Site-specific antibody conjugation and antibody-drug conjugate as a specific example thereof

Abstract

The present application relates to a technology that enables a specific number of chemical functional groups or load moieties to be labeled at specific sites of an antibody. The present application can provide an antibody product with a high degree of homogeneity. In addition, the present application can provide an antibody product in which the function of the antibody does not decrease. That is, an antibody product can be provided in which its affinity and half-life do not decrease. The present application is the first technology that can site-specifically label antibodies without any complex process, and is therefore very meaningful.

Description

Technical Field

[0001] This invention relates to techniques for selectively labeling specific amino acid residues of antibodies with substances (low molecular weight compounds, synthetic polymers, biopolymers (i.e., peptides, carbohydrates, proteins, etc.)) or for linking molecules (hereinafter referred to as "cargo") to antibodies to be delivered to specific target cells or tissues. Furthermore, this invention relates to techniques for labeling specific sites of antibodies with a desired amount of substance or for linking a desired amount of cargo to specific amino acid residues of antibodies. Additionally, this invention covers antibody complexes prepared by this method or by using antibody fragment complexes. Background Technology

[0002] Antibodies are biomolecules that have the function of recognizing specific molecules and have been used in a variety of industrial applications. For example, antibodies can be used to detect or screen specific substances and to examine the pathways by which specific substances move in the body or cells. In addition, antibodies can be used for therapeutic purposes by inducing an immune response against a specific substance.

[0003] To expand the functionality of such antibodies, attempts have been made to enhance their capabilities. Typically, attempts are made to label antibodies with foreign substances to supplement or extend their functionality. Generally, when antibodies are labeled with fluorescent substances, they can be used for fluorescence assays, or when antibodies are labeled with reagents for treating specific diseases, they can be used to maximize the therapeutic effect of the antibody. These attempts and techniques are generally referred to as "antibody labeling." This invention relates to a novel method for labeling antibodies.

[0004] Initially, antibody labeling was achieved by randomly attaching foreign substances to antibodies. However, this method has many problems. The problem with antibodies prepared in this way is their poor homogeneity. The problem with these antibodies is that their effects remain uneven because the amount of substance attached to each antibody varies, and the binding sites of the antibodies also differ. This problem is a major obstacle to the development of antibody-drug conjugate (ADC) technologies that require high safety and reproducibility.

[0005] Furthermore, this method suffers from the problem of potentially significantly reduced antibody recognition ability. Antibodies consist of a Fab domain, which includes an antigen-binding domain that recognizes the antigen, and an Fc domain that participates in antibody crystallization. Random labeling, by allowing foreign substances to bind to the antibody's antigen-binding domain or sites adjacent to it, leads to a highly degraded antibody recognition ability.

[0006] Therefore, there is a need in the relevant field for techniques to uniformly label antibodies in a site-specific manner. Although some techniques have been developed, such as genetically regulated or modified antibodies, most of them are technically and economically inefficient. Therefore, the present invention is designed to address these problems and thus targets techniques capable of specifically linking a particular substance (or "part") to a specific site of an antibody (a technique that does not require any additional regulation of the antibody), as well as techniques that also use antibodies to deliver a particular substance (a drug or labeled substance) into cells or tissues. Summary of the Invention

[0007] [Technical Issues]

[0008] This invention provides techniques for the specific transfer of chemical functional groups to specific sites on antibodies. In one embodiment, the invention provides a technique for the specific transfer of a chemical functional group to lysine 246 in an antibody. In another embodiment, the invention provides a technique for the specific transfer of a chemical functional group to lysine 248 in an antibody. In yet another embodiment, the invention provides techniques for the specific transfer of chemical functional groups to lysines 246 and lysine 248 in antibodies.

[0009] This invention provides a technique for linking a desired number of chemical functional groups to an antibody. In one specific embodiment, the invention provides an antibody or a fragment thereof having two specific portions thereto. In another specific embodiment, the invention provides an antibody or a fragment thereof having four specific portions thereto.

[0010] This invention provides techniques for specifically linking a payload portion to a specific site on an antibody. In one embodiment, the invention provides a technique for specifically linking a payload portion to lysine 246 in an antibody. In another embodiment, the invention provides a technique for specifically linking a payload portion to lysine 248 in an antibody. In yet another embodiment, the invention provides techniques for specifically linking a payload portion to both lysine 246 and lysine 248 in an antibody.

[0011] This invention provides a technique for linking a desired number of payload portions to an antibody. In one embodiment, the invention provides an antibody or a fragment thereof having two payload portions thereto bound. In another embodiment, the invention provides an antibody or a fragment thereof having four payload portions thereto bound.

[0012] This invention provides a method for using the aforementioned antibody or antibody fragment. In one specific embodiment, this invention provides a method for treating a specific disease using an antibody-drug complex.

[0013] [Technical Solution]

[0014] This application provides compounds of Formula 2:

[0015] [Equation 2]

[0016]

[0017] Wherein, H1 is the first-click chemical functional group, and D1 is selected from covalent bonds, C 1～4 Alkylene, C 2～4 imidene group, C 2～4 etyne and C 3～8 Cycloalkylene, X1 is S, D2 is selected from C 1～7 Alkylene, C 2～7 imidene group, C 2～7 etyne and C 3～8 Cycloalkylene, where X2 is O and R2′ is N-succinimide, p-nitrophenyl or pentafluorophenyl.

[0018] This application also provides compounds in which H1 is selected from terminal alkynes, azides, strained alkynes, dienes, dienophiles, alkenes, thiols, and tetrazines. Furthermore, this application provides compounds in which H1 is selected from norbornene, tetrazines, azides, and dibenzocyclooctynylamines.

[0019] In addition, this application provides compounds in which R2′ is N-succinimide.

[0020] Furthermore, this application provides compounds in which formula 2 is formula 2-1, formula 2-2, or formula 2-3:

[0021] [Equation 2-1]

[0022]

[0023] [Equation 2-2]

[0024]

[0025] [Equation 2-3]

[0026]

[0027] This application provides a peptide having the structure of Formula 4-2:

[0028] [Equation 4-2]

[0029] (Xaa) 1-3 -C-(Xaa)2-H-Xa1-G-Xa2-LV-Xa3-C-(Xaa) 1-3

[0030] In this context, each Xaa is independently any amino acid residue other than cysteine, C is a cysteine ​​residue, H is a histidine residue, G is a glycine residue, Xa2 is a glutamic acid residue or asparagine residue, L is a leucine residue, V is a valine residue, Xa3 is selected from tryptophan residues, naphthylalanine residues, and phenylalanine residues, and Xa1 is... Where D3 is a covalent bond or C 1～3 The peptide is an alkylene group, and X3 is NH2, wherein the peptide consists of 13 to 17 amino acid residues, wherein the peptide exhibits activity of binding human immunoglobulin G (IgG), and wherein a cysteine ​​residue 2 to 4 amino acids from the N-terminus is optionally linked to a cysteine ​​residue 2 to 4 amino acids from the C-terminus.

[0031] This application also provides peptides in which D3 is covalently bonded, methylene or ethylene.

[0032] Furthermore, this application provides a peptide in which formula 4-2 is a derivative of formula 4-6:

[0033] [Equation 4-6]

[0034] DCAWH-Xa1-GELVWCT

[0035] Where 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.

[0036] This application provides peptide-compound conjugates having the structure of Formula 6-2:

[0037] [Equation 6-2]

[0038] (Xaa) 1-3 -C-(Xaa)2-H-(Xa1)′-G-Xa2-LV-Xa3-C-(Xaa) 1-3

[0039] In this context, each Xaa is independently any amino acid residue other than cysteine, C is a cysteine ​​residue, H is a histidine residue, G is a glycine residue, Xa2 is a glutamic acid residue or asparagine residue, L is a leucine residue, V is a valine residue, and Xa3 is selected from tryptophan residues, naphthylalanine residues, and phenylalanine residues.

[0040] (Xa1)′ is Where H1 is the first-click chemical functional group, and D1 is selected from covalent bonds, C 1～4 Alkylene, C 2～4 imidene group, C 2～4 etyne and C 3～8 Cycloalkylene, X1 is S, D2 is selected from C1～7 Alkylene, C 2～7 imidene group, C 2～7 etyne and C 3～8 Cycloalkylene, D3 is covalent or C 1～3 The peptide is an alkylene group, and X3 is NH, wherein the peptide consists of 13 to 17 amino acid residues, wherein the peptide exhibits activity in binding 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.

[0041] This application also provides peptide-compound conjugates wherein the distance from the β carbon of (Xa1)′ to the first carbonyl carbon of (Xa1)′ is less than approximately

[0042] Furthermore, this application provides peptide-compound conjugates, wherein D2 is C y Alkylene, C y imide or C y The acetylenic group, D3 is C x Alkylene, where y is an integer greater than or equal to 1, and 1 ≤ x + y ≤ 5.

[0043] Furthermore, this application provides peptide-compound conjugates wherein the distance from the β carbon of (Xa1)′ to the first carbonyl carbon of (Xa1)′ is greater than approximately

[0044] Furthermore, this application provides peptide-compound conjugates, wherein D2 is C y Alkylene, C y imide or C y Ethyne group, and D3 is C x Alkylene, wherein y is an integer greater than or equal to 1, and 9 ≤ x + y ≤ 12. Furthermore, this application provides peptide-compound conjugates, wherein D2 is a C y imide or C y Alynyl group.

[0045] Furthermore, this application provides peptide-compound conjugates wherein the distance from the β carbon of (Xa1)′ to the first carbonyl carbon of (Xa1)′ is approximately to approximately

[0046] In addition, this application provides peptide-compound conjugates, wherein D2 is C y Alkylene, C y imide or C y Alkyne group, and D3 is C x Alkylene, where y is an integer greater than or equal to 1, and 6 ≤ x + y ≤ 8.

[0047] Furthermore, this application provides peptide-compound conjugates in which formula 6-2 is equivalent to formula 6-3:

[0048] [Equation 6-3]

[0049] DCAWH-(Xa1)′-GELVWCT

[0050] Where 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.

[0051] In addition, this application provides peptide-compound conjugates, wherein D1 is a covalent bond and D2 is a methylene group.

[0052] This application provides a method for preparing a reagent for transferring a first-click chemical functional group to an antibody, the method comprising reacting a compound of formula 2 with a peptide having a structure of formula 4-2:

[0053] [Equation 2]

[0054]

[0055] Wherein, H1 is the first-click chemical functional group, and D1 is selected from covalent bonds, C 1～4 Alkylene, C 2～4 imidene group, C 2～4 etyne and C 3～8 Cycloalkylene, X1 is S, D2 is selected from C 1～7 Alkylene, C 2～7 imidene group, C 2～7 etyne and C 3～8 Cycloalkylene, where X2 is O and R2′ is N-succinimide, p-nitrophenyl or pentafluorophenyl;

[0056] [Equation 4-2]

[0057] (Xaa) 1-3 -C-(Xaa)2-H-Xa1-G-Xa2-LV-Xa3-C-(Xaa) 1-3

[0058] In this context, each Xaa is independently any amino acid residue other than cysteine, C is a cysteine ​​residue, H is a histidine residue, G is a glycine residue, Xa2 is a glutamic acid residue or asparagine residue, L is a leucine residue, V is a valine residue, and Xa3 is selected from tryptophan residues, naphthylalanine residues, and phenylalanine residues.

[0059] Xa1 is Where D3 is a covalent bond or C 1～3The alkylene group, and X3 is NH2, wherein the peptide consists of 13 to 17 amino acid residues, wherein the peptide exhibits activity of binding human immunoglobulin G (IgG), and wherein a cysteine ​​residue 2 to 4 amino acids from the N-terminus is optionally linked to a cysteine ​​residue 2 to 4 amino acids from the C-terminus.

[0060] This application also provides a method for preparing reagents for transferring first-click chemical functional groups to antibodies, wherein D2 is C y Alkylene, C y imide or C y Alkyne group, 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 prepared by the method for transferring a first click chemical functional group to an antibody can react with the antibody to specifically deliver the first click chemical functional group to lysine residue 248 of the Fc domain of the antibody.

[0061] Furthermore, this application provides a method for preparing reagents for transferring a first-click chemical functional group to an antibody, wherein D2 is C y Alkylene, C y imide or C y Ethyne group, 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 prepared by the method for transferring a first click chemical functional group to an antibody can react with the antibody to specifically deliver the first click chemical functional group to lysine residue 248 of the Fc domain of the antibody.

[0062] Furthermore, this application provides a method for preparing reagents for transferring a first-click chemical functional group to an antibody, wherein D2 is C y Alkylene, C y imide or C y Alkyne group, 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 prepared by the method for transferring a first click chemical functional group to an antibody can react with the antibody to specifically deliver the first click chemical functional group to lysine residue 246 of the Fc domain of the antibody.

[0063] Furthermore, this application provides a method for preparing reagents for transferring a first-click chemical functional group to an antibody, wherein D2 is C y Alkylene, C y imide or C y Alkyne group, and D3 is C xAlkylene, wherein y is an integer greater than or equal to 1 and 6 ≤ x + y ≤ 8, characterized in that the reagent prepared by the method for transferring a first click chemical functional group to an antibody can react with the antibody to selectively deliver the first click chemical functional group to lysine 246 residue or lysine 248 residue of the Fc domain of the antibody.

[0064] This application provides a kit for preparing a first-click chemical functional group transferred to an antibody, the kit comprising a compound of formula 2 and a peptide having a structure of formula 4-2:

[0065] [Equation 2]

[0066]

[0067] Wherein, H1 is the first-click chemical functional group, and D1 is selected from covalent bonds, C 1～4 Alkylene, C 2～4 imidene group, C 2～4 etyne and C 3～8 Cycloalkylene, X1 is S, D2 is selected from C 1～7 Alkylene, C 2～7 imidene group, C 2～7 etyne and C 3～8 Cycloalkylene, where X2 is O and R2′ is N-succinimide, p-nitrophenyl or pentafluorophenyl;

[0068] [Equation 4-2]

[0069] (Xaa) 1-3 -C-(Xaa)2-H-Xa1-G-Xa2-LV-Xa3-C-(Xaa) 1-3

[0070] In this context, each Xaa is independently any amino acid residue other than cysteine, C is a cysteine ​​residue, H is a histidine residue, G is a glycine residue, Xa2 is a glutamic acid residue or asparagine residue, L is a leucine residue, V is a valine residue, and Xa3 is selected from tryptophan residues, naphthylalanine residues, and phenylalanine residues.

[0071] Xa1 is Where D3 is a covalent bond or C 1～3 The alkylene group, and X3 is NH2, wherein the peptide consists of 13 to 17 amino acid residues, wherein the peptide exhibits activity of binding human immunoglobulin G (IgG), and wherein a cysteine ​​residue 2 to 4 amino acids from the N-terminus is optionally linked to a cysteine ​​residue 2 to 4 amino acids from the C-terminus.

[0072] This application provides antibodies or fragments thereof comprising one or more amino acid sequences selected from Formula 8-1, Formula 8-2, and Formula 8-3:

[0073] [Equation 8-1]

[0074] GPSVFLFPP-(K)′-PKDTLMI

[0075] [Equation 8-2]

[0076] GPSVFLFPPKP-(K)′-DTLMI

[0077] [Equation 8-3]

[0078] GPSVFLFPP-(K)′-P-(K)′-DTLMI

[0079] Wherein, G represents glycine residue, P represents proline residue, S represents serine residue, V represents valine residue, F represents phenylalanine residue, L represents leucine residue, K represents lysine residue, D represents aspartic acid residue, T represents threonine residue, M represents methionine residue, and I represents isoleucine residue.

[0080] (K)′ is Where H1 is the first-click chemical functional group, and D1 is selected from covalent bonds, C 1～4 Alkylene, C 2～4 imidene group, C 2～4 etyne and C 3～8 Cycloalkylene.

[0081] This application also provides antibodies or fragments thereof in which D1 is a covalent bond.

[0082] Furthermore, this application provides an antibody or fragment thereof comprising the amino acid sequence of Formula 8-1 but excluding the amino acid sequences of Formulas 8-2 and 8-3. Additionally, this application provides an antibody or fragment thereof comprising the amino acid sequence of Formula 8-1 in both Fc domains.

[0083] Furthermore, this application provides an antibody or fragment thereof comprising the amino acid sequence of Formula 8-2 but excluding the amino acid sequences of Formulas 8-1 and 8-3. Additionally, this application provides an antibody or fragment thereof comprising the amino acid sequence of Formula 8-2 in both Fc domains.

[0084] Furthermore, this application provides antibodies or fragments thereof comprising the amino acid sequence of Formula 8-3 but excluding the amino acid sequences of Formulas 8-1 and 8-2. Additionally, this application provides antibodies or fragments thereof comprising the amino acid sequence of Formula 8-3 in both Fc domains.

[0085] This application provides a method for preparing an antibody or fragment thereof comprising a first click chemical functional group, the method comprising reacting a peptide-compound conjugate having a structure of formula 6-2 with an antibody or fragment thereof:

[0086] [Equation 6-2]

[0087] (Xaa) 1-3 -C-(Xaa)2-H-(Xa1)′-G-Xa2-LV-Xa3-C-(Xaa) 1-3

[0088] In this context, each Xaa is independently any amino acid residue other than cysteine, C is a cysteine ​​residue, H is a histidine residue, G is a glycine residue, Xa2 is a glutamic acid residue or asparagine residue, L is a leucine residue, V is a valine residue, and Xa3 is selected from tryptophan residues, naphthylalanine residues, and phenylalanine residues.

[0089] (Xa1)′ is Where H1 is the first-click chemical functional group, and D1 is selected from covalent bonds, C 1～4 Alkylene, C 2～4 imidene group, C 2～4 etyne and C 3～8 Cycloalkylene, X1 is S, D2 is selected from C 1～7 Alkylene, C 2～7 imidene group, C 2～7 etyne and C 3～8 Cycloalkylene, D3 is covalent or C 1～3 The peptide is an alkylene group, and X3 is NH, wherein the peptide consists of 13 to 17 amino acid residues, wherein the peptide exhibits activity of binding human immunoglobulin G (IgG), and wherein a cysteine ​​residue 2 to 4 amino acids from the N-terminus is optionally linked to a cysteine ​​residue 2 to 4 amino acids from the C-terminus.

[0090] This application provides a kit for preparing antibodies or fragments thereof containing a first click chemical functional group, the kit comprising a peptide-compound conjugate having a formula 6-2 structure and an antibody or fragment thereof:

[0091] [Equation 6-2]

[0092] (Xaa) 1-3 -C-(Xaa)2-H-(Xa1)′-G-Xa2-LV-Xa3-C-(Xaa) 1-3

[0093] Wherein, each Xaa is independently any amino acid residue other than cysteine, C is a cysteine ​​residue, H is a histidine residue, G is a glycine residue, Xa2 is a glutamic acid residue or asparagine residue, L is a leucine residue, V is a valine residue, and W is a tryptophan residue, and

[0094] (Xa1)′ is Where H1 is the first-click chemical functional group, and D1 is selected from covalent bonds, C 1～4 Alkylene, C 2～4 imidene group, C 2～4 etyne and C 3～8 Cycloalkylene, X1 is S, D2 is selected from C 1～7 Alkylene, C 2～7 imidene group, C 2～7 etyne and C 3～8 Cycloalkylene, D3 is covalent or C 1～3 The peptide is an alkylene group, and X3 is NH, wherein the peptide consists of 13 to 17 amino acid residues, wherein the peptide exhibits activity of binding human immunoglobulin G (IgG), and wherein a cysteine ​​residue 2 to 4 amino acids from the N-terminus is optionally linked to a cysteine ​​residue 2 to 4 amino acids from the C-terminus.

[0095] This application provides compounds having the structure of Formula 9:

[0096] [Formula 9]

[0097] C m ——————H2

[0098] Among them, C m For the loading part, H2 is the second click chemical functional group.

[0099] This application provides a method for preparing antibody-drug conjugates, the method comprising reacting an antibody or 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:

[0100] [Equation 8-1]

[0101] GPSVFLFPP-(K)′-PKDTLMI

[0102] [Equation 8-2]

[0103] GPSVFLFPPKP-(K)′-DTLMI

[0104] [Equation 8-3]

[0105] GPSVFLFPP-(K)′-P-(K)′-DTLMI

[0106] Wherein, G represents glycine residue, P represents proline residue, S represents serine residue, V represents valine residue, F represents phenylalanine residue, L represents leucine residue, K represents lysine residue, D represents aspartic acid residue, T represents threonine residue, M represents methionine residue, and I represents isoleucine residue.

[0107] (K)′ is Where H1 is the first-click chemical functional group, and D1 is selected from covalent bonds, C 1～4 Alkylene, C 2～4 imidene group, C 2～4 etyne and C 3～8 Cycloalkylene;

[0108] [Formula 9]

[0109] C m ——————H2

[0110] Among them, C m For the load portion, H2 is a second click chemical functional group that is complementary to the first click chemical functional group.

[0111] This application provides a kit for preparing antibody-drug conjugates, the kit comprising an antibody or a fragment thereof and a compound having a structure 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:

[0112] [Equation 8-1]

[0113] GPSVFLFPP-(K)′-PKDTLMI

[0114] [Equation 8-2]

[0115] GPSVFLFPPKP-(K)′-DTLMI

[0116] [Equation 8-3]

[0117] GPSVFLFPP-(K)′-P-(K)′-DTLMI

[0118] Wherein, G represents glycine residue, P represents proline residue, S represents serine residue, V represents valine residue, F represents phenylalanine residue, L represents leucine residue, K represents lysine residue, D represents aspartic acid residue, T represents threonine residue, M represents methionine residue, and I represents isoleucine residue.

[0119] (K)′ is Where H1 is the first-click chemical functional group, and D1 is selected from covalent bonds, C 1～4 Alkylene, C 2～4 imidene group, C 2～4 etyne and C 3～8 Cycloalkylene;

[0120] [Formula 9]

[0121] C m —————H2

[0122] Among them, C m For the load portion, H2 is a second click chemical functional group that is complementary to the first click chemical functional group.

[0123] This application provides antibodies or fragments thereof comprising one or more amino acid sequences selected from Formula 10-1, Formula 10-2, and Formula 10-3:

[0124] [Equation 10-1]

[0125] GPSVFLFPP-(K)″-PKDTLMI

[0126] [Equation 10-2]

[0127] GPSVFLFpPKP-(K)″-DTLMI

[0128] [Equation 10-3]

[0129] GPSVFLFPP-(K)″-P-(K)″-DTLMI

[0130] Wherein, G represents glycine residue, P represents proline residue, S represents serine residue, V represents valine residue, F represents phenylalanine residue, L represents leucine residue, K represents lysine residue, D represents aspartic acid residue, T represents threonine residue, M represents methionine residue, and I represents isoleucine residue.

[0131] (K)” is Where C m For the load portion, B is selected from

[0132] A1 and A2 are connected to the load section or D1, and they are not all connected to the same section, R x Selected from H, halogens and C 1～3 Alkyl group, D1 is selected from covalent, C 1～4Alkylene, C 2～4 imidene group, C 2～4 etyne and C 3～8 Cycloalkylene.

[0133] This application also provides antibodies or fragments thereof comprising the amino acid sequence of Formula 10-1 but excluding the amino acid sequences of Formulas 10-2 and 10-3. Furthermore, this application provides antibodies or fragments thereof comprising the amino acid sequence of Formula 10-1 in both Fc domains.

[0134] Furthermore, this application provides antibodies or fragments thereof comprising the amino acid sequence of Formula 10-2 but excluding the amino acid sequences of Formulas 10-1 and 10-3. Additionally, this application provides antibodies or fragments thereof comprising the amino acid sequence of Formula 10-2 in both Fc domains.

[0135] Furthermore, this application provides antibodies or fragments thereof comprising the amino acid sequence of Formula 10-3 but excluding the amino acid sequences of Formulas 10-1 and 10-2. Additionally, this application provides antibodies or fragments thereof comprising the amino acid sequence of Formula 10-3 in both Fc domains.

[0136] Furthermore, this application provides antibodies or fragments thereof in which the loading portion includes a drug portion. Additionally, this application provides antibodies or fragments thereof in which the loading portion includes two or more drug portions. Alternatively, this application provides antibodies or fragments thereof in which the drug portion is an anticancer agent. In addition, this application provides antibodies or fragments thereof, wherein the anticancer agent is selected from at least one of 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, cytokines, apoptosis agents, anti-angiogenic agents, lymphokines, taxanes, DNA alkylating agents, anthracyclines, tubulysin analogs, pyruvic acid analogs, auristatin E, auristatin F, maytansinoids, cytotoxic agents containing reactive polyethylene glycol moieties, taxon, cytochalasin B, bacitracin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, t. colchicine (t. Doxorubicin, daunorubicin, dihydroxyanthraquinone, mitoxantrone, sclerosomycin, actinomycin D, 1-dihydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin, methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil aminoimide, nitrogen mustard, thiotepa, chlorambucil, melphalan, carmustine, lomustine, cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, cisplatin, sclerosomycin, bleomycin, amiodarone, kacisplatin Calicutamine, Abiraterone, Bendamustine, Bortezomib, Carboplatin, Cabazitaxel, Dasatinib, Docetaxel, Epirubicin, Erlotinib, Everolimus, Gemcitabine, Gefitinib, Idarubicin, Imatinib, Hydroxyurea, Lapatinib, Leuprorelin, Melphalan, Nedaplatin, Nilotinib, Oxaliplatin, Pazopanib, Pemetrexed, Pyrapoplatin, Romidixin, Saplatin, Sorafenib, Vemurafenib, Sunitinib, Teniposide, Triplatin, and Vinorelbine.

[0137] This application provides a pharmaceutical composition for treating cancer, the pharmaceutical composition comprising the antibody-drug conjugate described above.

[0138] This 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.

[0139] In addition, this application provides a pharmaceutical composition for treating cancer, wherein the cancer is breast cancer.

[0140] This application provides a method for treating cancer, the method comprising administering to a subject a pharmaceutical composition comprising the antibody-drug conjugate described above.

[0141] This 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, stomach 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.

[0142] In addition, this application provides a method for treating cancer, wherein the cancer is breast cancer.

[0143] [Beneficial Effects]

[0144] The antibody products according to the present invention may have a specific number of chemical functional groups or loading moieties labeled at specific sites. Therefore, the present invention can provide antibody products with high uniformity. The present invention can also provide antibody products whose antibody function does not degrade. That is, the present invention can provide antibody products whose antibody binding affinity and half-life are not reduced. As the first technology to perform site-specific labeling of antibodies without any complex processes, the present invention is of great significance. Attached Figure Description

[0145] Figure 1 The partial sequence of the Fc structural domain and the numbering in the sequence according to the EU numbering system are shown.

[0146] Figure 2 The positions of lysine residues in the Fc domain, including lysine 246 and lysine 248, are shown.

[0147] Figure 3 and Figure 4 The topology between the SSFI and Fc domains is shown.

[0148] Figure 5 Lysine 246 and lysine 248 in the Fc domain and Xa1 of SSFI are shown.

[0149] Figure 6 The distance between the amino group of lysine 246 and the β carbon of Xa1 in SSFI is shown.

[0150] Figure 7 The distance between the amino group of lysine 248 and the β carbon of Xa1 in SSFI is shown.

[0151] Figure 8 The structure of R1′-L2-SSFI and the distance (L) between the first carbonyl carbon and the β carbon in (Xa1)′ are shown. c ).

[0152] Figure 9 The topology between the R1′-L2-SSFI and Fc structural domains is shown, such that the side chains of (Xa1)′ have a direction parallel to the x-axis in the figure (dashed arrow).

[0153] Figure 10 The conditions for allowing the first carbonyl carbon to react well with lysine 248 are shown.

