissolved in water containing 0.1% TFA-30% ACN, stirred for 6 hours, and then concentrated.

[1186] The concentrate was dissolved in a 0.01M ammonium acetate buffer (pH 6.5) solution containing 5%-DMSO-20%-ACN at a concentration of 0.1 mg / mL, and then stirred for 3 days while exposed to air. The progress of the disulfide bond formation reaction was monitored by HPLC. When it was found that the reaction was no longer progressing, the reaction solution was lyophilized to obtain a peptide precipitate.

[1187] (d) Purification

[1188] The peptide precipitate obtained by lyophilization in step (c) was separated under the preparative LC primary purification conditions listed in Table 10 below, further purified under the preparative LC secondary purification conditions listed in Table 11 below, and then lyophilized. The obtained peptide was confirmed to have a purity of 90% or more by analytical HPLC, and the molecular weight of the synthetic peptide was confirmed using an LC / MS mass spectrometer.

[1189] H-PEG8-Asp-Cys*-Ala-Trp-His-Dap-Glv-Glu-Leu-Va1-Trp-Cys*-Thr-NH2 (Cys*: disulfide bond site)

[1190] Preparative LC primary purification conditions

[1191] Preparative LC secondary purification conditions

[1192] Confirmation of the structure of SSFI (6Dap) Measurement equipment: Quattro Premier Xe (Waters)

[1193] Calculated molecular weight: 1968.21 g / mol

[1194] Measured molecular weight (M+2H) 2+ :984.71g / mol

[1195] The mass spectrometry results of SSFI (6Dap) were Fig. 27 Shown in.

[1196] 1.3. Synthesis and structure confirmation of VC linker

[1197] 1.3.1. Synthesis and confirmation of the VC linker (DD2) structure

[1198] [Scheme 10]

[1199] Synthesis of DD2

[1200]

[1201] Synthesis of VC linker (DD2)

[1202] Synthesis of compound 11

[1203] 5 g (14.7 mmol, 1.0 eq.) of Fmoc-Val-OH and 1.7 g (14.7 mmol, 1.0 eq.) of N-hydroxysuccinimide (NHS) were dissolved in 140 mL of dimethoxyethane (DME) and stirred. 2.5 mL (16.2 mmol, 1.1 eq.) of N,N'-diisopropylcarbodiimide (DIC) was added dropwise at 0°C and stirred for 16 hours. The reaction solution was filtered under reduced pressure to remove floating matter, and the filtrate was concentrated under reduced pressure. The residue was dissolved in acetone and stored at low temperature in a refrigerator for 4 hours. Thereafter, the resulting solution was filtered under reduced pressure to remove the re-formed floating matter, and used in the next reaction without any purification (crude yield: 5.5 g, 86%). TLC (EA: Hex = 1: 1); R f =0.5.

[1204] Synthesis of compound 12

[1205] 2.0 g (11.5 mmol, 1.0 eq.) of L-citrulline was dissolved in 100 mL of a 1:1 mixed solution of tetrahydrofuran (THF) and water and stirred. 988 mg of sodium bicarbonate was added thereto and stirred. Subsequently, 5.0 g (11.5 mmol, 1.0 eq.) of compound 8 was dissolved in 80 mL of acetone, added dropwise to the reaction solution and stirred. The resulting mixture was stirred for 21 hours and then concentrated under reduced pressure to remove the organic solvent. The aqueous layer was washed with ethyl acetate (EA) and then titrated to pH 3 by slowly dropping 2N HCl. EA was added thereto to extract the precipitate in the organic layer. The organic layer was then dried with a saturated saline solution and sodium sulfate and used in the next reaction without any purification (crude yield: 5.6 g, 98%). TLC (DCM: MeOH = 10: 1, one drop of formic acid); R f =0.1.

[1206] Synthesis of compound 13

[1207] 50 mL of 10% piperidine in N,N-dimethylformamide (DMF) was added dropwise to 2.84 g (5.72 mmol, 1.0 eq.) of compound 12 and stirred. After 4 hours, the resulting mixture was concentrated under reduced pressure to remove the reaction solution, and the residue was dissolved in water and filtered under reduced pressure to remove the floating matter formed. The aqueous layer was concentrated to obtain the target compound, which was used for the next step without any purification. TLC (DCM: MeOH = 10: 1); R f =0.01.

