Site-specific antibody conjugation and antibody-drug conjugate as concrete example of site-specific antibody conjugation

Abstract

To provide a technique for specifically shifting a chemical functional group to a specific site of an antibody.SOLUTION: The application relates to a technique for achieving labelling of a specific site of an antibody with a specific number of chemical functional groups or cargo parts. An antibody product material having high uniformity can be provided according to the application. In addition, an antibody product material in which function of an antibody is not reduced, is also provided according to the application. Namely, an antibody product material in which avidity and half life period are not reduced, can be provided. The application provides a first technique and extremely significant technique for achieving labelling of a specific site of an antibody without a complicated process.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to the modification of certain amino acid residues of an antibody by a substance (e.g., a low molecular weight compound, a synthetic polymer, selectively labeling with biopolymers (i.e., peptides, carbohydrates, proteins, etc.); or The molecule to be delivered to a specific target cell or tissue (hereinafter referred to as "cargo") is attached to the antibody. Furthermore, the present invention relates to a technique for linking a specific portion of an antibody to a desired number of Labeling with a substance or linking a desired number of cargoes to specific amino acid residues of an antibody In addition, the present invention relates to a technique for using this method or antibody fragment complex. The present invention also encompasses antibody conjugates prepared by the method. [Background technology]

[0002] Antibodies are biomolecules that have the ability to recognize specific molecules and are used in a variety of industrial applications. For example, antibodies are used to detect or screen for certain substances, It is possible to check the route that a particular substance takes within the body or cells. Antibodies can be used for therapeutic purposes by inducing an immune response against a specific substance. This can be done.

[0003] Attempts have been made to improve the performance of such antibodies in order to extend their functionality. Typically, antibodies are labeled with exogenous substances to supplement or enhance antibody function. Typically, when an antibody is labeled with a fluorophore, the antibody is attached to a fluorescent antibody. The antibodies can be used in assays or as drugs to treat certain diseases. When labeled, the antibody can be used to maximize the therapeutic effect of the antibody. These approaches and techniques are generally referred to as "antibody labeling." The present invention provides a method for labeling antibodies. It concerns a new method.

[0004] Initially, antibody labeling was achieved by randomly attaching exogenous substances to antibodies. However, this method has many problems. The antibodies prepared in this way have poor homogeneity. These antibodies have the problem that they are different in the number of substances attached to each antibody, and The issue of unequal effects remains due to differences in the binding sites of antibodies. This issue is being addressed by antibody-drug conjugates (ADCs), which require high safety and reproducibility. This is a major barrier to the development of the technology.

[0005] Furthermore, this method has the problem that the ability of the antibody to recognize the target protein may be significantly reduced. The antibody is composed of the Fab domain, which contains the antigen-binding domain that recognizes the antigen, and the Fab domain, which is involved in antibody crystallization. By random labeling, foreign substances can be attached to the antigen-binding domain or the Fc domain of the antibody. By allowing binding to sites adjacent to the antigen-binding domain, the ability of the antibody to recognize This results in a very low force.

[0006] Therefore, there is a need in the related art for techniques to uniformly label antibodies in a site-specific manner. There is a need for such technologies. For example, several techniques have been developed to genetically alter or modify antibodies. However, most of these are not effective from the technical and economical aspects. The present invention is designed to solve the problem by incorporating a specific substance (or "portion") into an antibody. Specific binding to a specific site on an antibody without any additional modifications, and similar Using antibodies to deliver a specific substance (drug or labeled substance) to cells or tissues It concerns technologies that make this possible. [Prior art documents] [Patent documents]

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(2001), Molecular Cell, Vol. 7, pp. 867-877, April 2001 [Non-licensed Document 10] Edelman GM et al., The covalent structure of an entire gamma-G immunoglobulin molecule; Proc. Natl. Acad. Sci. USA., May 1969, 63(1): pages 78~85 [Non-licensed Document 11] 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), pages 2726~2732 [Non-licensed Document 12] DeLano, WL et al., Convergent solutions to binding at a protein-protein interface; Science 2000, 287, pages 1279~1283 [Non-licensed Document 13] Ying T, Ju TW, Wang Y, Prabakaran P, Dimitrov DS., Interactions of IgG1 CH2 and CH3 Domains with FcRn; Front Immunol. 2014; 5:146 pages [Non-licensed Document 14] 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. 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[0009] The present invention provides techniques for specifically transferring chemical functional groups to specific sites on antibodies. In one specific embodiment, the present invention provides a method for specifically transferring a chemical functional group to lysine 246 of an antibody. In another specific embodiment, the present invention provides techniques for attaching chemical functional groups to the antibody. Techniques are provided for specifically transferring lysine 248. In yet another specific embodiment, The present invention provides a technique for specifically transferring chemical functional groups to lysines 246 and 248 of antibodies. To provide.

[0010] The present invention provides techniques that allow for the attachment of any desired number of chemical functional groups to an antibody. In a specific embodiment, the present invention provides an antibody or fragment thereof that binds two specific sites. In another specific embodiment, the present invention provides an antibody having four specific sites bound thereto. Provide the body or a fragment thereof.

[0011] The present invention provides techniques for specifically attaching cargo moieties to specific sites on antibodies. In a specific embodiment, the present invention provides a cargo moiety specifically linked to lysine 246 of an antibody. In another specific embodiment, the present invention provides techniques for incorporating cargo moieties into antibodies. Techniques are provided for specifically binding to lysine 248. In yet another specific embodiment, The present invention provides a technique for specifically attaching cargo moieties to lysines 246 and 248 of an antibody. To provide.

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

[0013] The present invention provides methods of using the aforementioned antibodies or fragments thereof. The present invention provides methods for treating certain diseases using antibody-drug conjugates. [Means for solving the problem]

[0014] The present application relates to a compound of formula 2:

[0015] [ka]

[0016] where H1 is a first click-reactive functional group, D1 is a covalent bond, C 1~4 Alkylene, C 2~4 Alkenylene, C 2~4 Alkynlene, and C 3~8 Selected from cycloalkylene X1 is S and D2 is C 1~7 Alkylene, C 2~7 Alkenylene, C 2~7 Alkin N, and C 3~8 cycloalkynlene, X2 is O, and R2' is N-succinimido, p-nitrophenyl, or pentafluorophenyl) The present invention provides a compound of the formula:

[0017] Moreover, the present application provides that H1 is a terminal alkyne, an azide, a strained alkyne, a diene, a dienophenanthate, a hydroxybenzo ... The compound is selected from the group consisting of olefins, alkenes, thiols, and tetrazines. The present application provides a method for preparing a compound in which H1 is norbornene, tetrazine, azide, and dibenzocyclooctyne-azide. The present invention provides a compound selected from the group consisting of:

[0018] Additionally, the present application provides compounds wherein R2' is N-succinimide.

[0019] Additionally, the present application provides a compound according to the present invention, wherein Formula 2 is Formula 2-1, 2-2, or 2-3:

[0020] [ka]

[0021] The present invention provides a compound,

[0022] The present application relates to a compound of formula 4-2: [Formula 4-2] (Xaa) 1-3 -C-(Xaa)2-H-Xa1-G-Xa2-LV-Xa3-C-(Xaa) 1-3 wherein each Xaa is independently any amino acid residue that is not a cysteine ​​residue; and C is is a cysteine ​​residue, H is a histidine residue, G is a glycine residue, and Xa2 is , a glutamic acid residue or an asparagine residue, L is a leucine residue, and V is a valency residue. Xa3 is a tryptophan residue, a naphthylalanine residue, and a phenylalanine residue. Xa1 is selected from the group consisting of:

[0023] [ka]

[0024] (Wherein, D3 is a covalent bond or C 1~3 and X3 is NH2). It is a peptide consisting of 13 to 17 amino acid residues, and binds to human immunoglobulin G (IgG). and has a cysteine ​​residue located 2 to 4 amino acids from the N-terminus of Formula 4-2, and 4-2, a peptide in which cysteine ​​residues located 2 to 4 amino acids from the C-terminus are optionally linked Provide the

[0025] Moreover, the present application provides a peptide, wherein D3 is a covalent bond, methylene, or ethylene. do.

[0026] Additionally, the present application provides a compound according to the present invention, wherein Formula 4-2 is a compound according to Formula 4-6: [Formula 4-6] DCAWH-Xa1-GELVWCT (wherein D is an aspartic acid residue, A is an alanine residue, and E is glutamic acid) residue, W is a tryptophan residue, and T is a threonine residue). The present invention provides a peptide,

[0027] The present application relates to a compound of formula 6-2: [Formula 6-2] (Xaa) 1-3 -C-(Xaa)2-H-(Xa1)'-G-Xa2-LV-Xa3-C-(Xaa) 1-3 wherein each Xaa is independently any amino acid residue that is not a cysteine ​​residue; and C is is a cysteine ​​residue, H is a histidine residue, G is a glycine residue, and Xa2 is , a glutamic acid residue or an asparagine residue, L is a leucine residue, and V is a valency residue. Xa3 is a tryptophan residue, a naphthylalanine residue, and a phenylalanine residue. is selected from nin residues, (Xa1)' is

[0028] [ka]

[0029] where H1 is a first click-reactive functional group, D1 is a covalent bond, C 1~4 Alkylene, C 2~4 Alkenylene, C 2~4 Alkynylene, and C 3~8 cycloalkylenes, X1 is S, and D2 is C 1~7 Alkylene, C 2~7 Alkenylene, C 2~7 Alkynylene and C3 ~8 cycloalkylene; D3 is a covalent bond or C 1~3 alkylene, and X3 is , NH) is] The peptide-compound conjugate of claim 1, wherein the peptide is composed of 13 to 17 amino acid residues. It exhibits binding activity to human immunoglobulin G (IgG), and has 2 to 4 amino acids from the N-terminus of formula 6-2. cysteine ​​residues in the amino acids of formula 6-2 and cysteines in the 2 to 4 amino acids from the C-terminus of formula 6-2 The residues are optionally linked to provide peptide-compound conjugates.

[0030] Furthermore, the present application provides a method for preparing a compound in which the distance from the β carbon of (Xa1)' to the first carbonyl carbon of (Xa1)' is: A peptide-compound conjugate is provided that is less than approximately 11.668 Å.

[0031] In addition, the present application provides that D2 is y Alkylene, C y Alkenylene, or C y Alkynylene , D3, C x Peptide compounds in which x is alkylene, y is an integer of 1 or greater, and 1≦x+y≦5. A conjugate is provided.

[0032] In addition, the present application provides a method for preparing a compound in which the distance from the β carbon of (Xa1)' to the first carbonyl carbon of (Xa1)' is: A peptide-compound conjugate is provided that is greater than approximately 16.208 Å.

[0033] In addition, the present application provides that D2 is y Alkylene, C y Alkenylene, or C y Alkynylene , D3, C x alkylene, y is an integer of 1 or more, and 9≦x+y≦12, Further, the present application provides a compound conjugate, wherein D2 is C y Alkenylene, or C y Alkini The present invention provides a peptide-compound conjugate, wherein the peptide-compound conjugate is a phenylene.

[0034] In addition, the present application provides a method for preparing a compound in which the distance from the β carbon of (Xa1)' to the first carbonyl carbon of (Xa1)' is: The peptide-compound conjugates are provided having a cross-sectional area of ​​approximately 11.668 Å to approximately 16.208 Å.

[0035] In addition, the present application provides that D2 is y Alkylene, C y Alkenylene, or C y Alkynylene , D3, C x alkylene, y is an integer of 1 or greater, and 6≦x+y≦8, A conjugate is provided.

[0036] Additionally, the present application provides a compound according to the present invention, wherein Formula 6-2 is a compound according to Formula 6-3: [Formula 6-3] DCAWH-(Xa1)'-GELVWCT (wherein D is an aspartic acid residue, A is an alanine residue, and E is glutamic acid) residue, W is a tryptophan residue, and T is a threonine residue). The present invention provides a peptide-compound conjugate, wherein

[0037] Moreover, the present application relates to a peptide-compound wherein D1 is a covalent bond and D2 is methylene. A conjugate is provided.

[0038] The present application provides a method for preparing an agent for transferring a first click-reactive functional group to an antibody. 1. A method comprising:

[0039] [ka]

[0040] where H1 is a first click-reactive functional group, D1 is a covalent bond, C 1~4 Alkylene, C 2~4Alkenylene, C 2~4 Alkynylene, and C 3~8 cycloalkylenes, X1 is S, and D2 is C 1~7 Alkylene, C 2~7 Alkenylene, C 2~7 Alkynylene and C3 ~8 cycloalkylene, X2 is O, and R2' is N-succinimide, p-nitro (e.g., pentafluorophenyl, tetrafluorophenyl, or pentafluorophenyl) The compound of formula 4-2: [Formula 4-2] (Xaa) 1-3 -C-(Xaa)2-H-Xa1-G-Xa2-LV-Xa3-C-(Xaa) 1-3 wherein each Xaa is independently any amino acid residue that is not a cysteine ​​residue; and C is is a cysteine ​​residue, H is a histidine residue, G is a glycine residue, and Xa2 is , a glutamic acid residue or an asparagine residue, L is a leucine residue, and V is a valency residue. Xa3 is a tryptophan residue, a naphthylalanine residue, and a phenylalanine residue. is selected from nin residues, Xa1 is

[0041] [ka]

[0042] (Wherein, D3 is a covalent bond or C 1~3 and X3 is NH2). The peptide is composed of 13 to 17 amino acid residues and is isolated from human immunoglobulin G. It exhibits binding activity to IgG (IgG), and the cysteine ​​residues located 2 to 4 amino acids from the N-terminus of Formula 4-2 are and a cysteine ​​residue located 2 to 4 amino acids from the C-terminus of Formula 4-2, optionally linked together The method includes reacting the peptide with a soluble form of the ...

[0043] Moreover, the present application provides a method for preparing an agent for transferring a first click-reactive functional group to an antibody. A method for producing a fluorine-containing compound, comprising: y Alkylene, C y Alkenylene, or C y Alkynylene D3 is C x alkylene, y is an integer of 1 or more, and 1≦x+y≦5, The first click-reactive functional group is transferred to the antibody by a reagent that reacts with the antibody. , the first click-reactive functional group is delivered specifically to the 248 lysine residue of the Fc domain of the antibody. The present invention provides a method for producing a fluororesin comprising the steps of:

[0044] In addition, the present application provides a method for preparing an agent for transferring a first click-reactive functional group to an antibody. A method for producing a fluorine-containing compound, comprising: y Alkylene, C y Alkenylene, or C y Alkynylene D3 is C x alkylene, y is an integer of 1 or more, and 1≦x+y≦5, The first click-reactive functional group is transferred to the antibody by a reagent that reacts with the antibody. , the first click-reactive functional group is delivered specifically to the 248 lysine residue of the Fc domain of the antibody. The present invention provides a method for producing a fluororesin comprising the steps of:

[0045] In addition, the present application provides a method for preparing an agent for transferring a first click-reactive functional group to an antibody. A method for producing a fluorine-containing compound, comprising: y Alkylene, C y Alkenylene, or C y Alkynylene D3 is Cx alkylene, y is an integer of 1 or more, and 9≦x+y≦12, The first click-reactive functional group is transferred to the antibody by a reagent that reacts with the antibody. , a first click-reactive functional group is delivered specifically to the lysine residue 246 of the Fc domain of the antibody. The present invention provides a method for producing a fluororesin comprising the steps of:

[0046] In addition, the present application provides a method for preparing an agent for transferring a first click-reactive functional group to an antibody. A method for producing a fluorine-containing compound, comprising: y Alkylene, C y Alkenylene, or C y Alkynylene D3 is C x alkylene, y is an integer of 1 or more, and 6≦x+y≦8, The first click-reactive functional group is transferred to the antibody by a reagent that reacts with the antibody. The first click-reactive functional group is selectively attached to the 246th or 248th lysine residue of the Fc domain of the antibody. The present invention provides a method for delivering a medicament to a medicament residue.