[0154] Figure 11 The conditions for allowing the first carbonyl carbon to react well with lysine 246 are shown.

[0155] Figure 12The conditions for allowing the first carbonyl carbon to react selectively with lysine 246 or lysine 248 are shown.

[0156] Figure 13 The FcRn binding site and lysine 246 and lysine 248 in the Fc domain are shown.

[0157] Figure 14 Analysis of the binding structure between the SSFI and Fc domains is shown compared to the binding site between Fc and FcRn.

[0158] Figure 15 A method for synthesizing compound I is shown.

[0159] Figure 16 The results of confirming the structure of compound I by mass spectrometry are shown.

[0160] Figure 17 A method for synthesizing compound II is shown.

[0161] Figure 18 The results of confirming the structure of compound II by mass spectrometry are shown.

[0162] Figure 19 A method for synthesizing compound III is shown.

[0163] Figure 20 The results of confirming the structure of compound III by mass spectrometry are shown.

[0164] Figure 21 and Figure 22 A method for synthesizing compound IV is shown.

[0165] Figure 23 The results of confirming the structure of compound IV by mass spectrometry are shown.

[0166] Figure 24 The results of confirming the structure of SSFI(6Lys) by mass spectrometry are shown.

[0167] Figure 25 The results of confirming the structure of SSFI(6Orn) by mass spectrometry are shown.

[0168] Figure 26 The results of confirming the structure of SSFI(6Dab) by mass spectrometry are shown.

[0169] Figure 27 The results of confirming the structure of SSFI(6Dap) by mass spectrometry are shown.

[0170] Figure 28 The results of confirming the structure of DD2 by mass spectrometry are shown.

[0171] Figure 29 The results of confirming the structure of DD3 by mass spectrometry are shown.

[0172] Figure 30 The results of confirming the structure of DD4 by mass spectrometry are shown.

[0173] Figure 31 The results of confirming the structure of DD5 by mass spectrometry are shown.

[0174] Figure 32 The results of confirming the structure of DD6 by mass spectrometry are shown.

[0175] Figure 33 The results of confirming the structure of compound I-SSFI(6Lys) by mass spectrometry are shown.

[0176] Figure 34 The results of confirming the structure of compound II-SSFI(6Lys) by mass spectrometry are shown.

[0177] Figure 35 The results of confirming the structure of compound III-SSFI(6Lys) by mass spectrometry are shown.

[0178] Figure 36 The results of confirming the structure of compound III-SSFI(6Orn) by mass spectrometry are shown.

[0179] Figure 37 The results of confirming the structure of compound III-SSFI(6Dab) by mass spectrometry are shown.

[0180] Figure 38 The results of confirming the structure of compound III-SSFI(6Dap) by mass spectrometry are shown.

[0181] Figure 39 The results of confirming the structure of compound IV-SSFI(6Dap) by mass spectrometry are shown.

[0182] Figure 40 The results of observing the binding reaction of trastuzumab and compound I-SSFI (6Lys) by HIC-HPLC are shown.

[0183] Figure 41 The results of observing the binding reaction of trastuzumab and compound II-SSFI (6Lys) by HIC-HPLC are shown.

[0184] Figure 42 The reaction of the antibody with compound III-SSFI (6Dap, Dab, Orn, or Lys) is shown.

[0185] Figure 43 The structure of Ab(246 / 248)-norbornene as the final product is shown.

[0186] Figure 44 The results of observing the binding reaction of trastuzumab and compound III-SSFI (6Dap) by HIC-HPLC are shown.

[0187] Figure 45 The results of observing the binding reaction of trastuzumab and compound III-SSFI (6Dab) by HIC-HPLC are shown.

[0188] Figure 46 The results of observing the binding reaction of trastuzumab and compound III-SSFI (6Orn) by HIC-HPLC are shown.

[0189] Figure 47 The results of observing the binding reaction of trastuzumab and compound III-SSFI (6Lys) by HIC-HPLC are shown.

[0190] Figure 48 The results of observing the binding reaction of trastuzumab and compound IV-SSFI (6Dap) by HIC-HPLC are shown.

[0191] Figure 49 and Figure 50 The increase in molecular weight spectrum caused by antibody-norbornene binding is shown.

[0192] Figures 51 to 54 MS / MS chromatographic results of trastuzumab and antibody-norbornene complex are shown.

[0193] Figure 55 The sequence matching results using MS / MS spectra are shown.

[0194] Figure 56 The mass spectrum of trastuzumab measured to confirm the structure of the trastuzumab-azide compound is shown.

[0195] Figure 57 The mass spectrum of the trastuzumab-azide complex, measured to confirm the structure of the trastuzumab-azide, is shown.

[0196] Figure 58 The results of confirming the structure of tetrazine-DM1 by mass spectrometry are shown.

[0197] Figure 59 The structure of the trastuzumab-DM1 conjugate based on compound III-SSFI(6Dap) is shown.

[0198] Figure 60 The results of observing the formation reaction of trastuzumab-DM1 conjugate by HIC-HPLC are shown.

[0199] Figure 61 and Figure 62 The increase in molecular weight spectrum caused by norbornene-tetraazine-DM1 binding is shown.

[0200] Figure 63 The structures of three designs for the payload bound to norbornene-labeled antibodies are shown.

[0201] Figure 64 The results of analyzing the antigen-binding affinity of antibody-drug conjugates are shown.

[0202] Figures 65 to 67 The results of the analysis of serum stability of antibody-drug conjugates are shown.

[0203] Figures 68 to 70 The results of evaluating the efficacy of antibody-drug conjugates at the cellular level are shown.

[0204] Figure 71 and Figure 72 The results of evaluating the efficacy of antibody-drug conjugates at the animal level are shown.

[0205] Figure 73 The results of pharmacokinetic tests for the antibody-drug conjugate are shown. Detailed Implementation

[0206] definition

[0207] Unless otherwise defined, all technical and scientific terms used in this invention shall have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The following references are provided to provide, to those skilled in the art, many common definitions of the terms used in this invention: Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd edition, 1994); The Cambridge Dictionary of Science and Technology (Walker, ed., 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 as used in this invention shall have the meanings described herein.

[0208] In some implementations, the chemical structure is disclosed along with the corresponding chemical name. When the terms contradict or conflict, the chemical structure takes precedence over the chemical name so that the meaning of the compound can be understood through the chemical structure.

[0209] As used in this invention, the term "heteroatom" refers to a compound or group of compounds containing at least one heteroatom. The term "heteroatom" refers to an atom other than a carbon or hydrogen atom, and includes, for example, B, Si, N, P, O, S, and Se. Preferably, the heteroatom comprises multivalent elements such as N, O, and S, or monovalent elements such as F, Cl, Br, and I, but the invention is not limited thereto.

[0210] The term "lower" as used in this invention is used to modify hydrocarbons, such as alkylene compounds, and therefore refers to hydrocarbons having six or fewer carbon atoms. For example, C 1-6 Straight-chain or branched alkyl groups are referred to by another name, such as "lower alkyl" groups.

[0211] The term "oxygen group" as used in this invention refers to the secondary radical (-O-) of an oxygen atom.

[0212] The terms "alkyl" or "alkane" refer to fully saturated straight-chain or branched non-aromatic hydrocarbons. Unless otherwise defined, straight-chain or branched alkyl groups typically have 1 to about 20 carbon atoms, preferably 1 to about 10 carbon atoms. Straight-chain and branched alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, pentyl, hexyl, pentyl, and octyl.

[0213] The term "alkenyl" or "olefin" refers to a straight-chain or branched non-aromatic hydrocarbon containing at least one double bond. Unless otherwise defined, straight-chain or branched alkenyl groups typically have 1 to about 20 carbon atoms, preferably 1 to about 10 carbon atoms.

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

[0215] The terms "cycloalkane" or "cycloalkyl" refer to a fully saturated cyclic hydrocarbon. "Cycloalkyl" includes monocyclic and polycyclic compounds. 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 in a polycyclic cycloalkyl group can be selected from saturated, unsaturated, and aromatic rings. The cycloalkyl group includes bicyclic molecules containing one, two, three, or more atoms shared between two rings. The term "fused cycloalkyl" refers to a polycyclic cycloalkyl group in which one ring shares two adjacent atoms with another ring. The rings other than the first ring in a fused polycyclic cycloalkyl group can be selected from saturated, unsaturated, and aromatic rings.

[0216] The term "cycloalkynyl" or "cycloalkynyl group" refers to a cyclic hydrocarbon containing at least one triple bond, also known as a "stressed alkyne." The "cycloalkynyl group" includes monocyclic and polycyclic compounds. Unless otherwise defined, monocyclic cycloalkynyl groups typically have 3 to approximately 10 carbon atoms, more commonly 3 to 8 carbon atoms. In polycyclic cycloalkynyl groups, the rings other than the first ring can be selected from saturated, unsaturated, and aromatic rings. The cycloalkynyl group is a bicyclic molecule containing one, two, three, 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. In fused polycyclic cycloalkynyl groups, the rings other than the first ring can be selected from saturated, unsaturated, and aromatic rings.

[0217] The term "alkylene" as used as the molecule itself or as part of another molecule refers to a divalent radical derived from an alkane. For example, this group includes -CH2CH2- and -CH2CH2CH2CH2-, both containing 10 or fewer carbon atoms, but the invention is not limited thereto. The term "lower alkylene" refers to a short alkylene group that typically has 6 or fewer carbon atoms. Unless otherwise stated, the term "alkylene" is intended to cover groups represented by "heteroalkylene" in this invention.

[0218] The term "heteroalkylene" refers to a divalent radical derived from a heteroalkyl group, and includes, for example, -CH2-CH2-S-CH2-CH2- and -CH2-S-CH2-CH2-NH-CH2-, but the invention is not limited thereto. The heteroalkylene group may contain the same or different heteroatoms (including alkyleneoxy, alkylenedioxy, alkyleneamino, alkylenediamino, aminooxyalkylene, etc., but the invention is not limited thereto) at each or all ends of its chain. Furthermore, the indication of the connection at both ends of the chain is independent of the group arrangement of the formula. For example, the formula -C(O)2R′- refers to both -C(O)2R′- and -R′(O)2C-.

[0219] The term "alkenyl" as used as the molecule itself or as part of another molecule refers to a divalent radical derived from an olefin. For example, this group includes -CH=CH-, -CH2CH=CHCH2-, and -CH=CH-CH=CH-, all having 10 or fewer carbon atoms, but the invention is not limited thereto. Unless otherwise stated, the term "alkenyl" is intended to cover heteroalkenyl groups as used in this invention.

[0220] The term "hypoynyl" as used as the molecule itself or as part of another molecule refers to a divalent radical derived from an alkyne. For example, this group includes -C≡C-, -CH2C≡CCH2-, and -C≡CC≡C-, each having 10 or fewer carbon atoms, but the invention is not limited thereto. Unless otherwise stated, the term "hypoynyl" is intended to cover heterohypoynyl groups as used in this invention.

[0221] The term "cycloalkylene" as used as a molecule itself or as part of another molecule refers to a divalent radical derived from a cycloalkane. Unless otherwise stated, "cycloalkylene" is intended to cover heterocycloalkylene compounds in this invention.

[0222] The term "alkylene" as used in this specification, examples, and claims is intended to cover both "unsubstituted alkylene" and "substituted alkylene." The latter refers to an alkylene group having a substituent having a hydrogen atom on one or more carbon atoms of the substituted hydrocarbon. Unless otherwise expressly indicated, the substituent may include, for example, halogens, hydroxyl groups, carbonyl groups (e.g., carboxyl, alkoxycarbonyl, formyl, or acyl), thiocarbonyl groups (e.g., thioesters, thioacetates, or thiocarbamates), alkoxy groups, phosphoryl groups, phosphate groups, phosphonate groups, hypophosphonate groups, amino groups, amide groups, amido groups, imine groups, cyano groups, nitro groups, azide groups, mercapto groups, alkylthio groups, sulfate groups, sulfonic acid groups, aminosulfonyl groups, sulfinylamino groups, sulfonyl groups, heterocyclic groups, aralkyl groups, or aromatic or heteroaromatic groups. When appropriately substituted, those skilled in the art will understand that the substituted residues on the hydrocarbon chain themselves can be substituted. For example, substituents in substituted alkylene groups may include substituted and unsubstituted amino, azide, imino, amide, phosphoryl (including phosphonates / esters and hypophosphonates / esters), sulfonyl (including sulfates / esters, sulfinamides, aminosulfonyls, and sulfonates / esters), and silyl groups, and may also include ethers, alkylthio, carbonyl (including ketones, aldehydes, carboxyl groups, and esters), -CF3, -CN, and their equivalents. Exemplary substituted alkyl groups are described below. Cycloalkylene groups may be further substituted with alkyl, alkenyl, alkoxy, alkylthio, aminoalkyl, carbonyl-substituted alkyl groups, -CF3, -CN, and their equivalents. This also applies to alkenyl and alkyneyl groups.

[0223] When used with residues such as alkylene, alkenylene, or ynylene, the term "C" is used. x-y "This aims to cover residues, for example, that contain x to y carbon atoms in their chain. For example, the term 'C'..." x-y "Alkylene" refers to a substituted or unsubstituted, straight-chain or branched alkylene group containing x to y carbon atoms in its chain. For example, it is intended to include, exemplarily, haloalkylene groups such as difluoromethylene, 2,2,2-trifluoroethylene, etc. CO alkylene refers to a covalent bond. The term "C"... 2-y "Ideinyl" and "C" 2-y "Alynyl" refers to a substituted or unsubstituted unsaturated aliphatic residue, applying the definitions of length and possible substitutions as described in the definition of alkylene to that residue. However, this means that each contains at least one double or triple bond.

[0224] The term "click chemistry" is used as a chemical concept introduced by K. Barry Sharpless of the Scripps Research Institute to explain chemical reactions and complementary chemical functional groups designed to enable two molecules to rapidly and stably form covalent bonds. Click chemistry does not refer to a specific reaction, but rather to the concept of such a rapid and stable reaction. In any embodiment, the click chemistry must be modular, broad-ranging, high-yield, produce only negligible byproducts, be stereooriented, physiologically stable, thermodynamically driven (e.g., greater than 84 kJ / mol), and / or have high atom economy. Some reactions are known to satisfy the following requirements:

[0225] (1) Huisgen 1,3-dipolar cycloaddition (e.g., including Cu(I)-catalyzed cycloaddition reactions, often referred to as “click reactions”; see Tornee et al., Journal of Organic Chemistry (2002) 67:3057-3064): copper or ruthenium is usually used as a catalyst;

[0226] [Schematic diagram of Huisgen 1,3-dipolar cycloaddition]

[0227]

[0228] (2) Diels-Alder reactions, such as cycloadditions (e.g. strain-promoted cycloadditions (SPAAC)), include normal electron-demanding Diels-Alder reactions and anti-electron-demanding Diels-Alder reactions, but the present invention is not limited thereto.

[0229] [Schematic diagram of the Diels-Alder reaction]

[0230]

[0231] [Examples of the Diels-Alder reaction; TCO and tetrazine]

[0232]

[0233] [Schematic diagram of tension-promoted cycloaddition]

[0234]

[0235] [Examples of strain-promoted cycloaddition; azides and DBCO]

[0236]

[0237] (3) Nucleophilic addition to low-strain rings of epoxides and aziridines, etc.;

[0238] (4) Nucleophilic addition to activated carbonyl groups;

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

[0240] [Addition to thiols and alkenes]

[0241]

[0242] The term "click chemistry functional group" as used in this invention refers to a functional group that participates in a click chemistry reaction. For example, a strained alkyne (e.g., cyclooctylene) corresponds to a click chemistry functional group. Generally, the click chemistry reaction requires at least two molecules, each containing a click chemistry functional group that is complementary to the other. Thus, pairs of click chemistry functional groups that are reactive with each other are generally referred to as "partner click chemistry functional groups" in this invention. For example, in the strain-promoted cycloaddition of cyclooctylene with an azide, the azide is the partner click chemistry functional group of cyclooctylene and other alkynes. Exemplary click chemistry functional groups used in this invention include terminal alkynes, azides, strained alkynes, dienes, dienophiles, trans-cyclooctene, alkenes, thiols, and tetrazines, but the invention is not limited thereto. Other click chemistry functional groups are known to those skilled in the art.

[0243] The term "leaving group" as used in this invention is the same as the concept well known to those skilled in the art (Advanced Organic Chemistry: reactions, mechanisms and structure, 4th edition, Jerry March, John Wiley and Sons, eds.; 1992, pp. 351-357), referring to a chemical functional group attached to any reactant that moves when the reactant is subjected to a substitution reaction (displacement reaction), such as a nucleophilic substitution reaction. A good leaving group is a leaving group that moves readily during a nucleophilic substitution reaction. Exemplary good leaving groups include halogens (F, Cl, Br and I), toluenesulfonates / esters, methanesulfonates / esters, trifluoromethanesulfonates / esters, acetates, trifluoromethyl acetates, camphorsulfonates / esters, 2-thionylbenzo[d]thiazolyl-3(2H)-yl, N-hydrogenated succinimide, N-aryl oxides, and aryl oxides substituted with one or more electron-withdrawing groups (EWG), but the 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-dinitrophenylene oxide, pentafluorophenylene oxide, 2-chloro-4-nitrobenzene oxide, 2,4-dichlorophenylene oxide, and 2,4,6-chlorophenylene oxide, and the electron-withdrawing groups include, for example, halogens (F, Cl, Br, or I), -NO2, -CN, -C(O)(C 1-6Alkyl), -C(O)(aryl), -C(O)O(C 1-6 Alkyl groups, -C(O)O (aryl groups), etc.

[0244] As used in this invention, the term "interaction set" refers to the proteins or peptides involved in a protein-protein interaction (PPI) when such interactions exist between proteins or peptides. For example, a chaperone protein and its passenger protein are an interaction set. "Protein-protein interaction" refers to the interaction of two or more protein or peptide molecules to achieve highly specific physical contact with each other. In this context, causal interactions include electromagnetic forces, hydrogen bonds, and hydrophobic interactions, but the invention is not limited thereto.

[0245] The term "antibody interactome" as used in this invention refers to the interaction set of antibodies, including immunoglobulins. Exemplary antibody interactomes are listed in Table 1. Among them, peptides known to bind to the Fc domains of immunoglobulins are also referred to as "Fc interactomes".

[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 bind specifically to a particular antigen. However, because the concept of an antibody according to the present invention also includes its fragments, the antibody does not necessarily exhibit the binding ability to a specific antigen as in the case of an Fc fragment. In addition to naturally occurring immunoglobulins, the antibody is also intended to cover all recombinant proteins, fusion proteins, chimeric proteins, human immunoglobulins, non-human animal immunoglobulins, etc., having the same or similar structures.

[0250] According to the present invention, the term "conjugate" refers to heterogeneous molecules produced when conjugates are paired together to form a covalent bond. The covalent bond can preferably be formed via a click chemistry reaction. The term "conjugate chaperone" refers to each molecule intended to form the conjugate. In this context, for convenience, when someone wishing to perform the conjugation intends to link a particular molecule to any other molecule, that particular molecule may generally be referred to as the "target molecule" or "target protein," while any other molecule may generally be referred to as the "loaded molecule" or "loaded portion."

[0251] According to the present invention, the term "carrier portion" refers to a portion of the molecule constituting the conjugate, i.e., a molecule that enhances the serum stability of the molecule to which it is attached or prolongs the half-life of the molecule. Molecules that can be used as carrier portions are well known in the relevant art. Representative examples of carrier portions include albumin, gelatin, elastin (including proelastin), polypeptides derived from elastin (α-elastin and elastin-like polypeptides (ELP)), gliadin, ginsenosides, zein, soy protein (e.g., soy protein isolate (SPI)), milk protein, 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 cover dyes or dye reagents used for fluorescence. Molecules that can be used as dyes and dye reagents are well known in the relevant fields. Representative examples of fluorescent moieties are listed in Table 2, but the invention is not limited thereto (Immunotech-Coulter Corp. catalog, "Cytometry Monoclonal Reagent Guide", 8 / 95, p. 3).

[0253] Table 2 List of Fluorescent Components

[0254]

[0255] According to the present invention, the term "pharmaceutical fraction" refers to a molecule that has a therapeutic effect on any disease. Pharmaceutical fractions according to the present invention include those known to those skilled in the art as being effective against any disease. Typically, pharmaceutical fractions with anticancer activity include 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, cytokines, apoptosis inducers, anti-angiogenic agents, lymphokines, taxanes, DNA alkylating agents, anthracyclines, tubulysin analogs, pyruvic analogs, auristatin E, and auristatin. F. Maytansinoids, cytotoxic agents containing reactive polyethylene glycol residues, taxon, cytochalasin B, bacitracin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthraquinone, mitoxantrone, scintillans, actinomycin D, 1-dihydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin, methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil aminoimide, nitrogen mustard, thiotepa, chlorambucil, melphalan, carmustine Lomustine, cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, cisplatin, cytosine, bleomycin, amiodarone, chachiin, abiraterone, bendamustine, bortezomib, carboplatin, carbamate, dasatinib, docetaxel, epirubicin, erlotinib, everolimus, gemcitabine, gefitinib, idarubicin, imatinib, hydroxyurea, lapatinib, leuprorelin, melphalan, nedaplatin, nilotinib, oxaliplatin, pazopanib, pemetrexed, pyrplatin, romidesin, saplatin, sorafenib, vemurafenib, sunitinib, teniposide, triplatinin, and vinorelbine, but the invention is not limited thereto.

[0256] According to the present invention, the term "radioactive portion" refers to a portion comprising a radioactive isotope. Radioactive isotope labeling can be used in diagnostic imaging and radiotherapy. Representative radioactive portions 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 excipients, diluents, or adjuvants. The carrier may be selected, for example, from 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, physiological saline, buffers such as PBS, methylparaben, propylparaben, talc, magnesium stearate, and mineral oil. The carrier may include fillers, anti-agglutinants, lubricants, wetting agents, flavoring agents, emulsifiers, preservatives, or combinations thereof.

[0258] The term "pharmaceutically acceptable salt" refers to a salt in which the proteome and the biological efficacy and properties of the compounds according to the invention are preserved and are not undesirable in biological or other respects. In many cases, the proteome 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. 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 a method for achieving a beneficial or desired clinical outcome. For the purposes of this invention, non-limiting examples of beneficial or desired clinical outcomes include symptom relief, reduction of disease extent, stabilization of the disease state (i.e., no worsening), delay of progression or reduction of the rate of disease progression, (partial or complete) improvement or temporary relief and mitigation of the disease condition, and whether it is detectable. The term "treatment" refers to all therapeutic treatments as well as preventative or preventative methods. The treatment includes treatment required for disorders to be prevented and for disorders that have already occurred. "Relief" of disease refers to a reduction in a range of disease conditions and / or undesirable clinical signs and / or a delay or prolongation of the time course of disease progression compared to an untreated disease state.

[0260] "Therapeutic effective amount" (or "effective amount") refers to the amount of an active ingredient, such as the reagent according to the invention, sufficient to achieve therapeutic effect when administered to a subject or patient. Therefore, those skilled in the art can readily determine the content of the therapeutic effective amount constituting the composition according to the invention. In the context of vision therapy, "therapeutic effective amount" is a quantity that causes an objectively measured change in one or more parameters related to the treatment of an eye disease or condition, including an increase or decrease in the expression of one or more genes related to the eye disease or condition, induction of apoptosis or other cell death pathways, clinical improvement of symptoms, reduction of abnormal angiogenesis or inflammation, etc. Of course, the therapeutic effective amount can vary depending on the condition to be treated and the specific subject, the subject's weight and age, the severity of the disease or condition, the specific compound to be selected, the subsequent dosing regimen, adjustments to the dosing time, the dosing pattern, etc., all of which can be readily determined by those skilled in the art. In the context of combination therapy, it should be understood that the therapeutic effective amount constituting a certain active ingredient may differ from the therapeutic effective amount constituting an active ingredient administered as a single therapy (i.e., a treatment regimen using a chemical entity as the active ingredient).

[0261] "Subject" or "patient" refers to an animal that requires treatment achievable through the molecules of the present invention. Animals to be treated according to the present invention include vertebrates. Particularly preferred examples of animals include mammals, such as cattle, dogs, horses, cats, sheep, pigs, and primates (including humans and non-human primates).

[0262] The term "about" or "approximately" refers to a quantity, level, value, number, frequency, percentage, size, 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.

[0263] 1. Antibody

[0264] It should be understood that the description of this invention is intended to provide a description of the structure, academic system, and physiological function of the provided antibodies to aid in understanding the invention, and antibodies falling within the scope of the claims of this invention are not intended to limit the description of the invention.

[0265] Antibodies consist of two heavy chains and two light chains, as is known in the art. When antibodies are simply classified functionally, they are divided into a fragment (Fab) containing an antigen-binding variable region of the light chain and a crystallizable region that is part of the heavy chain. The Fab includes complementary sites for antigen binding and refers to the region, as is known in the art, that allows the antibody to have specific binding activity against the antigen. Because the Fc domain is a ligand for the Fc receptor (FcR) in the cell, it plays an important role in inducing the immune response. The Fc domain also plays an important role in prolonging the half-life of the antibody by binding to the neonatal Fc receptor, thereby allowing the antibody to be repeatedly internalized into the cell.

[0266] From these facts, some desired directions for antibody labeling can be deduced: (1) First, it is desirable to label antibodies at locations separated from their complementary sites. When labeled at or adjacent to complementary sites, the binding affinity of the antibody to the antigen may be significantly reduced. (2) Second, it is desirable to label antibodies at locations separated from FcR recognition sites, including FcRn. When labeled at or adjacent to the receptor recognition site, the antibody's immune response induction function may be reduced, or the antibody's half-life may be shortened. Information on Fc domain binding to active 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 this invention, when referring to the amino acid sequence of the Fc domain of an antibody, unless otherwise stated, the sequence numbering follows the EU numbering system. Following the study of IgG sequences, the EU numbering system has been widely used as a sequencing system for the Fc domain, as described in Edelman GM et al., The covalent structure of an entire gammaG immunoglobulin molecule; Proc. Natl. Acad. Sci. USA, May 1969; 63(1):78-85.

[0268] 1.1. Searching for the desired marker site

[0269] The labeling sites of antibodies can be designed based on the criteria of the labeling sites, namely (1) sites separated from complementary sites; and (2) sites separated from FcR recognition sites including FcRn. Examples of amino acids used in bioconjugation reactions typically include lysine, cysteine, and tyrosine. Lysine 246 (Lys) is present in the Fc domain of the antibody. 246 ) and lysine 248 (Lys 248 ) are residues that meet all the requirements and are therefore the desired marker sites. Containing Lys 246 and Lys 248 The sequence of the Fc domain of the residues is GPSVFLFPPKPKDTLMI, and... Figure 1 The diagram shows the sequence and number in the sequence numbered according to the EU numbering system. Figure 1 (SEQ ID NO:1).

[0270] In specific embodiments, 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 wherein lysine 246 is substituted. Furthermore, the antibody according to the present invention may include a derivative of SEQ ID NO:1 wherein lysine 248 is substituted. Additionally, the antibody according to the present invention may include a derivative of SEQ ID NO:1 wherein both lysine 246 and lysine 248 are substituted.

[0271] The sequence of SEQ ID NO:1 or a derivative thereof includes a sequence mutated within an acceptable range. In specific embodiments, the mutated sequence may have greater than or equal to about 90%, about 85%, about 80%, about 75%, or about 70% homology relative to SEQ ID NO:1 or a derivative thereof. In the specific embodiments specified below, it should be understood that the derivatives of SEQ ID NO:1 represented by Formulas 7-1 to 7-3, 8-1 to 8-3, and 10-1 to 10-3 also include sequences 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] This invention discloses novel compounds that can be used to label antibodies. For convenience, such compounds are referred to herein as linkers, denoted by the symbol "R1′-L1".