[1208] Synthesis of compound 14

[1209] 54 mg (0.22 mmol, 1.0 eq.) of compound 10 was dissolved in 2 mL of DMF, and 0.05 mL (0.264 mmol, 1.2 eq.) of N, N-diisopropylethylamine (DIPEA) was added dropwise thereto. Compound 13 was completely dissolved by adding 2 mL of water, and 0.05 mL (0.528 mmol, 2.4 eq.) of acetic anhydride was added dropwise thereto at room temperature. After 3 hours, the resulting mixture was concentrated under reduced pressure to remove the reaction solution, and the concentrate was purified by reverse phase column chromatography to obtain the target compound. TLC (DCM: MeOH = 10: 1); R f =0.05.

[1210] Synthesis of compound 15

[1211] 1 g (3.16 mmol, 1.0 eq.) of compound 14 was dissolved in 30 mL of a 2:1 mixed solution of DCM and methanol, and 868 mg (3.48 mmol, 1.1 eq.) of N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) was added thereto and stirred. 451 mg (3.66 mmol, 1.16 eq.) of 4-aminobenzyl alcohol was added thereto and stirred for 5 hours. The resulting mixture was concentrated under reduced pressure to remove the reaction solution, and the concentrate was purified by column chromatography (10% MeOH in DCM) to obtain 419 mg of the target compound (yield: 32%). TLC (DCM: MeOH = 10: 1); R f =0.1.

[1212] Synthesis of compound DD2

[1213] 109 mg (0.3 mmol, 1.2 eq.) of phenol-activated SN38 and 0.06 mL (0.33 mmol, 1.3 eq.) of DIPEA were added dropwise to a solution obtained by dissolving 105 mg (0.25 mmol, 1.0 eq.) of compound 15 in 10 mL of DMF. The resulting mixture was stirred for 20 hours and then concentrated under reduced pressure to remove the reaction solution. The concentrate was purified by column chromatography (10% MeOH in DCM) to obtain the target compound. TLC (DCM: MeOH = 10: 1); R f =0.2.

[1214] Confirmation of the structure of the VC connector (DD2)

[1215] LRMS(ESI):m / z 840.4[M+H + ]

[1216] The result is Fig.28 Shown in.

[1217] 1.3.2. Synthesis and structure confirmation of VC linker (DD3)

[1218] [Scheme 11]

[1219]

[1220]

[1221] Synthesis of VC linker (DD3)

[1222] Synthesis of compound 16

[1223] 5 g (14.7 mmol, 1.0 eq.) of Fmoc-Val-OH and 1.7 g (14.7 mmol, 1.0 eq.) of N-hydroxysuccinimide (NHS) were dissolved in 140 mL of dimethoxyethane (DME) and stirred. 2.5 mL (16.2 mmol, 1.1 eq.) of N,N'-diisopropylcarbodiimide (DIC) was added dropwise at 0°C and stirred for 16 hours. The reaction solution was filtered under reduced pressure to remove floating matter, and the filtrate was concentrated under reduced pressure. The residue was dissolved in acetone and stored at low temperature in a refrigerator for 4 hours. Thereafter, the resulting solution was filtered under reduced pressure to remove the re-formed floating matter, and used for the next step reaction without any purification (crude yield: 5.5 g, 86%). TLC (EA: Hex = 1: 1); R f =0.5.

[1224] Synthesis of compound 17

[1225] 2.0 g (11.5 mmol, 1.0 eq.) of L-citrulline was dissolved in 100 mL of a 1:1 mixed solution of tetrahydrofuran (THF) and water and stirred. 988 mg of sodium bicarbonate was added thereto and stirred. Subsequently, 5.0 g (11.5 mmol, 1.0 eq.) of compound 16 was dissolved in 80 mL of acetone, added dropwise to the reaction solution and stirred. The resulting mixture was stirred for 21 hours and then concentrated under reduced pressure to remove the organic solvent. The aqueous layer was washed with ethyl acetate (EA) and then titrated to pH 3 by dropping 2N HCl. EA was added thereto to extract the precipitate in the organic layer. The organic layer was then dried with a saturated saline solution and sodium sulfate and used in the next step of the reaction without any purification (crude yield: 5.6 g, 98%). TLC (DCM: MeOH = 10: 1, one drop of formic acid); R f =0.1.