[0047] This application relates to a kit for preparing a first click-reactive functional group to be transferred to an antibody. So, Equation 2:

[0048] [ka]

[0049] where H1 is a first click-reactive functional group, D1 is a covalent bond, C 1~4 Alkylene, C 2~4 Alkenylene, C 2~4 Alkynylene, and C 3~8 cycloalkylenes, X1 is S, and D2 is C 1~7Alkylene, C 2~7 Alkenylene, C 2~7 Alkynylene and C3 ~8 cycloalkylene, X2 is O, and R2' is N-succinimide, p-nitro (e.g., pentafluorophenyl, tetrafluorophenyl, or pentafluorophenyl) and compounds of formula 4-2: [Formula 4-2] (Xaa) 1-3 -C-(Xaa)2-H-Xa1-G-Xa2-LV-Xa3-C-(Xaa) 1-3 wherein each Xaa is independently any amino acid residue that is not a cysteine ​​residue; and C is is a cysteine ​​residue, H is a histidine residue, G is a glycine residue, and Xa2 is , a glutamic acid residue or an asparagine residue, L is a leucine residue, and V is a valency residue. Xa3 is a tryptophan residue, a naphthylalanine residue, and a phenylalanine residue. is selected from nin residues, Xa1 is

[0050] [ka]

[0051] (Wherein, D3 is a covalent bond or C 1~3 and X3 is NH2). The peptide is composed of 13 to 17 amino acid residues and is isolated from human immunoglobulin G. It exhibits binding activity to IgG (IgG), and the cysteine ​​residues located 2 to 4 amino acids from the N-terminus of Formula 4-2 are and a cysteine ​​residue located 2 to 4 amino acids from the C-terminus of Formula 4-2, optionally linked together The present invention provides a kit comprising a peptide comprising:

[0052] The present application relates to compounds of formula 8-1, formula 8-2, and formula 8-3: [Formula 8-1] GPSVFLFPP-(K)'-PKDTLMI [Formula 8-2] GPSVFLFPPKP-(K)'-DTLMI [Formula 8-3] GPSVFLFPP-(K)'-P-(K)'-DTLMI wherein G is a glycine residue, P is a proline residue, and S is a serine residue; V is a valine residue, F is a phenylalanine residue, and L is a leucine residue. K is a lysine residue, D is an aspartic acid residue, and T is a threonine residue. where M is a methionine residue and I is an isoleucine residue; (K)' is

[0053] [ka]

[0054] where H1 is a first click-reactive functional group, D1 is a covalent bond, C 1~4 Alkylene, C 2~4 Alkenylene, C 2~4 Alkynylene, and C 3~8 cycloalkylene) is] The present invention provides an antibody or fragment thereof comprising one or more amino acid sequences selected from:

[0055] Moreover, the present application provides an antibody or fragment thereof, wherein D1 is a covalent bond.

[0056] In addition, the present application includes the amino acid sequence of formula 8-1, and the amino acid sequences of formulas 8-2 and 8-3. Furthermore, the present application provides an antibody or fragment thereof having an amino acid sequence of Formula 8-1 in both of its two Fc domains. The present invention provides an antibody or fragment thereof comprising the amino acid sequence.

[0057] In addition, the present application includes the amino acid sequence of formula 8-2, and the amino acid sequences of formulas 8-1 and 8-3. Furthermore, the present application provides an antibody or fragment thereof having an amino acid sequence of formula 8-2 in both of its two Fc domains. The present invention provides an antibody or fragment thereof comprising the amino acid sequence.

[0058] In addition, the present application includes the amino acid sequence of formula 8-3, and the amino acid sequences of formulas 8-1 and 8-2. Furthermore, the present application provides an antibody or fragment thereof having an amino acid sequence of formula 8-3 in both of its two Fc domains. The present invention provides an antibody or fragment thereof comprising the amino acid sequence.

[0059] The present application relates to a method for preparing an antibody or fragment thereof comprising a first click-reactive functional group. and Equation 6-2: [Formula 6-2] (Xaa) 1-3 -C-(Xaa)2-H-(Xa1)'-G-Xa2-LV-Xa3-C-(Xaa) 1-3 wherein each Xaa is independently any amino acid residue that is not a cysteine ​​residue; and C is is a cysteine ​​residue, H is a histidine residue, G is a glycine residue, and Xa2 is , a glutamic acid residue or an asparagine residue, L is a leucine residue, and V is a valency residue. Xa3 is a tryptophan residue, a naphthylalanine residue, and a phenylalanine residue. is selected from nin residues, (Xa1)' is

[0060] [ka]

[0061] where H1 is a first click-reactive functional group, D1 is a covalent bond, C1~4 Alkylene, C 2~4 Alkenylene, C 2~4 Alkynylene, and C 3~8 cycloalkylenes, X1 is S, and D2 is C 1~7 Alkylene, C 2~7 Alkenylene, C 2~7 Alkynylene and C3 ~8 cycloalkylene; D3 is a covalent bond or C 1~3 alkylene, and X3 is , NH) is] The peptide-compound conjugate of claim 1, wherein the peptide is composed of 13 to 17 amino acid residues. It exhibits binding activity to human immunoglobulin G (IgG), and has 2 to 4 amino acids from the N-terminus of formula 6-2. cysteine ​​residues in the amino acids of formula 6-2 and cysteines in the 2 to 4 amino acids from the C-terminus of formula 6-2 The peptide-compound conjugate, in which the residues are optionally linked, is reacted with an antibody or a fragment thereof. The method includes the step of reacting the

[0062] The present application provides a kit for preparing an antibody or fragment thereof comprising a first click-reactive functional group. It is Equation 6-2: [Formula 6-2] (Xaa) 1-3 -C-(Xaa)2-H-(Xa1)'-G-Xa2-LV-Xa3-C-(Xaa) 1-3 wherein each Xaa is independently any amino acid residue that is not a cysteine ​​residue; and C is is a cysteine ​​residue, H is a histidine residue, G is a glycine residue, and Xa2 is , a glutamic acid residue or an asparagine residue, L is a leucine residue, and V is a valency residue. W is a tryptophan residue; (Xa1)' is

[0063] [ka]

[0064] where H1 is a first click-reactive functional group, D1 is a covalent bond, C 1~4 Alkylene, C 2~4 Alkenylene, C 2~4 Alkynylene, and C 3~8 cycloalkylenes, X1 is S, and D2 is C 1~7 Alkylene, C 2~7 Alkenylene, C 2~7 Alkynylene and C3 ~8 cycloalkylene; D3 is a covalent bond or C 1~3 alkylene, and X3 is , NH) is] The peptide-compound conjugate of claim 1, wherein the peptide is composed of 13 to 17 amino acid residues. It exhibits binding activity to human immunoglobulin G (IgG), and has 2 to 4 amino acids from the N-terminus of formula 6-2. cysteine ​​residues in the amino acids of formula 6-2 and cysteines in the 2 to 4 amino acids from the C-terminus of formula 6-2 and peptide-compound conjugates, wherein the residue is optionally linked; and antibodies or fragments thereof. Piece A kit comprising:

[0065] The present application provides a compound of formula 9: [Formula 9] C m -H2 (In the formula, C m is the cargo moiety and H2 is the second click-reactive functional group) The present invention provides a compound of the formula:

[0066] The present application provides a method for preparing an antibody-drug conjugate, comprising the steps of: and Equation 8-3: [Formula 8-1] GPSVFLFPP-(K)'-PKDTLMI [Formula 8-2] GPSVFLFPPKP-(K)'-DTLMI [Formula 8-3] GPSVFLFPP-(K)'-P-(K)'-DTLMI wherein G is a glycine residue, P is a proline residue, and S is a serine residue; V is a valine residue, F is a phenylalanine residue, and L is a leucine residue. K is a lysine residue, D is an aspartic acid residue, and T is a threonine residue. where M is a methionine residue and I is an isoleucine residue; (K)' is

[0067] [ka]

[0068] where H1 is a first click-reactive functional group, D1 is a covalent bond, C 1~4 Alkylene, C 2~4 Alkenylene, C 2~4 Alkynylene, and C 3~8 cycloalkylene) is] and (c) a fragment thereof comprising one or more amino acid sequences selected from the group consisting of: [Formula 9] C m -H2 (In the formula, C m is the cargo moiety, and H2 is the second click-reactive functional group that is complementary to the first. (click-reactive functional group of with a compound of formula (I).

[0069] The present application relates to compounds of formula 8-1, formula 8-2, and formula 8-3: [Formula 8-1] GPSVFLFPP-(K)'-PKDTLMI [Formula 8-2] GPSVFLFPPKP-(K)'-DTLMI [Formula 8-3] GPSVFLFPP-(K)'-P-(K)'-DTLMI wherein G is a glycine residue, P is a proline residue, and S is a serine residue; V is a valine residue, F is a phenylalanine residue, and L is a leucine residue. K is a lysine residue, D is an aspartic acid residue, and T is a threonine residue. where M is a methionine residue and I is an isoleucine residue; (K)' is

[0070] [ka]

[0071] where H1 is a first click-reactive functional group, D1 is a covalent bond, C 1~4 Alkylene, C 2~4 Alkenylene, C 2~4 Alkynylene, and C 3~8 cycloalkylene) is] an antibody or fragment thereof comprising one or more amino acid sequences selected from the group consisting of: [Formula 9] C m -H2 (In the formula, C m is the cargo moiety, and H2 is the second click-reactive functional group that is complementary to the first. (click-reactive functional group of The present invention provides a kit for preparing an antibody-drug conjugate comprising the compound of formula (I).

[0072] The present application relates to Formula 10-1, Formula 10-2, and Formula 10-3: [Formula 10-1] GPSVFLFPP-(K)"-PKDTLMI [Formula 10-2] GPSVFLFPPKP-(K)"-DTLMI [Formula 10-3] GPSVFLFPP-(K)"-P-(K)"-DTLMI wherein G is a glycine residue, P is a proline residue, and S is a serine residue; V is a valine residue, F is a phenylalanine residue, and L is a leucine residue. K is a lysine residue, D is an aspartic acid residue, and T is a threonine residue. where M is a methionine residue and I is an isoleucine residue; (K)" is

[0073] [ka]

[0074] (In the formula, C m is the cargo moiety and B is

[0075] [ka]

[0076] where A1 and A2 are attached to a cargo moiety or D1, and they are not both attached to the same thing. , R x is H, halogen, and C 1~3 alkyl) is selected from D1 is a covalent bond, C 1~4 Alkylene, C 2~4 Alkenylene, C 2~4Alkynylene, and C 3~8 cycloalkylene). The present invention provides an antibody or fragment thereof comprising one or more amino acid sequences selected from:

[0077] Moreover, the present application includes the amino acid sequence of formula 10-1, and the amino acid sequences of formulas 10-2 and 10-3. Furthermore, the present application provides an antibody or fragment thereof having the structure of Formula 10-1 in both of its two Fc domains. An antibody or fragment thereof comprising the amino acid sequence is provided.

[0078] In addition, the present application includes the amino acid sequence of formula 10-2, and the amino acid sequences of formulas 10-1 and 10-3. Furthermore, the present application provides an antibody or fragment thereof having the structure of Formula 10-2 in both of its Fc domains. An antibody or fragment thereof comprising the amino acid sequence is provided.

[0079] In addition, the present application includes the amino acid sequence of formula 10-3, and the amino acid sequences of formulas 10-1 and 10-2. Furthermore, the present application provides an antibody or fragment thereof having the structure of Formula 10-3 in both of its two Fc domains. An antibody or fragment thereof comprising the amino acid sequence is provided.

[0080] Additionally, the present application provides antibodies or fragments thereof wherein the cargo moiety comprises a drug moiety. The present application provides antibodies or fragments thereof in which the cargo moiety comprises two or more drug moieties. The present application further provides an antibody or fragment thereof, wherein the drug moiety is an anti-cancer drug. The present application relates to a method for treating cancer, comprising administering to a subject a cancer treatment agent selected from the group consisting of DM1, DM3, DM4, abrin, ricin A, Pseudomonas exotoxin, and comatinib. Relatin, diphtheria toxin, tumor necrosis factor, α-interferon, β-interferon , nerve growth factor, platelet-derived growth factor, tissue plasminogen activator, cytokines, Apoptotic agents, antiangiogenic agents, lymphokines, taxanes, DNA-alkoxyl Dehydrogenating agents, anthracyclines, tubulysin analogues, duocarmycin analogues, auris maytansinoids, auristatin E, auristatin F, and maytansinoids containing reactive polyethylene glycol moieties. Cytotoxic agents including taxon, cytochalasin B, gramicidin D, ethidium bromide, emetine , mitomycin, etoposide, tenoposide, vincristine, vinblastine, t.col Anthracin, doxorubicin, daunorubicin, dihydroxyanthracin methicone, mitoxantrone, mithramycin, actinomycin D, 1-dihydrotestosterone 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine in, propranolol, puromycin, methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine, mechlorethamine, thio Tepa, chlorambucil, meiphalan, carmustine, lomustine, cyclo Phosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin Syn C, cisplatin, dactinomycin, bleomycin, anthramycin, caliche Amicin, Gemcitabine, Bendamustine, Bortezomib, Carboplatin cetaxel, epirubicin, erlotinib, everonib, dasatinib, docetaxel, epirubicin, erlotinib, everonib Rimus, gemcitabine, gefitinib, idarubicin, imatinib, hydrochloride Roxiurea, lapatinib, leuprorelin, melphalan, nedaplatin, nilotinib , oxaliplatin, pazopanib, pemetrexed, picoplatin, romidepsin, sat Laplatin, sorafenib, vemurafenib, sunitinib, teniposide, triplatin, and vinorelbine.

[0081] The present application relates to a pharmaceutical composition for treating cancer, comprising the aforementioned antibody-drug conjugate. to provide.

[0082] Moreover, the present application relates to a pharmaceutical composition for treating cancer, wherein the cancer is bladder cancer, bone cancer, Cancer, brain tumor, breast cancer, heart cancer, cervical cancer, colorectal cancer, rectal cancer, esophageal cancer, Fibrosarcoma, stomach cancer, digestive 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 reproductive The present invention provides a pharmaceutical composition for treating thymic carcinoma, wherein the thymic carcinoma is selected from the group consisting of thymoma, thymus carcinoma, and thymic carcinoma.