[0275] This invention provides compounds having the structure of Formula 1 below:

[0276] [Formula 1]

[0277]

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

[0279] D1 can be any alkylene, alkenylene, or ynylene group.

[0280] X1 is an element that is more electronegative than carbon.

[0281] D2 can be any alkylene, alkenylene, or ynylene group.

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

[0283] R2′ is the second chemical functional group.

[0284] In Formula 1, the carbonyl group attached to D1 is referred to as the first carbonyl group. Furthermore, the carbonyl group attached to D2 is referred to as the second carbonyl group.

[0285] In a specific embodiment, R1′ may include a click chemical functional group. The click chemical functional group may further include one or more selected from alkynes, azides, strained alkynes, dienes, dienophiles, alkenes, thiols, and tetrazines. Furthermore, the click chemical functional group may be selected from azides or strained alkynes. Furthermore, the click chemical functional group may be selected from azides or dibenzocyclooctynylamine. Furthermore, the click chemical functional group may be selected from dienes or dienophiles. Furthermore, the click chemical functional group may be selected from tetrazines or norbornene. Alternatively, the click chemical functional group may be selected from tetrazines or transcyclooctene. Furthermore, R1′ may contain two or more click chemical functional groups.

[0286] In other embodiments, R1′ may include a carrier portion, a fluorescent portion, a drug portion, or a radioactive portion. R1′ may also include a drug portion. Furthermore, R1′ may include a VC linker. In other embodiments, R1′ may include an antibody or an analogue thereof, said antibody or analogue thereof including complementary sites.

[0287] In a specific implementation, D1 may include components selected from covalent bonds, C... 1-4 Alkylene, C 2-4 imidene group, C 2-4 etyne and C 3-8Any of the cycloalkylene groups. D1 can also be -CH2OCH2-. Furthermore, D1 can be covalently bonded. When D1 is covalently bonded, R1′ is directly connected to the carbon atom of the first carbonyl group. In the following description of the structure of the compound, all alkylene, alkenylene, ynynylene, and cycloalkylene groups are intended to include heteroalkylene, heteroalkenylene, heteroynynylene, and heterocycloalkylene groups, respectively, the meanings of which are specified in the "Definitions" section.

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

[0289] In a specific implementation, D2 may include components selected from C. 1-7 Alkylene, C 2-7 imidene group, C 2-7 etyne and C 3-8 Any of the cycloalkylene groups. D2 can also be C. 1-2 Alkylene. Furthermore, D2 can be methylene.

[0290] In a specific implementation, X2 can be NR1, S, or O, where R1 can be H, a halogen, or a substituted or unsubstituted C. 1-3 Alkylene. 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 implementation, R2′ can be a 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-succinimidyl, p-nitrophenyl, or pentafluorophenyl. R2′ can also be N-succinimidyl. When a favorable leaving group is attached to the second carbonyl group, the reactivity of the second carbonyl group can be enhanced. For example, N-hydroxysuccinimidyl ester (NHS ester) is known to exhibit very high reactivity.

[0293] This invention provides compounds having the structures of the following formulas 1-2:

[0294] [Equation 1-2]

[0295]

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

[0297] D1 can be any alkylene, alkenylene, or ynylene group, and

[0298] R2′ is the second chemical functional group.

[0299] In a specific embodiment, R1′ may include a click chemical functional group. The click chemical functional group may further include one or more selected from alkynes, azides, strained alkynes, dienes, dienophiles, alkenes, thiols, and tetrazines. Furthermore, the click chemical functional group may be selected from azides or strained alkynes. Furthermore, the click chemical functional group may be selected from azides or dibenzocyclooctynylamine. Furthermore, the click chemical functional group may be selected from dienes or dienophiles. Furthermore, the click chemical functional group may be selected from tetrazines or norbornene. Alternatively, the click chemical functional group may be selected from tetrazines or transcyclooctene. Furthermore, R1′ may contain two or more click chemical functional groups.

[0300] In other embodiments, R1′ may include a carrier portion, a fluorescent portion, a drug portion, or a radioactive portion. R1′ may also include a drug portion. Furthermore, R1′ may include a VC linker. In other embodiments, R1′ may include an antibody or an analogue thereof, said antibody or analogue thereof including complementary sites.

[0301] In a specific implementation, D1 may include components selected from C. 1-7 Alkylene, C 2-7 imidene group, C 2-7 etyne and C 3-8 Any of the cycloalkylene groups. D1 can also be C. 1-2 Alkylene. Additionally, D1 can be methylene.

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

[0303] In specific embodiments, the compounds represented by formulas 1-2 may have the structures of formulas 1-3 as follows:

[0304] [Equation 1-3]

[0305]

[0306] 2.1. Connectors including first-click chemical functional groups (H1-L1)

[0307] This invention provides compounds having the structure of Formula 2 below:

[0308] [Equation 2]

[0309]

[0310] H1 is the first-click chemical functional group.

[0311] D1 can be any alkylene, alkenylene, or ynylene group.

[0312] X1 is an element that is more electronegative than carbon.

[0313] D2 can be any alkylene, alkenylene, or ynylene group.

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

[0315] R2′ represents the second chemical functional group. The connector with the structure of Formula 2 is referred to herein as a "connector containing the first click chemical functional group", which is indicated by the symbol "H1-L1".

[0316] In Formula 2, the carbonyl group attached to D1 is referred to as the first carbonyl group. Furthermore, the carbonyl group attached to D2 is referred to as the second carbonyl group.

[0317] In specific embodiments, H1 may include any one selected from alkynes, azides, strained alkynes, dienes, dienophiles, alkenes, thiols, and tetrazides. Furthermore, H1 may be an azide or a strained alkyne. Furthermore, H1 may be an azide or a dibenzocyclooctynylamine. Furthermore, H1 may be a diene or a dienophile. Furthermore, H1 may be a tetrazide or norbornene. Alternatively, H1 may be a tetrazide or transcyclooctene.

[0318] In a specific implementation, D1 may include components selected from covalent bonds, C... 1-4 Alkylene, C 2-4 imidene group, C 2-4 etyne and C 3-8 Any of the cycloalkylene groups. Furthermore, D1 can be -CH2OCH2-. Furthermore, D1 can be a covalent bond.

[0319] In a specific implementation, X1 can be NR1, S, or O, where R1 can be H, a halogen, or a substituted or unsubstituted C. 1-3 Alkylene. Furthermore, X1 can be S.

[0320] In a specific implementation, D2 may include components selected from C. 1-7 Alkylene, C 2-7 imidene group, C 2-7 etyne and C 3-8 Any of the cycloalkylene groups. D2 can also be C. 1-2 Alkylene. Additionally, D2 can be methylene.

[0321] In a specific implementation, X2 can be NR1, S, or O, where R1 can be H, a halogen, or a substituted or unsubstituted C. 1-3Alkylene. Furthermore, X2 can be O.

[0322] In a specific implementation, R2′ can be a 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-succinimidyl, p-nitrophenyl, or pentafluorophenyl. R2′ can also be N-succinimidyl.

[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] [Equation 2-1]

[0326]

[0327] [Equation 2-2]

[0328]

[0329] [Equation 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 the connectors and their sub-instances of Formulas 1 and 2, the sites of substitution reactions can be indicated based on the design of X1, X2, and R2′.

[0333] The linker disclosed in this invention functions to transfer R1′ to the target molecule via a substitution reaction occurring between activated first and / or second carbonyl groups. Such a substitution reaction is schematically shown in Scheme 1 below.

[0334] [Option 1]

[0335]

[0336] In Scheme 1, for convenience, the target molecule is designated as "Nu:". Nu: acts as a nucleophile because it possesses a non-shared electron pair, inducing a nucleophilic acyl substitution reaction with the first and / or second carbonyl groups to form a bond with the linker. In nucleophilic acyl substitution reactions, the reactivity of the carbonyl group can be determined by the basicity of the leaving group. Therefore, the reactivity of carbonyl groups is known to increase in the order of carboxylates / esters, amides, carboxylic acids, esters, thioesters, and acyl phosphates / esters. Carbonyl groups such as NHS esters are also known to be highly reactive because they form very stable leaving groups.

[0337] In specific embodiments, X1 and X2 can be elements that are more electronegative than carbon. X1 and X2 can also be NR1, S, or O, where R1 can be H, a halogen, or substituted or unsubstituted C. 1-3 Alkylene. The carbonyl group can be activated because X1 and X2, together with the residues they are attached to, can form leaving groups.

[0338] In this case, the 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 can be determined based on the difference in reactivity between the first and second carbonyl groups. For example, the first carbonyl group may react first when the basicity of the leaving group comprising X1 is lower than that of the leaving group comprising X2. In another embodiment, the second carbonyl group may react first when the basicity of the leaving group comprising X2 is lower than that of the leaving group comprising X1.

[0339] In a preferred embodiment, the reactivity of the second carbonyl group in the connector according to the invention is preferably higher than that of the first carbonyl group. According to the invention, selective reaction is achieved by allowing X2-R2′ to attach to the second carbonyl group to form a favorable leaving group, and by designing the first carbonyl group to exhibit mild reactivity, such as an amide, thioester, or ester (exhibiting moderate reactivity). For example, in the case of connectors of formulas 1-3, the second carbonyl group can be an NHS ester, thereby reacting faster than the first carbonyl group (thioester).

[0340] The prior art disclosed in US Publications 2018 / 0141976 A1 and WO 2018 / 199337 A1 is similar to the present invention in terms of reagent formation for transferring a first chemical functional group to an antibody (see Section 4 below), but differs from the present invention in that the crosslinking agent comprises two NHS esters. The two carbonyl groups of the crosslinking 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 the NHS esters, the crosslinking 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 or the like with mild reactivity.

[0341] 3. Site-specific antibody-interactome (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-interactomes, denoted by the symbol "SSAI".

[0343] The SSAI provided by the present invention can have binding activity to specific sites of antibodies.

[0344] In a specific embodiment, the SSAI can have binding activity to the Fab domain of the antibody. In this case, the SSAI can preferably have binding activity to sites separated from the complementary site 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 separated 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 that have specific binding activity to the Fc domain are referred to herein as "site-specific Fc interacting groups", which are represented by the symbol "SSFI".

[0348] In a specific implementation, 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] Each Xaa can be any amino acid except cysteine.

[0352] 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.

[0353] Xa1 is Where D3 is a covalent bond or C 1-3 Alkylene, and X3 is NH2, OH, or SH. As an analogue of the sequence AWHLGELVW (SEQ ID NO:2), this sequence was found to be active in binding to the Fc domain in the articles “Dias, RLA et al. (2006), Protein Ligand Design: From Phage Display to Synthetic Protein Epitope Mimetics 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 active motif according to the invention is characterized by: 1) First, specifying the key residues with Fc-binding activity; 2) The motif is also designed to allow nucleophilic substitution reactions with the linker by changing the fourth leucine residue of SEQ ID NO:2 to Xa1 with a free electron pair. (X) n This indicates that it consists of n X's, and (X) n-m This indicates a composition consisting of n or more but less than m X atoms. The carbon atom bonded to D3 is referred to as "β-carbon".

[0354] The amino acid sequence of Formula 3 can also have a structure represented by Formula 3-1 below:

[0355] [Equation 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.

[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 a peptide comprising each amino acid sequence of Formula 3 and its sub-examples is in the form of a cyclic peptide with its internal residues linked together, the peptide is known to have better binding activity. This invention provides cyclic peptides comprising the amino acid sequence of Formula 3.

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

[0361] [Equation 4-1]

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

[0363] Among them, the N-terminus 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, and Xa3 is selected from tryptophan, naphthylalanine, and phenylalanine.

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

[0366] 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.

[0367] This invention provides cyclic peptides having the structure of the following formula 4-2:

[0368] [Equation 4-2]

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

[0370] Each Xaa can be 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, and Xa3 is selected from tryptophan, naphthylalanine, and phenylalanine.

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

[0373] In a specific embodiment, the peptide may consist of 13 or more but no more than 17 amino acid residues.

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

[0375] 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.

[0376] In a specific implementation, an N-terminal (X) is formed. 1-3 One of the residues forms the C-terminus (X). 1-3 One of the residues can bind to each other. In an exemplary embodiment, the peptide of formula 4-2 may have the structure of formula 4-3 as follows:

[0377] [Equation 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-terminus 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 formula 4-4 as follows:

[0381] [Equation 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 ​​can be linked together.

[0384] This invention provides cyclic peptides having the structures of the following formulas 4-5:

[0385] [Equation 4-5]

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

[0387] Where D stands for aspartic acid and T stands for threonine.

[0388] Each Xaa can be 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, and Xa3 is selected from tryptophan, naphthylalanine, and phenylalanine.

[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 13 or more but no more than 17 amino acid residues.

[0392] In a specific embodiment, the cysteine ​​residues located two amino acids from the N-terminus and the cysteine ​​residues located two amino acids from the C-terminus can optionally be linked together. 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 implementation, equation 4-5 can be the same as equation 4-6 below:

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

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

[0396] In this context, A represents alanine, and E represents glutamic acid.

[0397] Peptides having structures of formulas 4-1 to 4-6 may possess antibody-binding activity. Furthermore, the peptides may have antibody-binding activity against immunoglobulin G (IgG). Additionally, the peptides may have antibody-binding activity against the Fc domain.

[0398] In a specific embodiment, the N-terminus of the SSFI according to the present invention can be succinylated. In a specific embodiment, the SSFI according to the present invention can have its N-terminus (X) [e.g., X-terminus]. 1-3 This includes polar amino acid residues. In other specific embodiments, the SSFI according to the invention may have polar amino acid residues at its C-terminus (X). 1-3 The residue includes polar amino acid residues. In this case, 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 interacting set according to the present invention can be arranged together with the Fc domain of an antibody having a specific topological structure.

[0400] In this document, compounds of formulas 4-6 are provided as examples to aid in understanding the invention, but the scope of the invention is not limited thereto. It should be noted that the following description also applies to compounds of formulas 3, 3-1, and 4-1 to 4-6, and for convenience, compounds of formulas 4-6 are provided only as examples.

[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 amino acid residues, the SSFI can be aligned with the Fc domain having a specific topology. 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 topologies formed therefrom 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 invention, it is important that the SSFI forms a stable topological structure with the Fc domain. This is because the reagents used to transfer the first chemical functional group to the antibody according to the invention, and the labeling process using those reagents, are designed based on the topological structure between the SSFI and the Fc domain as discovered in studies (see Sections 5.2, 5.3, and 5.4 below). It is disadvantageous when the interaction between the SSFI and the Fc domain becomes unstable during the design of the SSFI, thus interfering with its molecular topology, because the interaction may negatively impact the labeling process.

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

[0404] [Equation 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, etc. Figure 3 and Figure 4 This is illustrated (see DeLano, WL et al., Convergent solutions to binding at a protein-protein interface; Science 2000, 287, 1279-1283). In this case, the histidine residue at position 5 of the SSFI forms a salt bond with glutamic acid 380 in the Fc domain, indicating that this salt bond has a significant impact on the topology between the SSFI and the Fc domain (see...). Figure 4 (The dashed line in the diagram). Therefore, it is expected that the histidine residues and their positions will remain unchanged during the design of the SSFI (see the "H" immediately following residue Xa1 in Equations 3, 3-1, and 4-1 to 4-6). Furthermore, it was confirmed that glutamic acid 8 is an electronegative residue and therefore forms a salt bond with electronegative arginine 255 in the Fc domain, thus significantly affecting the topology between the SSFI and the Fc domain (see...). Figure 4 (dashed lines in the diagram). Therefore, the corresponding residues are preferably acidic amino acids that can correspond to glutamic acid and can be replaced by asparagine (see residue Xa2 in Formulas 3, 3-1, and 4-1 to 4-6). When amino acid residues are replaced by other amino acid residues or by any functional group, this may affect the intermolecular interactions, thereby affecting the topology between the SSFI and the Fc domain.

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

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

[0409] The distance between the amino group of lysine 246 and the β carbon of Xa1 was measured. The results confirmed that the minimum distance measured during bond rotation forming the lysine side chain was approximately [value missing]. (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 amino group of lysine 248 and the β carbon of Xa1 was measured. The results confirmed that the minimum distance measured during bond rotation forming the lysine side chain was approximately [value missing]. (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 may be an important consideration in designing the connector and the SSFI, as well as in the reagents for transferring the first chemical functional group to the antibody.

[0412] 4. Reagents for transferring the first chemical functional group to the antibody; conjugates 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 indicated herein by the symbol "R1′-L2-SSAI". Based on its structure, the compound is also referred to as a conjugate of R1′-L1 and SSAI (R1′-L2-SSAI conjugate).

[0414] This invention provides an R1′-L2-SSAI having the structure of Formula 5 below:

[0415] [Formula 5]

[0416]

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

[0418] D1 can be any alkylene, alkenylene, or ynylene group.

[0419] X1 is an element that is more electronegative than carbon.

[0420] D2 can be any alkylene, alkenylene, or ynylene group.

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

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

[0423] SSAI stands for site-specific antibody-antibody interactionome.

[0424] In a specific embodiment, R1′ may include a click chemical functional group. Furthermore, the click chemical functional group may include one or more selected from alkynes, azides, strained alkynes, dienes, dienophiles, alkenes, thiols, and tetrazines. Furthermore, the click chemical functional group may be selected from azides or strained alkynes. Furthermore, the click chemical functional group may be selected from azides or dibenzocyclooctynylamine. Furthermore, the click chemical functional group may be selected from dienes or dienophiles. Furthermore, the click chemical functional group may be selected from tetrazines or norbornene. Alternatively, the click chemical functional group may be selected from tetrazines or transcyclooctene. Furthermore, R1′ may contain two or more click chemical functional groups.

[0425] In other embodiments, R1′ may include a carrier portion, a fluorescent portion, a drug portion, or a radioactive portion. R1′ may also include a drug portion. Furthermore, R1′ may include a VC linker. In other embodiments, R1′ may include an antibody or an analogue thereof, said antibody or analogue thereof including complementary sites.

[0426] In a specific implementation, D1 may include components selected from covalent bonds, C... 1-4 Alkylene, C 2-4 imidene group, C 2-4 etyne and C 3-8 Any of the cycloalkylene groups. D1 can also be -CH2OCH2-. Furthermore, D1 can be a covalent bond.

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

[0428] In a specific implementation, D2 may include components selected from C. 1-7 Alkylene, C 2-7 imidene group, C 2-7 etyne and C 3-8 Any of the cycloalkylene groups. D2 can also be C 1-2 Alkylene. Additionally, D2 can be methylene.

[0429] In a specific implementation, X3 can be NH.

[0430] In a specific embodiment, the SSAI can be a peptide sequence that binds to the Fab domain. In other specific embodiments, the SSAI can be a peptide sequence that binds to the Fc domain.

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

[0432] This invention provides R1′-L2-SSFI comprising the amino acid sequence of Formula 5-1 below:

[0433] [Equation 5-1]

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

[0435] Each Xaa can be any amino acid except cysteine.

[0436] 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.

[0437] (Xa1)′ is

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

[0439] D1 can be any alkylene, alkenylene, or ynylene group.

[0440] X1 is an element that is more electronegative than carbon.

[0441] D2 can be any alkylene, alkenylene, or ynylene group.

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

[0443] X3 can be NH, O, or S.

[0444] In a specific embodiment, R1′ may include a click chemical functional group. The click chemical functional group may further include one or more selected from alkynes, azides, strained alkynes, dienes, dienophiles, alkenes, thiols, and tetrazines. Furthermore, the click chemical functional group may be selected from azides or strained alkynes. Furthermore, the click chemical functional group may be selected from azides or dibenzocyclooctynylamine. Furthermore, the click chemical functional group may be selected from dienes or dienophiles. Furthermore, the click chemical functional group may be selected from tetrazines or norbornene. Alternatively, the click chemical functional group may be selected from tetrazines or transcyclooctene. Furthermore, R1′ may contain two or more click chemical functional groups.

[0445] In other embodiments, R1′ may include a carrier portion, a fluorescent portion, or a drug portion. R1′ may also include a VC linker. Furthermore, R1′ may include a radioactive portion. R1′ may also include a drug portion. In other embodiments, R1′ may include an antibody or an analogue thereof, said antibody or analogue thereof including complementary sites.

[0446] In a specific implementation, D1 may include components selected from covalent bonds, C... 1-4 Alkylene, C 2-4 imidene group, C 2-4 etyne and C 3-8 Any of the cycloalkylene groups. Furthermore, D1 can be -CH2OCH2-. Furthermore, D1 can be a covalent bond.

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

[0448] In a specific implementation, D2 may include components selected from C. 1-7 Alkylene, C 2-7 imidene group, C 2-7 etyne and C 3-8 Any of the cycloalkylene groups. D2 can also be C 1-2 Alkylene. Additionally, D2 can be methylene.

[0449] In a specific implementation, X3 can be NH.

[0450] 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.

[0451] This invention provides an R1′-L2-SSFI having the structure of the following formula 5-2:

[0452] [Equation 5-2]

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

[0454] Each Xaa can be 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 (Xa1)′ is...

[0456] Wherein, R1′ is the first chemical functional group.

[0457] D1 can be any alkylene, alkenylene, or ynylene group.

[0458] X1 is an element that is more electronegative than carbon.

[0459] D2 can be any alkylene, alkenylene, or ynylene group.

[0460] D3 is a covalent bond or C 1-3 Alkylene, and

[0461] X3 can be NH, O, or S.

[0462] In a specific embodiment, R1′ may include a click chemical functional group. The click chemical functional group may further include one or more selected from alkynes, azides, strained alkynes, dienes, dienophiles, alkenes, thiols, and tetrazines. Furthermore, the click chemical functional group may be selected from azides or strained alkynes. Furthermore, the click chemical functional group may be selected from azides or dibenzocyclooctynylamine. Furthermore, the click chemical functional group may be selected from dienes or dienophiles. Furthermore, the click chemical functional group may be selected from tetrazines or norbornene. Alternatively, the click chemical functional group may be selected from tetrazines or transcyclooctene. Furthermore, R1′ may contain two or more click chemical functional groups.

[0463] In other embodiments, R1′ may include a carrier portion, a fluorescent portion, a drug portion, or a radioactive portion. R1′ may also include a drug portion. Furthermore, R1′ may include a VC linker. In other embodiments, R1′ may include an antibody or an analogue thereof, said antibody or analogue thereof including complementary sites.

[0464] In a specific implementation, D1 may include components selected from covalent bonds, C... 1-4 Alkylene, C 2-4 imidene group, C2-4 etyne and C 3-8 Any of the cycloalkylene groups. D1 can also be -CH2OCH2-. Furthermore, D1 can be covalently bonded.

[0465] In a specific implementation, X1 can be NR1, S, or O, where R1 can be H, a halogen, or a substituted or unsubstituted C. 1-3 Alkylene.

[0466] In a specific implementation, D2 may include components selected from C. 1-7 Alkylene, C 2-7 imidene group, C 2-7 etyne and C 3-8 Any of the cycloalkylene groups. D2 can also be C. 1-2 Alkylene. Additionally, D2 can be methylene.

[0467] In a specific implementation, Formula 5-2 may consist of 13 or more but less than 17 amino acid residues (including (Xa1)′).

[0468] In a specific implementation, cysteine ​​residues located 2 to 4 amino acids from the N-terminus and cysteine ​​residues located 2 to 4 amino acids from the C-terminus can optionally be linked together.

[0469] In a specific embodiment, X3 can be NH. 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.

[0470] In a specific implementation, an N-terminal (X) is formed. 1-3 One of the residues forms the C-terminus (X). 1-3 One of the residues can bind to the other.

[0471] In a specific implementation, equation 5-2 can be the same as equation 5-3:

[0472] [Equation 5-3]

[0473] DCAWH-(Xa1)′-GELVWCT (SEQ ID NO:13)

[0474] In this case, A is alanine and E is glutamic acid.

[0475] R1′-L2-SSAI or R1′-L2-SSFI having the structures of Formula 5 and Formulas 5-1 to 5-3 may have antibody-binding activity. R1′-L2-SSAI or R1′-L2-SSFI may also have immunoglobulin G (IgG)-binding activity. Furthermore, R1′-L2-SSAI or R1′-L2-SSFI may have binding activity to the Fc domain of the antibody.

[0476] 4.1. Reagents for transferring the first-click chemical functional groups to the antibody; conjugates of H1-L1 and SSAI (H1-L2-SSAI)

[0477] According to the present invention, a reagent for transferring a first click chemical functional group to an antibody is disclosed. This compound is indicated herein by the symbol "H1-L2-SSAI". Based on 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 indicated by the symbol "H1-L2-SSFI".

[0478] The H1-L2-SSAI or H1-L2-SSFI according to the present invention includes those wherein R1′ contains the click chemical functional group in section “4. Reagent for transferring a first chemical functional group to an antibody”, but the invention is not limited to the following exemplary embodiments.

[0479] This invention provides H1-L2-SSFI comprising the amino acid sequence of Formula 6-1 below:

[0480] [Equation 6-1]

[0481] (Xaa)2-H-(Xa1)'-G-Xa2-LV-Xa3 (SEQ ID NO:14)

[0482] Each Xaa can be any amino acid except cysteine.

[0483] 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.

[0484] (Xa1)′ is

[0485] H1 is the first-click chemical functional group.

[0486] D1 can be any alkylene, alkenylene, or ynylene group.

[0487] X1 is an element that is more electronegative than carbon.

[0488] D2 can be any alkylene, alkenylene, or ynylene group.

[0489] D3 is a covalent bond or C 1-3 Alkylene, and

[0490] X3 can be NH, O, or S.

[0491] In a specific embodiment, H1 may include any one selected from alkynes, azides, strained alkynes, dienes, dienophiles, alkenes, thiols, and tetrazides. Furthermore, H1 may be an azide or a strained alkyne. Furthermore, H1 may be an azide or a dibenzocyclooctynylamine. Furthermore, H1 may be a diene or a dienophile. Furthermore, H1 may be a tetrazide or norbornene. Alternatively, H1 may be a tetrazide or transcyclooctene.

[0492] In a specific implementation, D1 may include components selected from covalent bonds, C... 1-4 Alkylene, C 2-4 imidene group, C 2-4 etyne and C 3-8 Any of the cycloalkylene groups. D1 can also be -CH2OCH2-. Furthermore, D1 can be a covalent bond.

[0493] In a specific implementation, X1 can be NR1, S, or O, where R1 can be H, a halogen, or a substituted or unsubstituted C. 1-3 Alkylene.

[0494] In a specific implementation, D2 may include components selected from C. 1-7 Alkylene, C 2-7 imidene group, C 2-7 etyne and C 3-8 Any of the cycloalkylene groups. Furthermore, D2 can be C. 1-2 Alkylene. Additionally, D2 can be methylene.

[0495] In a specific implementation, X3 can be NH.

[0496] 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.

[0497] This invention provides an H1-L2-SSFI having the structure of the following formula 6-2:

[0498] [Equation 6-2]

[0499] (Xaa) 1-3 -C-(Xaa)2-H-(Xa1)′-G-Xa2-LV-Xa3-C-(Xaa) 1-3 (SEQ ID NO:15)

[0500] Each Xaa can be any amino acid except cysteine.

[0501] 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 (Xa1)′ is...

[0502] H1 is the first-click chemical functional group.

[0503] D1 can be any alkylene, alkenylene, or ynylene group.

[0504] X1 is an element that is more electronegative than carbon.

[0505] D2 can be any alkylene, alkenylene, or ynylene group.

[0506] D3 is a covalent bond or C 1-3 Alkylene, and

[0507] X3 can be NH, O, or S.