[1226] Synthesis of compound 18

[1227] 50 mL of 10% piperidine in N,N-dimethylformamide (DMF) was added dropwise to 2.84 g (5.72 mmol, 1.0 eq.) of compound 17 and stirred. After 4 hours, the resulting mixture was concentrated under reduced pressure to remove the reaction solution, and the residue was dissolved in water and filtered under reduced pressure to remove the floating matter formed. The aqueous layer was concentrated to obtain the target compound, which was used for the next step without any purification. TLC (DCM: MeOH = 10: 1); R f =0.01.

[1228] Synthesis of compound 19

[1229] 54 mg (0.22 mmol, 1.0 eq.) of compound 18 was dissolved in 2 mL of DMF, and 0.05 mL (0.264 mmol, 1.2 eq.) of N,N-diisopropylethylamine (DIPEA) was added dropwise thereto. Compound 15 was completely dissolved by adding 2 mL of water, and 0.05 mL (0.528 mmol, 2.4 eq.) of acetic anhydride was added dropwise thereto at room temperature. After 3 hours, the resulting mixture was concentrated under reduced pressure to remove the reaction solution, and the concentrate was purified by reverse phase column chromatography to obtain the target compound. TLC (DCM: MeOH = 10: 1); R f =0.05.

[1230] Synthesis of compound 20

[1231] 1 g (3.16 mmol, 1.0 eq.) of compound 19 was dissolved in 30 mL of a 2:1 mixed solution of DCM and methanol, and 868 mg (3.48 mmol, 1.1 eq.) of N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) was added thereto and stirred. 451 mg (3.66 mmol, 1.16 eq.) of 4-aminobenzyl alcohol was added thereto and stirred for 5 hours. The resulting mixture was concentrated under reduced pressure to remove the reaction solution, and the concentrate was purified by column chromatography (10% MeOH in DCM) to obtain 419 mg of the target compound (yield: 32%). TLC (DCM: MeOH = 10: 1); R f =0.1.

[1232] Synthesis of compound 21

[1233] 240mg (0.55mmol, 1.0eq.) of compound 20 was dissolved in 10mL of DMF, and 0.3mL (1.65mmol, 3.0eq.) of DIPEA was added thereto and stirred. Subsequently, 509mg (1.65mmol, 3.0eq.) of bis-(4-aminophenyl) carbonate was added to react. After 3 hours, the resulting mixture was concentrated under reduced pressure to remove the reaction solution, and ether was added thereto to precipitate the target compound, and the compound was filtered under reduced pressure. The resulting target compound was used for the next step reaction without any purification.

[1234] Synthesis of compound 22

[1235] 33 mg (0.06 mmol, 1.0 eq.) of compound 21 was dissolved in 5 mL of DMF, and 16 mg (0.08 mmol, 1.3 eq.) of 1-[(tert-butoxycarbonyl)amino]-2-aminoethane and 0.02 mL (0.08 mmol, 1.3 eq.) of DIPEA were added dropwise thereto and stirred. The resulting mixture was stirred for 7 hours, then concentrated under reduced pressure to remove the reaction solution, and ether was added thereto to precipitate the target compound, which was filtered under reduced pressure. The target compound obtained was used for the next step without any purification. TLC (DCM: MeOH = 10: 1); R f =0.1.

[1236] Synthesis of compound 23

[1237] 17 mg (0.03 mmol, 1.0 eq.) of compound 22 was dissolved in 1 mL of DCM, and then 1 mL of trifluoroacetic acid (TFA) was added dropwise thereto at 0°C. The reaction was carried out by stirring the resulting mixture at 0°C for 30 minutes and then at room temperature for 30 minutes. Subsequently, the mixture was concentrated under reduced pressure to remove the reaction solution such as DCM, and the concentration was repeated 3 times. The concentrated material was dried under high vacuum and purified by reverse phase column chromatography to obtain 13 mg of the target compound. TLC (DCM: MeOH = 10: 1); R f =0.01.

[1238] Synthesis of compound DD3

[1239] 0.01 mL (0.04 mmol, 1.5 eq.) of DIPEA was added dropwise to a solution obtained by dissolving 13 mg (0.03 mmol, 1.0 eq.) of compound 23 in 3 mL of DMF, and 22 mg (0.04 mmol, 1.5 eq.) of phenol-activated SN38 was added thereto. The resulting mixture was stirred for 1 hour and then concentrated under reduced pressure to remove the reaction solution. The concentrate was purified by column chromatography (10% MeOH in DCM) to obtain the target compound. TLC (DCM: MeOH = 10: 1); R f =0.2.