[0083] In addition, the present application provides a pharmaceutical composition for treating cancer, wherein the cancer is breast cancer. , a pharmaceutical composition is provided.

[0084] The present application relates to a method for treating cancer, comprising the antibody-drug conjugate described above. The method includes administering to a subject a pharmaceutical composition comprising the compound.

[0085] Additionally, the present application provides a method for treating cancer, wherein the cancer is bladder cancer, bone cancer, Brain cancer, breast cancer, heart cancer, cervical cancer, colorectal cancer, rectal cancer, esophageal cancer, fibromuscular cancer tumor, stomach cancer, digestive 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, and testicular germ cell cancer The method further provides a method for treating a thymic carcinoma, wherein the thymic carcinoma is selected from the group consisting of thymoma, thymoma, and thymic carcinoma.

[0086] Furthermore, the present application relates to a method for treating cancer, wherein the cancer is breast cancer. provide. [Effects of the Invention]

[0087] The antibody product of the present invention has a specific number of chemical functional groups labeled at certain sites on the antibody product. Thus, the present invention provides a method for producing antibodies with high uniformity. Furthermore, the present invention provides a composition capable of producing antibodies without reducing the function of the antibodies. That is, the present invention can provide an antibody having a reduced binding affinity and half-life. The present invention can provide an antibody product without any complicated process. This is of great importance as it is the first technique that allows site-specific labeling of antibodies in vivo. [Brief explanation of the drawings]

[0088] [Figure 1] The partial sequence of the Fc domain is shown, with the sequence numbered according to the EU numbering system. [Figure 2] The positions of the lysine residues in the Fc domain, including lysines 246 and 248, are indicated. [Figure 3] The topology between the SSFI and Fc domains is shown. [Figure 4] The topology between the SSFI and Fc domains is shown. [Figure 5] Xa1 and lysines 246 and 248 of SSFI in the Fc domain are indicated. [Figure 6] The distance between the amine group of lysine 246 and the β carbon of Xa1 of SSFI is shown. [Figure 7] The distance between the amine group of lysine 248 and the β carbon of Xa1 of SSFI is shown. [Figure 8]The structure of R1'-L2-SSFI and the distance (Lc) between the β carbon of (Xa1)' and the first carbonyl carbon are shown. [Figure 9] The topology between R1'-L2-SSFI and the Fc domain is shown, with the side chain of (Xa1)' oriented parallel to the x-axis of the diagram (dotted arrow). [Figure 10] The conditions used are shown to allow the first carbonyl carbon to react fully with lysine 248. [Figure 11] The conditions used are shown to allow the first carbonyl carbon to react fully with lysine 246. [Figure 12] The conditions used are shown to allow the first carbonyl carbon to react selectively with lysine 246 or lysine 248. [Figure 13] The FcRn binding site and lysines 246 and 248 of the Fc domain are indicated. [Figure 14] 1 shows an analysis of the binding structure between SSFI and the Fc domain compared to the binding site between Fc and FcRn. [Figure 15] A method for synthesizing Compound I is shown below. [Figure 16] The structure of Compound I was confirmed by mass spectrometry. [Figure 17] A method for synthesizing compound II is shown below. [Figure 18] The structure of Compound II was confirmed by mass spectrometry. [Figure 19] A method for synthesizing compound III is shown below. [Figure 20] The structure of Compound III was confirmed by mass spectrometry. [Figure 21] A method for synthesizing compound IV is shown below. [Figure 22] A method for synthesizing compound IV is shown below. [Figure 23] The structure of Compound IV was confirmed by mass spectrometry. [Figure 24] The structure of SSFI(6Lys) was confirmed by mass spectrometry. [Figure 25] The structure of SSFI(6Orn) was confirmed by mass spectrometry. [Figure 26] The structure of SSFI (6Dab) was confirmed by mass spectrometry. [Figure 27] The structure of SSFI (6Dap) was confirmed by mass spectrometry. [Figure 28] The structure of DD2 was confirmed by mass spectrometry. [Figure 29] The structure of DD3 was confirmed by mass spectrometry. [Figure 30] The structure of DD4 was confirmed by mass spectrometry. [Figure 31] The structure of DD5 was confirmed by mass spectrometry. [Figure 32] The structure of DD6 was confirmed by mass spectrometry. [Figure 33] The structure of compound I-SSFI(6Lys) was confirmed by mass spectrometry. [Figure 34] The structure of compound II-SSFI(6Lys) was confirmed by mass spectrometry. [Figure 35] The structure of compound III-SSFI(6Lys) was confirmed by mass spectrometry. [Figure 36] The structure of compound III-SSFI(6Orn) was confirmed by mass spectrometry. [Figure 37] The structure of compound III-SSFI (6Dab) was confirmed by mass spectrometry. [Figure 38] The structure of compound III-SSFI (6Dap) was confirmed by mass spectrometry. [Figure 39] The structure of compound IV-SSFI (6Dap) was confirmed by mass spectrometry. [Figure 40] 1 shows the results of observing the binding reaction using trastuzumab and compound I-SSFI(6Lys) by HIC-HPLC. [Figure 41]1 shows the results of observing the binding reaction using trastuzumab and Compound II-SSFI(6Lys) by HIC-HPLC. [Figure 42] 1 shows the reaction of antibodies with Compound III-SSFI (6Dap, Dab, Orn, or Lys). [Figure 43] The structure of the final product, Ab(246 / 248)-norbornene, is shown. [Figure 44] 1 shows the results of observing the binding reaction using trastuzumab and Compound III-SSFI (6Dap) by HIC-HPLC. [Figure 45] 1 shows the results of observing the binding reaction using trastuzumab and Compound III-SSFI (6 Dabs) by HIC-HPLC. [Figure 46] 1 shows the results of observing the binding reaction using trastuzumab and Compound III-SSFI(6Orn) by HIC-HPLC. [Figure 47] 1 shows the results of observing the binding reaction using trastuzumab and compound III-SSFI(6Lys) by HIC-HPLC. [Figure 48] 1 shows the results of observing the binding reaction using trastuzumab and Compound IV-SSFI (6Dap) by HIC-HPLC. [Figure 49] The increase in molecular weight spectrum due to antibody-norbornene binding is shown. [Figure 50] The increase in molecular weight spectrum due to antibody-norbornene binding is shown. [Figure 51] 1 shows the MS / MS chromatogram results of trastuzumab and an antibody-norbornene conjugate. [Figure 52] 1 shows the MS / MS chromatogram results of trastuzumab and an antibody-norbornene conjugate. [Figure 53] 1 shows the MS / MS chromatogram results of trastuzumab and an antibody-norbornene conjugate. [Figure 54] 1 shows the MS / MS chromatogram results of trastuzumab and an antibody-norbornene conjugate. [Figure 55]The results of sequence matching based on MS / MS spectra are shown. [Figure 56] 1 shows the mass spectrum of trastuzumab taken to confirm the trastuzumab-azide structure. [Figure 57] 1 shows the mass spectrum of the trastuzumab-azide conjugate measured to confirm the trastuzumab-azide structure. [Figure 58] The structure of tetrazine-DM1 was confirmed by mass spectrometry. [Figure 59] 1 shows the structure of a trastuzumab-DM1 conjugate based on compound III-SSFI(6Dap). [Figure 60] 1 shows the results of observing the formation reaction of a trastuzumab-DM1 conjugate by HIC-HPLC. [Figure 61] 1 shows the increase in molecular weight spectrum due to norbornene-tetrazine-DM1 binding. [Figure 62] 1 shows the increase in molecular weight spectrum due to norbornene-tetrazine-DM1 binding. [Figure 63] 1 shows the structures of three designs of payload attached to a norbornene-labeled antibody. [Figure 64] 1 shows the results of analyzing the antigen-binding affinity of antibody-drug conjugates. [Figure 65] 1 shows the results of analyzing the serum stability of antibody-drug conjugates. [Figure 66] 1 shows the results of analyzing the serum stability of antibody-drug conjugates. [Figure 67] 1 shows the results of analyzing the serum stability of antibody-drug conjugates. [Figure 68] 1 shows the results of evaluating the efficacy of antibody-drug conjugates at the cellular level. [Figure 69] 1 shows the results of evaluating the efficacy of antibody-drug conjugates at the cellular level. [Figure 70] 1 shows the results of evaluating the efficacy of antibody-drug conjugates at the cellular level. [Figure 71]1 shows the results of evaluating the efficacy of antibody-drug conjugates at the animal level. [Figure 72] 1 shows the results of evaluating the efficacy of antibody-drug conjugates at the animal level. [Figure 73] 1 shows the results of a pharmacokinetic study of antibody-drug conjugates. DETAILED DESCRIPTION OF THE INVENTION

[0089] definition Unless otherwise defined, all technical and scientific terms used herein are intended to be understood as meaning the invention as defined herein. The following citations have the same meaning as commonly understood by one of ordinary skill in the art: is provided to provide those skilled in the art with general definitions of some of the terms used in this invention. See: Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd ed., 1994) ; The Cambridge Dictionary of Science and Technology (ed. Walker, 1988); The Glo Syllabus of Genetics, 5th ed., R. Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, The Harper Collins Dictionary of Biology (1991). Not otherwise specifically identified Insofar as used herein, the following terms have the meanings ascribed to them as follows:

[0090] In some embodiments, chemical structures are disclosed along with the corresponding chemical names. If there are contradictions or conflicts between the chemical structures, the chemical structure is used to understand the meaning of the compound. takes precedence over chemical names.

[0091] As used herein, the term "hetero" refers to a compound containing at least one heteroatom. or a group of compounds. The term "heteroatom" refers to an atom other than a carbon or hydrogen atom; Examples include B, Si, N, P, O, S, and Se. Preferably, heteroatoms are, among others, N, O and S, or monovalent elements such as F, Cl, Br, and I, but in the present invention are not limited to these.

[0092] The term "lower" as used herein modifies hydrocarbons, such as alkylenes. When used in the above example, it means that the corresponding hydrocarbon has 6 or fewer carbon atoms. For example, C 1~6 The straight or branched chain alkyl groups refer to alternative names such as "lower alkyl" groups.

[0093] As used herein, the term "oxy" refers to a secondary radical of an oxygen atom (-O-).

[0094] The term "alkyl" or "alkane" refers to a straight or branched chain non-aromatic alkyl group that is fully saturated. Unless otherwise defined, straight or branched chain alkyl groups typically include: It has 1 to about 20 carbon atoms, preferably 1 to about 10 carbon atoms. Chain alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, and sec-butyl. , tert-butyl, pentyl, hexyl, pentyl, and octyl.

[0095] The term "alkenyl" or "alkene" refers to a straight-chain alkyl group containing at least one double bond. or branched chain non-aromatic hydrocarbons. Unless otherwise defined, straight or branched chain alkenyl The group typically has from 1 to about 20 carbon atoms, preferably from 1 to about 10 carbon atoms. do.

[0096] The term "alkynyl" or "alkyne" refers to a straight-chain or alkynyl group having at least one triple bond. refers to a branched chain non-aromatic hydrocarbon. Unless otherwise defined, straight or branched chain alkynyl groups Typically, the alkyl group has 1 to about 20 carbon atoms, preferably 1 to about 10 carbon atoms. do.

[0097] The term "cycloalkane" or "cycloalkyl" refers to a cyclic group that is fully saturated. "Cycloalkyl" refers to a hydrocarbon. "Cycloalkyl" includes monocyclic and polycyclic rings. Unless otherwise defined, Monocyclic cycloalkyl groups typically contain from 3 to about 10 carbon atoms, more typically from 3 to 8 carbon atoms. The rings other than the first ring of a polycyclic cycloalkyl may be saturated, unsaturated, and The cycloalkyl may be selected from 1, 2, or 3 aromatic rings shared between two rings. includes bicyclic molecules containing three or more atoms. The term "fused cycloalkyl" refers to a polycyclic cycloalkyl in which one ring shares two adjacent atoms with another ring. The rings other than the first ring of the fused polycyclic cycloalkyl are selected from saturated, unsaturated, and aromatic rings. It can be done.

[0098] The term "cycloalkyne" or "cycloalkynyl" refers to a cycloalkynyl group containing at least one triple bond. It refers to a cyclic hydrocarbon containing a cycloalkynyl group, also known as a "strained alkyne." Unless otherwise defined, a monocyclic cycloalkynyl is generally a cycloalkynyl group. Polycyclic cycloaliphatic rings have from 3 to about 10 carbon atoms, more commonly from 3 to 8 carbon atoms. The rings other than the first ring of the alkynyl may be selected from saturated, unsaturated, and aromatic rings. Alkynyl contains 1, 2, 3, or more atoms shared between two rings. The term "fused cycloalkynyl" refers to a bicyclic molecule in which one ring is bonded to another ring. Polycyclic cycloalkynyls that share adjacent atoms. The rings other than the first ring may be selected from saturated, unsaturated, and aromatic rings.

[0099] The term "alkylene" used as a molecule by itself or as part of another molecule "Alkane" refers to a divalent radical derived from an alkane. For example, the radicals are -CH2CH2- and -CH2CH2CH2CH2- all of which contain 10 or fewer carbon atoms, but the present invention is not limited to these. The term "lower alkylene" refers to a shorter alkylene, generally having six or fewer carbon atoms. Unless otherwise specified, the term "alkylene" refers to the "heteroalkylene" group in the present invention. " is intended to encompass groups represented by the formula ".

[0100] The term "heteroalkylene" refers to a divalent group derived from heteroalkyl, e.g., -CH2-C H2-S-CH2-CH2- and -CH2-S-CH2-CH2-NH-CH2-, but the present invention is not limited to these. Heteroalkylene groups may have the same or different heteroatoms at each or all of the chain ends. may contain alkylene groups (such as alkyleneoxy, alkylenedioxy, alkyleneamino, alkylene (The present invention is not limited to these.) Furthermore, the designation of the connections at both ends of the chain is independent of the placement of the groups in the formula. For example, the formula -C(O)2R'- and refers to both -C(O)2R'- and -R'(O)2C-.

[0101] The term "alkenyl" used as a molecule by itself or as part of another molecule "Alkene" refers to a divalent radical derived from an alkene. For example, the radicals are -CH=CH-, -CH2CH=CHCH2-, and and -CH=CH-CH=CH-, all of which contain 10 or fewer carbon atoms, but for purposes of the present invention: Unless otherwise specified, the term "alkenylene" refers to any heterocyclic group in the present invention. It is intended to include alkylenes.

[0102] The term "alkynyl" used as a molecule by itself or as part of another molecule "Alkyne" refers to a divalent radical derived from an alkyne. For example, groups include -C≡C-, -CHC≡CCH-, and -C≡CC≡C-, all of which contain 10 or fewer carbon atoms, Unless otherwise stated, the term "alkynylene" refers to any heteroaryl group in the present invention. It is intended to include alkylene.