[0508] In a specific embodiment, H1 may include any one selected from alkynes, azides, strained alkynes, dienes, dienophiles, alkenes, thiols, and tetrazides. Furthermore, H1 may be an azide or a strained alkyne. Furthermore, H1 may be an azide or a dibenzocyclooctynylamine. Furthermore, H1 may be a diene or a dienophile. Furthermore, H1 may be a tetrazide or norbornene. Alternatively, H1 may be a tetrazide or transcyclooctene.

[0509] In a specific implementation, D1 may include components selected from covalent bonds, C... 1-4 Alkylene, C 2-4 imidene group, C 2-4 etyne and C 3-8 Any of the cycloalkylene groups. Furthermore, D1 can be -CH2OCH2-. Furthermore, D1 can be a covalent bond.

[0510] In a specific implementation, X1 can be NR1, S, or O, where R1 can be H, a halogen, or a substituted or unsubstituted C. 1-3 Alkylene.

[0511] In a specific implementation, D2 may include components selected from C. 1-7 Alkylene, C 2-7 imidene group, C 2-7 etyne and C 3-8 Any of the cycloalkylene groups. Furthermore, D2 can be C. 1-2 Alkylene. Additionally, D2 can be methylene.

[0512] In a specific implementation, Formula 6-2 may consist of 13 or more but less than 17 amino acid residues (including (Xa1)′).

[0513] In a specific implementation, cysteine ​​residues located 2 to 4 amino acids from the N-terminus and cysteine ​​residues located 2 to 4 amino acids from the C-terminus can optionally be linked together.

[0514] In a specific embodiment, X3 can be NH. 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.

[0515] In a specific implementation, an N-terminal (X) is formed. 1-3 One of the residues forms the C-terminus (X). 1-3 One of the residues can bind to the other.

[0516] In a specific implementation, equation 6-2 can be the same as equation 6-3:

[0517] [Formula 6-3](SEQ ID NO:16)

[0518] DCAWH-(Xa1)′-GELVWCT (SEQ ID NO:16)

[0519] In this case, A is alanine and E is glutamic acid.

[0520] H1-L2-SSAI or H1-L2-SSFI having structures of formulas 6-1 to 6-3 may have binding activity to antibodies. H1-L2-SSAI or H1-L2-SSFI may also have binding activity to immunoglobulin G (IgG). Furthermore, H1-L2-SSAI or H1-L2-SSFI may have binding activity to the Fc domain of the antibody.

[0521] 4.2. Method for preparing reagents for transferring first chemical functional groups to antibodies

[0522] 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 methods is provided to aid in understanding the present invention.

[0523] For example, a method for preparing compounds of formula 5 will be described. In a specific embodiment, compounds of formula 5 can be prepared by the reaction of scheme 2 below.

[0524] [Option 2]

[0525]

[0526] The R1′-L2-SSAI according to the invention can be prepared by reacting a site-specific antibody-interacting assembly (SSAI) with a linker (R1′-L1) according to the invention. The SSAI according to the invention is designed to include a nucleophile X3. X3 can attack an activated carbonyl group contained in the linker to induce a nucleophilic substitution reaction. In this case, the R1′-L2-SSAI according to the invention is prepared because X3 attacks the carbon of the second carbonyl group.

[0527] As a more specific example, a method for preparing compounds of formula 6-3 will be described. In a specific embodiment, compounds of formula 6-3 can be prepared by the reaction described in scheme 3 below.

[0528] [Option 3]

[0529]

[0530] SSAI comprising the amino acid sequence of Formula 3 or Formula 3-1 according to the invention and having the structure of each of Formulas 4-1 to 4-6 includes an Xa1 residue containing the nucleophile X3. R1′-L2-SSAI according to the invention is thus prepared because X3 attacks the second carbonyl group contained in the linker.

[0531] In a specific embodiment, in the connector according to the invention, the basicity of the leaving group including X2 can be lower than that of the leaving group including X1. In a specific embodiment, the connector according to the invention allows X2-R2′ attached to the second carbonyl group to form a favorable 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 that of the first carbonyl group. Therefore, X3 of SSAI can specifically attack the second carbonyl group of the connector.

[0532] According to the present invention, a method for preparing a reagent for transferring a first chemical functional group to an antibody is disclosed.

[0533] This invention provides a method for preparing R1′-L2-SSAI, the method comprising: reacting a linker according to the invention with a site-specific antibody interactome according to the invention.

[0534] In a specific implementation, the connector can be any one selected from Formula 1, Formula 2, and Formulas 2-1 to 2-3.

[0535] In a specific implementation, the site-specific antibody interactome may include or have any one of the structures selected from Formula 3, Formula 3-1 and Formulas 4-1 to 4-6.

[0536] The linker may also have the structure of Formula 2, and the site-specific antibody interacting group may have any structure selected from Formula 4-2 to Formula 4-6. Furthermore, the site-specific antibody interacting group may have the structure of Formula 4-6.

[0537] According to the present invention, a method for preparing a reagent for transferring a first click chemical functional group to an antibody is disclosed.

[0538] This invention provides a method for preparing H1-L2-SSAI, the method comprising: reacting a linker according to the invention with a site-specific antibody interactome according to the invention.

[0539] In a specific implementation, the connector can be any one selected from Formula 2, Formula 2-1 to Formula 2-3.

[0540] In a specific implementation, the site-specific antibody interactome may include or have any one of the structures selected from Formula 3, Formula 3-1 and Formulas 4-1 to 4-6.

[0541] The linker may also have the structure of Formula 2, and the site-specific antibody interactome may have any structure selected from Formulas 4-2 to 4-6. Furthermore, the site-specific antibody interactome may have the structure of Formula 4-6.

[0542] According to the present invention, a kit for preparing reagents for transferring a first chemical functional group to an antibody is disclosed.

[0543] The present invention provides a kit for preparing reagents 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 interactome according to the present invention.

[0544] In a specific implementation, the connector can be any one selected from Formula 1, Formula 2, and Formulas 2-1 to 2-3.

[0545] In a specific implementation, the site-specific antibody interactome may include or have any one of the structures selected from Formula 3, Formula 3-1 and Formulas 4-1 to 4-6.

[0546] The linker may also have the structure of Formula 2, and the site-specific antibody interacting group may have any structure selected from Formula 4-2 to Formula 4-6. Furthermore, the site-specific antibody interacting group may have the structure of Formula 4-6.

[0547] According to the present invention, a kit for preparing reagents for transferring a first click chemical functional group to an antibody is disclosed.

[0548] The present invention provides a kit for preparing reagents for transferring a first click chemical functional group to an antibody, the kit comprising a linker and a site-specific antibody interactome containing the first click chemical functional group according to the present invention.

[0549] In a specific implementation, the connector can be any one selected from Formula 2, Formula 2-1 to Formula 2-3.

[0550] In a specific implementation, the site-specific antibody interactome may include or have any one of the structures selected from Formula 3, Formula 3-1 and Formulas 4-1 to 4-6.

[0551] The linker may also have the structure of Formula 2, and the site-specific antibody interacting group may have any structure selected from Formula 4-2 to Formula 4-6. Furthermore, the site-specific antibody interacting group may have the structure of Formula 4-6.

[0552] 5. Antibodies containing the first functional group (R1′-Ab)

[0553] According to the present invention, an antibody containing a first chemical functional group is disclosed. Such a compound is indicated herein by the symbol "R1′-Ab".

[0554] This invention provides R1′-Ab as represented by Equation 7:

[0555] [Formula 7]

[0556]

[0557] Wherein, R1′ is the first chemical functional group.

[0558] D1 can be any alkylene, alkenylene, or ynylene group.

[0559] X4 is NH, O, or S, and

[0560] Ab stands for antibody.

[0561] In specific embodiments, R1′ can be a click chemical functional group. R1′ may also include any one selected from alkynes, azides, strained alkynes, dienes, dienophiles, alkenes, thiols, and tetrazines. Furthermore, R1′ can be an azide or a strained alkyne. Furthermore, R1′ can be an azide or a dibenzocyclooctynylamine. Furthermore, R1′ can be a diene or a dienophile. Furthermore, R1′ can be a tetrazine or norbornene. Alternatively, R1′ can be a tetrazine or transcyclooctene.

[0562] In other embodiments, R1′ may include a carrier portion, a fluorescent portion, a drug portion, or a radioactive portion. R1′ may also include a drug portion. Furthermore, R1′ may include a VC linker. In other embodiments, R1′ may include an antibody or an analogue thereof, said antibody or analogue thereof including complementary sites.

[0563] In a specific implementation, D1 may include components selected from covalent bonds, C... 1-4 Alkylene, C 2-4 imidene group, C 2-4 etyne and C 3-8 Any of the cycloalkylene groups. Furthermore, D1 can be -CH2OCH2-. Furthermore, D1 can be a covalent bond.

[0564] In one specific implementation, X4 may be NH.

[0565] In a specific embodiment, Ab can be a human antibody. In other embodiments, Ab can be a non-human animal antibody. In a specific embodiment, Ab can be immunoglobulin G (IgG). In a specific embodiment, Ab can be a complete antibody. In other embodiments, Ab can be a fragment of an antibody.

[0566] In a specific embodiment, X4 and Ab can be connected through the Fab domain of Ab. In other 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. Furthermore, X4 and Ab can be connected through lysine 246 in the Fc domain of Ab. Alternatively, X4 and Ab can be connected through both lysine 246 and lysine 248 in the Fc domain of Ab. In a specific embodiment, X4 and Ab can be connected through only one of the two Fc domains of Ab. In other embodiments, X4 and Ab can be connected through both Fc domains of Ab.

[0567] This invention provides antibodies or fragments thereof comprising the amino acid sequence of Formula 7-1 below:

[0568] [Equation 7-1]

[0569] GPSVFLFPP-(K)′-PKDTLMI(SEQ ID NO:17)

[0570] 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.

[0571] (K)′ is

[0572] Wherein, R1′ is the first chemical functional group, and

[0573] D1 can be any alkylene, alkenylene, or ynylene group. The antibody or its fragment has an R1′ linked via lysine 246 or a site corresponding to lysine 246 in its Fc domain.

[0574] In a specific embodiment, R1′ may include a click chemical functional group. The click chemical functional group may further include one or more selected from alkynes, azides, strained alkynes, dienes, dienophiles, alkenes, thiols, and tetrazines. Furthermore, the click chemical functional group may be selected from azides or strained alkynes. Furthermore, the click chemical functional group may be selected from azides or dibenzocyclooctynylamine. Furthermore, the click chemical functional group may be selected from dienes or dienophiles. Furthermore, the click chemical functional group may be selected from tetrazines or norbornene. Alternatively, the click chemical functional group may be selected from tetrazines or transcyclooctene. Furthermore, R1′ may contain two or more click chemical functional groups.

[0575] In other embodiments, R1′ may include a carrier portion, a fluorescent portion, a drug portion, or a radioactive portion. Furthermore, R1′ may include a drug portion. Additionally, R1′ may include a VC linker. In other embodiments, R1′ may include an antibody or an analogue thereof, said antibody or analogue thereof including complementary sites.

[0576] In a specific implementation, D1 may include components selected from covalent bonds, C... 1-4 Alkylene, C 2-4 imidene group, C 2-4 etyne and C 3-8 Any of the cycloalkylene groups. D1 can also be -CH2OCH2-. Furthermore, D1 can be a covalent bond.

[0577] In a specific embodiment, the antibody may be a human antibody. In other specific embodiments, the antibody may be a non-human animal antibody. In a specific embodiment, the antibody may be immunoglobulin G (IgG). In a specific embodiment, the antibody may be a complete antibody. In other specific embodiments, the antibody may be a fragment of an antibody.

[0578] In a specific embodiment, the antibody may contain the amino acid sequence of Formula 7-1 in only one of its two Fc domains. In other specific embodiments, the antibody may contain the amino acid sequence of Formula 7-1 in both of its Fc domains.

[0579] This invention provides antibodies or fragments thereof comprising the amino acid sequence of Formula 7-2 below:

[0580] [Equation 7-2]

[0581] GPSVFLFPPKP-(K)′-DTLMI(SEQ ID NO:18)

[0582] 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.

[0583] (K)′ is

[0584] Wherein, R1′ is the first chemical functional group, and

[0585] D1 can be any alkylene, alkenylene, or ynylene group. The antibody or its fragment has an R1′ linked via lysine 248 or a site corresponding to lysine 248 in its Fc domain.

[0586] In a specific embodiment, R1′ may include a click chemical functional group. The click chemical functional group may further include one or more selected from alkynes, azides, strained alkynes, dienes, dienophiles, alkenes, thiols, and tetrazines. Furthermore, the click chemical functional group may be selected from azides or strained alkynes. Furthermore, the click chemical functional group may be selected from azides or dibenzocyclooctynylamine. Furthermore, the click chemical functional group may be selected from dienes or dienophiles. Furthermore, the click chemical functional group may be selected from tetrazines or norbornene. Alternatively, the click chemical functional group may be selected from tetrazines or transcyclooctene. Furthermore, R1′ may contain two or more click chemical functional groups.

[0587] In other embodiments, R1′ may include a carrier portion, a fluorescent portion, a drug portion, or a radioactive portion. R1′ may also include a drug portion. Furthermore, R1′ may include a VC linker. In other embodiments, R1′ may include an antibody or an analogue thereof, said antibody or analogue thereof including complementary sites.

[0588] In a specific implementation, D1 may include components selected from covalent bonds, C... 1-4 Alkylene, C 2-4 imidene group, C2-4 etyne and C 3-8 Any of the cycloalkylene groups. Furthermore, D1 can be -CH2OCH2-. Furthermore, D1 can be covalently bonded.

[0589] In a specific embodiment, the antibody may be a human antibody. In other specific embodiments, the antibody may be a non-human animal antibody. In a specific embodiment, the antibody may be immunoglobulin G (IgG). In a specific embodiment, the antibody may be a complete antibody. In other specific embodiments, the antibody may be a fragment of an antibody.

[0590] In a specific embodiment, the antibody may contain the amino acid sequence of Formula 7-2 in only one of its two Fc domains. In other specific embodiments, the antibody may contain the amino acid sequence of Formula 7-2 in both of its Fc domains.

[0591] This invention provides antibodies or fragments thereof comprising the amino acid sequences of Formulas 7-3 below:

[0592] [Formula 7-3](SEQ ID NO:19)

[0593] GPSVFLFPP-(K)′-P-(K)′-DTLMI(SEQ ID NO:19)

[0594] 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.

[0595] (K)′ is

[0596] Wherein, R1′ is the first chemical functional group, and

[0597] D1 can be any alkylene, alkenylene, or ynylene group. The antibody or its fragment has an R1′ linked through lysine 246 and lysine 248 or a site corresponding to lysine 246 and lysine 248 in its Fc domain.

[0598] In a specific embodiment, R1′ may include a click chemical functional group. The click chemical functional group may further include one or more selected from alkynes, azides, strained alkynes, dienes, dienophiles, alkenes, thiols, and tetrazines. Furthermore, the click chemical functional group may be selected from azides or strained alkynes. Furthermore, the click chemical functional group may be selected from azides or dibenzocyclooctynylamine. Furthermore, the click chemical functional group may be selected from dienes or dienophiles. Furthermore, the click chemical functional group may be selected from tetrazines or norbornene. Alternatively, the click chemical functional group may be selected from tetrazines or transcyclooctene. Furthermore, R1′ may contain two or more click chemical functional groups.

[0599] In other embodiments, R1′ may include a carrier portion, a fluorescent portion, a drug portion, or a radioactive portion. R1′ may also include a drug portion. Furthermore, R1′ may include a VC linker. In other embodiments, R1′ may include an antibody or an analogue thereof, said antibody or analogue thereof including complementary sites.

[0600] In a specific implementation, D1 may include components selected from covalent bonds, C... 1-4 Alkylene, C 2-4 imidene group, C 2-4 etyne and C 3-8 Any of the cycloalkylene groups. D1 can also be -CH2OCH2-. Furthermore, D1 can be a covalent bond.

[0601] In a specific embodiment, the antibody may be a human antibody. In other specific embodiments, the antibody may be a non-human animal antibody. In a specific embodiment, the antibody may be immunoglobulin G (IgG). In a specific embodiment, the antibody may be a complete antibody. In other specific embodiments, the antibody may be a fragment of an antibody.

[0602] In a specific embodiment, the antibody may contain the amino acid sequence of Formula 7-3 in only one of its two Fc domains. In other specific embodiments, the antibody may contain the amino acid sequence of Formula 7-3 in both of its Fc domains.

[0603] The present invention provides an antibody or a fragment thereof comprising 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.

[0604] In a specific implementation, D1 can be a covalent bond.

[0605] In specific embodiments, the antibody or its fragment may comprise only the amino acid sequence of Formula 7-1, and may exclude the amino acid sequences of Formulas 7-2 and 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. Furthermore, the antibody may include the amino acid sequence of Formula 7-1 in both of its Fc domains.

[0606] In other specific embodiments, the antibody or fragment thereof may comprise only the amino acid sequence of Formula 7-2, and may exclude the amino acid sequences of Formulas 7-1 and 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. Furthermore, the antibody may include the amino acid sequence of Formula 7-2 in both of its Fc domains.

[0607] In specific embodiments, the antibody or its fragment may comprise only the amino acid sequence of Formula 7-3, and may exclude the amino acid sequences of Formulas 7-1 and 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. Furthermore, the antibody may include the amino acid sequence of Formula 7-3 in both of its Fc domains.

[0608] 5.1. Antibodies containing first-click chemical functional groups

[0609] According to the present invention, an antibody containing a first click chemical functional group is disclosed. Such a compound is indicated herein by the symbol "H1-Ab".

[0610] This invention provides H1-Ab represented by Equation 8:

[0611] [Formula 8]

[0612]

[0613] H1 is the first-click chemical functional group.

[0614] D1 can be any alkylene, alkenylene, or ynylene group.

[0615] X4 is NH, O, or S, and

[0616] Ab stands for antibody.

[0617] In specific embodiments, H1 may include any one selected from alkynes, azides, strained alkynes, dienes, dienophiles, alkenes, thiols, and tetrazines. Furthermore, H1 may be an azide or a strained alkyne. Furthermore, H1 may be an azide or a dibenzocyclooctynylamine. Furthermore, H1 may be a diene or a dienophile. Furthermore, H1 may be a tetrazine or norbornene. Alternatively, H1 may be a tetrazine or transcyclooctene.

[0618] In a specific implementation, D1 may include components selected from covalent bonds, C... 1-4 Alkylene, C 2-4 imidene group, C 2-4 etyne and C 3-8 Any of the cycloalkylene groups. D1 can also be -CH2OCH2-. Furthermore, D1 can be a covalent bond.

[0619] In one specific implementation, X4 may be NH.

[0620] In a specific embodiment, Ab can be a human antibody. In other embodiments, Ab can be a non-human animal antibody. In a specific embodiment, Ab can be immunoglobulin G (IgG). In a specific embodiment, Ab can be a complete antibody. In other embodiments, Ab can be a fragment of an antibody. In a specific embodiment, Ab can be a wild-type antibody. In other embodiments, Ab can be a manipulated antibody.

[0621] In a specific embodiment, X4 and Ab can be linked through the Fab domain of Ab. In other embodiments, X4 and Ab can be linked through the Fc domain of Ab. X4 and Ab can also be linked through lysine 246 or lysine 248 in the Fc domain of Ab. Furthermore, X4 and Ab can be linked through lysine 246 in the Fc domain of Ab. Alternatively, X4 and Ab can be linked through both lysine 246 and lysine 248 in the Fc domain of Ab. In a specific embodiment, X4 and Ab can be linked through only one of the two Fc domains of Ab. In other embodiments, X4 and Ab can be linked through both Fc domains of Ab.

[0622] This invention provides antibodies or fragments thereof comprising the amino acid sequence of Formula 8-1 below:

[0623] [Equation 8-1]

[0624] GPSVFLFPP-(K)′-PKDTLMI(SEQ ID NO:20)

[0625] 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.

[0626] (K)′ is

[0627] Wherein, H1 is the first-click chemical functional group, and

[0628] D1 can be any alkylene, alkenylene, or ynylene group. The antibody or its fragment has an H1 linked through lysine 246 or a site corresponding to lysine 246 in its Fc domain.

[0629] In specific embodiments, H1 may include any one selected from alkynes, azides, strained alkynes, dienes, dienophiles, alkenes, thiols, and tetrazines. Furthermore, H1 may be an azide or a strained alkyne. Furthermore, H1 may be an azide or a dibenzocyclooctynylamine. Furthermore, H1 may be a diene or a dienophile. Furthermore, H1 may be a tetrazine or norbornene. Alternatively, H1 may be a tetrazine or transcyclooctene.

[0630] In a specific implementation, D1 may include components selected from covalent bonds, C... 1-4 Alkylene, C 2-4 imidene group, C 2-4 etyne and C 3-8 Any of the cycloalkylene groups. D1 can also be -CH2OCH2-. Furthermore, D1 can be a covalent bond.

[0631] In a specific embodiment, the antibody may be a human antibody. In other specific embodiments, the antibody may be a non-human animal antibody. In a specific embodiment, the antibody may be immunoglobulin G (IgG). In a specific embodiment, the antibody may be a complete antibody. In other specific embodiments, the antibody may be a fragment of an antibody. In a specific embodiment, the antibody may be a wild-type antibody. In other specific embodiments, the antibody may be a manipulated antibody.

[0632] In a specific embodiment, the antibody may contain the amino acid sequence of Formula 8-1 in only one of its two Fc domains. In other specific embodiments, the antibody may contain the amino acid sequence of Formula 8-1 in both of its Fc domains.

[0633] This invention provides antibodies or fragments thereof comprising the amino acid sequence of Formula 8-2 below:

[0634] [Equation 8-2]

[0635] GPSVFLFPPKP-(K)′-DTLMI(SEQ ID NO:21)

[0636] 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.

[0637] (K)′ is

[0638] Wherein, H1 is the first-click chemical functional group, and

[0639] D1 can be any alkylene, alkenylene, or ynylene group. The antibody or its fragment has an H1 linked through lysine 248 or a site corresponding to lysine 248 in its Fc domain.

[0640] In specific embodiments, H1 may include any one selected from alkynes, azides, strained alkynes, dienes, dienophiles, alkenes, thiols, and tetrazines. Furthermore, H1 may be an azide or a strained alkyne. Furthermore, H1 may be an azide or a dibenzocyclooctynylamine. Furthermore, H1 may be a diene or a dienophile. Furthermore, H1 may be a tetrazine or norbornene. Alternatively, H1 may be a tetrazine or transcyclooctene.

[0641] In a specific implementation, D1 may include components selected from covalent bonds, C... 1-4 Alkylene, C 2-4 imidene group, C 2-4 etyne and C 3-8 Any of the cycloalkylene groups. D1 can also be -CH2OCH2-. Furthermore, D1 can be a covalent bond.

[0642] In a specific embodiment, the antibody may be a human antibody. In other specific embodiments, the antibody may be a non-human animal antibody. In a specific embodiment, the antibody may be immunoglobulin G (IgG). In a specific embodiment, the antibody may be a complete antibody. In other specific embodiments, the antibody may be a fragment of an antibody. In a specific embodiment, the antibody may be a wild-type antibody. In other specific embodiments, the antibody may be a manipulated antibody.

[0643] In a specific embodiment, the antibody may contain the amino acid sequence of Formula 8-1 in only one of its two Fc domains. In other specific embodiments, the antibody may contain the amino acid sequence of Formula 8-2 in both of its Fc domains.

[0644] This invention provides antibodies or fragments thereof comprising the amino acid sequences of Formulas 8-3 below:

[0645] [Formula 8-3](SEQ ID NO:22)

[0646] GPSVFLFPP-(K)′-P-(K)′-DTLMI(SEQ ID NO:22)

[0647] 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.

[0648] (K)′ is

[0649] Wherein, H1 is the first-click chemical functional group, and

[0650] D1 can be any alkylene, alkenylene, or ynylene group. The antibody or its fragment has an H1 linked through lysine 246 and lysine 248 or a site corresponding to lysine 246 and lysine 248 in its Fc domain.

[0651] In a specific embodiment, H1 may include any one selected from alkynes, azides, strained alkynes, dienes, dienophiles, alkenes, thiols, and tetrazides. Furthermore, H1 may be an azide or a strained alkyne. Furthermore, H1 may be an azide or a dibenzocyclooctynylamine. Furthermore, H1 may be a diene or a dienophile. Furthermore, H1 may be a tetrazide or norbornene. Alternatively, H1 may be a tetrazide or transcyclooctene.

[0652] In a specific implementation, D1 may include components selected from covalent bonds, C... 1-4 Alkylene, C 2-4 imidene group, C 2-4 etyne and C 3-8 Any of the cycloalkylene groups. D1 can also be -CH2OCH2-. Furthermore, D1 can be a covalent bond.

[0653] In a specific embodiment, the antibody may be a human antibody. In other specific embodiments, the antibody may be a non-human animal antibody. In a specific embodiment, the antibody may be immunoglobulin G (IgG). In a specific embodiment, the antibody may be a complete antibody. In other specific embodiments, the antibody may be a fragment of an antibody. In a specific embodiment, the antibody may be a wild-type antibody. In other specific embodiments, the antibody may be a manipulated antibody.

[0654] In a specific embodiment, the antibody may contain the amino acid sequence of Formula 8-3 in only one of its two Fc domains. In other specific embodiments, the antibody may contain the amino acid sequence of Formula 8-3 in both of its Fc domains.

[0655] The present invention provides 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. In this case, the contents of the sequences of Formula 8-1 to Formula 8-3 are as described above.

[0656] In a specific implementation, D1 can be a covalent bond.

[0657] In specific embodiments, the antibody or its fragment may include only the amino acid sequence of Formula 8-1 and may exclude the amino acid sequences of Formulas 8-2 and 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. Furthermore, the antibody may include the amino acid sequence of Formula 8-1 in both of its Fc domains.

[0658] In other specific embodiments, the antibody or fragment thereof may comprise only the amino acid sequence of Formula 8-2, and may exclude the amino acid sequences of Formulas 8-1 and 8-3. The antibody or fragment thereof may also include the amino acid sequence of Formula 8-2 only in one of its two Fc domains. Furthermore, the antibody may include the amino acid sequence of Formula 8-2 in both of its Fc domains.

[0659] In specific embodiments, the antibody or its fragment may comprise only the amino acid sequence of Formula 8-3, and may exclude the amino acid sequences of Formulas 8-1 and 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. Furthermore, the antibody may include the amino acid sequence of Formula 8-3 in both of its Fc domains.

[0660] In a specific embodiment, the antibody may be a human antibody. In other specific embodiments, the antibody may be a non-human animal antibody. In a specific embodiment, the antibody may be immunoglobulin G (IgG). In a specific embodiment, the antibody may be a complete antibody. In other specific embodiments, the antibody may be a fragment of an antibody. In a specific embodiment, the antibody may be a wild-type antibody. In other specific embodiments, the antibody may be a manipulated antibody.

[0661] 5.2. Method for preparing antibodies containing a first functional group

[0662] According to the present invention, a method for preparing R1′-Ab and H1-Ab (hereinafter generally referred to as "R1′-Ab") is disclosed. It should be noted that the following description of the preparation method is provided to aid in understanding the present invention.

[0663] For example, a method for preparing compounds of formula 7 will be described. In a specific embodiment, compounds of formula 7 can be prepared by the reaction described in scheme 4 below.