[1240] Confirmation of the structure of the VC connector (DD3)

[1241] LRMS(ESI):m / z 926.4[M+H + ]

[1242] The result is Fig.29 Shown in.

[1243] 1.3.3. Synthesis and structure confirmation of VC linker (DD4)

[1244] [Scheme 12]

[1245] Synthesis of DD4

[1246]

[1247] Synthesis of VC linker (DD4)

[1248] Synthesis of compound 24

[1249] 5 g (14.7 mmol, 1.0 eq.) of Fmoc-Val-OH and 1.7 g (14.7 mmol, 1.0 eq.) of N-hydroxysuccinimide (NHS) were dissolved in 140 mL of dimethoxyethane (DME) and stirred. 2.5 mL (16.2 mmol, 1.1 eq.) of N,N'-diisopropylcarbodiimide (DIC) was added dropwise at 0°C and stirred for 16 hours. The reaction solution was filtered under reduced pressure to remove floating matter, and the filtrate was concentrated under reduced pressure. The residue was dissolved in acetone and stored at low temperature in a refrigerator for 4 hours. Thereafter, the resulting solution was filtered under reduced pressure to remove the re-formed floating matter, and used for the next step reaction without any purification (crude yield: 5.5 g, 86%). TLC (EA: Hex = 1: 1); R f =0.5.

[1250] Synthesis of compound 25

[1251] 2.0 g (11.5 mmol, 1.0 eq.) of L-citrulline was dissolved in 100 mL of a 1:1 mixed solution of tetrahydrofuran (THF) and water and stirred. 988 mg of sodium bicarbonate was added thereto and stirred. Subsequently, 5.0 g (11.5 mmol, 1.0 eq.) of compound 21 was dissolved in 80 mL of acetone, added dropwise to the reaction solution and stirred. The resulting mixture was stirred for 21 hours and then concentrated under reduced pressure to remove the organic solvent. The aqueous layer was washed with ethyl acetate (EA) and then titrated to pH 3 by slowly dropping 2N HCl. EA was added thereto to extract the precipitate in the organic layer. The organic layer was then dried with a saturated saline solution and sodium sulfate and used in the next reaction without any purification (crude yield: 5.6 g, 98%). TLC (DCM:MeOH=10:1, one drop of formic acid); R f =0.1.

[1252] Synthesis of compound 26

[1253] 50 mL of 10% piperidine in N,N-dimethylformamide (DMF) was added dropwise to 2.84 g (5.72 mmol, 1.0 eq.) of compound 25 and stirred. After 4 hours, the resulting mixture was concentrated under reduced pressure to remove the reaction solution, and the residue was dissolved in water and filtered under reduced pressure to remove the floating material formed. The aqueous layer was concentrated to obtain the target compound, which was used for the next step without any purification. TLC (DCM: MeOH = 10: 1); R f =0.01.

[1254] Synthesis of compound 27

[1255] 54 mg (0.22 mmol, 1.0 eq.) of compound 26 was dissolved in 2 mL of DMF, and 0.05 mL (0.264 mmol, 1.2 eq.) of N,N-diisopropylethylamine (DIPEA) was added dropwise thereto. Compound 23 was completely dissolved by adding 2 mL of water, and 0.05 mL (0.528 mmol, 2.4 eq.) of acetic anhydride was added dropwise thereto at room temperature. After 3 hours, the resulting mixture was concentrated under reduced pressure to remove the reaction solution, and the concentrate was purified by reverse phase column chromatography to obtain the target compound. TLC (DCM: MeOH = 10: 1); R f =0.05.

[1256] Synthesis of compound 28

[1257] 300 mg (0.95 mmol, 1.0 eq.) of compound 27 was dissolved in 6 mL of DMF, and then 550 mg (1.43 mmol, 1.5 eq.) of 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (HATU) was added thereto. Subsequently, 0.25 mL (1.43 mmol, 1.5 eq.) of DIPEA was added dropwise, and 230 mg (1.43 mmol, 1.5 eq.) of 1-[(tert-butoxycarbonyl)amino]-2-aminoethane was dissolved in 3.5 mL of DMF and added dropwise at room temperature. After 7 hours, the reaction solution was diluted with ethyl acetate (EA), and the organic layer was washed with a saturated sodium bicarbonate solution. Then, the organic layer was dried with a saturated saline solution and sodium sulfate, and filtered to obtain the target compound. The obtained target compound was used for the next step reaction without any purification. TLC (DCM:MeOH=10:1); R f =0.5.