[0103] The term "cycloaliphatic" used as a molecule by itself or as part of another molecule "Alkylene" refers to a divalent group derived from a cycloalkene. The term "alkylene" is intended to encompass heterocycloalkylene in the present invention.

[0104] The term "alkylene" as used in the specification, examples, and claims means "unsubstituted The term "alkylene" is intended to encompass both "alkylene" and "substituted alkylene." The latter is an alkyl group having substituents replacing hydrogen atoms on one or more carbon atoms of a hydrocarbon. Unless specifically specified otherwise, substituents may include, for example, halogen, hydrochloride, and the like. an alkoxyl group, a carbonyl group (e.g., carboxyl, alkoxycarbonyl, formyl, or acyl), thiocarbonyl groups (e.g., thioesters, thioacetates, or thioformates), phosphate), alkoxy group, phosphoryl group, phosphate group, phosphonate group, phosphine Aromatic groups, amino groups, amide groups, amidine groups, imine groups, cyano groups, nitro groups, azide groups, alkylhydryl group, alkylthio group, sulfate group, sulfonate group, sulfamoyl group , sulfonamide group, sulfonyl group, heterocyclyl group, aralkyl group, or aromatic or When appropriately substituted, the hydrocarbon chain may contain a substituent group such as a substituted or heteroaromatic group. It will be understood by those skilled in the art that the substituents of the substituted alkylene may themselves be substituted. are substituted and unsubstituted amino, azido, imino, amide, phosphoryl (phosphonate and phosphinates), sulfonyl (sulfates, sulfonamides, sulfamoyl, and sulfonates), and silyl groups, and may include ether, alkylthio, Carbonyls (including ketones, aldehydes, carboxylates, and esters), -CF3, -CN and equivalents thereof. Exemplary substituted alkyls are described below. Cycloalkyl The alkyl group includes alkyl, alkenyl, alkoxy, alkylthio, aminoalkyl, carbonyl, and the like. The invention can be further substituted with alkyl, -CF3, -CN, and the like. This also applies equally to alkenylene and alkynylene.

[0105] When used with a residue such as alkylene, alkenylene, or alkynylene, The word “C” x~y " is intended to encompass, for example, residues containing x to y carbon atoms in the chain. For example, the term "C x~y"Alkylene" means a group containing x to y carbon atoms in its chain. , substituted or unsubstituted, straight or branched chain alkylene groups. For example, difluoromethylene Typically, this is meant to include haloalkylene groups such as 2,2,2-trifluoroethylene and the like. C0 alkylene refers to a covalent bond. 2~y alkenylene" and "C 2~y Archi "Nylene" refers to a substituted or unsubstituted unsaturated aliphatic residue, with definitions of length and possible substitution. , applies as described in the definition of alkylene. However, this also applies if each is at least one This means that the compound contains two double or triple bonds.

[0106] The term "click chemistry" refers to the rapid and stable synthesis of two molecules using complementary chemical functional groups. Describes chemical reactions designed to form covalent bonds between molecules. Scripps Research It is used as a chemical concept introduced by K. Barry Sharpless of the Arch Institute. Click chemistry does not refer to a specific reaction, but rather to the general idea of ​​such a rapid and stable reaction. In any embodiment, click chemistry is modular and wide-ranging. It is a method that gives high yields, produces few by-products, is stereospecific, and is physiologically stable. constant, driven by a thermodynamic driving force (e.g., greater than 84 kJ / mol) and / or with high atomic energy Several reactions are known to meet the requirements. R: (1) Huisgen 1,3-dipolar cycloaddition (e.g., Cu(I)-catalyzed cycloaddition, usually referred to as "click") "Reaction," Tornoe et al., Journal of Organic Chemistry (2002) 67: 3057-3064 (see ): Copper and ruthenium are commonly used as catalysts; [Scheme of Huisgen 1,3-dipolar cycloaddition]

[0107] [ka]

[0108] (2) Diels-Alder reactions, such as normal electron demand Diels-Alder reactions, and and inverse electron demand Diels-Alder reactions, but the present invention is not limited to these. , cycloaddition (e.g., strain-promoted cycloaddition (SPAAC)); [Schematic diagram of the Diels-Alder reaction]

[0109] [ka]

[0110] [Example of Diels-Alder reaction: TCO and tetrazine]

[0111] [ka]

[0112] [Schematic diagram of strain-promoted cycloaddition]

[0113] [ka]

[0114] [Examples of strain-promoted cycloaddition: azide and DBCO]

[0115] [ka]

[0116] (3) nucleophilic addition to small strained rings such as epoxides and aziridines; (4) nucleophilic addition to activated carbonyl groups; (5) Addition to a carbon-carbon double or triple bond. [Addition of thiols and alkenes]

[0117] [ka]

[0118] The term "click-reactive functional group" as used herein refers to a functional group that participates in a click chemistry reaction. For example, strained alkynes (e.g., cyclooctyne) are Click-reactive functional groups. Generally, click chemistry requires at least two molecules, Each contains a click-reactive functional group that is complementary to the other. In many cases, a pair of click-reactive functional groups having the same functional groups is referred to as a "partner click reaction" in the present invention. In the strain-promoted cycloaddition of cyclooctyne with azide, for example, The cyclooctyne is a partner click-reactive functional group for cyclooctyne and other alkynes. Exemplary click-reactive functional groups for use in the present invention include terminal alkynes, azides, strained Alkynes, dienes, dienophiles, trans-cyclooctene, alkenes, thiols, and Other click-reactive functional groups include, but are not limited to, tetrazines. Functional groups are known to those skilled in the art.

[0119] The term "leaving group" as used in the present invention has the same concept as that well known to those skilled in the art (Advan ced Organic Chemistry: reactions, mechanisms and structure-Jerry March, John Will (Eds. W. Beck, J. M., and Sons, 4th ed.; 1992, pp. 351-357), refers to a chemical functional group that bonds with any reactant. This is because the reactants undergo a substitution reaction, e.g. For example, a good leaving group is one that migrates easily during a nucleophilic substitution reaction. Exemplary good leaving groups are halogens (F, Cl, Br, and I), trimethylsilyl groups (Trimethylsilyl), and methyl groups (F, Cl, Br, and I). Sylate, mesylate, triflate, acetate, trifluoromethyl acetate, Benzosulfonate, 2-thioxobenzo[d]thiazol-3(2H)-yl, N-hydrosuccinic acid Imides, N-aryloxides, and aryl groups substituted with one or more electron-withdrawing groups (EWGs) Examples of suitable amines include, but are not limited to, amines and amines of the present invention. Aryloxides substituted with several electron-withdrawing groups (EWG) include 2-nitrophenoxide, 4-nitrophenoxide, 2,4-dinitrophenoxide, pentafluorophenoxide, 2-chloro-4 -nitrophenoxide, 2,4-dichlorophenoxide, and 2,4,6-chlorophenoxide The electron-withdrawing group may be, for example, a halogen (F, Cl, Br, or I), —NO2, —CN, —C(O)(C 1~6 Al -C(O)(aryl), -C(O)O(C 1~6 -C(O)O(aryl), etc.

[0120] The term "interactome" as used herein refers to the interactions between proteins or peptides. Protein-protein interactions in the presence of protein-protein interactions (PPIs) For example, chaperone proteins and their pathways The interactome of proteins is a protein-protein interaction. The term "action" refers to the interaction of two or more proteins or peptide molecules with high specificity. In this case, the causative interaction is electrical These include, but are not limited to, magnetic forces, hydrogen bonds, and hydrophobic interactions. stomach.

[0121] As used herein, the term "antibody interactome" refers to the interaction of antibodies, including immunoglobulins. Exemplary antibody interactomes are listed in Table 1. Among these, peptides such as Fc-III bind to the Fc domain of immunoglobulins. In this case, the peptides are also part of the "Fc interactome." It is called.

[0122] [Table 1-1]

[0123] [Table 1-2]

[0124] According to the present invention, the term "antibody" refers to an immunoglobulin molecule or a fragment thereof. Globulins are generally well known and have the ability to specifically bind to a particular antigen. However, since the antibody according to the present invention is a concept that also encompasses its fragments, the antibody may be an Fc fragment. It is not necessary for the antibody to exhibit the ability to bind to a specific antigen, as in the case of naturally occurring immunoglobulins. In addition to antibodies, antibodies are also available for all recombinant proteins, fusion proteins, chimeric proteins, and the like. , human immunoglobulins, non-human animal immunoglobulins, etc., which are have the same or similar structure.

[0125] According to the present invention, the term "conjugate" refers to a conjugate in which the conjugate partners are covalently linked together. Covalent bonds are preferably formed by cleaving or cleaving molecules. The term "conjugate partner" refers to a conjugate that can be formed by a conjugate chemical reaction. The term "conjugate" refers to each of the molecules intended to form the conjugate. The person intending to carry out the experiment intends to bind a particular molecule to any other molecule. When illustrating a target molecule, that particular molecule is generally referred to as a "target molecule" or "target protein." Any other molecule may be generally referred to for convenience as a "cargo molecule" or "cargo moiety." It is possible.

[0126] According to the present invention, the term "carrier moiety" refers to the molecule that constitutes the conjugate, i.e. Molecules that function to improve the serum stability of the molecule to which they are linked or to extend the half-life of the molecule. Molecules that can be used as carrier moieties are well known in the relevant art. Representative examples of carrier moieties include albumin, gelatin, elastin (tropoelastin), and elastin-derived polypeptides (including α-elastin and elastin-like polypeptides) ELP), gliadin, legumin, zein, soy protein (e.g., soy protein Protein isolate (SPI), milk protein, whey protein, bilirubin, etc. The present invention is not limited to these.

[0127] According to the present invention, the term "fluorescent moiety" refers to a dye or dye reagent used for fluorescence. Molecules that can be used as dyes or dye reagents are intended to encompass related molecules. Representative examples of fluorescent moieties are listed in Table 2, and are well known in the art. Examples of suitable antibodies include, but are not limited to, those listed in the Immunotech-Coulter Corp. catalog, "Cytometry Monoclonal Antibodies" and "Cytometry Monoclonal Antibodies" (Cytometry Monoclonal Antibodies). nal Reagent Guide”, 8 / 95, p. 3).

[0128] [Table 2]

[0129] According to the present invention, the term "drug moiety" refers to a molecule that has a therapeutic effect against any disease. The drug moieties according to the present invention are known to those skilled in the art as being effective against any disease. Typically, the drug moieties with anti-cancer activity include DM1, DM3, DM4, Ab Phosphorus, ricin A, Pseudomonas exotoxin, cholera toxin, diphtheria toxin, tumor necrosis factor, α-interferon, β-interferon, nerve growth factor, platelet-derived growth factor, tissue Plasminogen activators, cytokines, apoptosis inducers, antiangiogenic agents, lympho Caine, taxane, DNA-alkylating agent, anthracycline, tubulysin analogue, duo Carmycin analogues, auristatin E, auristatin F, maytansinoids, reactive poly(amycin) Cytotoxic agents containing polyethylene glycol residues, such as taxon, cytochalasin B, and gramicidin. D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine vinblastine, colchicine, doxorubicin, daunorubicin, dihydrochloride Cyanthracindione, mitoxantrone, mithramycin, actinomycin D, 1-di Hydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, Propranolol, puromycin, methotrexate, 6-mercaptopurine, 6-thio Guanine, cytarabine, 5-fluorouracil decarbazine, mechlorethamine, thiotepa, Chlorambucil, melphalan, carmustine, lomustine, cyclophosphamide, busulfan Irfan, dibromomannitol, streptozotocin, mitomycin C, cisplatin dactinomycin, bleomycin, anthramycin, calicheamicin, abirate Ron, bendamustine, bortezomib, carboplatin, cabazitaxel, dasatinib, Docetaxel, epirubicin, erlotinib, everolimus, gemcitabine , gefitinib, idarubicin, imatinib, hydroxyurea, lapatinib, leuproreductase Relin, melphalan, nedaplatin, nilotinib, oxaliplatin, pazopanib, Methotrexed, picoplatin, romidepsin, satraplatin, sorafenib, Bemurafenib These include benzodiazepine, sunitinib, teniposide, triplatin, and vinorelbine, but the present invention , but not limited to these.

[0130] According to the present invention, the term "radioactive moiety" refers to a moiety that contains a radioisotope. Labeling of radioactive moieties is useful in diagnostic imaging and radiotherapy. 18 F, 11 C. 125 I, 123 I, 124 I, 131 I, and 99m Tc, but in the present invention, Not determined.

[0131] The term "pharmaceutically acceptable carrier" is meant to include an additive, excipient, or adjuvant. Carriers that can be used include, for example, lactose, dextrose, sucrose, Rubitol, mannitol, xylitol, erythritol, maltitol, starch, Acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose cellulose, methylcellulose, polyvinylpyrrolidone, water, saline, buffers, e.g., PB S, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, stearyl The carrier may be selected from the group consisting of a filler, magnesium phosphate, and mineral oil. anti-agglomerating agents, lubricants, wetting agents, flavoring agents, emulsifiers, preservatives, or combinations thereof. can.

[0132] The term "pharmaceutically acceptable salts" refers to salts of proteomes and biologically active compounds according to the present invention. It refers to salts that preserve the effects and properties and are not biologically or otherwise undesirable. In many cases, the proteome and the compounds according to the invention contain charged groups, e.g., charged amino groups. and / or in the presence of carbonyl groups, etc., acid and / or base salts can be formed. Pharmaceutically acceptable acid addition salts can be prepared from inorganic and organic acids, and are pharmaceutically acceptable. Such base addition salts can be prepared from inorganic and organic bases.

[0133] The term "treatment" refers to an approach for obtaining a beneficial or desired clinical outcome. For purposes of this disclosure, non-limiting examples of beneficial or desirable clinical outcomes include symptomatic relief, reduction in the extent of disease, and a decrease in the level of the disease, a stabilization of the disease state (i.e., no worsening), a delay in the progression of the disease or a decrease in the rate of progression, Improvement (partial or total) or temporal remission and alleviation of the disease state, and whether it is detectable Treatment refers to all therapeutic treatments as well as prophylactic or preventative measures. Treatment includes disorders to be prevented as well as treatments required for disorders already occurring. "To understand" means to improve the extent and / or severity of a disease state compared to when the disease is not treated. Undesirable clinical signs are reduced and / or the time course of disease progression is slowed or means that it is stretched.