[0664] [Option 4]

[0665]

[0666] Because the first carbonyl group of the connector according to the invention has mild reactivity, cross-linking can be achieved when the first carbonyl group of the connector has a close positional relationship with the amino group of the antibody. First, R1′-L2-SSAI represented by Formula 5 is brought into close contact with a specific site of the antibody to create an environment in which a reaction may occur. Because R1′-L2-SSAI according to the invention has an activated carbonyl group corresponding to the first carbonyl group of the connector (i.e., a carbonyl group attached to X1), R1′-L2-SSAI of the invention can initiate 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, through the design of the connector, the site-specific antibody interaction group (SSAI) leaves when 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.

[0667] In a specific embodiment, X4 can be NH2. X4 can also be NH2 of a lysine residue. In other specific embodiments, X4 can be SH. X4 can also be SH of a cysteine ​​residue. In other specific embodiments, X4 can be OH.

[0668] As a more specific example, a method for preparing antibodies or fragments thereof comprising the amino acid sequence of Formula 8-3 will be described. In a specific embodiment, compounds comprising the amino acid sequence of Formula 8-3 can be prepared by the reaction of Scheme 5 below.

[0669] [Option 5]

[0670]

[0671] Compounds having the structure of Formula 6-3 (where two cysteine ​​residues are optionally linked) target the Fc domain of an antibody because the compound has an SSFI sequence (see Section 3.2). In this case, an antibody comprising 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 amino group of lysine 246 attacks the first carbonyl group of Formula 6-3 to produce a compound of Formula 8-1. Thus, 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.

[0672] According to the present invention, a method for preparing antibodies containing a first chemical functional group is disclosed.

[0673] The present invention provides a method for preparing R1′-Ab, the method comprising: reacting an antibody or a fragment thereof with a reagent for transferring a first chemical functional group to an antibody according to the present invention.

[0674] In a specific embodiment, the reagent used to transfer 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.

[0675] In a specific embodiment, 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 a specific embodiment, the antibody or fragment thereof may be immunoglobulin G (IgG). In a specific embodiment, 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 a specific embodiment, 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.

[0676] In a specific embodiment, the present invention provides a method for preparing an antibody having a first chemical functional group that is transferred to a specific lysine residue in 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.

[0677] According to the present invention, a method for preparing antibodies containing a first click chemical functional group is disclosed.

[0678] The present invention provides a method for preparing H1-Ab, comprising: reacting a reagent for transferring a first click chemical functional group to an antibody according to the present invention with the antibody or a fragment thereof.

[0679] In a specific embodiment, the reagent used to transfer the first click chemical functional group to the antibody can be any one selected from Formulas 6-1 to 6-3. The reagent used to transfer the first click chemical functional group to the antibody may also have the structure of Formula 6-3.

[0680] In a specific embodiment, 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 a specific embodiment, the antibody or fragment thereof may be immunoglobulin G (IgG). In a specific embodiment, 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 a specific embodiment, 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.

[0681] In a specific embodiment, the present invention provides a method for preparing an antibody having a first click chemical functional group that transfers to a specific lysine residue in its Fc domain. In this case, those aspects described in Section 5.4 below can be used as reagents for transferring the first click chemical functional group to the antibody.

[0682] According to the present invention, a kit for preparing antibodies or fragments thereof containing a first chemical functional group is disclosed.

[0683] 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 an antibody according to the present invention, and the antibody or a fragment thereof.

[0684] In a specific embodiment, the reagent used to transfer 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.

[0685] In a specific embodiment, 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 a specific embodiment, the antibody or fragment thereof may be immunoglobulin G (IgG). In a specific embodiment, 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 a specific embodiment, 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.

[0686] In addition, the present invention provides a kit for preparing antibodies or fragments thereof containing a first chemical functional group, the kit comprising:

[0687] The connector (R1′-L1) according to the present invention;

[0688] According to the site-specific antibody-antibody interaction group of the present invention; and

[0689] Antibody or fragment thereof.

[0690] In a specific implementation, the connector can be any one selected from Formula 1, Formula 2, and Formulas 2-1 to 2-3.

[0691] In a specific implementation, the site-specific antibody interaction group can be any one selected from Formula 3, Formula 3-1 and Formulas 4-1 to 4-6.

[0692] In a specific embodiment, 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 a specific embodiment, the antibody or fragment thereof may be immunoglobulin G (IgG). In a specific embodiment, 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 a specific embodiment, 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.

[0693] This invention provides a kit for preparing antibodies having a first chemical functional group that is transferred to a specific lysine residue in 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 the antibody, linker, and site-specific antibody-antibody interaction group.

[0694] According to the present invention, a kit for preparing antibodies or fragments thereof containing a first click chemical functional group is disclosed.

[0695] The present invention provides a kit for preparing an antibody or a fragment thereof containing a first click chemical functional group, the kit comprising a reagent for transferring the first click chemical functional group to an antibody according to the present invention, and an antibody or a fragment thereof.

[0696] In a specific embodiment, the reagent used to transfer the first click chemical functional group to the antibody can be any one selected from Formulas 6-1 to 6-3. The reagent used to transfer the first click chemical functional group to the antibody may also have the structure of Formula 6-3.

[0697] In a specific embodiment, 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 a specific embodiment, the antibody or fragment thereof may be immunoglobulin G (IgG). In a specific embodiment, 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 a specific embodiment, 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.

[0698] The present invention also provides a kit for preparing antibodies or fragments thereof containing a first click chemical functional group, the kit comprising:

[0699] The connector (H1-L1) according to the present invention;

[0700] According to the site-specific antibody-antibody interaction group of the present invention; and

[0701] Antibody or fragment thereof.

[0702] In a specific implementation, the connector can be any one selected from Formula 2, Formula 2-1 to Formula 2-3.

[0703] In a specific implementation, the site-specific antibody interaction group can be any one selected from Formula 3, Formula 3-1 and Formulas 4-1 to 4-6.

[0704] In a specific embodiment, 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 a specific embodiment, the antibody or fragment thereof may be immunoglobulin G (IgG). In a specific embodiment, 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 a specific embodiment, 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.

[0705] This invention provides a kit for preparing antibodies having a first chemical functional group that transfers to a specific lysine residue in 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 the antibody, linker, and site-specific antibody-antibody interaction group.

[0706] 5.3. The function of (Xa1)′ and the design principle of its position on R1′-L2-SSFI

[0707] In this document, compounds of Formula 6-3 are provided as examples to aid in understanding the invention, but the scope of the invention is not limited thereto. It should be noted that the following description also applies to compounds of Formula 5, Formulas 5-1 to 5-3, and Formulas 6-1 to 6-3, and for convenience, compounds of Formula 6-3 are provided only as one example of the compounds.

[0708] As discussed in Section 5.2, the role of (Xa1)′ in R1′-L2-SSFI is to transfer R1′ to the antibody via a nucleophilic substitution reaction. According to the invention, the conditions for promoting the nucleophilic substitution reaction are considered to be: (1) (Xa1)′ is adjacent to a lysine residue in the Fc domain, and (2) the side chain to which R1′ is bound points towards the lysine residue. It is anticipated that as the position of (Xa1)′ and the orientation of the side chain become further away from the lysine residue in the Fc domain, the yield and uniformity of the process will decrease. (3) It will also be preferred that the substitution position of (Xa1)′ has little effect on the interaction between the SSFI and the Fc domain (see Section 3.2).

[0709] like Figure 3 and Figure 5 Indirectly, based on Equation 6-3, it was confirmed that the positions of lysine 246 and lysine 248 in the Fc domain that satisfy the requirement of (1) in the SSFI are positions 5, 6, 7 and 8.

[0710] [Equation 6-3]

[0711] DCAWH-(Xa1)′-GELVWCT

[0712] In this case, since the histidine at position 5 forms a salt bond with glutamic acid 380 in the Fc domain, the substitution of histidine may affect the interaction between the SSFI and the Fc domain. Therefore, this does not satisfy requirement (3). Since the orientation of this side chain is not close to lysine 246 and lysine 248, this also does not satisfy requirement (2). Since the glycine at 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 position 8 forms a salt bond with arginine 255 in the Fc domain, the substitution of glutamic acid may affect the interaction between the SSFI and the Fc domain. Therefore, this does not satisfy requirement (3) (see Section 3.2). Position 6 is determined to be the most suitable position for (Xa1)′ because position 6 satisfies all the requirements of (1), (2) and (3). Therefore, based on these facts, the R1′-L2-SSFI according to the present invention is completed.

[0713] 5.4. The location of the R1′ transferred to the antibody can vary depending on the length of D2 in R1′-L1 and D3 in SSFI.

[0714] This document aims to explain the preferred design principles of the R1′-L2-SSFI according to the present invention. According to the design of the R1′-L2-SSFI, R1′ can be specifically transferred to a specific lysine residue in the Fc domain. Furthermore, this document aims to explain the design principles of R1′-L1 and SSFI to prepare the preferred R1′-L2-SSFI.

[0715] Those skilled in the art who have read Section 5.2 above will recognize that a nucleophilic substitution reaction may occur when the first carbonyl carbon of (Xa1)′ of R1′-L2-SSFI is positioned adjacent to the amino group of lysine in the Fc domain. Based on the description in Section 3.2 above, those skilled in the art will also recognize that R1′-L2-SSFI is arranged with an Fc domain having a specific topology, wherein the amino groups of lysine 246 and lysine 248 in the Fc domain are spaced at a certain distance from the β carbon of (Xa1)′. Figure 5 By using this combination to design R1′-L2-SSFI, it is expected that the distance between the β carbon of (1)(Xa1)′ and the first carbonyl carbon (hereinafter generally referred to as "L") will be... c When the distance between the β carbon of (2)(Xa1)′ and the amino groups of lysine 246 and lysine 248 in the Fc domain of the specific topology is the same or similar, it can specifically label the desired lysine residue.

[0716] According to the structure of R1′-L2-SSFI and the L according to the present invention c exist Figure 8 As shown in the image. 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′.

[0717] For convenience, this invention is embodied under the following assumptions: D3 is C x Alkylene, X3 is N, D2 is C y Alkylene, C y imide or C y The alkylene group, and X1 is S, where y is an integer greater than or equal to 1. In this case, it is assumed that the alkylene, alkenylene, and alkylene groups have the same length. Figure 8 The structure of the connector shown was modeled using Discovery Studio software to calculate L. c Value. As a result, L is determined relative to the value of x+y.c The values ​​are listed in Table 3. During the calculation, the chain with the longest length was modeled.

[0718] L relative to x+y values c value

[0719] x+y Lc(A) 1(x=0,y=1) 6.616 2 7.784 3 9.102 4 10.299 5 11.599 6 12.816 7 14.100 8 15.334 9 16.601 10 17.848 11 19.099 12 20.357 13 21.592 14 22.857

[0720] In the following text, based on Figure 9 A diagram is provided to illustrate the topological structure between the R1′-L2-SSFI and the lysine residues of the Fc domain. Figure 9 The topology between the R1′-L2-SSFI and Fc domains is shown, such that the direction of the (Xa1)′ sidechain (dashed arrow) is parallel to the x-axis in the figure. Figure 9 As shown, it can be seen that the side chain of (Xa1)′ points to the amino 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)′ and the side chain may specifically react with lysine 246 or lysine 248. Figures 10-12 It is along the exemplary L c The length is observed in a direction parallel to the y-axis in the diagram (thick solid arrow). Figure 9 The chart is an illustration.

[0721] The distance (D) between the β carbon of (Xa1)′ and the amino groups of lysine 246 and lysine 248 in the Fc domain. 246,min D 246,max D 248,min and D 248,max As described in Section 3.2, the β-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 ( Figure 10 When the first carbonyl carbon reacts well with lysine 248, L c Value longer than D 248,max ( Figure 11 When the first carbonyl carbon reacts well with lysine 246, and when L c The value is longer than or equal to D 246,min And shorter than or equal to D 248,max ( Figure 12 When the first carbonyl carbon reacts selectively with lysine 246 and lysine 248, the first carbonyl carbon will react with lysine 246 and lysine 248.

[0722] According to the present invention, R1′-L2-SSFI for specifically transferring a first chemical functional group to lysine 248 in the Fc domain of an antibody is disclosed (see [link to invention]). Figure 11 ).

[0723] This invention provides a reagent for transferring a first chemical functional group to an antibody, characterized in that the distance (L) between the β carbon of (Xa1)′ and the first carbonyl carbon is... c Shorter than D 246,min (about In this case, L c It can have approximately about about about about about or about The value of .

[0724] In a specific implementation, when the R1′-L2-SSFI has any one of the structures selected from Equation 5, Equations 5-1 to 5-3, and Equations 6-1 to 6-3, D3 is C. x Alkylene, X3 is N, D2 is C y Alkylene, C y imide or C y The alkynyl group, X1 being S, can be an integer greater than or equal to 1, and the sum of x and y can be 1 ≤ x + y ≤ 5. The sum of x and y can also be 1, 2, 3, 4, or 5. For example, x = 0, and y can be 1 ≤ y ≤ 5. In another exemplary embodiment, x = 1, and y can be 1 ≤ y ≤ 4. In yet another exemplary embodiment, x = 2, and y can be 1 ≤ y ≤ 3. In yet another exemplary embodiment, x = 3, and y can be 1 ≤ y ≤ 2. The corresponding numerical ranges are determined based on the values ​​listed in Table 3.

[0725] 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.

[0726] According to one aspect of the invention, the invention provides a reagent for transferring a first chemical functional group to an antibody, characterized in that the distance (L) between the β carbon of (Xa1)′ and the first carbonyl carbon is... c Longer than D 248,max (about For example, L c It can have approximately about about about about or about The value of .

[0727] In a specific implementation, when R1′-L2-SSFI has any structure selected from Equation 5, Equations 5-1 to 5-3, and Equations 6-1 to 6-3, D3 is C. x Alkylene, X3 is N, D2 is C y Alkylene, C y imide or C y The alkynyl group, X1, is S, where y can be an integer greater than or equal to 1, and the sum of x and y can be greater than or equal to 9. The sum of x and y can also be 9, 10, 11, or 12. For example, x = 0, and y can be 9 ≤ y ≤ 12. In another exemplary embodiment, x = 1, and y can be 8 ≤ y ≤ 11. In yet another exemplary embodiment, x = 2, and y can be 7 ≤ y ≤ 10. In yet another exemplary embodiment, x = 3, and y can be 6 ≤ y ≤ 9. Optionally, D2 can 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.

[0728] 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.

[0729] According to one aspect of the invention, the invention provides a reagent for transferring a first chemical functional group to an antibody, characterized in that the distance (L) between the β 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 The value of L. In this case, c It can have approximately about about about about about about or about The value of .

[0730] In a specific implementation, when R1′-L2-SSFI has any structure selected from Equation 5, Equations 5-1 to 5-3, and Equations 6-1 to 6-3, D3 is C. x Alkylene, X3 is N, D2 is C y Alkylene, C y imide or C yThe alkynyl group, X1 being S, where y can be an integer greater than or equal to 1, and the sum of x and y can be 6 ≤ x + y ≤ 8. In this case, the sum of x and y can be 6, 7, or 8. For example, x = 0, and y can be 6 ≤ y ≤ 8. In another exemplary embodiment, x = 1, and y can be 5 ≤ y ≤ 7. In yet another exemplary embodiment, x = 2, and y can be 4 ≤ y ≤ 6. In yet another exemplary embodiment, x = 3, and y can be 3 ≤ y ≤ 5.

[0731] According to the present invention, a method for preparing R1′-L2-SSFI is disclosed, wherein the R1′-L2-SSFI is used to specifically transfer a first chemical functional group to lysine 248 in the Fc domain of an antibody.

[0732] As an example of the method for preparing reagents 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 by comprising: reacting a linker according to the present invention with a site-specific antibody interacting group according to the present invention, wherein D2 in the linker is C y Alkylene, C y imide or C y etyne group, and

[0733] D3 in the site-specific antibody-interactiform group is C x Alkylene

[0734] Where 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.

[0735] According to the present invention, a kit for preparing R1′-L2-SSFI is disclosed, the kit being used to specifically transfer a first chemical functional group to lysine 248 in the Fc domain of an antibody.

[0736] As an example of the method for preparing reagents 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:

[0737] The connector according to the invention; and

[0738] According to the site-specific antibody interactome of the present invention,

[0739] Wherein, D2 in the connector is C y Alkylene, C y imide or C yalkyne group, and

[0740] D3 in the site-specific antibody-interactiform group is C x Alkylene

[0741] Where y is an integer greater than or equal to 1, and the sum of x and y is 1 ≤ x + y ≤ 5.

[0742] According to the present invention, a method for preparing R1′-L2-SSFI is disclosed, wherein R1′-L2-SSFI is used to specifically transfer a first chemical functional group to lysine 246 in the Fc domain of an antibody.

[0743] As an example of the method for preparing reagents 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 by comprising:

[0744] The adapter according to the invention is reacted with the site-specific antibody interactant group according to the invention, wherein D2 in the adapter is C. y Alkylene, C y imide or C y alkyne group, and

[0745] D3 in the site-specific antibody-interactiform group is C x Alkylene

[0746] Where 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.

[0747] According to the present invention, a kit for preparing R1′-L2-SSFI is disclosed, the kit being used to specifically transfer a first chemical functional group to lysine 246 in the Fc domain of an antibody.

[0748] As an example of the method for preparing reagents 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:

[0749] The connector according to the invention; and

[0750] According to the site-specific antibody interactome of the present invention,

[0751] Wherein, D2 in the connector is C y Alkylene, C y imide or C y alkyne group, and

[0752] D3 in the site-specific antibody-interactiform group is C x Alkylene

[0753] Where y is an integer greater than or equal to 1, and the sum of x and y is 9 ≤ x + y ≤ 12.

[0754] According to the present invention, a method for preparing R1′-L2-SSFI is disclosed, wherein the R1′-L2-SSFI is used to selectively transfer a first chemical functional group to lysine 246 or lysine 248 in the Fc domain of an antibody.

[0755] As an example of the method for preparing reagents 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 by comprising:

[0756] The adapter according to the invention is reacted with the site-specific antibody interactant group according to the invention, wherein D2 in the adapter is C. y Alkylene, C y imide or C y etyne group, and

[0757] D3 in the site-specific antibody-interactiform group is C x Alkylene

[0758] Where 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.

[0759] According to the present invention, a kit for preparing R1′-L2-SSFI is disclosed, the kit being used to selectively transfer a first chemical functional group to lysine 246 or lysine 248 in the Fc domain of an antibody.

[0760] As an example of the method for preparing reagents 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:

[0761] The connector according to the invention; and

[0762] According to the site-specific antibody interactome of the present invention,

[0763] Wherein, D2 in the connector is C y Alkylene, C y imide or C yalkyne group, and

[0764] D3 in the site-specific antibody-interactiform group is C x Alkylene

[0765] Where y is an integer greater than or equal to 1, and the sum of x and y is 6 ≤ x + y ≤ 8.

[0766] According to the present invention, a method for preparing an antibody having a first chemical functional group of lysine 248 specifically translocated to its Fc domain is disclosed.

[0767] As an example of the method for preparing antibodies containing a first chemical functional group as described in Section 5.2, the present invention provides a method for preparing R1′-Ab, characterized by comprising:

[0768] The reagent used to transfer the first chemical functional group to the antibody according to the invention reacts with the antibody or a fragment thereof.

[0769] 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 is... c Shorter than approximately

[0770] In a specific implementation, in the reagent used to transfer the first chemical functional group to the antibody, D3 is C. x Alkylene, and D2 is C y Alkylene, C y imide or C y Imyethynyl,

[0771] Where y can be an integer greater than or equal to 1, and the sum of x and y can be 1 ≤ x + y ≤ 5.

[0772] According to the present invention, a kit for preparing an antibody having a first chemical functional group of lysine 248 specifically translocated to its Fc domain is disclosed.

[0773] As an example of the method for preparing antibodies 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:

[0774] A reagent for transferring a first chemical functional group to the antibody according to the invention; and

[0775] Antibodies or fragments thereof,

[0776] In the reagent used to transfer the first chemical functional group to the antibody, the distance (L) between the β carbon of (Xa1)′ and the first carbonyl carbon is... c Shorter than approximately

[0777] In a specific implementation, in the reagent used to transfer the first chemical functional group to the antibody, D3 is C x Alkylene, and D2 is C y Alkylene, C y imide or C y Imyethynyl,

[0778] Where y can be an integer greater than or equal to 1, and the sum of x and y can be 1 ≤ x + y ≤ 5.

[0779] Optionally, as an example of the method for preparing antibodies 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:

[0780] The connector according to the invention;

[0781] According to the site-specific antibody-antibody interaction group of the present invention; and

[0782] Antibodies or fragments thereof,

[0783] Wherein, D2 in the connector is C y Alkylene, C y imide or C y etymynyl, and

[0784] D3 in the site-specific antibody-interactiform group is C x Alkylene

[0785] Where y is an integer greater than or equal to 1, and the sum of x and y is 1 ≤ x + y ≤ 5.

[0786] According to the present invention, a method for preparing an antibody having a first chemical functional group specifically transferred to lysine 246 in its Fc domain is disclosed.

[0787] As an example of the method for preparing antibodies containing a first chemical functional group as described in Section 5.2, the present invention provides a method for preparing R1′-Ab, characterized by comprising:

[0788] The antibody or a fragment thereof is permitted to react with reagents used to transfer a first chemical functional group to the antibody according to the invention.

[0789] In the reagent used to transfer the first chemical functional group to the antibody, the distance (L) between the β carbon of (Xa1)′ and the first carbonyl carbon is... c Longer than about

[0790] In a specific implementation, in the reagent used to transfer the first chemical functional group to the antibody, D3 is C. x Alkylene, and D2 is C y Alkylene, C y imide or C y Imyethynyl,

[0791] Where y can be an integer greater than or equal to 1, and the sum of x and y can be 9 ≤ x + y ≤ 12.

[0792] 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.

[0793] As an example of the method for preparing antibodies 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:

[0794] Reagents for transferring the first chemical functional group to the antibody of the present invention; and

[0795] Antibodies or fragments thereof,

[0796] In the reagent used to transfer the first chemical functional group to the antibody, the distance (L) between the β carbon of (Xa1)′ and the first carbonyl carbon is... c Longer than about

[0797] In a specific implementation, in the reagent used to transfer the first chemical functional group to the antibody, D3 is C. x Alkylene, and D2 is C y Alkylene, C y imide or C y Imyethynyl,

[0798] Where y can be an integer greater than or equal to 1, and the sum of x and y can be 9 ≤ x + y ≤ 12.

[0799] Optionally, as an example of the method for preparing antibodies 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:

[0800] The connector according to the invention;

[0801] According to the site-specific antibody-antibody interaction group of the present invention; and

[0802] Antibodies or fragments thereof,

[0803] Wherein, D2 in the connector is C y Alkylene, C yimide or C y alkyne group, and

[0804] D3 in the site-specific antibody-interactiome is C. x Alkylene

[0805] Where y is an integer greater than or equal to 1, and the sum of x and y is 9 ≤ x + y ≤ 12.

[0806] According to the present invention, a method for preparing an antibody having a first chemical functional group that selectively transfers to lysine 246 or lysine 248 in its Fc domain is disclosed.

[0807] As an example of the method for preparing antibodies containing a first chemical functional group as described in Section 5.2, the present invention provides a method for preparing R1′-Ab, characterized by comprising:

[0808] The reagent used to transfer the first chemical functional group to the antibody according to the invention reacts with the antibody or a fragment thereof.

[0809] In the reagent used to transfer the first chemical functional group to the antibody, the distance (L) between the β carbon of (Xa1)′ and the first carbonyl carbon is... c Longer than or equal to approximately And shorter than or equal to approximately

[0810] In a specific implementation, in the reagent used to transfer the first chemical functional group to the antibody, D3 is C. x Alkylene, and D2 is C y Alkylene, C y imide or C y Imyethynyl,

[0811] Where y can be an integer greater than or equal to 1, and the sum of x and y can be 6≤x+y≤8.

[0812] According to the present invention, a kit for preparing antibodies having a first chemical functional group, lysine 246 or lysine 248, that is selectively transferred into its Fc domain is disclosed.

[0813] As an example of the method for preparing antibodies 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:

[0814] A reagent for transferring a first chemical functional group to the antibody according to the invention; and

[0815] Antibodies or fragments thereof,

[0816] In the reagent used to transfer the first chemical functional group to the antibody, the distance (L) between the β carbon of (Xa1)′ and the first carbonyl carbon is... c Longer than or equal to approximately And shorter than or equal to approximately

[0817] In a specific implementation, in the reagent used to transfer the first chemical functional group to the antibody, D3 is C. x Alkylene, and D2 is C y Alkylene, C y imide or C y Imyethynyl,

[0818] Where y can be an integer greater than or equal to 1, and the sum of x and y can be 6≤x+y≤8.

[0819] Optionally, as an example of the method for preparing antibodies 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:

[0820] The connector according to the invention;

[0821] According to the site-specific antibody-antibody interaction group of the present invention; and

[0822] Antibodies or fragments thereof,

[0823] Wherein, D2 in the connector is C y Alkylene, C y imide or C y alkyne group, and

[0824] D3 in the site-specific antibody-interactiome is C. x Alkylene

[0825] Where y is an integer greater than or equal to 1, and the sum of x and y is 6 ≤ x + y ≤ 8.

[0826] According to the present invention, a method for preparing an antibody having a first chemical functional group having both lysines 246 and 248 transferred to its Fc domain is disclosed.

[0827] This invention provides a method for preparing R1′-Ab, the method comprising:

[0828] A first reagent for transferring a first chemical functional group to an antibody reacts with the antibody or a fragment thereof; and

[0829] A second reagent used to transfer a first chemical functional group to an antibody reacts with the antibody or a fragment thereof.

[0830] In a specific embodiment, the first reagent used to transfer the first chemical functional group to the antibody may be R1′-L2-SSFI, wherein R1′-L2-SSFI is used to specifically transfer the first chemical functional group to lysine 248 in the Fc domain as described above, and the second reagent used to transfer the first chemical functional group to the antibody may be R1′-L2-SSFI, wherein R1′-L2-SSFI is used to specifically transfer the first chemical functional group to lysine 246 in the Fc domain as described above.

[0831] In a specific embodiment, the first reagent used to transfer the first chemical functional group to the antibody may be R1′-L2-SSFI, wherein R1′-L2-SSFI is used to specifically transfer the first chemical functional group to lysine 246 in the Fc domain as described above, and the second reagent used to transfer the first chemical functional group to the antibody may be R1′-L2-SSFI, wherein R1′-L2-SSFI is used to specifically transfer the first chemical functional group to lysine 248 in the Fc domain as described above.

[0832] 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, may be carried out sequentially.

[0833] According to the present invention, a kit for preparing an antibody having a first chemical functional group, namely lysine 246 and lysine 248, which are transferred into its Fc domain, is disclosed.

[0834] This invention provides a kit for preparing R1′-Ab, the kit comprising:

[0835] The first reagent used to transfer the first chemical functional group to the antibody;

[0836] A second reagent for transferring a first chemical functional group to an antibody; and

[0837] Antibody or fragment thereof.

[0838] In a specific embodiment, the first reagent used to transfer the first chemical functional group to the antibody may be R1′-L2-SSFI, wherein R1′-L2-SSFI is used to specifically transfer the first chemical functional group to lysine 248 in the Fc domain as described above, and the second reagent used to transfer the first chemical functional group to the antibody may be R1′-L2-SSFI, wherein R1′-L2-SSFI is used to specifically transfer the first chemical functional group to lysine 246 in the Fc domain as described above.

[0839] In a specific embodiment, the first reagent used to transfer the first chemical functional group to the antibody may be R1′-L2-SSFI, wherein R1′-L2-SSFI is used to specifically transfer the first chemical functional group to lysine 246 in the Fc domain as described above, and the second reagent used to transfer the first chemical functional group to the antibody may be R1′-L2-SSFI, wherein R1′-L2-SSFI is used to specifically transfer the first chemical functional group to lysine 248 in the Fc domain as described above.