[1258] Synthesis of compound 29

[1259] Compound 28 was dissolved in a 2:1 mixed solution of DCM and TFA, and then reacted at 0°C. Subsequently, the reaction mixture was concentrated under reduced pressure to remove the reaction solution (such as DCM), and the concentration was repeated 3 times. The residue was dried under high vacuum and purified by reverse phase column chromatography to obtain 370 mg of the target compound. TLC (DCM: MeOH = 10: 1); R f =0.01.

[1260] Synthesis of compound DD4

[1261] 0.03 mL (0.17 mmol, 1.2 eq.) of DIPEA was added dropwise to a solution obtained by dissolving 50 mg (0.14 mmol, 1.0 eq.) of compound 26 in 3 mL of DMF, and 107 mg (0.21 mmol, 1.5 eq.) of phenol-activated SN38 was added thereto. The resulting mixture was stirred for 1 hour and then concentrated under reduced pressure to remove the reaction solution. The concentrate was purified by column chromatography (10% MeOH in DCM) to obtain the target compound. TLC (DCM: MeOH = 10: 1); R f =0.1.

[1262] Confirmation of the structure of the VC connector (DD4)

[1263] LRMS(ESI):m / z 777.3[M+H + ]

[1264] The result is Fig.30 Shown in.

[1265] 1.3.4. Synthesis and structure confirmation of VC linker (DD5)

[1266] [Scheme 13] Synthesis of DD5

[1267]

[1268]

[1269] Synthesis of VC linker (DD5)

[1270] Synthesis of compound 30

[1271] 5 g (14.7 mmol, 1.0 eq.) of Fmoc-Val-OH and 1.7 g (14.7 mmol, 1.0 eq.) of N-hydroxysuccinimide (NHS) were dissolved in 140 mL of dimethoxyethane (DME) and stirred. 2.5 mL (16.2 mmol, 1.1 eq.) of N,N'-diisopropylcarbodiimide (DIC) was added dropwise at 0°C and stirred for 16 hours. The reaction solution was filtered under reduced pressure to remove floating matter, and the filtrate was concentrated under reduced pressure. The residue was dissolved in acetone and stored at low temperature in a refrigerator for 4 hours. Thereafter, the resulting solution was filtered under reduced pressure to remove the re-formed floating matter, and used for the next step reaction without any purification (crude yield: 5.5 g, 86%). TLC (EA: Hex = 1: 1); R f =0.5.

[1272] Synthesis of compound 31

[1273] 2.0 g (11.5 mmol, 1.0 eq.) of L-citrulline was dissolved in 100 mL of a 1:1 mixed solution of tetrahydrofuran (THF) and water and stirred. 988 mg of sodium bicarbonate was added thereto and stirred. Subsequently, 5.0 g (11.5 mmol, 1.0 eq.) of compound 30 was dissolved in 80 mL of acetone, added dropwise to the reaction solution and stirred. The resulting mixture was stirred for 21 hours and then concentrated under reduced pressure to remove the organic solvent. The aqueous layer was washed with ethyl acetate (EA) and then titrated to pH 3 by slowly dropping 2N HCl. EA was added thereto to extract the precipitate in the organic layer. The organic layer was then dried with a saturated saline solution and sodium sulfate and used in the next step of the reaction without any purification (crude yield: 5.6 g, 98%). TLC (DCM: MeOH = 10: 1, one drop of formic acid); R f =0.1.

[1274] Synthesis of compound 32

[1275] 50 mL of 10% piperidine in N,N-dimethylformamide (DMF) was added dropwise to 2.84 g (5.72 mmol, 1.0 eq.) of compound 31 and stirred. After 4 hours, the resulting mixture was concentrated under reduced pressure to remove the reaction solution, and the residue was dissolved in water and filtered under reduced pressure to remove the floating matter formed. The aqueous layer was concentrated to obtain the target compound, which was used for the next step without any purification. TLC (DCM: MeOH = 10: 1); R f =0.01.

[1276] Synthesis of compound 33

[1277] 54 mg (0.22 mmol, 1.0 eq.) of compound 29 was dissolved in 2 mL of DMF, and 0.05 mL (0.264 mmol, 1.2 eq.) of N,N-diisopropylethylamine (DIPEA) was added dropwise thereto. Compound 32 was completely dissolved by adding 2 mL of water, and 0.05 mL (0.528 mmol, 2.4 eq.) of acetic anhydride was added dropwise thereto at room temperature. After 3 hours, the resulting mixture was concentrated under reduced pressure to remove the reaction solution, and the concentrate was purified by reverse phase column chromatography to obtain the target compound. TLC (DCM: MeOH = 10: 1); R f =0.05.