[0134] A "therapeutically effective amount" (or "effective amount") is an amount that, when administered to a subject or patient, achieves treatment. Therefore, the amount of the active ingredient, e.g., the drug according to the present invention, sufficient to achieve the therapeutic effect of the present invention. What constitutes an effective amount of a composition can be readily determined by one of ordinary skill in the art. In this context, a "therapeutically effective amount" refers to an amount that is effective to treat one or more parameters associated with the treatment of an ocular disease or condition. The amount that produces an objectively measurable change in the eye that is associated with an ocular disease or condition. Increase or decrease in expression of one or more genes involved in the induction of apoptosis or other cell death pathways, The therapeutic effect includes clinical improvement of symptoms, reduction of abnormal angiogenesis or inflammation, etc. The amount will depend on the particular subject and condition being treated, the subject's weight and age, the severity of the disease state, the type of Depending on the particular compound selected, the subsequent administration schedule, the timing of administration, the mode of administration, etc. These may vary depending on the type of protein, all of which can be readily determined by one skilled in the art. In the context of combination therapy, a therapeutically effective amount of a particular active ingredient is used in combination with a monotherapy ( a therapeutically effective amount administered in a treatment regimen using one chemical entity as the active ingredient It is to be understood that the active ingredient may differ from what is comprised.

[0135] A "subject" or "patient" refers to an animal in need of treatment that can be achieved by a molecule of the invention. The animals to be treated according to the present invention include vertebrates. Particularly preferred examples of animals include: , mammals, such as bovine, canine, equine, feline, ovine, porcine, and primate animals (humans and (including non-human primates).

[0136] The term "about" or "approximately" refers to a reference amount, level, value, number, frequency, percentage, 30, 25, 20, 25, 10, 9, 8, 7, 6 for dimension, size, quantity, mass, or length , 5, 4, 3, 2, or 1% change in amount, level, value, number, frequency, percentage, size, size It refers to the size, quantity, mass, or length.

[0137] 1. Antibodies The description of the present invention will help to understand the structure of antibodies, academic systems, and It should be understood that the intent is to provide a description of biological activity and is not intended to be limiting of the scope of the invention. The antibodies in scope are not intended to limit the description of the present invention.

[0138] Antibodies are comprised of two heavy chains and two light chains as known in the art. When classified in terms of their basic aspects, antibodies can be divided into fragments containing light chains, antigen-binding variable regions (Fab), and portions of heavy chains. The Fab fragment contains the paratope that binds to the antigen. The domains that enable the antibody to have specific binding activity for the antigen known in the art are The Fc domain is a ligand for intracellular Fc receptors (FcRs), and therefore plays a key role in immune responses. Furthermore, the Fc domain plays an important role in inducing a response to antibodies. It is important for extending the half-life of antibodies by binding to the neonatal Fc receptor in a manner that enhances their activity. Take on a role.

[0139] From these facts, it is possible to deduce several desirable directions for labeling antibodies. (1) First, it is desirable to label the antibody at a position distant from the antibody paratope. When labeling is performed at the paratope or adjacent to the paratope, the antigen (2) Second, the binding affinity of the antibody to the FcR, including the FcRn, may be significantly reduced. It is desirable to label the antibody at a position spaced from the receptor recognition site or If performed adjacent to the recognition site, the antibody's ability to induce an immune response may be reduced. Alternatively, the half-life of the antibody may be shortened. Information on the binding activity motif of the Fc domain is available from DeLano et al. , WL (2000): Convergent Solutions to Binding at a Protein-Protein Interface; Science, 287(5456), pp. 1279-1283, and W. Lance Martin et al. (2001), Molecular Cell, 7, pp. 867-877, April 2001.

[0140] In the present invention, when referring to the amino acid sequence of the Fc domain of an antibody, the sequence number is used as indicated otherwise. Unless otherwise stated, they are numbered according to the EU numbering system. The system has been widely used as a sequencing system for the Fc domain after examining the IgG sequence. Edelman GM et al., The covalent structure of an entire gamma-G immunoglobulin m olecule; as described in Proc. Natl. Acad. Sci. USA., May 1969, 63(1): 78-85 is.

[0141] 1.1. Search for desirable labeling sites The labeling site of the antibody is the labeling site, i.e., (1) a site distant from the paratope; and (2) the FcR. The design can be carried out taking into consideration the criteria for the site distant from the FcR recognition site, including n. Examples of amino acids used in the conjugation reaction are typically lysine, cis- tyrosine, and lysine 246 (Lys) present in the Fc domain of antibodies. 246 ) and and lysine 248 (Lys 248 ) are residues that satisfy all the requirements, so both are desirable labeling moieties. Lys 246 and Lys 248 The sequence of the Fc domain containing the residues is GPSVFLFPPKPKDTLMI. , sequences, and the numbers of the sequences numbered according to the EU numbering system are shown in Figure 1 ( Figure 1, SEQ ID NO:1).

[0142] In a specific embodiment, the antibody according to the invention comprises the sequence of SEQ ID NO: 1, or a derivative thereof. Furthermore, the antibody according to the present invention may contain a derivative of SEQ ID NO: 1 in which lysine 246 is substituted. In addition, the antibody according to the present invention may comprise a derivative of SEQ ID NO: 1 in which lysine 248 is substituted. Furthermore, the antibody according to the invention may comprise SEQ ID NO: 1, in which lysines 246 and 248 are substituted. It may include derivatives of:

[0143] The sequence of SEQ ID NO: 1 or its derivatives includes sequences that have been mutated within the permissible range. In one embodiment, the mutated sequence is approximately 9 to 10 times the sequence of SEQ ID NO: 1, or a derivative thereof. The homology may be 0%, about 85%, about 80%, about 75%, or about 70% or more. In specific embodiments described below, the compounds represented by formulas 7-1 to 7-3, 8-1 to 8-3, and 10-1 to 10-3 are It should be understood that derivatives of SEQ ID NO: 1 also include sequences that are mutated to the extent permitted. be.

[0144] FIG. 2 shows the locations of the lysine residues in the Fc domain, including lysines 246 and 248.

[0145] 2. Linker (R1'-L1) The present invention discloses novel compounds that can be used to label antibodies. Such compounds are referred to herein as linkers and are represented by the symbol "R1'-L1."

[0146] The present invention relates to a compound of formula 1:

[0147] [ka]

[0148] wherein R is a first chemical functionality; D1 is any alkylene, alkenylene, or alkynylene; X1 is an element that is more electronegative than carbon, D2 is any alkylene, alkenylene, or alkynylene; X2 is an element that is more electronegative than carbon, R2' is a second chemical functional group The present invention provides a compound having the structure:

[0149] In Formula 1, the carbonyl group connected to D1 refers to the first carbonyl group. The carbonyl group refers to a second carbonyl group.

[0150] In specific embodiments, R1' can comprise a click-reactive functional group. Click-reactive functional groups include alkynes, azides, strained alkynes, dienes, dienophiles, It may comprise one or more selected from alkenes, thiols, and tetrazines. The carboxylic reactive functional group can be selected from an azide or a strained alkyne. The click-reactive functional group can be selected from azide or dibenzocyclooctyne-amine. In addition, the click-reactive functional group can be selected from dienes or dienophiles. Furthermore, the click-reactive functional group can be selected from tetrazine or norbornene. Alternatively, the click-reactive functional group can be a tetrazine or a trans-cyclopentadiene. In addition, R1' may contain two or more click-reactive functional groups. It can be done.

[0151] In other specific embodiments, R1' is a carrier moiety, a fluorescent moiety, a drug moiety, or a radioactive moiety. Furthermore, R1' may include a drug moiety. In addition, R1' may include a VC linker. In another specific embodiment, R1' may comprise an antibody or analog thereof comprising a paratope. do.

[0152] In specific embodiments, D1 is a covalent bond, C 1~4 Alkylene, C 2~4 Alkenylene, C 2~ 4 alkynylene, and C 3~8 cycloalkylene. In the above, D1 can be -CH2OCH2-. Additionally, D1 can be a covalent bond. In this case, R1' and the carbon of the first carbonyl group are directly connected to each other. When describing the structure of a compound, all alkylenes, alkenylenes, alkynylenes, and cycloalkylenes are The alkylene includes heteroalkylene, heteroalkenylene, and heteroalkynylene, respectively. and heterocycloalkylene, the contents of which are also described in the "Definitions" section. will be done.

[0153] In specific embodiments, X1 can be NR1, S, or O, and R1 can be H, halogen, or substituted. or unsubstituted C 1~3 Additionally, X1 can be S. X1 can be a first It can attract electrons from the carbonyl carbon, activating the first carbonyl group. do.

[0154] In specific embodiments, D2 is C 1~7 Alkylene, C 2~7 Alkenylene, C 2~7 Alkini Ren, and C. 3~8 Furthermore, D2 may include any one selected from the group consisting of: C 1~2 It can be alkylene. Furthermore, D2 can be methylene.

[0155] In specific embodiments, X2 can be NR1, S, or O, and R1 is H, halogen, or substituted. or unsubstituted C 1~3 Additionally, X2 can be O. X2 can be a second alkylene. It can attract electrons from the carbonyl carbon, activating a second carbonyl group. do.

[0156] In specific embodiments, R2' is halogen, N-succinimide, p-nitrophenyl, or can be pentafluorophenyl.

[0157] In specific embodiments, R2' and X2 together can form a leaving group, for example , X2 can be O, and R2' can be N-succinimido, p-nitrophenyl, or pentafluoro. Additionally, R2' can be N-succinimide. A good leaving group is When connecting with a second carbonyl group, the reactivity of the second carbonyl group can be increased. For example, N-hydroxysuccinimide esters (NHS esters) are highly reactive. It is known to show

[0158] The present invention relates to a compound of the following formula 1-2:

[0159] [ka]

[0160] wherein R is a first chemical functionality; D1 is any alkylene, alkenylene, or alkynylene; R2' is a second chemical functional group The present invention provides a compound having the structure:

[0161] In specific embodiments, R1' can comprise a click-reactive functional group. Click-reactive functional groups include alkynes, azides, strained alkynes, dienes, dienophiles, It may comprise one or more selected from alkenes, thiols, and tetrazines. The carboxylic reactive functional group can be selected from an azide or a strained alkyne. The click-reactive functional group can be selected from azide or dibenzocyclooctyne-amine. In addition, the click-reactive functional group can be selected from dienes or dienophiles. Furthermore, the click-reactive functional group can be selected from tetrazine or norbornene. Alternatively, the click-reactive functional group can be a tetrazine or a trans-cyclopentadiene. In addition, R1' may contain two or more click-reactive functional groups. It can be done.

[0162] In other specific embodiments, R1' is a carrier moiety, a fluorescent moiety, a drug moiety, or a radioactive moiety. Furthermore, R1' may include a drug moiety. In addition, R1' may include a VC linker. In another specific embodiment, R1' may comprise an antibody or analog thereof comprising a paratope. do.

[0163] In specific embodiments, D1 is C 1~7 Alkylene, C 2~7 Alkenylene, C 2~7 Alkini Ren, and C. 3~8 Furthermore, D1 may include any one selected from the group consisting of: C 1~2 It can be alkylene. Furthermore, D1 can be methylene.

[0164] In specific embodiments, R2' and O together can form a leaving group, in which case: R2' can be N-succinimido, p-nitrophenyl, or pentafluorophenyl Additionally, R2' can be N-succinimide.

[0165] In a specific embodiment, the compound of formula 1-2 has the following formula 1-3:

[0166] [ka]

[0167] It may have the structure:

[0168] 2.1. Linker (H1-L1) Containing a First Click-Reactive Functional Group The present invention relates to a compound of formula 2:

[0169] [ka]

[0170] wherein H1 is a first click-reactive functional group; D1 is any alkylene, alkenylene, or alkynylene; X1 is an element that is more electronegative than carbon, D2 is any alkylene, alkenylene, or alkynylene; X2 is an element that is more electronegative than carbon, R2' is a second chemical functional group In the present invention, a linker having the structure of Formula 2 is referred to as a "first The linker is referred to as a "linker containing a click-reactive functional group" and is represented by the symbol "H1-L1."

[0171] In Formula 2, the carbonyl group connected to D1 refers to the first carbonyl group. The carbonyl group refers to a second carbonyl group.

[0172] In specific embodiments, H1 is an alkyne, an azide, a strained alkyne, a diene, a dienophenoxy group, or a hydroxy group. The compound may include any one selected from the group consisting of olefins, alkenes, thiols, and tetrazines. , H1 can be an azide or a strained alkyne. Additionally, H1 can be a diene or a dienophile. Furthermore, H1 can be tetrazine or norbornene. Alternatively, H1 can be tetrazine, Or it can be trans-cyclooctene.

[0173] In specific embodiments, D1 is a covalent bond, C 1~4 Alkylene, C 2~4 Alkenylene, C 2~ 4 alkynylene, and C 3~8 cycloalkylene. where D1 can be -CH2OCH2-. Additionally, D1 can be a covalent bond.

[0174] In specific embodiments, X1 can be NR1, S, or O, and R1 can be H, halogen, or substituted. or unsubstituted C 1~3 Additionally, X1 can be S.

[0175] In specific embodiments, D2 is C 1~7 Alkylene, C 2~7 Alkenylene, C 2~7 Alkini Ren, and C. 3~8 Furthermore, D2 may include any one selected from the group consisting of: C 1~2 It can be alkylene. Furthermore, D2 can be methylene.

[0176] In specific embodiments, X2 can be NR1, S, or O, and R1 is H, halogen, or substituted. or unsubstituted C 1~3 Additionally, X2 can be O.

[0177] In specific embodiments, R2' is halogen, N-succinimide, p-nitrophenyl, or can be pentafluorophenyl.

[0178] In specific embodiments, R2' and X2 together can form a leaving group, for example , X2 can be O, and R2' can be N-succinimido, p-nitrophenyl, or pentafluoro. Additionally, R2' can be N-succinimide.

[0179] In a specific embodiment, the compound represented by formula 2 is represented by the following formulas 2-1 to 2-3:

[0180] [ka]

[0181] It may have any one structure selected from:

[0182] 2.2. The position at which the substitution reaction occurs depends on the reactivity between the first and second carbonyl groups. It can be specifically determined by the difference When linkers of formula 1 and 2, and sub-examples thereof, are designed, the sites for substitution reactions are: X1, X2, and R2' can be specified based on the design.

[0183] The linkers disclosed in the present invention are those which are formed between activated first and / or second carbonyl groups. This substitution reaction functions to transfer R1' to the target molecule. is shown schematically in Scheme 1 below.

[0184] [ka]

[0185] In Scheme 1, the target molecule is conveniently designated "Nu:". Nu: acts as a nucleophile and is a non- Since it has a shared electron pair, the first carbonyl group and / or the second carbonyl group and the nucleophilic acyl group In a nucleophilic acyl substitution reaction, a bond is formed with the linker. The reactivity of the carbonyl group can be determined by the basicity of the leaving group. The reactivity of the aryl group can be carboxylate, amide, carboxylic acid, ester, thioester, It is known that the concentration of NHS esters increases in the order of acyl phosphate and hydroxyl phosphate. Such carbonyl groups are highly reactive because they form very stable leaving groups. It is known to have

[0186] In specific embodiments, X1 and X2 can be elements that are more electronegative than carbon. where X1 and X2 can be NR1, S, or O, and R1 is H, halogen, or substituted or unsubstituted. C 1~3 X1 and X2, together with the residue to which they are attached, form a leaving group. When this occurs, the carbonyl group can be activated.