[0840] 5.5. By adjusting the reactivity of the first carbonyl group, the first chemical functional group can be transferred more site-specifically.

[0841] As described in Section 5.2 above, antibodies containing a first chemical functional group can be prepared by attacking the first carbonyl group of R1′-L2-SSFI with the nucleophile of the antibody. The first carbonyl group is characterized by having a milder reactivity than the second carbonyl group of the linker (see Section 2.2). The reactivity of the first carbonyl group can be tuned such that specific amines of the antibody react with the first carbonyl group only when a specific amine group is in proximity to the first carbonyl group. Thus, the present invention allows the first carbonyl group to not react well with any amine group through vicinity conditions of the reaction, and results in high position specificity of the reaction.

[0842] The prior art disclosed in US Publications 2018 / 0141976A1 and WO 2018 / 199337A1 aims to selectively modulate lysine 246 or lysine 248 in the Fc domain using Fc-III analog sites. Because the prior art uses a bis(succinimide) crosslinking agent, both the first and second carbonyl groups have equally high reactivity (see Section 2.2). Therefore, due to the high reactivity of the first carbonyl group and the lack of reactive proximity conditions, any lysine residue is highly likely to be labeled.

[0843] 5.6. The antibody containing the first chemical functional group prepared according to the present invention has high uniformity and yield.

[0844] The technical problem and solution for specifically transferring (1) a “desired number” of first chemical functional groups to (2) a “specific site on the antibody” has 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 the antibodies containing the first chemical functional groups prepared by this 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.

[0845] As shown in the background art, conjugates with high homogeneity due to antibody labeling have 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 in antibody function can be avoided because the functional region of the antibody can be avoided while labeling the antibody.

[0846] The prior art disclosed in US Publications 2018 / 0141976A1 and WO 2018 / 199337A1 aims to selectively modulate lysine 246 or lysine 248 in the Fc domain using Fc-III analog sites. However, according to the prior art, antibody uniformity and yield may inevitably be low for two reasons. Firstly, due to the distance (L) between the β carbon of (Xa1)′ and the first carbonyl carbon... c ) for Therefore, lysine residues at positions 246 or 248 can be selectively labeled, but it is not possible to specifically select one of the lysine residues 246 and 248 to be labeled. Secondly, the first carbonyl group of the crosslinking agent according to the prior art lacks reactive proximity conditions due to its high reactivity (see Section 5.5). As a result, the first carbonyl group is highly likely to react with any lysine residue. Therefore, the prior art has the disadvantage that the uniformity and yield of the antibody-labeled product are inferior to those of the present invention.

[0847] 5.7. Because 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.

[0848] The design principles for the antibody labeling sites are: (1) to be separated from the complementary site; and (2) to be separated from the FcR recognition site, which includes FcRn, as described in Section 1.1 above. Since lysine 246 and lysine 248 are contained within the Fc domain, and are separated from the complementary site, these sites may overlap with the FcRn recognition site, which could pose a problem.

[0849] FcRn is known to have multiple functions, and plays a particularly important role in prolonging the half-life of antibodies by participating in IgG recycling. When antibodies are used in vivo, such as as therapeutic agents or contrast agents, the interaction between FcRn and the antibody may not occur smoothly, thus preventing the antibody from functioning properly due to its short half-life.

[0850] 5.7.1. Lysine 246 and lysine 248 are spaced apart from the FcRn binding site of the antibody.

[0851] The distance between the lysine residues in the Fc domain and the FcRn binding site of Fc is an important factor to consider when selecting lysine residues in the Fc domain. Figure 13 The binding structure between the Fc domain and FcRn is shown, along with the FcRn binding site and the positions of lysine 246 and lysine 248 in the Fc domain. Papers and computer modeling were used to visualize the binding structure between the Fc domain and FcRn (Ying T, Ju TW, Wang Y, Prabakaran P, Dimitrov DS., Interactions of IgG1 CH2 and CH3 Domains with FcRn, Front Immunol. 2014; 5:146; Monnet C, Jorieux S, Urbain R et al., Selection of IgG Variants with Increased FcRn Binding Using Random and Directed Mutagenesis: Impact on Effector Functions. Front Immunol. 2015; 6:39) (see also...) Figure 13 (Left figure). The FcRn binding site and lysine 246 and lysine 248 in the Fc domain, as disclosed in the same article's data, are shown (see [reference]). Figure 13 (See the right figure). 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.

[0852] From these facts, it is expected that when an antibody labeling site is selected, the antibody labeling site will not affect the interaction between the FcRn and the Fc domain. In fact, it has been confirmed that the half-life of the prepared antibody is not shortened, so that the antibody labeling site does not affect the binding of FcRn as intended (see Experimental Example 5).

[0853] 5.8. The method for preparing antibodies containing a first chemical functional group according to the present invention is characterized by SSAI departure.

[0854] As can be seen from Schemes 4 and 5 in Section 5.2, the process for preparing R1′-Ab according to the invention is characterized by the departure of SSAI from the final product R1′-Ab, because SSAI is contained in the leaving group of the nucleophilic substitution reaction. The binding structure between SSFI and the Fc domain according to the invention was analyzed (see Section 3.2) compared to the binding site between Fc and FcRn. The results confirmed that a portion of the FcRn binding site is covered by SSFI ( Figure 14In this regard, it can be expected that if the SSFI does not leave during the preparation of R1′-Ab, the SSFI will have a negative impact on the physical properties of the antibody (e.g., half-life, etc.).

[0855] The prior art disclosed in US Publication No. 2018 / 0141976A1 aims to selectively modulate lysine 246 or lysine 248 in the Fc domain using Fc-III analog sites. However, the corresponding prior art differs from the present invention in that the SSFI is included in the final antibody-labeled product because the SSFI does not leave during the preparation process.

[0856] The prior art disclosed in Publication No. WO 2018 / 199337A1 aims to selectively modulate lysine 246 or lysine 248 in the Fc domain using an analog site of Fc-III, wherein the SSFI is not included in the final antibody-labeled product. However, the corresponding prior art further includes cleaving a "cleavable linker, which is a divalent group" during preparation to remove the SSFI, but the advantage of the present invention is that the SSFI leaves during the coupling reaction without any additional process. Cleavage of the divalent linker according to the corresponding invention has the disadvantage that the disulfide bonds forming the antibody structure can be cleaved. Since the present invention uses a nucleophilic substitution reaction, which can be easily carried out without any special conditions, this problem can be significantly improved.

[0857] 5.9. Because the antibody containing the first click chemical functional group according to the present invention does not contain highly bioreactive chemical functional groups, no side reactions will occur except for the conjugate formation reaction.

[0858] The antibody prepared by the method for preparing R1′-Ab according to the present invention has the structure represented by Formula 7:

[0859] [Formula 7]

[0860]

[0861] When R1′ is transferred to lysine 246 or lysine 248, X4 can be NH, and D1 can be any alkylene, alkenyl, or alkyneyl group. In this case, the -NH-(CO)- bond linked to the antibody is an amide bond that is generally stable in vivo. D1 also typically has a less bioreactive structure. Therefore, the R1′-Ab prepared according to the present invention is expected to have high safety because a highly bioreactive structure is not added to a molecule other than the labeled molecule (R1′).

[0862] Specifically, when R1′-Ab is an H1-Ab with click chemistry residues transferred thereto, a highly bioreactive structure is not added to R1′-Ab. Therefore, the yield of the click chemistry reaction can be increased because no secondary reactions occur other than the click chemistry reaction itself.

[0863] The prior art disclosed in Publication No. WO 2018 / 199337A1 aims to use Fc-III analogs and employ biorthogonal functional groups to site-selectively modulate lysine 246 or lysine 248 in the Fc domain. However, the corresponding prior art differs from the present invention in that, during the cleavage process as a "cleavable linker with a divalent group," the antibody product according to the corresponding prior art is allowed to have additional chemical functional groups, such as thiols, hydroxyl groups, carboxylic acids, phosphates, amines, etc. These additional chemical functional groups are bioreactive functional groups and may therefore affect the safety of the antibody product.

[0864] 6. Payload (C) m -H2)

[0865] According to the present invention, the payload is disclosed. This compound is referred to herein by the symbol "C". m -H2” is shown.

[0866] This invention provides C represented by Equation 9 m -H2:

[0867] [Formula 9]

[0868] C m —————H2

[0869] Among them, C m For the load-bearing part,

[0870] H2 is the second click chemical functional group.

[0871] In a specific implementation, C m It may include a carrier portion, a fluorescent portion, a drug portion, or a radioactive portion. R1′ may also include a drug portion. Furthermore, R1′ may include a VC linker. In other embodiments, R1′ may include an antibody or an analogue thereof, said antibody or analogue thereof including complementary sites.

[0872] In a specific implementation, when C mWhen a pharmaceutical component is included, the pharmaceutical component may be an anticancer drug. The anticancer drug may further include one or more of 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, cytokines, apoptosis inducers, anti-angiogenic agents, lymphokines, taxanes, DNA alkylating agents, anthracyclines, tubulysin analogs, pyruvic analogs, auristatin E, and auristatin. F. Maytansinoids, cytotoxic agents containing reactive polyethylene glycol residues, taxon, cytochalasin B, bacitracin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthraquinone, mitoxantrone, sclerosomycin, actinomycin D, 1-dihydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin, methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil aminoimide, nitrogen mustard, thiotepa, chlorambucil, melphalan. Carmustine, lomustine, cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, cisplatin, styromycin, bleomycin, amiodarone, chachiin, abiraterone, bendamustine, bortezomib, carboplatin, cabazitaxel, dasatinib, docetaxel, epirubicin, erlotinib, everolimus, gemcitabine, gefitinib, idarubicin, imatinib, hydroxyurea, lapatinib, leuprorelin, melphalan, nedaplatin, nilotinib, oxaliplatin, pazopanib, pemetrexed, pyrplatin, romidixin, saplatin, sorafenib, vemurafenib, sunitinib, teniposide, triplatin, and vinorelbine.

[0873] In a specific implementation, C m It may include multiple carrier portions, fluorescent portions, drug portions, or radioactive portions. m It may also include two or more drug components. Furthermore, C m It may include a VC connector.

[0874] In specific embodiments, H2 may include any one selected from alkynes, azides, strained alkynes, dienes, dienophiles, alkenes, thiols, and tetrazides. Furthermore, H2 may be an azide or a strained alkyne. Furthermore, H2 may be an azide or a dibenzocyclooctynylamine. Furthermore, H2 may be a diene or a dienophile. Furthermore, H2 may be a tetrazide or norbornene. Alternatively, H2 may be a tetrazide or transcyclooctene.

[0875] In a specific embodiment, when H2 reacts with H1-Ab according to the present invention, H2 can be complementary to the first click chemical functional group (H1).

[0876] 7. Antibody-payload conjugate (C m -Ab)

[0877] 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.

[0878] This invention provides C represented by Equation 10. m -Ab:

[0879] [Formula 10]

[0880]

[0881] Among them, C m For the load-bearing part,

[0882] B is a structure formed through a click chemical reaction between the first and second click chemical functional groups.

[0883] D1 can be any alkylene, alkenylene, or ynylene group.

[0884] X4 is NH, O, or S, and

[0885] Ab stands for antibody.

[0886] The contents of the load section apply to the contents disclosed in Section 6. Payload.

[0887] In a specific embodiment, B can be a structure formed by a click chemical reaction between any of the alkynes, azides, strained alkynes, dienes, dienophiles, alkenes, thiols, and tetrazides and their partner "click chemical" functional groups. B can also be selected from any of the following:

[0888] Both A1 and A2 are connected to the load section or D1, such that A1 and A2 are not connected to the same section, and R x It can be selected from H, halogens and C 1-3 Alkyl group. Furthermore, B can be... In addition, B can be

[0889] In a specific implementation, D1 may include components selected from covalent bonds, C... 1-4 Alkylene, C 2-4 imidene group, C2-4 etyne and C 3-8 Any of the cycloalkylene groups. Furthermore, D1 can be -CH2OCH2-. Furthermore, D1 can be covalently bonded.

[0890] In one specific implementation, X4 may be NH.

[0891] 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 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.

[0892] In a specific embodiment, X4 and Ab can be connected through the Fab domain of Ab. In other embodiments, X4 and Ab can be connected through the Fc domain of Ab. Furthermore, X4 and Ab can be connected through lysine 246 or lysine 248 in the Fc domain of Ab. Alternatively, X4 and Ab can be connected through both lysine 246 and lysine 248 in the Fc domain of Ab. In a specific embodiment, X4 and Ab can be connected through only one of the two Fc domains of Ab. In other embodiments, X4 and Ab can be connected through both Fc domains of Ab.

[0893] This invention provides antibodies or fragments thereof comprising the amino acid sequence of Formula 10-1:

[0894] [Equation 10-1]

[0895] GPSVFLFPP-(K)″-PKDTLMI(SEQ ID NO:23)

[0896] 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.

[0897] (K)” is

[0898] Among them, C m For the load-bearing part,

[0899] B is a structure formed through a click chemical reaction between a first click chemical functional group and a second click chemical functional group, and

[0900] D1 is any alkylene, alkenylene, or ynylene group. The antibody or its fragment has a loading portion coupled to lysine 246 in its Fc domain, or a site corresponding to lysine 246.

[0901] The contents of the load section apply to the contents disclosed in Section 6. Payload.

[0902] In a specific embodiment, B can be a structure formed by a click chemical reaction between any of the alkynes, azides, strained alkynes, dienes, dienophiles, alkenes, thiols, and tetrazides and their partner "click chemical functional groups". B can also be selected from any of the following:

[0903] Both A1 and A2 are connected to the load section or D1, ensuring that A1 and A2 are not connected to the same section, and R x It can be selected from H, halogens and C 1-3 Alkyl group. Furthermore, B can be... In addition, B can be

[0904] In a specific implementation, D1 may include components selected from covalent bonds, C... 1-4 Alkylene, C 2-4 imidene group, C 2-4 etyne and C 3-8 Any of the cycloalkylene groups. D1 can also be -CH2OCH2-. Furthermore, D1 can be a covalent bond.

[0905] In a specific embodiment, the antibody may be a human antibody. In other specific embodiments, the antibody may be a non-human animal antibody. In a specific embodiment, the antibody may be immunoglobulin G (IgG). In a specific embodiment, the antibody may be a complete antibody. In other specific embodiments, the antibody may be a fragment of an antibody.

[0906] In a specific embodiment, the antibody may contain an amino acid sequence of Formula 10-1 in only one of its two Fc domains. In other specific embodiments, the antibody may contain an amino acid sequence of Formula 10-1 in both of its Fc domains.

[0907] This invention provides antibodies or fragments thereof comprising the amino acid sequence of Formula 10-2:

[0908] [Equation 10-2]

[0909] GPSVFLFPPKP-(K)″-DTLMI(SEQ ID NO:24)

[0910] 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.

[0911] (K)” is

[0912] Among them, C m For the load-bearing part,

[0913] B is a structure formed through a click chemical reaction between a first click chemical functional group and a second click chemical functional group, and

[0914] D1 is any alkylene, alkenylene, or ynylene group. The antibody or its fragment has a loading portion coupled to lysine 246 in its Fc domain, or a site corresponding to lysine 246.

[0915] The contents of the load section apply to the contents disclosed in Section 6. Payload.

[0916] In a specific embodiment, B can be a structure formed by a click chemical reaction between any of the alkynes, azides, strained alkynes, dienes, dienophiles, alkenes, thiols, and tetrazides and their partner "click chemical functional groups". B can also be selected from any of the following:

[0917] Both A1 and A2 are connected to the load section or D1, ensuring that A1 and A2 are not connected to the same section, and R x It can be selected from H, halogens and C 1-3 Alkyl group. Furthermore, B can be... In addition, B can be

[0918] In a specific implementation, D1 may include components selected from covalent bonds, C... 1-4 Alkylene, C 2-4 imidene group, C 2-4 etyne and C 3-8 Any of the cycloalkylene groups. D1 can also be -CH2OCH2-. Furthermore, D1 can be a covalent bond.

[0919] In a specific embodiment, the antibody may be a human antibody. In other specific embodiments, the antibody may be a non-human animal antibody. In a specific embodiment, the antibody may be immunoglobulin G (IgG). In a specific embodiment, the antibody may be a complete antibody. In other specific embodiments, the antibody may be a fragment of an antibody.

[0920] In a specific embodiment, the antibody may contain an amino acid sequence of formula 10-2 in only one of its two Fc domains. In other specific embodiments, the antibody may contain an amino acid sequence of formula 10-2 in both of its Fc domains.

[0921] This invention provides antibodies or fragments thereof comprising the amino acid sequence of Formula 10-3:

[0922] [Equation 10-3]

[0923] GPSVFLFPP-(k)″-P-(K)″-DTLMI(SEQ ID NO:25)

[0924] 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.

[0925] (K)” each independently Where C m For the load-bearing part,

[0926] B is a structure formed through a click chemical reaction between a first click chemical functional group and a second click chemical functional group, and

[0927] D1 is any alkylene, alkenylene, or ynylene group. The antibody or its fragment has a loading portion coupled to lysine 246 in its Fc domain, or a site corresponding to lysine 246.

[0928] The contents of the load section apply to the contents disclosed in Section 6. Payload.

[0929] In a specific embodiment, B can be a structure formed by a click chemical reaction between any of the alkynes, azides, strained alkynes, dienes, dienophiles, alkenes, thiols, and tetrazides and their partner "click chemical" functional groups. B can also be selected from any of the following:

[0930] Both A1 and A2 are connected to the load section or D1, ensuring that A1 and A2 are not connected to the same section, and R x It can be selected from H, halogens and C 1-3 Alkyl group. Furthermore, B can be... In addition, B can be

[0931] In a specific implementation, D1 may include components selected from covalent bonds, C... 1-4 Alkylene, C 2-4 imidene group, C 2-4 etyne and C 3-8 Any of the cycloalkylene groups. D1 can also be -CH2OCH2-. Furthermore, D1 can be a covalent bond.

[0932] In a specific embodiment, the antibody may be a human antibody. In other specific embodiments, the antibody may be a non-human animal antibody. In a specific embodiment, the antibody may be immunoglobulin G (IgG). In a specific embodiment, the antibody may be a complete antibody. In other specific embodiments, the antibody may be a fragment of an antibody.

[0933] In a specific embodiment, the antibody may contain an amino acid sequence of formula 10-3 in only one of its two Fc domains. In other specific embodiments, the antibody may contain an amino acid sequence of formula 10-3 in both of its Fc domains.

[0934] The present invention provides an antibody or 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 Formulas 10-1 to 10-3 are as described above.

[0935] In a specific implementation, D1 can be a covalent bond.

[0936] In specific embodiments, the antibody or its fragment may include only the amino acid sequence of Formula 10-1 and may exclude the amino acid sequences of Formulas 10-2 and 10-3. Furthermore, the antibody or its fragment may include the amino acid sequence of Formula 10-1 only in one of its two Fc domains. Alternatively, the antibody may include the amino acid sequence of Formula 10-1 in both of its Fc domains.

[0937] In other specific embodiments, the antibody or fragment thereof may comprise only the amino acid sequence of Formula 10-2, and may exclude the amino acid sequences of Formulas 10-1 and 10-3. The antibody or fragment thereof may also include the amino acid sequence of Formula 10-2 only in one of its two Fc domains. Furthermore, the antibody may include the amino acid sequence of Formula 10-2 in both of its Fc domains.

[0938] In specific embodiments, the antibody or its fragment may comprise only the amino acid sequence of Formula 10-3, and may exclude the amino acid sequences of Formulas 10-1 and 10-2. The antibody or its fragment may also include the amino acid sequence of Formula 10-3 only in one of its two Fc domains. Furthermore, the antibody may include the amino acid sequence of Formula 10-3 in both of its Fc domains.

[0939] 7.1. Antibody-Drug Conjugates (ADCs)

[0940] According to the present invention, novel antibody-drug conjugates (ADCs) are disclosed. An ADC according to the present invention refers to an antibody-payload conjugate comprising a drug component.

[0941] This invention provides, according to the present invention, C m -Ab, wherein the load portion includes a drug portion.

[0942] In a specific implementation, the load portion may include two or more drug portions.

[0943] In a specific embodiment, the drug component may be an anticancer drug. The anticancer drug may further include one or more of 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, cytokines, apoptosis inducers, anti-angiogenic agents, lymphokines, taxanes, DNA alkylating agents, anthracyclines, tubulysin analogs, pyruvic analogs, auristatin E, and auristatin. F. Maytansinoids, cytotoxic agents containing reactive polyethylene glycol residues, taxon, cytochalasin B, bacitracin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthraquinone, mitoxantrone, sclerosomycin, actinomycin D, 1-dihydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin, methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil aminoimide, nitrogen mustard, thiotepa, chlorambucil, melphalan. Carmustine, lomustine, cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, cisplatin, styromycin, bleomycin, amiodarone, chachiin, abiraterone, bendamustine, bortezomib, carboplatin, cabazitaxel, dasatinib, docetaxel, epirubicin, erlotinib, everolimus, gemcitabine, gefitinib, idarubicin, imatinib, hydroxyurea, lapatinib, leuprorelin, melphalan, nedaplatin, nilotinib, oxaliplatin, pazopanib, pemetrexed, pyrplatin, romidixin, saplatin, sorafenib, vemurafenib, sunitinib, teniposide, triplatin, and vinorelbine.

[0944] In a specific implementation, the load portion may include a VC connector.

[0945] 7.2. Methods for preparing antibody-load-conjugates

[0946] According to the present invention, a method for preparing antibody-load conjugates is disclosed.

[0947] The antibody-load conjugate of the present invention can be made by using C m -H2 reacts with H1-Ab to prepare it. In this case, the content of H1-Ab applies to the disclosure in Section 5.1, and the content disclosed in Section 6 applies to the C... m -H2. When the first click chemical functional group of H1-Ab and C mWhen the second click chemical functional groups of -H2 are complementary to each other, that is, when the first click chemical functional group of H1-Ab and C m When the second click chemical functional group of -H2 acts as a chaperone click chemical functional group, a click chemical reaction can occur to prepare the antibody-load conjugate according to the invention. The details of the click chemical reaction are fully described in the "Definitions" section above. The click chemical reaction is a biathogonal reaction, and has the advantage of occurring at a very fast rate and being used to form strong binding structures. Therefore, the click chemical reaction has the same advantages as those described in Sections 7.3 and 7.4 below.

[0948] The present invention provides a method for preparing antibody-load conjugates, characterized in that the method comprises reacting an antibody containing a first click chemical functional group according to the present invention with a load according to the present invention, wherein the H2 of the load is a second click chemical functional group complementary to the first click chemical functional group.

[0949] In this case, the antibody provided in Section 5.1 is applied to an antibody containing the first click chemical functional group, and the payload provided in Section 6 is applied to the payload. Because all antibodies containing the first click chemical functional group in lysine 246 and / or lysine 248 are disclosed in Section 5.1, it is desirable that antibody-payload conjugates in which the payload portion is coupled to lysine 246 and / or lysine 248 be disclosed to a fully reproducible degree (see Formulas 10 and 10-1 to 10-3 in Section 7).

[0950] The present invention provides a kit for preparing antibody-load-conjugates, characterized in that the kit comprises an antibody containing a first click chemical functional group according to the present invention; and a load according to the present invention, wherein the H2 of the load is a second click chemical functional group complementary to the first click chemical functional group.

[0951] In this case, the antibody provided in Section 5.1 is applied to the antibody containing the first click chemical functional group, and the payload provided in Section 6 is applied to the payload.

[0952] Optionally, the present invention provides a kit for preparing antibody-payload conjugates, the kit comprising:

[0953] Antibody;

[0954] A reagent for transferring a first-click chemical functional group to the antibody according to the invention;

[0955] The payload according to the present invention.

[0956] In a specific embodiment, the reagent used to transfer the first click chemical functional group to the antibody may be H1-L2-SSFI, which is used to specifically transfer the first click chemical 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 used to transfer the first click chemical functional group to the antibody may be H1-L2-SSFI, which is used to specifically transfer the first click chemical functional group to lysine 246 in the Fc domain of the antibody according to the present invention. In yet another specific embodiment, the reagent used to transfer the first click chemical functional group to the antibody may be H1-L2-SSFI, which is used to specifically transfer the first click chemical functional group to lysine 248 in the Fc domain of the antibody according to the present invention.

[0957] Optionally, the present invention provides a kit for preparing antibody-payload conjugates, the kit comprising:

[0958] Antibody;

[0959] The connector according to the invention;

[0960] According to the site-specific antibody-antibody interaction group of the present invention; and

[0961] The payload according to the present invention.

[0962] In a specific implementation, D2 in the connector is C. y Alkylene, C y imide or C y The acetylinyl group, and D3 in the site-specific antibody interactome is C. x Alkylene, where y can be an integer greater than or equal to 1. The sum of x and y can also be 1 ≤ x + y ≤ 5. Additionally, the sum of x and y can be 6 ≤ x + y ≤ 8. Further, the sum of x and y can be 9 ≤ x + y ≤ 12.

[0963] 7.3. The antibody-load conjugate according to the present invention uses a biorthogonal reaction to form a stable bond.

[0964] The antibody-load conjugate according to the present invention is formed via a click chemistry reaction. This click chemistry reaction is a biathogonal reaction, which does not affect naturally occurring biochemical phenomena in vivo. The bonds formed by the biathogonal reaction are also not recognized by lysing enzymes in vivo. Therefore, the advantage of the antibody-load conjugate according to the present invention is that the antibody and the load moiety form a very stable bond in vivo.

[0965] 7.4. The antibody-load conjugate prepared according to the present invention has high uniformity and yield.

[0966] As described in Section 5.6 above, the antibodies containing the first click chemical functional group according to the present invention exhibit high uniformity and yield. Therefore, the antibody-load conjugates prepared from the antibodies according to the present invention also possess high uniformity and yield. m - The homogeneity of the Ab must be very high in order for the antibody conjugate to have uniform properties.

[0967] In the case of antibody conjugates prepared using the prior art disclosed in US 2018 / 0141976A1 and WO 2018 / 199337A1, the poor yield and uniformity of the antibody conjugates may negatively affect the uniformity of performance, as described in Section 5.6 above.

[0968] 7.5. Because the antibody-load conjugate according to the invention does not have a blocked FcRn binding site, the function of the antibody is not reduced.

[0969] According to the present invention C m -Ab has the same advantages as described in Sections 5.7 and 5.8. In particular, the pharmacokinetic (PK) characteristics of ADCs used in vivo may be greatly improved because ADCs have an increased half-life.

[0970] The FcRn binding site of the ADC prepared using the prior art disclosed in US 20180141976A1 is blocked because the final antibody-labeled product contains SSFI. Therefore, the ADC according to the present invention has superior pharmacokinetic properties compared to the ADC according to the corresponding prior art.

[0971] 7.6. The antibody-drug conjugates according to the present invention are stable because they do not contain highly bioreactive chemical functional groups.

[0972] 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 are able to achieve stable antibody drug action.

[0973] In the case of ADCs prepared using the prior art disclosed in Publication No. WO 2018199337A1, the final antibody-labeled product contains additional chemical functional groups, such as thiols, hydroxyl groups, carboxylic acids, phosphates, amines, etc. Therefore, the ADC according to the present invention exhibits superior PD properties compared to ADCs based on the corresponding prior art.

[0974] 8. Composition

[0975] According to the present invention, compositions comprising antibodies are 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-load conjugate according to the present invention. The antibody may also be an antibody-drug conjugate according to the present invention.