[1278] Synthesis of compound 34

[1279] 1 g (3.16 mmol, 1.0 eq.) of compound 33 was dissolved in 30 mL of a 2:1 mixed solution of DCM and methanol, and 868 mg (3.48 mmol, 1.1 eq.) of N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) was added thereto and stirred. 451 mg (3.66 mmol, 1.16 eq.) of 4-aminobenzyl alcohol was added thereto and stirred for 5 hours. The resulting mixture was concentrated under reduced pressure to remove the reaction solution, and the concentrate was purified by column chromatography (10% MeOH in DCM) to obtain 419 mg of the target compound (yield: 32%). TLC (DCM: MeOH = 10: 1); R f =0.1.

[1280] Synthesis of compound 35

[1281] 240mg (0.55mmol, 1.0eq.) of compound 34 was dissolved in 10mL of DMF, and 0.3mL (1.65mmol, 3.0eq.) of DIPEA was added thereto and stirred. Subsequently, 509mg (1.65mmol, 3.0eq.) of bis-(4-aminophenyl) carbonate was added to react. After 3 hours, the obtained mixture was concentrated under reduced pressure to remove the reaction solution, and ether was added thereto to precipitate the target compound, and the compound was filtered under reduced pressure. The target compound obtained was used for the next step reaction without any purification.

[1282] Synthesis of compound 36

[1283] 33 mg (0.06 mmol, 1.0 eq.) of compound 35 was dissolved in 5 mL of DMF, and 16 mg (0.08 mmol, 1.3 eq.) of N-[(tert-butoxy)carbonyl]-N,N'-dimethylethylenediamine and 0.02 mL (0.08 mmol, 1.3 eq.) of DIPEA were added dropwise thereto and stirred. The resulting mixture was stirred for 7 hours, then concentrated under reduced pressure to remove the reaction solution, and ether was added thereto to precipitate the target compound, which was filtered under reduced pressure. The target compound obtained was used for the next step without any purification. TLC (DCM: MeOH = 10: 1); R f =0.1.

[1284] Synthesis of compound 37

[1285] 17 mg (0.03 mmol, 1.0 eq.) of compound 36 was dissolved in 1 mL of DCM, and then 1 mL of trifluoroacetic acid (TFA) was added dropwise thereto at 0°C. The reaction was carried out by stirring the resulting mixture at 0°C for 30 minutes and then at room temperature for 30 minutes. Subsequently, the mixture was concentrated under reduced pressure to remove the reaction solution such as DCM, and the concentration was repeated 3 times. The concentrated material was dried under high vacuum and purified by reverse phase column chromatography to obtain 13 mg of the target compound. TLC (DCM: MeOH = 10: 1); R f =0.01.

[1286] Synthesis of compound DD5

[1287] 0.01 mL (0.04 mmol, 1.5 eq.) of DIPEA was added dropwise to a solution obtained by dissolving 13 mg (0.03 mmol, 1.0 eq.) of compound 37 in 3 mL of DMF, and 22 mg (0.04 mmol, 1.5 eq.) of phenol-activated SN38 was added thereto. The resulting mixture was stirred for 1 hour and then concentrated under reduced pressure to remove the reaction solution. The concentrate was purified by column chromatography (10% MeOH in DCM) to obtain the target compound. TLC (DCM: MeOH = 10: 1); R f =0.2.

[1288] 3-4-2. Confirmation of the structure of the VC connector (DD5)

[1289] LRMS(ESI):m / z 954.4[M+H + ]

[1290] The result is...

Claims

1. An antibody or a fragment thereof, comprising one or more amino acid sequences selected from Formula 8-1, Formula 8-2 and Formula 8-3: [Formula 8-1] GPSVFLFPP-(K)′-PKDTLMI [Formula 8-2] GPSVFLFPPKP-(K)′-DTLMI [Formula 8-3] GPSVFLFPP-(K)′-P-(K)′-DTLMI in, G is a glycine residue, P is a proline residue, S is a serine residue, V is a valine residue, F is a phenylalanine residue, L is a leucine residue, K is a lysine residue, D is an aspartic acid residue, T is a threonine residue, M is a methionine residue, and I is an isoleucine residue, and (K)' is in, H1 is the first click chemistry functional group, D1 is selected from covalent bonds, C 1～4 Alkylene, C 2～4 Alkenylene, C 2～4 Alkynylidene and C 3～8 Cycloalkylene.