[0187] In this case, activation of the carbonyl group may optionally be performed by Nu: i) preferentially activating the first carbonyl group with or ii) preferentially react with the second carbonyl group. The tendency depends on the difference in reactivity between the first and second carbonyl groups. For example, when the basicity of the leaving group containing X1 is lower than the basicity of the leaving group containing X2, In this case, the first carbonyl group may react first. In another embodiment, the salt of the leaving group containing X2 If the basicity of the leaving group containing X is lower than that of the second carbonyl group, the second carbonyl group will react first. do.

[0188] In a preferred embodiment, the reactivity of the second carbonyl group is preferably In the present invention, the selective reaction is By allowing X2-R2' to connect to the second carbonyl group to form a good leaving group, This is achieved by converting the first carbonyl group into a mildly reactive amide, thioester, or ester. For example, in the case of the linker of formula 1-3, the second carbonyl group is , which may be an NHS ester and react faster than the first carbonyl group (thioester). can be done.

[0189] The prior art disclosed in application numbers US 2018 / 0141976 A1 and WO 2018 / 199337 A1 is based on the first chemical Although this is similar to the present invention in terms of the form of the agent for transferring the chemical functional group to the antibody (see Section 4 below), (See section 1.) The crosslinker differs from the present invention in that it contains two NHS esters. The aryl group has the same reactivity and allows for the transfer of chemical functional groups to prepare the desired agent in high yield. Furthermore, the crosslinker reacts with two SSFIs due to the high reactivity of the NHS ester. According to the present invention, such a problem can be solved by using a mildly reactive thioester. This problem has been solved by designing a first carbonyl group that is a sterol.

[0190] 3. Site-specific antibody interactome (SSAI) According to the present invention, a novel method for attaching a molecule to be labeled to a specific site of an antibody is provided. Disclosed are peptides. For convenience, such peptides are referred to herein as site-specific antibodies. This is called the interactome and is represented by the symbol "SSAI."

[0191] The SSAIs provided by the present invention may have binding activity to a specific site of an antibody.

[0192] In a specific embodiment, the SSAI may have binding activity to the Fab domain of an antibody. In this case, the SSAI preferably has binding activity for a site that is distant from the paratope of the antibody. may have.

[0193] In a specific embodiment, the SSAI may have binding activity to the Fc domain of an antibody. In this case, the SSAI preferably affects the FcRn-binding site of the antibody or a polypeptide that does not affect the FcRn-binding site of the antibody. The antibody may have binding activity for a site distant from the target antibody residue.

[0194] 3.1. Site-specific Fc interactome (SSFI) In the SSAI, a peptide having specific binding activity to an Fc domain is herein defined as: This is referred to as the "site-specific Fc interactome" and is designated by the symbol "SSFI."

[0195] In a specific embodiment, the SSFI has the following formula 3: [Formula 3] (Xaa)2-H-Xa1-G-Xa2-LV-Xa3 (SEQ ID NO: 3) wherein each Xaa is independently any amino acid other than cysteine; H is histidine, G is glycine, and Xa2 is glutamic acid or asparagine L is leucine, V is valine, and Xa3 is tryptophan, naphthyl alanine, and phenylalanine; Xa1 is

[0196] [ka]

[0197] (Wherein, D3 is a covalent bond or C 1~3 alkylene, and X3 is NH2, OH, or SH. [ru] The amino acid sequence may be represented by the following formula: esign: From Phage Display to Synthetic Protein Epitope Mimetics in Human Antibod y Fc-Binding Peptidomimetics; Journal of the American Chemical Society, 128(8), pp. 2726-2732; and DeLano, WL et al., Convergent solutions to binding at a protein- protein interface; Science 2000, 287, 1279-1283, regarding the Fc domain As an analog of the sequence AWHLGELVW (SEQ ID NO: 2), a sequence found to have binding activity: This motif has binding activity to the Fc domain. It has the following features: 1) First, it identifies the important residues that have Fc binding activity. The motif also involves changing the fourth leucine of SEQ ID NO:2 to Xa1, which has a free electron pair. (X) is designed to allow nucleophilic substitution reaction with the linker. n Is there n X's? (X) n-m means that it consists of n or more and m or less Xs. The carbon connected to is called the "beta carbon (β carbon)."

[0198] Furthermore, the amino acid sequence of Formula 3 is represented by the following Formula 3-1: [Formula 3-1] AWH-Xa1-G-Xa2-LV-Xa3 (SEQ ID NO: 4) [Wherein A is alanine, H is histidine, G is glycine, and Xa2 is glutamic acid or asparagine, L is leucine, V is valine, and Xa3 is selected from tryptophan, naphthylalanine, and phenylalanine; Xa1 is

[0199] [ka]

[0200] (Wherein, D3 is a covalent bond or C 1~3 alkylene, and X3 is NH2, OH, or SH. [ru] In formulas 3 and 3-1, X3 can be NH2. Alternatively, Xa2 can be Alternatively, Xa3 can be tryptophan.

[0201] Peptides containing each of the amino acid sequences of Formula 3 and its subexamples are those in which the internal residues are connected to each other. It is known that peptides have better binding activity when in the form of a cyclic peptide that binds to the hydroxylase. The present invention provides a cyclic peptide comprising the amino acid sequence of formula 3.

[0202] The present invention relates to a compound of the following formula 4-1: [Formula 4-1] L P-(Xaa)2-H-Xa1-G-Xa2-LV-Xa3- D P (SEQ ID NO: 5) [In the formula, N-terminus L P and D P forms a D-proline-L-proline template, each Xaa is independently any amino acid other than cysteine; and H is histidine; G is glycine, Xa2 is glutamic acid or asparagine, and L is leucine. V is valine, and Xa3 is tryptophan, naphthylalanine, and phenylalanine. Selected from Lanin, Xa1 is

[0203] [ka]

[0204] (Wherein, D3 is a covalent bond or C 1~3 alkylene, and X3 is NH2, OH, or SH; be] The present invention provides a cyclic peptide having the structure:

[0205] In specific embodiments, X3 can be NH2. In specific embodiments, (X)2 is AW. In a specific embodiment, Xa2 can be glutamic acid. In the above, Xa3 can be tryptophan.

[0206] The present invention relates to a compound represented by the following formula 4-2: [Formula 4-2] (Xaa) 1-3 -C-(Xaa)2-H-Xa1-G-Xa2-LV-Xa3-C-(Xaa) 1-3 (SEQ ID NO: 6) wherein each Xaa is independently any amino acid other than cysteine; C is cysteine, H is histidine, G is glycine, and Xa2 is glutamate. L is leucine, V is valine, and Xa3 is thiamin or asparagine. is selected from tryptophan, naphthylalanine, and phenylalanine; Xa1 is

[0207] [ka]

[0208] (Wherein, D3 is a covalent bond or C 1~3 alkylene, and X3 is NH2, OH, or SH. [ru] The present invention provides a cyclic peptide having the structure:

[0209] In specific embodiments, the peptide may consist of at least 13 and at most 17 amino acid residues.

[0210] In a specific embodiment, a cysteine ​​located 2 to 4 amino acids from the N-terminus and a C-terminus The cysteines located 2 to 4 amino acids from the amino acid residue can be optionally connected to each other.

[0211] In specific embodiments, X3 can be NH2. In specific embodiments, (X)2 is AW. In a specific embodiment, Xa2 can be glutamic acid. In the above, Xa3 can be tryptophan.

[0212] In a specific embodiment, the N-terminus (X) 1-3 One of the residues constituting the C-terminus (X) 1-3 Configure One of the residues can be bonded to each other. In a specific embodiment, the peptide of formula 4-2 is expressed as follows: [Formula 4-3] L PDC-(Xaa)2-H-Xa1-G-Xa2-LV-Xa3-CT- D P (SEQ ID NO: 7) (In the formula, N-terminus L P and D P forms a D-proline-L-proline template) It may have the structure:

[0213] In another specific embodiment, the peptide of formula 4-2 has the following formula 4-4: [Formula 4-4] CDC-(Xaa)2-H-Xa1-G-Xa2-LV-Xa3-CTC (SEQ ID NO: 8) wherein the N-terminal cysteine ​​and the C-terminal cysteine ​​can be connected to each other. It may have the structure:

[0214] The present invention relates to a compound of the following formula 4-5: [Formula 4-5] DC-(Xaa)2-H-Xa1-G-Xa2-LV-Xa3-CT (SEQ ID NO: 9) wherein D is aspartic acid and T is threonine; each Xaa is independently any amino acid other than cysteine; C is cysteine, H is histidine, G is glycine, and Xa2 is glutamate. L is leucine, V is valine, and Xa3 is thiamin or asparagine. is selected from tryptophan, naphthylalanine, and phenylalanine; Xa1 is

[0215] [ka]

[0216] (Wherein, D3 is a covalent bond or C 1~3 alkylene, and X3 is NH2, OH, or SH. [ru] The present invention provides a cyclic peptide having the structure:

[0217] In specific embodiments, the peptide may consist of at least 13 and at most 17 amino acid residues.

[0218] In a specific embodiment, a cysteine ​​is located two amino acids from the N-terminus and one amino acid from the C-terminus. The cysteines located two amino acids from the first one can optionally be connected to each other. In certain embodiments, X3 can be NH2. In certain embodiments, (X)2 can be AW. In certain embodiments, Xa2 can be glutamic acid. It may be putophan.

[0219] In a specific embodiment, formula 4-5 is represented by the following formula 4-6: [Formula 4-6] (SEQ ID NO: 10) DCAWH-Xa1-GELVWCT (SEQ ID NO: 10) (wherein A is alanine and E is glutamic acid) may be identical to

[0220] The peptides having the structures of formulas 4-1 to 4-6 may have binding activity to antibodies. The peptide may have binding activity to immunoglobulin G (IgG). The antibody may have binding activity to the Fc domain of the antibody.

[0221] In a specific embodiment, the N-terminus of the SSFI according to the present invention may be succinylated. In one embodiment, the SSFI according to the present invention has at its N-terminus (X) 1-3 and may contain polar amino acid residues In another specific embodiment, the SSFI according to the present invention has a C-terminal (X) 1-3 And polar amino In this case, the polar amino acid residues may include acidic and basic amino acids. Moreover, the polar amino acid residue may include glutamic acid or aspartic acid.

[0222] 3.2. The site-specific Fc interactome according to the present invention is a specific topology of the Fc of an antibody. It can be co-located with the domain. In this context, compounds of formulas 4-6 are provided as examples to aid in understanding the invention. However, the scope of the present invention is not limited thereto. The following description also applies to Formulas 3, 3-1, and 4-1 to 4-6. It should be noted that the present invention applies to compounds of formulas 4-6 only, and compounds of formulas 4-6 are provided as examples for convenience. I want to be done that.

[0223] As described above, the SSFI according to the present invention has binding activity to the Fc domain of an antibody. In this case, SSFI is connected to the Fc domain in a specific topology by interactions between amino acid residues. Representative interactions between the SSFI sequence and the Fc domain according to the present invention include: (1) salt binding of SSFI with histidine 433 of the Fc domain, (2) hydrogen binding of SSFI with asparagine 434 (3) salt-bonding of SSFI with glutamic acid 380, and (4) salt-bonding of SSFI with arginine 255. These interactions and the specific topologies thus formed are well known in the art. This can be determined from existing research in the field (DeLano, WL et al., Convergen t solutions to binding at a protein-protein interface, Science 2000, 287, 1279 ~See page 1283).

[0224] When the SSFI according to the present invention is designed, it is necessary to form a stable topology with the Fc domain. This is important for SSFI, as it allows the transfer of the first chemical functional group to the antibody according to the present invention. The agent and the labeling process using it are the binding sites between the SSFI and Fc domains found in the study. (See Sections 5.2, 5.3, and 5.4 below.) The interaction between SSFI and the Fc domain was destabilized during the design of these SSFIs. If the intermolecular topology of the molecules is disturbed, the interaction may have a negative effect on the labeling process. Therefore, it is not desirable.

[0225] One embodiment of the design principle is described in the following exemplary compounds. SSFI represented by Formula 4-6 is , the following structure: [Formula 4-6] DCAWH-Xa1-GELVWCT (SEQ ID NO: 10) It has.

[0226] The topology between SSFI and the Fc domain shown in SEQ ID NO: 10 was simulated based on the data in the paper. The results are shown in Figures 3 and 4 (DeLano, WL et al., Convergent solutions to binary ding at a protein-protein interface; see Science 2000, 287, 1279-1283). In this case, the histidine residue at position 5 of SSFI forms a salt bond with glutamic acid 380 of the Fc domain. We show that this salt formation has a significant effect on the topology between the SSFI and Fc domains. (See the dotted line in Figure 4.) Therefore, the histidine residues and their positions were varied during the design of SSFI. It is desirable not to convert it into a hydroxy group (see "H" next to residue Xa1 in formulas 3, 3-1, and 4-1 to 4-6). Since glutamic acid 8 is an electronegative residue, it is salt-formed with arginine 255 of the Fc domain. It was confirmed that SSFI formed a bond with the Fc domain, which indicates electropositivity. (See the dotted line in Figure 4.) Therefore, the corresponding residues are Preferably, it is an acidic amino acid that can correspond to glutamic acid and replaces asparagine. (See residue Xa2 in Formula 3, Formula 3-1, and Formulas 4-1 to 4-6.) When the amino acid residue is replaced or substituted with any functional group, it can be used to improve intermolecular interactions. It has an effect on the topology between the SSFI and Fc domains. do.

[0227] In addition, the glycine at position 7 of the sequence of SEQ ID NO: 10 is required to form the bent structure of SSFI. Therefore, the glycine residue and its position were varied during the design of SSFI. (See "G" between residues Xa1 and Xa2 in formulas 3, 3-1, and 4-1 to 4-6.) ).

[0228] Figure 5 shows the topology between the lysine residues of the Fc domain and SSFI. The lysine residues of the Fc domain closest to residue Xa1 are lysines 246 and 248. This can be seen (Figure 5).

[0229] The distance between the amine group of lysine 246 and the β carbon of Xa1 was measured. Because the bonds that make up the chain rotate, the minimum distance is approximately 11.668 Å (hereafter referred to as "D 246,最小 (D2 46,min The maximum distance is measured to be approximately 20.765 Å (hereinafter referred to as "D 246,最大 (D 246,max ) was measured (Figure 6).

[0230] The distance between the amine group of lysine 248 and the β-carbon of Xa1 was measured. Because the bonds that make up the chain rotate, the minimum distance is approximately 6.723 Å (hereafter referred to as "D 248,最小 (D 24 8,min The maximum distance is measured to be approximately 16.208 Å (hereinafter referred to as "D248,最大 ( D 248,max ) was measured (Figure 7).

[0231] As described in Section 5.3 below, the distance relationship is determined by the linker and the SSFI, and the first chemical agent. This can be an important consideration in the design of an agent for transferring functional groups to an antibody.