[0976] Depending on the function of the antibody contained in the composition, the compositions according to the invention can be used in a variety of applications. For example, when the first chemical functional group or the loading portion includes a radioactive portion, the corresponding composition can be used as a radioactive contrast agent, etc. Alternatively, when the first chemical functional group or the loading portion includes a fluorescent portion, the corresponding composition can be used as a label used in enzyme-linked immunosorbent assays (ELISA), etc. Alternatively, when the first chemical functional group or the loading portion includes a pharmaceutical portion, the corresponding composition can be used as a pharmaceutical composition. In this case, the components of the compositions commonly used in the relevant fields fall within the scope of the invention. Moreover, the compositional proportions of the corresponding components commonly used in the art also fall within the scope of the invention.

[0977] 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 may 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.

[0978] The present invention also provides compositions comprising an antibody containing a first click chemical functional group according to the present invention. In a specific embodiment, the antibody containing the first click chemical functional group may be at least one selected from Formula 8 and Formulas 8-1 to 8-3.

[0979] Furthermore, the present invention provides compositions comprising the antibody-payload conjugate according to the present invention. In this case, the content described in Section 7 applies to the content of the antibody-payload conjugate.

[0980] Optionally, the present invention provides compositions comprising antibody-drug conjugates according to the present invention.

[0981] In this case, the content described in Section 7.1 applies to the content of the antibody-drug conjugate.

[0982] Pharmaceutical Composition

[0983] The following content relates to compositions, which are pharmaceutical compositions for diagnostic, preventive, and / or therapeutic purposes. Only within the scope of "pharmaceutical compositions" herein, the R1′-Ab, H1-Ab, and C... m -Ab and ADC are used interchangeably with the term "antibody or fragment thereof".

[0984] This invention provides pharmaceutical compositions comprising antibodies or fragments thereof according to the invention. The pharmaceutical compositions may also be compositions for treating cancer. Furthermore, the cancers may include any of 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 carcinoma. Additionally, the cancer may be breast cancer.

[0985] This invention provides a treatment method comprising administering to a subject a pharmaceutical composition comprising an antibody or a fragment thereof according to the invention. The treatment method may also be a method for treating cancer. Furthermore, the cancer may include any of the following: bladder cancer, bone cancer, brain cancer, breast cancer, heart cancer, cervical cancer, colon cancer, rectal cancer, esophageal cancer, fibrosarcoma, 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 carcinoma. Additionally, the cancer may be breast cancer.

[0986] To prepare a pharmaceutical composition or sterile composition comprising an antibody or a fragment thereof, the antibody or a fragment thereof according to the invention may be mixed with a pharmaceutically acceptable carrier or excipient. The composition may further comprise one or more other therapeutic agents suitable for treating or preventing cancers 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, glioblastoma, soft tissue clear cell sarcoma, malignant mesothelioma, neurofibromatosis, renal cancer, melanoma, prostate cancer, benign prostatic hyperplasia (BPH), gynecomastia, rhabdomyosarcoma, and endometriosis.

[0987] 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, for example, 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, 1990; Weiner and Kotkoskie, Excipient Toxicity and Safety, Marcel Dekker, Inc., New York, NY, 2000).

[0988] In a specific embodiment, the clinical service form (CSF) of the antibody-drug conjugate according to the invention is a lyophilized product contained in vials containing ADC, sodium succinate, and polysorbate 20. The lyophilized product can be reconstituted with water for injection, and the solution comprises ADC, sodium succinate, sucrose, and polysorbate 20, with a pH of approximately 5.0. For consecutive intravenous administration, the resulting solution is typically further diluted in a carrier solution.

[0989] The choice of treatment dosing regimen depends on a number of factors, including the serum or tissue replacement rate of the substance, symptom level, immunogenicity of the substance, and accessibility of target cells in the biological matrix. In a specific implementation, the dosing regimen maximizes the amount of therapeutic agent to be delivered to the patient to meet acceptable levels of side effects. Therefore, the amount of biological agent to be administered depends in part on the specific substance and the severity of the condition being treated. Guidelines can be used to select appropriate doses of antibodies, cytokines, and small molecules (see, for example, 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).

[0990] Appropriate dosage is determined by the clinician using parameters or factors known or suspected to affect treatment in the relevant field, or parameters or factors expected to affect treatment. Typically, the dosage is started at a slightly lower than optimal dose and then increased in small increments until the desired or optimal effect is achieved relative to any negative side effects. Important diagnostic measures include inflammatory symptoms or the levels of inflammatory cytokines produced.

[0991] The actual dose level of the active ingredient in the pharmaceutical composition according to the invention can be varied to achieve an effective amount of the active ingredient in a specific patient, composition, and route of administration without causing any toxicity to the patient. The selected dose level may be determined based on various pharmacokinetic factors, including: the activity of the composition of the invention used or its ester, salt, or amide, route of administration, time of administration, secretion rate of the specific compound used, duration of treatment, other drugs, substances and / or compounds used in combination with the specific compound used, age, sex, weight, disease condition, overall health status, and medical history of the patient to be treated, as well as other factors known in the medical field.

[0992] Compositions comprising antibodies or fragments thereof according to the present invention may be administered by continuous infusion or as a single dose, for example daily, weekly, once 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 dose may be administered intravenously, subcutaneously, topically, orally, intranasally, rectally, intramuscularly, intracerebrally, or by inhalation. Specific dosing regimens relate to maximum doses or dosing frequencies that avoid significant unwanted side effects.

[0993] For the antibody or fragment thereof according to the invention, the dose to be administered to the patient can be in the range of 0.0001 mg / kg to 100 mg / kg (patient body weight). The dosage may be in the following ranges: 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 weight). The dosage of the antibody or fragment thereof according to the present invention can be calculated by multiplying the patient's weight (kg) by the dose to be administered (mg / kg).

[0994] The antibody or fragment thereof according to the invention can be administered repeatedly 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, the antibody or fragment thereof according to the invention can be administered repeatedly every three weeks.

[0995] The effective dose 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, for example, 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).

[0996] The route of administration can be, for example, by topical or skin application, by intravenous, intraperitoneal, intracerebral, intramuscular, intraocular, intraarterial, intramedullary, intralesional injection or infusion, or by sustained-release systems or implants (see, for example, 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. Patent 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 formulated using an inhaler or nebulizer, or may be formulated for pulmonary administration using a nebulizer. 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 Publications WO92 / 19244, WO 97 / 32572, WO 97 / 44013, WO 98 / 31346, and WO 99 / 66903, the entire contents of which are incorporated herein by reference.

[0997] The compositions of the present invention can also be administered via one or more routes of administration using one or more methods known in the relevant art. As will be appreciated by those skilled in the art, the route and / or manner of administration will depend on the desired outcome. Routes of administration selected for antibodies or fragments thereof according to the present invention include intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, spinal, or other parenteral routes, such as administration by injection or infusion. Parenteral administration generally represents a method of administration by injection other than enteral and local administration, and includes intravenous, intramuscular, intraarticular, intrasheath, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, tracheal, subcutaneous, subepidermal, intra-articular, subcapsular, subarachnoid, spinal, epidural, and intrasternal injections and infusions, but the invention is not limited thereto. Alternatively, the compositions of the present invention can be administered via non-parenteral routes, such as local, intradermal, or intramucosal routes, and can be administered, for example, intranasally, or orally, vaginally, rectally, sublingually, or locally. In one exemplary embodiment, antibodies or fragments thereof according to the present invention can be administered by infusion. In another exemplary embodiment, the antibody or a fragment thereof according to the invention may be administered subcutaneously.

[0998] When administering the antibody or its fragment according to the invention using a controlled-release or delayed-release system, a pump can be used to achieve controlled-release or delayed-release (see Langer, ibid.; 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). Polymer materials can be used to achieve the controlled or delayed release in the treatments of the present invention (see, for example, 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; also see Levy et al., Science 228: 190, 1985; During et al., Ann. Neurol. 25: 351, 1989; Howard et al., J. Neurosurg. 7). 1:105,1989; U.S. Patent No. 5,679,377; U.S. Patent No. 5,916,597; U.S. Patent No. 5,912,015; U.S. Patent No. 5,989,463; U.S. Patent No. 5,128,326; PCT Publication No. WO 99 / 15154; and PCT Publication No. WO 99 / 20253). Examples of polymers for use in sustained-release formulations include poly(2-hydroxyethyl methacrylate), poly(methyl methacrylate), poly(acrylic acid), poly(ethylene-co-vinyl acetate), poly(methacrylic acid), polyglycolic acid (PLG), polyanhydride, poly(N-vinylpyrrolidone), poly(vinyl alcohol), polyacrylamide, poly(ethylene glycol), polylactide (PLA), poly(lactide-co-glycolic acid) (PLGA), and polyorthoesters, but the invention is not limited thereto. In one exemplary embodiment, the polymer used for the sustained-release formulation is inactive, free of filterable impurities, and stable and biodegradable during storage and sterilization. Controlled-release or delayed-release systems may be located near the preventive or therapeutic target and therefore require a portion of the systemic dose (see, for example, Goodson, in Medical Applications of Controlled Release, ibid., Vol. 2, pp. 115-138, 1984).

[0999] The controlled-release system is discussed in the commentary of the article "Langer, Science 249:1527-1533, 1990". Any prior art known to those skilled in the art can be used to produce sustained-release formulations comprising one or more antibodies or fragments thereof according to the present invention. See, for example, U.S. Patent 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.

[1000] When the antibody or fragment thereof according to the invention is administered topically, the antibody or fragment thereof may be formulated as an ointment, cream, transdermal patch, lotion, gel, shampoo, spray, aerosol, solution, or emulsion, or other forms known to those skilled in the art. See, for example, 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 having a higher kinematic viscosity than water are often used as topical dosage forms. Suitable formulations 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., osmotic pressure, if necessary), but the invention is not limited thereto. In some cases, other suitable topical dosage forms include sprayable aerosol formulations that are combined with an active ingredient and a solid or liquid inactive carrier and then filled in a mixture of compressed volatile substances (e.g., gaseous propellants such as Freon) or squeeze bottles. If necessary, humectants or wetting agents may also be added to the pharmaceutical composition and dosage form. Examples of such additional components are known in the prior art.

[1001] When a composition containing an antibody or fragment thereof is administered intranasally, the composition may be formulated as an aerosol, spray, nebulizer, or drops. In particular, the preventative or therapeutic agents used in this invention may be conveniently delivered in an aerosol spray form from a pressurized package or nebulizer using a suitable propellant (e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide, or other suitable gas). In the case of a pressurized aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges for inhalers or blowpipes (e.g., composed of gelatin) may be formulated as a powder mixture comprising the compound and a suitable powder matrix (e.g., lactose or starch).

[1002] Methods for co-administering or treating with a second therapeutic agent (e.g., cytokines, steroids, chemotherapy agents, antibiotics, or radiotherapy agents) 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. An effective amount of the therapeutic agent may reduce symptoms by at least 10%, at least 20%, at least about 30%, at least 40%, or at least 50%.

[1003] Additional therapies (e.g., prophylactic or therapeutic agents) that can be administered in combination with antibodies or fragments of the invention may be administered together with antibodies or fragments of the invention at the following intervals: less than 5 minutes, less than 30 minutes, 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, 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 treatments may be administered during the same patient visit.

[1004] In specific embodiments, antibodies or fragments thereof according to the invention can be formulated to ensure proper in vivo distribution. For example, the blood-brain barrier (BBB) ​​excludes many highly hydrophilic compounds. To ensure that the therapeutic compounds of the invention cross the blood-brain barrier (if necessary), they can be formulated, for example, in liposomes. For methods of preparing liposomes, see, for example, U.S. Patent Nos. 4,522,811, 5,374,548, and 5,399,331. The liposomes may include one or more portions 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 components include folic acid or biotin (see, for example, U.S. Patent No. 5,416,016 (granted 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), etc. See also K. Keinanen; ML Laukkanen (1994) FEBS Lett. 346: 123; JJ Killion; IJ Fidler (1994) Immunomethods 4: 273.

[1005] This invention provides a method for administering a pharmaceutical composition, alone or in combination with other therapies, to a subject in need, the pharmaceutical composition comprising an antibody or fragment thereof according to the invention. The therapeutic agents (e.g., prophylactic or therapeutic agents) of the invention for combination therapy can be administered to the subject simultaneously or sequentially. The therapeutic agents (e.g., prophylactic or therapeutic agents) for combination therapy of the invention can also be administered periodically. Periodic treatment includes administering a first therapeutic agent (e.g., a first prophylactic or therapeutic agent) for a predetermined period of time, administering a second therapeutic agent (e.g., a second prophylactic or therapeutic agent) for a predetermined period of time, and then repeating the administration steps sequentially, i.e., a predetermined period for reducing the development of resistance to one of the therapeutic agents (e.g., an agonist), and / or preventing or reducing the side effects of one of the therapeutic agents (e.g., an agonist), and / or improving the efficacy of the therapeutic agent.

[1006] The therapeutic agent (e.g., a preventative agent or a therapeutic agent) used in the combined treatment of the present invention can be administered to the subject simultaneously.

[1007] The term "simultaneously" means that the treatment (e.g., a prophylactic or therapeutic agent) does not need to be administered unrestricted at exactly the same time, but rather sequentially to the subject with a pharmaceutical composition comprising an antibody or a fragment thereof according to the invention, and at a time interval that can provide greater benefit compared to administering the antibody of the invention at a different time than other therapies. For example, the individual therapeutic agents may be administered to the subject sequentially at the same time or at different time points in any order; however, they should be administered at sufficiently short intervals to provide the desired therapeutic or preventative effect when administered at different times. The individual therapeutic agents may be administered to the subject via any suitable route of administration in any appropriate form. In various exemplary embodiments, the therapeutic agent (e.g., a preventative or therapeutic agent) may be administered to the subject at intervals of less than 15 minutes, less than 30 minutes, less than 1 hour, about 1 hour, about 1 to about 2 hours, about 2 to about 3 hours, about 3 to about 4 hours, about 4 to about 5 hours, about 5 to about 6 hours, about 6 to about 7 hours, about 7 to about 8 hours, about 8 to about 9 hours, about 9 to about 10 hours, about 10 to about 11 hours, about 11 to about 12 hours, 24 hours, 48 ​​hours, 72 hours, or one week. In other exemplary embodiments, two or more therapeutic agents (e.g., preventative or therapeutic agents) may be administered during the same patient visit.

[1008] The prophylactic or therapeutic agent used for combination therapy may be administered to the subject in the same pharmaceutical composition. Alternatively, the prophylactic or therapeutic agent used for combination therapy may be administered to the subject simultaneously in a single pharmaceutical composition. The prophylactic or therapeutic agent may be administered to the subject via the same or different routes of administration.

[1009] Experimental Examples

[1010] 1. Preparation Examples of Compounds for the Preparation of Reagents for Transferring Site-Specific First-Click Chemical Functional Groups to Antibodies

[1011] 1.1. Assembly and Validation of the Joint (H1-L1) Structure

[1012] 1.1.1. Compound I (SO1 connector: NHS & norbornene)

[1013] [Option 6]

[1014]

[1015] Synthesis of Compound I (Scheme 6) Figure 15 )

[1016] Synthesis of Compound 1

[1017] 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. 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 stirring the reaction solution for 3 hours, the solution was concentrated under reduced pressure to give 1.97 g of the target compound (90% yield).

[1018] Synthesis of Compound 2

[1019] 1.97 g (9.88 mmol, 1.0 eq.) of compound 1 was dissolved in 20 mL of acetonitrile (ACN), and then 0.68 mL (9.88 mmol, 1.0 eq.) of mercaptoacetic acid and 2.06 mL (14.82 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 three 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 give 2.05 g of the target compound (yield: 79%).

[1020] 1 H NMR (500MHz, 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).

[1021] Synthesis of Compound I

[1022] 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 three times with EA. The organic layer was recovered, dried over magnesium sulfate, and then concentrated under reduced pressure. The residue was then purified by chromatography (EA:Hex = 2:1) on a silica gel column to obtain 2.7 g of the target compound (yield: 98%).

[1023] 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).

[1024] Confirmation of the structure of compound I

[1025] 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, 73Hz, 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).

[1026] LRMS(ESI): m / z 371.2 [M+NH4] + ]

[1027] The structure of compound I was confirmed by mass spectrometry. The results are shown in... Figure 16 .

[1028] 1.1.2. Compound II (SO2 connector: NHS & norbornene)

[1029] [Option 7]

[1030]

[1031] Synthesis of Compound II (Scheme 7) Figure 17 )

[1032] Synthesis of Compound 3

[1033] 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 three 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 give 0.56 g of the target compound (yield: 89%).

[1034] Synthesis of Compound II

[1035] 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 three times with EA. The organic layer was dried over magnesium sulfate and concentrated under reduced pressure. The residue was then purified by silica gel column chromatography (EA:Hex = 2:1) to give 0.63 g of the target compound (yield: 83%).

[1036] Confirmation of the structure of compound II

[1037] 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).

[1038] LRMS(ESI): m / z 385.1 [M+NH4] + ]

[1039] The structure of compound II was confirmed by mass spectrometry. The results are shown in... Figure 18 .

[1040] 1.1.3. Compound III (SO3 connector: NHS & norbornene)

[1041] [Option 8]

[1042]

[1043] Synthesis of Compound III (Scheme 8) Figure 19 )

[1044] Synthesis of Compound 4

[1045] 0.5 g (3.62 mmol, 1.0 eq.) of exo-5-norbornenic 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 stirring the reaction solution for 3 hours, it was concentrated under reduced pressure to give 0.47 g of the target compound (83% yield).

[1046] Synthesis of Compound 5

[1047] 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 mercaptoacetic 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 three 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 give 0.3 g of the target compound (yield: 47%).

[1048] 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).

[1049] Synthesis of Compound III

[1050] 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 three times with EA. The organic layer was recovered, dried over magnesium sulfate, and then concentrated under reduced pressure. The residue was then purified by chromatography (EA:Hex = 2:1) on a silica gel column to obtain 0.32 g of the target compound (yield: 85%).

[1051] Confirmation of the structure of compound III

[1052] 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).

[1053] LCMS (ESI): m / z 332.16 [M+Na] + ]

[1054] The structure of compound III was confirmed by mass spectrometry. The results are shown in... Figure 20 .

[1055] 1.1.4. Compound IV (SO4 linker: NHS & azide)

[1056] [Option 9]

[1057]

[1058] Synthesis of Compound IV (Scheme 9) Figure 21 and Figure 22 )

[1059] Synthesis of Compound 6

[1060] 2-(2-chloroethoxy)ethanol (2 mL, 18.94 mmol) was dissolved in distilled water (12 mL), and NaN3 (3.08 g, 47.35 mmol, 2.5 eq.) was added. 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.) (20 mL), and then stirred for about 10 minutes. The reaction mixture was extracted three times with diethyl ether, and the organic layer was dried over magnesium sulfate and filtered. The filtrate was concentrated under reduced pressure to give 2.47 g of the target compound (99% yield).

[1061] Synthesis of Compound 7

[1062] 2.47 g (18.84 mmol) of compound 6 was dissolved in acetone (50 mL), and then 1 M Jone 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, then heated to room temperature and stirred for about 10 minutes. The reaction mixture was extracted three times with ethyl acetate (EA), and the organic layer was dried over magnesium sulfate and filtered. The filtrate was concentrated under reduced pressure to give 2.69 g of the target compound (98% yield).

[1063] Synthesis of Compound 8

[1064] 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 stirring the reaction solution for 3 hours, it was concentrated under reduced pressure to give 2.42 g of the target compound (80% yield).

[1065] Synthesis of Compound 9

[1066] 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 three 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 give 0.73 g of the target compound (yield: 49%).

[1067] Synthesis of Compound 10

[1068] 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. After stirring the reaction solution for 8 hours, it was concentrated under reduced pressure to give 0.40 g of the target compound (70% yield).

[1069] Synthesis of Compound IV

[1070] 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 three times with EA. The organic layer was recovered, dried over magnesium sulfate, and then concentrated under reduced pressure. The residue was then purified by chromatography (EA:Hex = 2:1) on a silica gel column to obtain 1.03 g of the target compound (yield: 36%).

[1071] Confirmation of the structure of compound IV

[1072] 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).

[1073] LRMS(ESI): m / z 334.0 [M+NH4] + ]

[1074] The structure of compound IV was confirmed by mass spectrometry. The results are shown in... Figure 23 .

[1075] 1.2. Synthesis and structural confirmation of site-specific Fc interactomes (SSFIs)

[1076] 1.2.1. Synthesis and structural confirmation of SSFI (where Xa1 is lysine)

[1077] Synthesis of SSFI(6Lys)

[1078]

[1079] List and order of the Fmoc amino acids used

[1080] 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.

[1081] Preparation method

[1082] (a) Introduction of amino acids

[1083] The amounts of reagents used in the following process are based on 0.25 mmol. 0.5 g (0.48 mmol / g) of transparent amide resin (Peptide International Inc., USA) was placed in the synthesis reactor, and 1 mmol of each Fmoc-amino acid block was weighed to prepare the peptide amino acid sequence from C-terminus to N-terminus in the order described above.

[1084] The reaction proceeds sequentially from the C-terminal amino acids to activate Fmoc-amino acids, thereby linking the activated residues to the transparent amide resin.

[1085] Fmoc removal was performed in DMF containing 20% ​​piperidine, and the activation and introduction of residues were carried out by mixing the amino acid corresponding to the sequence with 2 mL of 0.5 M DMF solution containing HOBt, 2 mL of 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 resin for 2 hours.

[1086] The introduction reaction was confirmed using the Kaiser assay. If incomplete introduction was confirmed, the reaction was repeated, or the resin was capped using a DMF solution containing 20% ​​Ac₂O. During each introduction reaction and Fmoc removal, the resin was thoroughly washed with DMF and DCM before proceeding to the next step. This process was repeated until the target peptide sequence was obtained.

[1087] (b) Introduction of H-PEG8-OH

[1088] To introduce H-PEG8-OH to the N-terminus of the sequence after the amino acid introduction is completed, 1 mL of 0.5 M Fmoc-N-amido-dPEG8-acid-in-DMF solution, 1 mL of 0.5 M DMF solution containing HBTU, 1 mL of 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 reactive resin for 2 hours.

[1089] The reaction progress was monitored using the Kaiser assay. If unreacted amine residue was found, the reaction time was extended by 1–3 hours, or the reaction solution was discarded, and the above reaction process was repeated. The N-terminal Fmoc protecting group was removed using DMF containing 20% ​​piperidine, and then the resin with attached peptides was dried and weighed.

[1090] (c) The 250 mg of peptide-attached resin prepared in step (b) was stirred with 2 mL of a mixture 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 approximately half its volume using nitrogen. Subsequently, diethyl ether was poured into the mixture to precipitate the peptide. The precipitated peptide was washed three times with diethyl ether and dried under nitrogen. The dried precipitate was dissolved in water containing 0.1% TFA and 30% ACN, stirred for 6 hours, and then concentrated.

[1091] The concentrate was dissolved at a concentration of 0.1 mg / mL in a 0.01 M ammonium acetate buffer solution (pH 6.5) containing 5% DMSO and 20% ACN, and then stirred for 3 days while exposed to air. The progress of the disulfide bond formation reaction was monitored by HPLC. When the reaction was found to have ceased to progress, the reaction solution was lyophilized to obtain the peptide precipitate.

[1092] (d) Purification

[1093] The peptide precipitate obtained by lyophilization in step (c) was separated under the preparative LC primary purification conditions listed in Table 4, and further purified under the preparative LC secondary purification conditions listed in Table 5, followed by lyophilization. The purity of the obtained peptide was confirmed to be 90% or higher by analytical HPLC, and the molecular weight of the synthesized peptide was confirmed by LC / MS mass spectrometry.

[1094] H-PEG8-Asp-Cys*-Ala-Trp-His-Lys-Gly-Glu-Leu-Val-Trp-Cys*-Thr-NH2 (Cys*: disulfide bond site)

[1095] Table 4. Preparative LC Primary Purification Conditions

[1096]

[1097] Table 5 <Preparative LC Secondary Purification Conditions>

[1098]

[1099] Confirmation of the structure of SSFI(6Lys)

[1100] The synthesis of SSFI(6Lys) was confirmed by molecular weight measurement using matrix-assisted laser desorption / ionization (MALDI) mass spectrometry.

[1101] Measuring equipment: Ultraflextreme (Bruker)

[1102] Measurement matrices: CHCA (α-cyano-4-hydroxycinnamic acid) and DHB (2,5-dihydroxybenzoic acid)

[1103] Calculated molecular weight: 2010.29 g / mol

[1104] Measured molecular weight (M+H) + 2011.89 g / mol

[1105] The SSFI(6Lys) mass spectrometry results were then... Figure 24 As shown in the image.

[1106] 1.2.2. Synthesis and structural confirmation of SSFI (where Xa1 is ornithine)

[1107]

[1108] Synthesis of SSFI(6Orn)

[1109] The order and list of Fmoc amino acids used

[1110] 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.

[1111] Preparation method

[1112] (a) Introduction of amino acids

[1113] The amounts of reagents used in the following process are based on 0.25 mmol. 0.5 g (0.48 mmol / g) of transparent amide resin (Peptide International Inc., USA) was placed in the synthesis reactor, and 1 mmol of each Fmoc-amino acid block was weighed to prepare the peptide amino acid sequence from C-terminus to N-terminus in the order described above.

[1114] The reaction proceeds sequentially from the C-terminal amino acids to activate Fmoc-amino acids, thereby linking the activated residues to the transparent amide resin.

[1115] Fmoc removal was performed in DMF containing 20% ​​piperidine, and the activation and introduction of residues were carried out by mixing the amino acid corresponding to the sequence with 2 mL of 0.5 M DMF solution containing HOBt, 2 mL of 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 resin for 2 hours.

[1116] The introduction reaction was confirmed using the Kaiser assay. If incomplete introduction was confirmed, the reaction was repeated, or the resin was capped using a DMF solution containing 20% ​​Ac₂O. During each introduction reaction and Fmoc removal, the resin was thoroughly washed with DMF and DCM before proceeding to the next step. This process was repeated until the target peptide sequence was obtained.

[1117] (b) Introduction of H-PEG8-OH

[1118] To introduce H-PEG8-OH to the N-terminus of the sequence after the amino acid introduction is completed, 1 mL of 0.5 M Fmoc-N-amido-dPEG8-acid-in-DMF solution, 1 mL of 0.5 M DMF solution containing HBTU, 1 mL of 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 reactive resin for 2 hours.

[1119] The reaction progress was monitored using the Kaiser assay. If unreacted amine residue was found, the reaction time was extended by 1–3 hours, or the reaction solution was discarded, and the above reaction process was repeated. The N-terminal Fmoc protecting group was removed using DMF containing 20% ​​piperidine, and then the resin with attached peptides was dried and weighed.

[1120] (c) The 250 mg of peptide-attached resin prepared in step (b) was stirred with 2 mL of a mixture 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 approximately half its volume using nitrogen. Subsequently, diethyl ether was poured into the mixture to precipitate the peptide. The precipitated peptide was washed three times with diethyl ether and dried under nitrogen. The dried precipitate was dissolved in water containing 0.1% TFA and 30% ACN, stirred for 6 hours, and then concentrated.

[1121] The concentrate was dissolved at a concentration of 0.1 mg / mL in a 0.01 M ammonium acetate buffer solution (pH 6.5) containing 5% DMSO and 20% ACN, and then stirred for 3 days while exposed to air. The progress of the disulfide bond formation reaction was monitored by HPLC. When the reaction was found to have ceased to progress, the reaction solution was lyophilized to obtain the peptide precipitate.

[1122] (d) Purification

[1123] The peptide precipitate obtained by lyophilization in step (c) was separated under the preparative LC primary purification conditions listed in Table 6, and further purified under the preparative LC secondary purification conditions listed in Table 7, followed by lyophilization. The purity of the obtained peptide was confirmed to be 90% or higher by analytical HPLC, and the molecular weight of the synthesized peptide was confirmed by LC / MS mass spectrometry.