2. The antibody or fragment thereof according to claim 1, wherein D1 is a covalent bond.

3. The antibody or fragment thereof according to claim 1, wherein the antibody or fragment thereof comprises the amino acid sequence of Formula 8-1 but does not comprise the amino acid sequences of Formula 8-2 and Formula 8-3.

4. The antibody or fragment thereof according to claim 1, comprising the amino acid sequence of Formula 8-1 in both Fc domains.

5. The antibody or fragment thereof according to claim 1, comprising the amino acid sequence of Formula 8-2 but not the amino acid sequences of Formula 8-1 and Formula 8-3.

6. The antibody or fragment thereof according to claim 1, comprising the amino acid sequence of Formula 8-2 in both Fc domains.

7. The antibody or fragment thereof according to claim 1, comprising the amino acid sequence of Formula 8-3 but not the amino acid sequences of Formula 8-1 and Formula 8-2.

8. The antibody or fragment thereof according to claim 7, comprising the amino acid sequence of Formula 8-3 in both Fc domains.

9. A method for preparing an antibody-drug conjugate, the method comprising reacting an antibody or a fragment thereof comprising one or more amino acid sequences selected from Formula 8-1, Formula 8-2 and Formula 8-3 with a compound of Formula 9: [Formula 8-1] GPSVFLFPP-(K)′-PKDTLMI [Formula 8-2] GPSVFLFPPKP-(K)′-DTLMI [Formula 8-3] GPSVFLFPP-(K)′-P-(K)′-DTLMI in, G is a glycine residue, P is a proline residue, S is a serine residue, V is a valine residue, F is a phenylalanine residue, L is a leucine residue, K is a lysine residue, D is an aspartic acid residue, T is a threonine residue, M is a methionine residue, and I is an isoleucine residue, and (K)' is in, H1 is the first click chemistry functional group, D1 is selected from covalent bonds, C 1～4 Alkylene, C 2～4 Alkenylene, C 2～4 Alkynylidene and C 3～8 Cycloalkylene; [Formula 9] C m -H2 in, C m is the load part, H2 is a second click chemistry functional group complementary to the first click chemistry functional group.

10. A kit for preparing an antibody-drug conjugate, the kit comprising an antibody or a fragment thereof and a compound of Formula 9, wherein the antibody or the fragment thereof comprises one or more amino acid sequences selected from Formula 8-1, Formula 8-2 and Formula 8-3: [Formula 8-1] GPSVFLFPP-(k)′-PKDTLMI [Formula 8-2] GPSVFLFPPKP-(K)′-DTLMI [Formula 8-3] GPSVFLFPP-(K)′-P-(K)′-DTLMI in, G is a glycine residue, P is a proline residue, S is a serine residue, V is a valine residue, F is a phenylalanine residue, L is a leucine residue, K is a lysine residue, D is an aspartic acid residue, T is a threonine residue, M is a methionine residue, and I is an isoleucine residue, and (K)' is in, H1 is the first click chemistry functional group, D1 is selected from covalent bonds, C 1～4 Alkylene, C 2～4 Alkenylene, C 2～4 Alkynylidene and C 3～8 Cycloalkylene; [Formula 9] C m -H2 in, C m is the load part, H2 is a second click chemistry functional group complementary to the first click chemistry functional group.

11. An antibody or a fragment thereof, comprising one or more amino acid sequences selected from Formula 10-1, Formula 10-2 and Formula 10-3: [Formula 10-1] GPSVFLFPP-(K)″-PKDTLMI [Formula 10-2] GPSVFLFPPKP-(K)″-DTLMI [Formula 10-3] GPSVFLFPP-(K)″-P-(K)″-DTLMI in, G is a glycine residue, P is a proline residue, S is a serine residue, V is a valine residue, F is a phenylalanine residue, L is a leucine residue, K is a lysine residue, D is an aspartic acid residue, T is a threonine residue, M is a methionine residue, and I is an isoleucine residue, and (K)” in, C m is the load part, B is selected from Where A1 and A2 are connected to the load part or D1 and they are not both connected to the same part, R x Selected from H, halogen and C 1～3 alkyl, D1 is selected from covalent bonds, C 1～4 Alkylene, C 2～4 Alkenylene, C 2～4 Alkynylidene and C 3～8 Cycloalkylene. 12 . The antibody or fragment thereof according to claim 11 , comprising the amino acid sequence of Formula 10-1 but not comprising the amino acid sequences of Formula 10-2 and Formula 10-3.