[0232] 4. Agents for transferring a first chemical functional group to an antibody; conjugates of R1'-L1 and SSAI (R1 '-L2-SSAI) According to the present invention, an agent for transferring a first chemical functional group to an antibody is disclosed. Such compounds are designated herein by the symbol "R1'-L2-SSAI." Compounds also include those Depending on the structure, it may also be referred to as a conjugate of R1'-L1 and SSAI (R1'-L2-SSAI conjugate). will be done.

[0233] The present invention relates to a compound of formula 5:

[0234] [ka]

[0235] wherein R is a first chemical functionality; D1 is any alkylene, alkenylene, or alkynylene; X1 is an element that is more electronegative than carbon, D2 is any alkylene, alkenylene, or alkynylene; D3 is a covalent bond or C 1~3 is alkylene, X3 is NH, O, or S; SSAI is site-specific antibody interactome) The present invention provides an R1'-L2-SSAI having the structure:

[0236] In specific embodiments, R1' can comprise a click-reactive functional group. Click-reactive functional groups include alkynes, azides, strained alkynes, dienes, dienophiles, It may comprise one or more selected from alkenes, thiols, and tetrazines. The carboxylic reactive functional group can be selected from an azide or a strained alkyne. The click-reactive functional group can be selected from azide or dibenzocyclooctyne-amine. In addition, the click-reactive functional group can be selected from dienes or dienophiles. Furthermore, the click-reactive functional group can be selected from tetrazine or norbornene. Alternatively, the click-reactive functional group can be a tetrazine or a trans-cyclopentadiene. In addition, R1' may contain two or more click-reactive functional groups. It can be done.

[0237] In other specific embodiments, R1' is a carrier moiety, a fluorescent moiety, a drug moiety, or a radioactive moiety. Furthermore, R1' may include a drug moiety. In addition, R1' may include a VC linker. In another specific embodiment, R1' may comprise an antibody or analog thereof comprising a paratope. do.

[0238] In specific embodiments, D1 is a covalent bond, C 1~4 Alkylene, C 2~4 Alkenylene, C 2~ 4 alkynylene, and C 3~8 cycloalkylene. where D1 can be -CH2OCH2-. Additionally, D1 can be a covalent bond.

[0239] In specific embodiments, X1 can be NR1, S, or O, and R1 can be H, halogen, or substituted. or unsubstituted C 1~3 Additionally, X1 can be S.

[0240] In specific embodiments, D2 is C 1~7 Alkylene, C 2~7 Alkenylene, C 2~7 Alkini Ren, and C. 3~8 Furthermore, D2 may include any one selected from the group consisting of: C 1~2 It can be alkylene. Furthermore, D2 can be methylene.

[0241] In specific embodiments, X3 can be NH.

[0242] In a specific embodiment, the SSAI can be a peptide sequence that has binding activity for a Fab. In another specific embodiment, the SSAI is a peptide having binding activity to an Fc domain. It can be an array.

[0243] When SSAI in Formula 5 is SSFI, it is represented by the symbol "R1'-L2-SSFI". The R1'-L2-SSFI is a nucleophilic substitution reaction with the second carbonyl group of R1'-L1 of Xa1 of the SSFI of the present invention. (See Section 4-2 and Scheme 2 below.) Therefore, the R1'-L2 -SSFI includes SSFIs of formula 3, formula 3-1, and formulas 4-1 to 4-6 in which Xa1 is replaced with (Xa1)'. However, the present invention is not limited to the following specific embodiments.

[0244] The present invention relates to a compound of the following formula 5-1: [Formula 5-1] (Xaa)2-H-(Xa1)'-G-Xa2-LV-Xa3 (SEQ ID NO: 11) wherein each Xaa is independently any amino acid other than cysteine; H is histidine, G is glycine, and Xa2 is glutamic acid or asparagine L is leucine, V is valine, and Xa3 is tryptophan, naphthyl alanine, and phenylalanine; (Xa1)' is

[0245] [ka]

[0246] wherein R is a first chemical functionality; D1 is any alkylene, alkenylene, or alkynylene; X1 is an element that is more electronegative than carbon, D2 is any alkylene, alkenylene, or alkynylene; D3 is a covalent bond or C 1~3 is alkylene, X3 is NH, O, or S). The present invention provides an R1'-L2-SSFI comprising the amino acid sequence:

[0247] In specific embodiments, R1' can comprise a click-reactive functional group. Click-reactive functional groups include alkynes, azides, strained alkynes, dienes, dienophiles, It may comprise one or more selected from alkenes, thiols, and tetrazines. The carboxylic reactive functional group can be selected from an azide or a strained alkyne. The click-reactive functional group can be selected from azide or dibenzocyclooctyne-amine. In addition, the click-reactive functional group can be selected from dienes or dienophiles. Furthermore, the click-reactive functional group can be selected from tetrazine or norbornene. Alternatively, the click-reactive functional group can be a tetrazine or a trans-cyclopentadiene. In addition, R1' may contain two or more click-reactive functional groups. It can be done.

[0248] In other specific embodiments, R1' can include a carrier moiety, a fluorescent moiety, or a drug moiety. Additionally, R1' may include a VC linker. Additionally, R1' may include a radioactive moiety. In another specific embodiment, R1' is an antibody containing a paratope. The compound may comprise a hydroxybenzoate or an analog thereof.

[0249] In specific embodiments, D1 is a covalent bond, C 1~4 Alkylene, C 2~4 Alkenylene, C 2~ 4 alkynylene, and C 3~8 cycloalkylene. where D1 can be -CH2OCH2-. Additionally, D1 can be a covalent bond.

[0250] In specific embodiments, X1 can be NR1, S, or O, and R1 can be H, halogen, or substituted. or unsubstituted C 1~3 It can be alkylene.

[0251] In specific embodiments, D2 is C 1~7 Alkylene, C 2~7 Alkenylene, C 2~7 Alkini Ren, and C. 3~8 Furthermore, D2 may include any one selected from the group consisting of: C 1~2 It can be alkylene. Furthermore, D2 can be methylene.

[0252] In specific embodiments, X3 can be NH.

[0253] In specific embodiments, (X)2 can be AW. In specific embodiments, Xa2 can be a group. In a specific embodiment, Xa3 can be tryptophan.

[0254] The present invention relates to a compound represented by the following formula 5-2: [Formula 5-2] (Xaa) 1-3 -C-(Xaa)2-H-(Xa1)'-G-Xa2-LV-Xa3-C-(Xaa) 1-3 (SEQ ID NO: 12) wherein each Xaa is independently any amino acid other than cysteine; C is cysteine, H is histidine, G is glycine, and Xa2 is glutamate. L is leucine, V is valine, and Xa3 is thiamin or asparagine. is selected from tryptophan, naphthylalanine, and phenylalanine; (Xa1)' is

[0255] [ka]

[0256] wherein R is a first chemical functionality; D1 is any alkylene, alkenylene, or alkynylene; X1 is an element that is more electronegative than carbon, D2 is any alkylene, alkenylene, or alkynylene; D3 is a covalent bond or C 1~3 is alkylene, X3 is NH, O, or S). Provided is an R1'-L2-SSFI having the structure:

[0257] In specific embodiments, R1' can comprise a click-reactive functional group. Click-reactive functional groups include alkynes, azides, strained alkynes, dienes, dienophiles, It may comprise one or more selected from alkenes, thiols, and tetrazines. The carboxylic reactive functional group can be selected from an azide or a strained alkyne. The click-reactive functional group can be selected from azide or dibenzocyclooctyne-amine. In addition, the click-reactive functional group can be selected from dienes or dienophiles. Furthermore, the click-reactive functional group can be selected from tetrazine or norbornene. Alternatively, the click-reactive functional group can be a tetrazine or a trans-cyclopentadiene. In addition, R1' may contain two or more click-reactive functional groups. It can be done.

[0258] In other specific embodiments, R1' is a carrier moiety, a fluorescent moiety, a drug moiety, or a radioactive moiety. Furthermore, R1' may include a drug moiety. In addition, R1' may include a VC linker. In another specific embodiment, R1' may comprise an antibody or analog thereof comprising a paratope. do.

[0259] In specific embodiments, D1 is a covalent bond, C 1~4 Alkylene, C 2~4 Alkenylene, C 2~ 4 alkynylene, and C 3~8 cycloalkylene. where D1 can be -CH2OCH2-. Additionally, D1 can be a covalent bond.

[0260] In specific embodiments, X1 can be NR1, S, or O, and R1 can be H, halogen, or substituted. or unsubstituted C 1~3 It can be alkylene.

[0261] In specific embodiments, D2 is C 1~7 Alkylene, C 2~7 Alkenylene, C 2~7 Alkini Ren, and C. 3~8 Furthermore, D2 may include any one selected from the group consisting of: C 1~2 It can be alkylene. Furthermore, D2 can be methylene.

[0262] In a specific embodiment, formula 5-2 can consist of 13 to 17 amino acid residues ((Xa1 )').

[0263] In a specific embodiment, a cysteine ​​located 2 to 4 amino acids from the N-terminus and a C-terminus The cysteines located 2 to 4 amino acids from the amino acid residue can be optionally connected to each other.

[0264] In specific embodiments, X3 can be NH. In specific embodiments, (X)2 can be AW. In a specific embodiment, Xa2 can be glutamic acid. Xa3 can be tryptophan.

[0265] In a specific embodiment, the N-terminus (X) 1-3 One of the residues constituting the C-terminus (X) 1-3 Configure One of the residues can bond to each other.

[0266] In specific embodiments, Formula 5-2 is Formula 5-3: [Formula 5-3] DCAWH-(Xa1)'-GELVWCT (SEQ ID NO: 13) (wherein A is alanine and E is glutamic acid) may be identical to

[0267] R1'-L2-SSAI or R1'-L2-SSFI having the structure of formula 5 and 5-1 to 5-3 are capable of binding to an antibody. Furthermore, R1'-L2-SSAI or R1'-L2-SSFI may have activity against immunoglobulin G (IgG). In addition, R1'-L2-SSAI or R1'-L2-SSFI may have binding activity to the Fc domain of an antibody. It may have a binding activity to

[0268] 4.1. Reagents for transferring the first click-reactive functional group to antibodies; H1-L1 and SSAI conjugates Jugate (H1-L2-SSAI) According to the present invention, an agent for transferring a first click-reactive functional group to an antibody is disclosed. Such compounds are designated herein by the symbol "H1-L2-SSAI." Also, depending on the structure, a conjugate of H1-L1 and SSAI (H1-L2-SSAI conjugate) In this case, if the SSAI is an SSFI, it is designated by the symbol "H1-L2-SSFI". can be.

[0269] The H1-L2-SSAI or H1-L2-SSFI according to the present invention is a compound in which R1' is a compound selected from the group consisting of those described in Section 4. The present invention also includes those containing a click-reactive functional group in the "agent for transferring" However, the present invention is not limited to the following specific embodiments.

[0270] The present invention relates to a compound of the following formula 6-1: [Formula 6-1] (Xaa)2-H-(Xa1)'-G-Xa2-LV-Xa3 (SEQ ID NO: 14) wherein each Xaa is independently any amino acid other than cysteine; H is histidine, G is glycine, and Xa2 is glutamic acid or asparagine L is leucine, V is valine, and Xa3 is tryptophan, naphthyl alanine, and phenylalanine; (Xa1)' is

[0271] [ka]

[0272] wherein H1 is a first click-reactive functional group; D1 is any alkylene, alkenylene, or alkynylene; X1 is an element that is more electronegative than carbon, D2 is any alkylene, alkenylene, or alkynylene; D3 is a covalent bond or C 1~3 is alkylene, X3 is NH, O, or S). The present invention provides an H1-L2-SSFI comprising the amino acid sequence:

[0273] In specific embodiments, H1 is an alkyne, an azide, a strained alkyne, a diene, a dienophenoxy group, or a hydroxy group. The compound may include any one selected from the group consisting of olefins, alkenes, thiols, and tetrazines. , H1 can be an azide or a strained alkyne. Additionally, H1 can be a diene or a dienophile. Furthermore, H1 can be tetrazine or norbornene. Alternatively, H1 can be tetrazine, Or it can be trans-cyclooctene.

[0274] In specific embodiments, D1 is a covalent bond, C1~4 Alkylene, C 2~4 Alkenylene, C 2~ 4 alkynylene, and C 3~8 cycloalkylene. where D1 can be -CH2OCH2-. Additionally, D1 can be a covalent bond.

[0275] In specific embodiments, X1 can be NR1, S, or O, and R1 can be H, halogen, or substituted. or unsubstituted C 1~3 It can be alkylene.

[0276] In specific embodiments, D2 is C 1~7 Alkylene, C 2~7 Alkenylene, C 2~7 Alkini Ren, and C. 3~8 Furthermore, D2 may include any one selected from the group consisting of: C 1~2 It can be alkylene. Furthermore, D2 can be methylene.

[0277] In specific embodiments, X3 can be NH.

[0278] In specific embodiments, (X)2 can be AW. In specific embodiments, Xa2 can be a group. In a specific embodiment, Xa3 can be tryptophan.

[0279] The present invention relates to a compound of the following formula 6-2: [Formula 6-2] (Xaa) 1-3 -C-(Xaa)2-H-(Xa1)'-G-Xa2-LV-Xa3-C-(Xaa) 1-3 (SEQ ID NO: 15) wherein each Xaa is independently any amino acid other than cysteine; C is cysteine, H is histidine, G is glycine, and Xa2 is glutamate. L is leucine, V is valine, and Xa3 is thiamin or asparagine. is selected from tryptophan, naphthylalanine, and phenylalanine; (Xa1)' is

[0280] [ka]

[0281] wherein H1 is a first click-reactive functional group; D1 is any alkylene, alkenylene, or alkynylene; X1 is an element that is more electronegative than carbon, D2 is any alkylene, alkenylene, or alkynylene; D3 is a covalent bond or C 1~3 is alkylene, X3 is NH, O, or S). The present invention provides an H1-L2-SSFI having the structure:

[0282] In specific embodiments, H1 is an alkyne, an azide, a strained alkyne, a diene, a dienophenoxy group, or a hydroxy group. The compound may include any one selected from the group consisting of olefins, alkenes, thiols, and tetrazines. , H1 can be an azide or a strained alkyne. Additionally, H1 can be a diene or a dienophile. Furthermore, H1 can be tetrazine or norbornene. Alternatively, H1 can be tetrazine, Or it can be trans-cyclooctene.

[0283] In specific embodiments, D1 is a covalent bond, C 1~4 Alkylene, C 2~4 Alkenylene, C 2~ 4 alkynylene, and C 3~8cycloalkylene. where D1 can be -CH2OCH2-. Additionally, D1 can be a covalent bond.

[0284] In specific embodiments, X1 can be NR1, S, or O, and R1 can be H, halogen, or substituted. or unsubstituted C 1~3 It can be alkylene.

[0285] In specific embodiments, D2 is C 1~7 Alkylene, C 2~7 Alkenylene, C 2~7 Alkini Ren, and C. 3~8 Furthermore, D2 may include any one selected from the group consisting of: C 1~2 It can be alkylene. Furthermore, D2 can be methylene.