[1124] H-PEG8-Asp-Cys*-Ala-Trp-His-Orn-Gly-Glu-Leu-Val-Trp-Cys*-Thr-NH2 (Cys*: disulfide bond site)

[1125] Table 6. Preparative LC Primary Purification Conditions

[1126]

[1127] Table 7 <Preparative LC Secondary Purification Conditions>

[1128]

[1129] Confirmation of the structure of SSFI(6Orn)

[1130] The synthesis of SSFI (6-ornithine) was confirmed by molecular weight measurement using matrix-assisted laser desorption / ionization (MALDI) mass spectrometry.

[1131] Measuring equipment: Ultraflextreme (Bruker)

[1132] Measurement matrices: CHCA (α-cyano-4-hydroxycinnamic acid) and DHB (2,5-dihydroxybenzoic acid)

[1133] Calculated molecular weight: 1996.26 g / mol

[1134] The measured molecular weight (M+2H) 2+ 999.13 g / mol

[1135] The mass spectrometry results of SSFI(6Orn) were used in Figure 25 As shown in the image.

[1136] 1.2.3. Synthesis and structural confirmation of SSFI (where Xa1 is diaminobutyric acid (Dab))

[1137]

[1138] Synthesis of SSFI(6Dab)

[1139] The order and list of Fmoc amino acids used

[1140] 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.

[1141] Preparation method

[1142] (a) Introduction of amino acids

[1143] The amounts of reagents used in the following process are based on 0.25 mmol. 0.5 g (0.48 mmol / g) of transparent amide resin (Peptide International Inc., USA) was placed in the synthesis reactor, and 1 mmol of each Fmoc-amino acid block was weighed to prepare the peptide amino acid sequence from C-terminus to N-terminus in the order described above.

[1144] The reaction proceeds sequentially from the C-terminal amino acids to activate Fmoc-amino acids, thereby linking the activated residues to the transparent amide resin.

[1145] Fmoc removal was performed in DMF containing 20% ​​piperidine, and the activation and introduction of residues were carried out by mixing the amino acid corresponding to the sequence with 2 mL of 0.5 M DMF solution containing HOBt, 2 mL of 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 resin for 2 hours.

[1146] The introduction reaction was confirmed using the Kaiser assay. If incomplete introduction was confirmed, the reaction was repeated, or the resin was capped using a DMF solution containing 20% ​​Ac₂O. During each introduction reaction and Fmoc removal, the resin was thoroughly washed with DMF and DCM before proceeding to the next step. This process was repeated until the target peptide sequence was obtained.

[1147] (b) Introduction of H-PEG8-OH

[1148] To introduce H-PEG8-OH to the N-terminus of the sequence after the amino acid introduction is completed, 1 mL of 0.5 M Fmoc-N-amido-dPEG8-acid-in-DMF solution, 1 mL of 0.5 M DMF solution containing HBTU, 1 mL of 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 reactive resin for 2 hours.

[1149] The reaction progress was monitored using the Kaiser assay. If unreacted amine residue was found, the reaction time was extended by 1–3 hours, or the reaction solution was discarded, and the above reaction process was repeated. The N-terminal Fmoc protecting group was removed using DMF containing 20% ​​piperidine, and then the resin with attached peptides was dried and weighed.

[1150] (c) The 250 mg of peptide-attached resin prepared in step (b) was stirred with 2 mL of a mixture 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 approximately half its volume using nitrogen. Subsequently, diethyl ether was poured into the mixture to precipitate the peptide. The precipitated peptide was washed three times with diethyl ether and dried under nitrogen. The dried precipitate was dissolved in water containing 0.1% TFA and 30% ACN, stirred for 6 hours, and then concentrated.

[1151] The concentrate was dissolved at a concentration of 0.1 mg / mL in a 0.01 M ammonium acetate buffer solution (pH 6.5) containing 5% DMSO and 20% ACN, and then stirred for 3 days while exposed to air. The progress of the disulfide bond formation reaction was monitored by HPLC. When the reaction was found to have ceased to progress, the reaction solution was lyophilized to obtain the peptide precipitate.

[1152] (d) Purification

[1153] The peptide precipitate obtained by lyophilization in step (c) was separated under the preparative LC primary purification conditions listed in Table 8, and further purified under the preparative LC secondary purification conditions listed in Table 9, followed by lyophilization. The purity of the obtained peptide was confirmed to be 90% or higher by analytical HPLC, and the molecular weight of the synthesized peptide was confirmed by LC / MS mass spectrometry.

[1154] H-PEG8-Asp-Cys*-Ala-Trp-His-Dab-Gly-Glu-Leu-Val-Trp-Cys*-Thr-NH2 (Cys*: disulfide bond site)

[1155] Table 8. Preparative LC Primary Purification Conditions

[1156]

[1157] Table 9. Preparative LC Secondary Purification Conditions

[1158]

[1159] Confirmation of the structure of SSFI (6Dab)

[1160] The synthesis of SSFI(6Dab) was confirmed by molecular weight measurement using matrix-assisted laser desorption / ionization (MALDI) mass spectrometry.

[1161] Measuring equipment: Ultraflextreme (Bruker)

[1162] Measurement matrices: CHCA (α-cyano-4-hydroxycinnamic acid) and DHB (2,5-dihydroxybenzoic acid)

[1163] Calculated molecular weight: 1982.24 g / mol

[1164] The measured molecular weight (M+2H) 2+ 992.33 g / mol

[1165] The mass spectrometry results of SSFI (6Dab) were used in Figure 26 As shown in the image.

[1166] 1.2.4. Synthesis and structural confirmation of SSFI (where Xa1 is diaminopropionic acid (Dap))

[1167]

[1168] Synthesis of SSFI (6Dap)

[1169] Sequence: Nor.-PEG8-DCAWHA (β-aminoalanine, Dap)GELVWCT-CONH2 Introduction sequence and list of Fmoc amino acids used

[1170] 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.

[1171] Preparation method

[1172] (a) Introduction of amino acids

[1173] The amounts of reagents used in the following process are based on 0.25 mmol. 0.5 g (0.48 mmol / g) of transparent amide resin (Peptide International Inc., USA) was placed in the synthesis reactor, and 1 mmol of each Fmoc-amino acid block was weighed to prepare the peptide amino acid sequence from C-terminus to N-terminus in the order described above.

[1174] The reaction proceeds sequentially from the C-terminal amino acids to activate Fmoc-amino acids, thereby linking the activated residues to the transparent amide resin.

[1175] Fmoc removal was performed in DMF containing 20% ​​piperidine, and the activation and introduction of residues were carried out by mixing the amino acid corresponding to the sequence with 2 mL of 0.5 M DMF solution containing HOBt, 2 mL of 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 resin for 2 hours.

[1176] The introduction reaction was confirmed using the Kaiser assay. If incomplete introduction was confirmed, the reaction was repeated, or the resin was capped using a DMF solution containing 20% ​​Ac₂O. During each introduction reaction and Fmoc removal, the resin was thoroughly washed with DMF and DCM before proceeding to the next step. This process was repeated until the target peptide sequence was obtained.

[1177] (b) Introduction of H-PEG8-OH

[1178] To introduce H-PEG8-OH to the N-terminus of the sequence after the amino acid introduction is completed, 1 mL of 0.5 M Fmoc-N-amido-dPEG8-acid-in-DMF solution, 1 mL of 0.5 M DMF solution containing HBTU, 1 mL of 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 reactive resin for 2 hours.

[1179] The reaction progress was monitored using the Kaiser assay. If unreacted amine residue was found, the reaction time was extended by 1–3 hours, or the reaction solution was discarded, and the above reaction process was repeated. The N-terminal Fmoc protecting group was removed using DMF containing 20% ​​piperidine, and then the resin with attached peptides was dried and weighed.

[1180] (c) The 250 mg of peptide-attached resin prepared in step (b) was stirred with 2 mL of a mixture 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 approximately half its volume using nitrogen. Subsequently, diethyl ether was poured into the mixture to precipitate the peptide. The precipitated peptide was washed three times with diethyl ether and dried under nitrogen. The dried precipitate was dissolved in water containing 0.1% TFA and 30% ACN, stirred for 6 hours, and then concentrated.

[1181] The concentrate was dissolved at a concentration of 0.1 mg / mL in a 0.01 M ammonium acetate buffer solution (pH 6.5) containing 5% DMSO and 20% ACN, and then stirred for 3 days while exposed to air. The progress of the disulfide bond formation reaction was monitored by HPLC. When the reaction was found to have ceased to progress, the reaction solution was lyophilized to obtain the peptide precipitate.

[1182] (d) Purification

[1183] The peptide precipitate obtained by lyophilization in step (c) was separated under the preparative LC primary purification conditions listed in Table 10, and further purified under the preparative LC secondary purification conditions listed in Table 11, and then lyophilized. The purity of the obtained peptide was confirmed to be 90% or higher by analytical HPLC, and the molecular weight of the synthesized peptide was confirmed by LC / MS mass spectrometry.

[1184] H-PEG8-Asp-Cys*-Ala-Trp-His-Dap-Gly-Glu-Leu-Val-Trp-Cys*-Thr-NH2 (Cys*: disulfide bond site)

[1185] Preparative LC First-Stage Purification Conditions

[1186]

[1187] Preparative LC Secondary Purification Conditions

[1188]

[1189] Confirmation of the structure of SSFI (6Dap)

[1190] Measuring equipment: Quattro Premier Xe (Waters)

[1191] Calculated molecular weight: 1968.21 g / mol

[1192] The measured molecular weight (M+2H) 2+ 984.71 g / mol

[1193] The mass spectrometry results of SSFI (6Dap) were used in Figure 27 As shown in the image.

[1194] 1.3. Assembly and structural confirmation of VC connectors

[1195] 1.3.1. Synthesis and Validation of the VC Connector (DD2) Structure

[1196] [Option 10]

[1197] Synthesis of DD2

[1198]

[1199] Synthesis of VC connector (DD2)

[1200] Synthesis of Compound 11

[1201] 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. The resulting solution was then filtered under reduced pressure to remove reformed floating matter and used in the next reaction without any purification (crude product yield: 5.5 g, 86%). TLC (EA:Hex = 1:1); R f =0.5.

[1202] Synthesis of Compound 12

[1203] 2.0 g (11.5 mmol, 1.0 eq.) of L-citrulline was dissolved in 100 mL of a 1:1 mixture of tetrahydrofuran (THF) and water and stirred. 988 mg of sodium bicarbonate was added and stirred. Subsequently, 5.0 g (11.5 mmol, 1.0 eq.) of compound 8 was dissolved in 80 mL of acetone and added dropwise to the reaction solution while stirring. 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 slow addition of 2N HCl. EA was added to extract the precipitate from the organic layer. The organic layer was then dried over a saturated brine solution and sodium sulfate and used in the next step of the reaction without any purification (crude product yield: 5.6 g, 98%). TLC (DCM:MeOH = 10:1, one drop of formic acid); R f =0.1.

[1204] Synthesis of Compound 13

[1205] 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 any floating matter. The aqueous layer was concentrated to give the target compound, which could be used for the next reaction without any purification. TLC (DCM:MeOH = 10:1); R f =0.01.

[1206] Synthesis of Compound 14

[1207] 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. 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 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 reversed-phase column chromatography to obtain the target compound. TLC (DCM:MeOH = 10:1); R f =0.05.

[1208] Synthesis of Compound 15

[1209] 1 g (3.16 mmol, 1.0 eq.) of compound 14 was dissolved in 30 mL of a 2:1 mixture of DCM and methanol, and 868 mg (3.48 mmol, 1.1 eq.) of N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) was added and stirred. 451 mg (3.66 mmol, 1.16 eq.) of 4-aminobenzyl alcohol was added 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.

[1210] Synthesis of compound DD2

[1211] 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.

[1212] Confirmation of the structure of the VC connector (DD2)

[1213] LRMS(ESI): m / z 840.4 [M+H + ]

[1214] The results are in Figure 28 As shown in the image.

[1215] 1.3.2. Assembly and structural confirmation of the VC connector (DD3)

[1216] [Option 11]

[1217]

[1218]

[1219] Synthesis of VC connector (DD3)

[1220] Synthesis of Compound 16

[1221] 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 for 4 hours. The resulting solution was then filtered under reduced pressure to remove reformed floating matter and used for the next reaction without any purification (crude product yield: 5.5 g, 86%). TLC (EA:Hex = 1:1); R f =0.5.

[1222] Synthesis of Compound 17

[1223] 2.0 g (11.5 mmol, 1.0 eq.) of L-citrulline was dissolved in 100 mL of a 1:1 mixture of tetrahydrofuran (THF) and water and stirred. 988 mg of sodium bicarbonate was added and stirred. Subsequently, 5.0 g (11.5 mmol, 1.0 eq.) of compound 16 was dissolved in 80 mL of acetone and added dropwise to the reaction solution while stirring. 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 dropwise addition of 2N HCl. EA was added to extract the precipitate from the organic layer. The organic layer was then dried over a saturated brine solution and sodium sulfate and used for the next reaction without any purification (crude product yield: 5.6 g, 98%). TLC (DCM:MeOH = 10:1, one drop of formic acid); R f =0.1.

[1224] Synthesis of Compound 18

[1225] 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 any floating matter. The aqueous layer was concentrated to give the target compound, which was used for the next reaction without any purification. TLC (DCM:MeOH = 10:1); R f =0.01.

[1226] Synthesis of Compound 19

[1227] 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. 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 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 reversed-phase column chromatography to obtain the target compound. TLC (DCM:MeOH = 10:1); R f =0.05.

[1228] Synthesis of Compound 20

[1229] 1 g (3.16 mmol, 1.0 eq.) of compound 19 was dissolved in 30 mL of a 2:1 mixture of DCM and methanol, and 868 mg (3.48 mmol, 1.1 eq.) of N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) was added and stirred. 451 mg (3.66 mmol, 1.16 eq.) of 4-aminobenzyl alcohol was added 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.

[1230] Synthesis of Compound 21

[1231] 240 mg (0.55 mmol, 1.0 eq.) of compound 20 was dissolved in 10 mL of DMF, and 0.3 mL (1.65 mmol, 3.0 eq.) of DIPEA was added dropwise with stirring. Subsequently, the reaction was initiated by adding 509 mg (1.65 mmol, 3.0 eq.) of bis-(4-aminophenyl) carbonate. After 3 hours, the resulting mixture was concentrated under reduced pressure to remove the reaction solution, and diethyl ether was added to precipitate the target compound, which was then filtered under reduced pressure. The resulting target compound was used in the next reaction without any purification.

[1232] Synthesis of Compound 22

[1233] 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 with stirring. The resulting mixture was stirred for 7 hours, then concentrated under reduced pressure to remove the reaction solution, and diethyl ether was added to precipitate the target compound, which was then filtered under reduced pressure. The obtained target compound was used for the next reaction without any purification. TLC (DCM:MeOH = 10:1); R f =0.1.

[1234] Synthesis of Compound 23

[1235] 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 at 0 °C. The reaction was initiated by stirring the resulting mixture at 0 °C for 30 min, followed by stirring at room temperature for 30 min. Subsequently, the mixture was concentrated under reduced pressure to remove the reaction solution, such as DCM, and this concentration was repeated three times. The concentrated substance was dried under high vacuum and purified by reversed-phase column chromatography to give 13 mg of the target compound. TLC (DCM:MeOH = 10:1); R f =0.01.

[1236] Synthesis of compound DD3

[1237] 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 to the solution. 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.

[1238] Confirmation of the structure of the VC connector (DD3)

[1239] LRMS(ESI): m / z 926.4 [M+H + ]

[1240] The results are in Figure 29 As shown in the image.

[1241] 1.3.3. Assembly and structural confirmation of VC connector (DD4)

[1242] [Option 12]

[1243]

[1244] Synthesis of VC connector (DD4)

[1245] Synthesis of Compound 24

[1246] 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 for 4 hours. The resulting solution was then filtered under reduced pressure to remove reformed floating matter and used for the next reaction without any purification (crude product yield: 5.5 g, 86%). TLC (EA:Hex = 1:1); R f =0.5.

[1247] Synthesis of Compound 25

[1248] 2.0 g (11.5 mmol, 1.0 eq.) of L-citrulline was dissolved in 100 mL of a 1:1 mixture of tetrahydrofuran (THF) and water and stirred. 988 mg of sodium bicarbonate was added and stirred. Subsequently, 5.0 g (11.5 mmol, 1.0 eq.) of compound 21 was dissolved in 80 mL of acetone and added dropwise to the reaction solution while stirring. 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 slow dropwise addition of 2N HCl. EA was added to extract the precipitate from the organic layer. The organic layer was then dried over a saturated brine solution and sodium sulfate and used for the next step of the reaction without any purification (crude product yield: 5.6 g, 98%). TLC (DCM:MeOH = 10:1, one drop of formic acid); R f =0.1.

[1249] Synthesis of Compound 26

[1250] 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 any floating matter. The aqueous layer was concentrated to give the target compound, which could be used for the next reaction without any purification. TLC (DCM:MeOH = 10:1); R f =0.01.

[1251] Synthesis of Compound 27

[1252] 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. 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 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 reversed-phase column chromatography to obtain the target compound. TLC (DCM:MeOH = 10:1); R f =0.05.

[1253] Synthesis of Compound 28

[1254] 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. 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 dissolved in 3.5 mL of DMF was added dropwise at room temperature. After 7 hours, the reaction solution was diluted with ethyl acetate (EA), and the organic layer was washed with saturated sodium bicarbonate solution. The organic layer was then dried over saturated salt solution and sodium sulfate, and filtered to obtain the target compound. The obtained target compound was used in the next reaction without any purification. TLC(DCM:MeOH=10:1); R f =0.5.

[1255] Synthesis of Compound 29

[1256] Compound 28 was dissolved in a 2:1 mixture of DCM and TFA, and the reaction was carried out at 0 °C. The reaction mixture was then concentrated under reduced pressure to remove the reaction solution (e.g., DCM), and this concentration was repeated three times. The residue was dried under high vacuum and purified by reversed-phase column chromatography to give 370 mg of the target compound. TLC (DCM:MeOH = 10:1); R f =0.01.

[1257] Synthesis of compound DD4

[1258] 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 to the solution. 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.

[1259] Confirmation of the structure of the VC connector (DD4)

[1260] LRMS(ESI): m / z 777.3 [M+H + ]

[1261] The results are in Figure 30 As shown in the image.

[1262] 1.3.4. Assembly and structural confirmation of the VC connector (DD5)

[1263] [Option 13]

[1264]

[1265]

[1266] Synthesis of VC connector (DD5)

[1267] Synthesis of Compound 30

[1268] 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 for 4 hours. The resulting solution was then filtered under reduced pressure to remove reformed floating matter and used for the next reaction without any purification (crude product yield: 5.5 g, 86%). TLC (EA:Hex = 1:1); R f =0.5.

[1269] Synthesis of Compound 31

[1270] 2.0 g (11.5 mmol, 1.0 eq.) of L-citrulline was dissolved in 100 mL of a 1:1 mixture of tetrahydrofuran (THF) and water and stirred. 988 mg of sodium bicarbonate was added and stirred. Subsequently, 5.0 g (11.5 mmol, 1.0 eq.) of compound 30 was dissolved in 80 mL of acetone and added dropwise to the reaction solution while stirring. 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 slow dropwise addition of 2N HCl. EA was added to extract the precipitate from the organic layer. The organic layer was then dried over a saturated brine solution and sodium sulfate and used for the next step of the reaction without any purification (crude product yield: 5.6 g, 98%). TLC (DCM:MeOH = 10:1, one drop of formic acid); R f =0.1.

[1271] Synthesis of Compound 32

[1272] 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 any floating matter. The aqueous layer was concentrated to give the target compound, which could be used for the next reaction without any purification. TLC (DCM:MeOH = 10:1); R f =0.01.

[1273] Synthesis of Compound 33

[1274] 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. 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 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 reversed-phase column chromatography to obtain the target compound. TLC (DCM:MeOH = 10:1); R f =0.05.

[1275] Synthesis of Compound 34

[1276] 1 g (3.16 mmol, 1.0 eq.) of compound 33 was dissolved in 30 mL of a 2:1 mixture of DCM and methanol, and 868 mg (3.48 mmol, 1.1 eq.) of N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) was added and stirred. 451 mg (3.66 mmol, 1.16 eq.) of 4-aminobenzyl alcohol was added 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.

[1277] Synthesis of Compound 35

[1278] 240 mg (0.55 mmol, 1.0 eq.) of compound 34 was dissolved in 10 mL of DMF, and 0.3 mL (1.65 mmol, 3.0 eq.) of DIPEA was added dropwise with stirring. Subsequently, the reaction was initiated by adding 509 mg (1.65 mmol, 3.0 eq.) of bis-(4-aminophenyl) carbonate. After 3 hours, the resulting mixture was concentrated under reduced pressure to remove the reaction solution, and diethyl ether was added to precipitate the target compound, which was then filtered under reduced pressure. The resulting target compound was used in the next reaction without any purification.

[1279] Synthesis of Compound 36

[1280] 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 with stirring. The resulting mixture was stirred for 7 hours, then concentrated under reduced pressure to remove the reaction solution, and diethyl ether was added to precipitate the target compound, which was then filtered under reduced pressure. The obtained target compound was used for the next reaction without any purification. TLC (DCM:MeOH = 10:1); R f =0.1.

[1281] Synthesis of Compound 37

[1282] 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 at 0 °C. The reaction was initiated by stirring the resulting mixture at 0 °C for 30 min, followed by stirring at room temperature for 30 min. Subsequently, the mixture was concentrated under reduced pressure to remove the reaction solution, such as DCM, and this concentration was repeated three times. The concentrated substance was dried under high vacuum and purified by reversed-phase column chromatography to give 13 mg of the target compound. TLC (DCM:MeOH = 10:1); R f =0.01.

[1283] Synthesis of compound DD5

[1284] 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 to the solution. 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.

[1285] 3-4-2. Confirmation of the structure of the VC connector (DD5)

[1286] LRMS(ESI): m / z 954.4 [M+H + ]

[1287] The results are in Figure 31 As shown in the image.

[1288] 1.3.5. Assembly and structural confirmation of VC connector (DD6)

[1289] [Option 14]

[1290]

[1291] Synthesis of VC connector (DD6)

[1292] Synthesis of Compound 38

[1293] 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 for 4 hours. The resulting solution was then filtered under reduced pressure to remove reformed floating matter and used for the next reaction without any purification (crude product yield: 5.5 g, 86%). TLC (EA:Hex = 1:1); R f =0.5.

[1294] Synthesis of Compound 39

[1295] 2.0 g (11.5 mmol, 1.0 eq.) of L-citrulline was dissolved in 100 mL of a 1:1 mixture of tetrahydrofuran (THF) and water and stirred. 988 mg of sodium bicarbonate was added and stirred. Subsequently, 5.0 g (11.5 mmol, 1.0 eq.) of compound 38 was dissolved in 80 mL of acetone and added dropwise to the reaction solution while stirring. 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 titrat...

Claims

1. A peptide-compound conjugate having the structure of Formula 6-3: [Formula 6-3] DCAWH-(Xa1)′-GELVWCT in, D is an aspartic acid residue, C is a cysteine ​​residue, A is an alanine residue, W is a tryptophan residue, H is a histidine residue, G is a glycine residue, E is a glutamic acid residue, L is a leucine residue, V is a valine residue, T is a threonine residue, and (Xa1)′ 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, X1 is S, D2 is selected from C 1～7 Alkylene, C 2～7 Alkenylene and C 2～7 Alkynylidene, D3 is a covalent bond or C 1～3 an alkylene group, and X3 is NH, and wherein the N-terminal cysteine ​​residue is optionally linked to the C-terminal cysteine ​​residue, The alkylene, alkenylene, alkynylene and cycloalkylene groups include heteroalkylene, heteroalkenylene, heteroalkynylene and heterocycloalkylene groups, respectively.

2. The peptide-compound conjugate according to claim 1, wherein D1 is a covalent bond or -(CH2) 1-4 -.

3. The peptide-compound conjugate according to claim 1, wherein D1 includes at least one heteroatom.

4. The peptide-compound conjugate according to claim 1, wherein H1 is selected from alkynes and alkenes.

5. The peptide-compound conjugate according to claim 1, wherein H1 is selected from strained alkynes and dienes.

6. The peptide-compound conjugate according to claim 1, wherein H1 is a dienophile.

7. The peptide-compound conjugate according to claim 1, wherein H1 is selected from the group consisting of azide, dibenzocyclooctyne-amine, trans-cyclooctene and norbornene.

8. The peptide-compound conjugate according to claim 1, wherein D2 is -CH2-.

9. 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 6-3 with an antibody or a fragment thereof: [Formula 6-3] DCAWH-(Xa1)′-GELVWCT in, D is an aspartic acid residue, C is a cysteine ​​residue, A is an alanine residue, W is a tryptophan residue, H is a histidine residue, G is a glycine residue, E is a glutamic acid residue, L is a leucine residue, V is a valine residue, T is a threonine residue, and (Xa1)′ 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, X1 is S, D2 is selected from C 1～7 Alkylene, C 2～7 Alkenylene and C 2～7 Alkynylidene, D3 is a covalent bond or C 1～3 an alkylene group, and X3 is NH, and wherein the N-terminal cysteine ​​residue is optionally linked to the C-terminal cysteine ​​residue, The alkylene, alkenylene, alkynylene and cycloalkylene groups include heteroalkylene, heteroalkenylene, heteroalkynylene and heterocycloalkylene groups, respectively.

10. The method according to claim 9, wherein: D1 is a covalent bond or -(CH2) 1-4 -.

11. The method according to claim 9, wherein: D1 includes at least one heteroatom.

12. The method according to claim 9, wherein: H1 is selected from alkynes and alkenes.

13. The method according to claim 9, wherein: H1 is selected from strained alkynes and dienes.

14. The method according to claim 9, wherein: H1 is a dienophile.

15. The method according to claim 9, wherein: H1 is selected from the group consisting of azide, dibenzocyclooctyne-amine, trans-cyclooctene and norbornene.

16. The method according to claim 9, wherein: D2 is -CH2-.

17. 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 6-3 and an antibody or a fragment thereof: [Formula 6-3] DCAWH-(Xa1)′-GELVWCT in, D is an aspartic acid residue, C is a cysteine ​​residue, A is an alanine residue, W is a tryptophan residue, H is a histidine residue, G is a glycine residue, E is a glutamic acid residue, L is a leucine residue, V is a valine residue, T is a threonine residue, (Xa1)′ 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, X1 is S, D2 is selected from C 1～7 Alkylene, C 2～7 Alkenylene and C 2～7 Alkynylidene, D3 is a covalent bond or C 1～3 an alkylene group, and X3 is NH, wherein the N-terminal cysteine ​​residue is optionally linked to the C-terminal cysteine ​​residue, The alkylene, alkenylene, alkynylene and cycloalkylene groups include heteroalkylene, heteroalkenylene, heteroalkynylene and heterocycloalkylene groups, respectively.

18. The kit according to claim 17, wherein D1 is a covalent bond or -(CH2) 1-4 -.

19. The kit according to claim 17, wherein D1 includes at least one heteroatom.

20. The kit according to claim 17, wherein H1 is selected from alkynes and alkenes.

21. The kit according to claim 17, wherein H1 is selected from strained alkynes and dienes.

22. The kit according to claim 17, wherein H1 is a dienophile.

23. The kit according to claim 17, wherein H1 is selected from the group consisting of azide, dibenzocyclooctyne-amine, trans-cyclooctene and norbornene.

24. The kit according to claim 17, wherein D2 is -CH2-.

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