13. The antibody or fragment thereof according to claim 12, comprising the amino acid sequence of Formula 10-1 in both Fc domains. 14 . The antibody or fragment thereof according to claim 11 , comprising the amino acid sequence of Formula 10-2 but not the amino acid sequences of Formula 10-1 and Formula 10-3.

15. The antibody or fragment thereof according to claim 14, comprising the amino acid sequence of Formula 10-2 in both Fc domains. 16 . The antibody or fragment thereof according to claim 11 , comprising the amino acid sequence of Formula 10-3 but not the amino acid sequences of Formula 10-1 and Formula 10-2.

17. The antibody or fragment thereof according to claim 16, comprising the amino acid sequence of Formula 10-3 in both Fc domains.

18. The antibody or fragment thereof according to claim 11, wherein The cargo portion comprises a drug portion.

19. The antibody or fragment thereof according to claim 18, wherein The loading portion comprises two or more drug portions.

20. The antibody or fragment thereof according to claim 18, wherein The drug moiety is an anticancer agent.

21. The antibody or fragment thereof according to claim 20, wherein The anticancer agent is at least one selected from the following: DM1, DM3, DM4, abrin, ricin A, Pseudomonas exotoxin, cholera toxin, diphtheria toxin, tumor necrosis factor, alpha-interferon, beta-interferon, nerve growth factor, platelet-derived growth factor, tissue plasminogen activator, cytokine, apoptotic agent, anti-angiogenic agent, lymphokine, taxane, DNA-alkylating agent, anthracycline, tubulysin analog, duocarmycin analog, auristatin E, auristatin F, maytansine compounds, cytotoxic agents containing a reactive polyethylene glycol portion, taxane, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenotoposide, vincristine, vinblastine, t. colchicine, doxorubicin, daunorubicin, dihydroxyanthraxandione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, Glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin, methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbamide, nitrogen mustard, thiotepa, chlorambucil, melphalan, carmustine, lomustine, cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, cisplatin, dactinomycin, bleomycin, anthramycin, calicheamicin, gemcitabine avena cava, bendamustine, bortezomib, carboplatin, cabazitaxel, dasatinib, docetaxel, epirubicin, erlotinib, everolimus, gemcitabine, gefitinib, idarubicin, imatinib, hydroxyurea, lapatinib, leuprolide, melphalan, nedaplatin, nilotinib, oxaliplatin, pazopanib, pemetrexed, picoplatin, romidepsin, satraplatin, sorafenib, vemurafenib, sunitinib, teniposide, triplatinib, and vinorelbine.

22. A pharmaceutical composition for treating cancer, comprising the antibody-drug conjugate according to claim 20.

23. The pharmaceutical composition for treating cancer according to claim 22, wherein: The cancer is selected from bladder cancer, bone cancer, brain tumor, breast cancer, heart cancer, cervical cancer, colorectal cancer, rectal cancer, esophageal cancer, fibrosarcoma, gastric cancer, gastrointestinal cancer, head and neck cancer, Kaposi's sarcoma, kidney cancer, leukemia, liver cancer, lung cancer, lymphoma, melanoma, myeloma, ovarian cancer, pancreatic cancer, penile cancer, prostate cancer, testicular germ cell cancer, thymoma and thymic carcinoma.

24. The pharmaceutical composition for treating cancer according to claim 23, wherein The cancer is breast cancer.

25. A method for treating cancer, comprising administering to a subject a pharmaceutical composition comprising the antibody-drug conjugate according to claim 20.

26. The method for treating cancer according to claim 25, wherein: The cancer is selected from bladder cancer, bone cancer, brain tumor, breast cancer, heart cancer, cervical cancer, colorectal cancer, rectal cancer, esophageal cancer, fibrosarcoma, gastric cancer, gastrointestinal cancer, head and neck cancer, Kaposi's sarcoma, kidney cancer, leukemia, liver cancer, lung cancer, lymphoma, melanoma, myeloma, ovarian cancer, pancreatic cancer, penile cancer, prostate cancer, testicular germ cell cancer, thymoma and thymic carcinoma.

27. A method for treating cancer according to claim 26, wherein: The cancer is breast cancer.

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