[0286] In a specific embodiment, formula 5-2 can consist of 13 to 17 amino acid residues ((Xa1 )').

[0287] In a specific embodiment, a cysteine ​​located 2 to 4 amino acids from the N-terminus and a C-terminus The cysteines located 2 to 4 amino acids from the amino acid residue can be optionally connected to each other.

[0288] In specific embodiments, X3 can be NH. In specific embodiments, (X)2 can be AW. In a specific embodiment, Xa2 can be glutamic acid. Xa3 can be tryptophan.

[0289] In a specific embodiment, the N-terminus (X) 1-3 One of the residues constituting the C-terminus (X) 1-3 Configure One of the residues can bond to each other.

[0290] In specific embodiments, Formula 6-2 is Formula 6-3: [Formula 6-3] (SEQ ID NO: 16) DCAWH-(Xa1)'-GELVWCT (SEQ ID NO: 16) (wherein A is alanine and E is glutamic acid) may be identical to

[0291] H1-L2-SSAI or H1-L2-SSFI having the structure of formulas 6-1 to 6-3 has binding activity to an antibody. Furthermore, H1-L2-SSAI or H1-L2-SSFI has binding activity to immunoglobulin G (IgG). In addition, H1-L2-SSAI or H1-L2-SSFI may have binding activity to the Fc domain of an antibody. It is possible.

[0292] 4.2. Methods for Preparing an Agent for Transferring a First Chemical Functional Group to an Antibody According to the present invention, R1'-L2-SSAI, R1'-L2-SSFI, H1-L2-SSAI, and H1-L2-SSFI are prepared. (hereinafter collectively referred to as "R1'-L2-SSAI"). The following description of the preparation method is Please note that this will help you understand the invention.

[0293] For example, methods for preparing compounds of formula 5 are described. In specific embodiments, the compounds of formula 5 The compound can be prepared through the reaction of Scheme 2 below.

[0294] [ka]

[0295] The R1'-L2-SSAI according to the present invention is a site-specific antibody interactome (SSAI) according to the present invention. The present invention can be prepared by allowing the reaction of the compound with a linker (R1'-L1) that is The SSAIs of the invention are designed to contain a nucleophilic X3. X3 is an activated nucleophilic group contained in a linker. In this case, X3 can attack the carbonyl group of the second By attacking the carbonyl group, the R1'-L2-SSAI according to the present invention is prepared.

[0296] As a more specific example, a method for preparing a compound of formula 6-3 is described. In the form, compounds of formula 6-3 can be prepared via the reaction of Scheme 3 below.

[0297] [ka]

[0298] SSAIs according to the present invention comprising the amino acid sequence of formula 3 or 3-1 and having the structures of formulas 4-1 to 4-6. contains a Xa1 residue containing a nucleophilic X3, which attacks a second carbonyl group in the linker. Thus, the R1'-L2-SSAI according to the present invention can be prepared.

[0299] In a specific embodiment, the basicity of the leaving group comprising X2 in a linker according to the invention is In a specific embodiment, the basicity of the leaving group, including X1, is lower than that of the leaving group, The linker allows X2-R2' to connect with the second carbonyl group to form a good leaving group. Additionally, X2 can be O and R2' can be N-succinimide, p-nitrophenyl, or can be pentafluorophenyl. In this case, the reactivity of the second carbonyl group is Therefore, X3 of SSAI is linked to the second carbonyl group of the linker. It can attack specifically.

[0300] In accordance with the present invention, a method for preparing an agent for transferring a first chemical functional group to an antibody is disclosed. is shown.

[0301] The present invention provides a method for preparing an R1'-L2-SSAI, comprising: Reacting a linker according to the present invention with a site-specific antibody interactome according to the present invention process The present invention provides a method comprising:

[0302] In specific embodiments, the linker is any one selected from formulas 1, 2, and 2-1 through 2-3. It is possible.

[0303] In specific embodiments, the site-specific antibody interactome is represented by Formulas 3, 3-1, and 4-1 through 4-6: It may comprise or have any one structure selected from:

[0304] Additionally, the linker may have the structure of Formula 2, and the site-specific antibody interactome may have the structure of Formula 4-2 The site-specific antibody interacts with the nucleotides of the present invention. The compound may have the structure of formula 4-6.

[0305] According to the present invention, a method for preparing an agent for transferring a first click-reactive functional group to an antibody is provided. A method is disclosed.

[0306] The present invention provides a method for preparing an H1-L2-SSAI, comprising the steps of: Reacting a linker according to the present invention with a site-specific antibody interactome according to the present invention process The present invention provides a method comprising:

[0307] In a specific embodiment, the linker is any one selected from formulas 2 and 2-1 to 2-3: Possible.

[0308] In specific embodiments, the site-specific antibody interactome is represented by Formulas 3, 3-1, and 4-1 through 4-6: It may comprise or have any one structure selected from:

[0309] Additionally, the linker may have the structure of Formula 2, and the site-specific antibody interactome may have the structure of Formula 4-2 The site-specific antibody interacts with the nucleotides of the present invention. The compound may have the structure of formula 4-6.

[0310] In accordance with the present invention, a method for preparing an agent for transferring a first chemical functional group to an antibody is provided. A set of methods is disclosed.

[0311] The present invention provides a kit for preparing an agent for transferring a first chemical functional group to an antibody. and comprising a linker according to the present invention and a site-specific antibody interactome according to the present invention. Provide a kit.

[0312] In a specific embodiment, the linker is any one selected from Formulas 1, 2, and 2-1 to 2-3. It could be one.

[0313] In specific embodiments, the site-specific antibody interactome is represented by Formulas 3, 3-1, and 4-1 through 4-6: It may comprise or have any one structure selected from:

[0314] Additionally, the linker may have the structure of Formula 2, and the site-specific antibody interactome may have the structure of Formula 4-2 The site-specific antibody interacts with the nucleotides of the present invention. The compound may have the structure of formula 4-6.

[0315] According to the present invention, a method for preparing an agent for transferring a first click-reactive functional group to an antibody is provided. A kit for the preparation of the compound is disclosed.

[0316] The present invention provides a method for preparing an agent for transferring a first click-reactive functional group to an antibody. a linker comprising a first click-reactive functional group according to the present invention, and Kits containing site-specific antibody interactomes are provided.

[0317] In a specific embodiment, the linker is any one selected from formulas 2 and 2-1 to 2-3: Possible.

[0318] In specific embodiments, the site-specific antibody interactome is represented by Formulas 3, 3-1, and 4-1 through 4-6: It may comprise or have any one structure selected from:

[0319] Additionally, the linker may have the structure of Formula 2, and the site-specific antibody interactome may have the structure of Formula 4-2 The site-specific antibody interacts with the nucleotides of the present invention. The compound may have the structure of formula 4-6.

[0320] 5. Antibody Containing a First Chemical Functional Group (R1'-Ab) In accordance with the present invention, an antibody is disclosed that contains a first chemical functionality. Such a compound is , which is denoted herein by the symbol "R1'-Ab."

[0321] The present application provides a compound of formula 7:

[0322] [ka]

[0323] wherein R is a first chemical functionality; D1 is any alkylene, alkenylene, or alkynylene; X4 is NH, O, or S; Ab is antibody) The present invention provides an R1'-Ab represented by the formula:

[0324] In specific embodiments, R1' can be a click-reactive functional group. Alkynes, azides, strained alkynes, dienes, dienophiles, alkenes, thiols, and tetracyclines Furthermore, R1' may include any one selected from azide, strained alkyl, Additionally, R1' can be azide or dibenzocyclooctyne-amine. Additionally, R1' can be a diene or a dienophile. Alternatively, R1' can be tetrazine or trans-cyclooctene. Possible.

[0325] In other specific embodiments, R1' is a carrier moiety, a fluorescent moiety, a drug moiety, or a radioactive moiety. Furthermore, R1' may include a drug moiety. In addition, R1' may include a VC linker. In another specific embodiment, R1' may comprise an antibody or analog thereof comprising a paratope. do.

[0326] In specific embodiments, D1 is a covalent bond, C 1~4 Alkylene, C 2~4 Alkenylene, C 2~ 4 alkynylene, and C 3~8 cycloalkylene. where D1 can be -CH2OCH2-. Additionally, D1 can be a covalent bond.

[0327] In one specific embodiment, X4 can be NH.

[0328] In specific embodiments, the Ab can be a human antibody. In other specific embodiments, the Ab can be In a specific embodiment, the Ab is an immunoglobulin G (IgG). In a specific embodiment, the Ab may be a complete antibody. Alternatively, the Ab may be a fragment of an antibody.

[0329] In a specific embodiment, X4 and Ab can be connected via the Fab domain of the Ab. In another specific embodiment, X4 and the Ab can be connected via the Fc domain of the Ab. Furthermore, X4 and Ab can be connected via lysine 246 or 248 in the Fc domain of Ab. Additionally, X4 and Ab can be connected via lysine 246 in the Fc domain of the Ab. Additionally, X4 and Ab can be connected via lysine 248 of the Fc domain of the Ab. X4 and Ab can be connected via lysines 246 and 248 of the Fc domain of the Ab. In a typical embodiment, X4 and the Ab may be connected via only one of the two Fc domains of the Ab. In another specific embodiment, X4 and Ab bind to each other via both of the two Fc domains of the Ab. can be connected.

[0330] The present invention relates to a compound of the formula 7-1: [Formula 7-1] GPSVFLFPP-(K)'-PKDTLMI (SEQ ID NO: 17) wherein G is glycine, P is proline, S is serine, and V is valine. where F is phenylalanine, L is leucine, K is lysine, and D is , is aspartic acid, T is threonine, M is methionine, and I is isoform is leucine, (K)' is

[0331] [ka]

[0332] wherein R is a first chemical functionality; D1 is any alkylene, alkenylene, or alkynylene. The antibody or fragment thereof comprises the amino acid sequence of the Fc domain. The amino acid sequence has R1' connected via lysine 246 of the amino acid sequence, or a site corresponding to lysine 246.

[0333] In specific embodiments, R1' can comprise a click-reactive functional group. Click-reactive functional groups include alkynes, azides, strained alkynes, dienes, dienophiles, It may comprise one or more selected from alkenes, thiols, and tetrazines. The carboxylic reactive functional group can be selected from an azide or a strained alkyne. The click-reactive functional group can be selected from azide or dibenzocyclooctyne-amine. In addition, the click-reactive functional group can be selected from dienes or dienophiles. Furthermore, the click-reactive functional group can be selected from tetrazine or norbornene. Alternatively, the click-reactive functional group can be a tetrazine or a trans-cyclopentadiene. In addition, R1' may contain two or more click-reactive functional groups. It can be done.

[0334] In other specific embodiments, R1' is a carrier moiety, a fluorescent moiety, a drug moiety, or a radioactive moiety. Furthermore, R1' may include a drug moiety. In addition, R1' may include a VC linker. In another specific embodiment, R1' may comprise an antibody or analog thereof comprising a paratope. do.

[0335] In specific embodiments, D1 is a covalent bond, C 1~4 Alkylene, C 2~4 Alkenylene, C 2~ 4 alkynylene, and C 3~8 cycloalkylene. where D1 can be -CH2OCH2-. Additionally, D1 can be a covalent bond.

[0336] In specific embodiments, the antibody may be a human antibody. The antibody may be a non-human animal antibody. In a specific embodiment, the antibody is an immunoglobulin G (Ig In specific embodiments, the antibody may be a whole antibody. In embodiments, the antibody may be a fragment of an antibody.

[0337] In a specific embodiment, the antibody contains the amino acid sequence of formula 7-1 in only one of its two Fc domains. In another specific embodiment, the antibody may comprise both of its two Fc domains. The amino acid sequence of formula 7-1 may be included.

[0338] The present invention relates to a compound of the following formula 7-2: [Formula 7-2] GPSVFLFPPKP-(K)'-DTLMI (SEQ ID NO: 18) wherein G is glycine, P is proline, S is serine, and V is valine. where F is phenylalanine, L is leucine, K is lysine, and D is , is aspartic acid, T is threonine, M is methionine, and I is isoform is leucine, (K)' is

[0339] [ka]

[0340] wherein R is a first chemical functionality; D1 is any alkylene, alkenylene, or alkynylene. The antibody or fragment thereof comprises the amino acid sequence of the Fc domain. The amino acid sequence has R1' connected via lysine 248 of the amino acid sequence, or a site corresponding to lysine 248.

[0341] In specific embodiments, R1' can comprise a click-reactive functional group. Click-reactive functional groups include alkynes, azides, strained alkynes, dienes, dienophiles, It may comprise one or more selected from alkenes, thiols, and tetrazines. The carboxylic reactive functional group can be selected from an azide or a strained alkyne. The click-reactive functional group can be selected from azide or dibenzocyclooctyne-amine. In addition, the click-reactive functional group can be selected from dienes or dienophiles. Furthermore, the click-reactive functional group can be selected from tetrazine or norbornene. Alternatively, the click-reactive functional group can be a tetrazine or a trans-cyclopentadiene. In addition, R1' may contain two or more click-reactive functional groups. It can be done.

[0342] In other specific embodiments, R1' is a carrier moiety, a fluorescent moiety, a drug moiety, or a radioactive moiety. Furthermore, R1' may include a drug moiety. In addition, R1' may include a VC linker. In another specific embodiment, R1' may comprise an antibody or analog thereof comprising a paratope. do.

[0343] In specific embodiments, D1 is a covalent bond, C 1~4 Alkylene, C 2~4 Alkenylene, C 2~ 4 alkynylene, and C 3~8 cycloalkylene. where D1 can be -CH2OCH2-. Additionally, D1 can be a covalent bond.

[0344] In specific embodiments, the antibody may be a human antibody. The antibody may be a non-human animal antibody. In a specific embodiment, the antibody is an immunoglobulin G (Ig In specific embodiments, the antibody may be a whole antibody. In embodiments, the antibody may be a fragment of an antibody.

[0345] In a specific embodiment, the antibody contains the amino acid sequence of formula 7-2 in only one of its two Fc domains. In another specific embodiment, the antibody may comprise both of its two Fc domains. The amino acid sequence of formula 7-2 may be included.

[0346] The present invention relates to a compound of formula 7-3: [Formula 7-3] (SEQ ID NO: 19) GPSVFLFPP-(K)'-P-(K)'-DTLMI (SEQ ID NO: 19) wherein G is glycine, P is proline, S is serine, and V is valine. where F is phenylalanine, L is leucine, K is lysine, and D is , is aspartic acid, T is threonine, M is methionine, and I is isoform is leucine, (K)' is

[0347] [ka]

[0348] wherein R is a first chemical functionality; D1 is any alkylene, alkenylene, or alkynylene. The antibody or fragment thereof comprises the amino acid sequence of the Fc domain. R1' connected via lysines 246 and 248 of the amino acid sequence, or the site corresponding to lysines 246 and 248 of the amino acid sequence Has.

[0349] In specific embodiments, R1' can comprise a click-reactive functional group. Click-reactive functional groups include alkynes, azides, strained alkynes, dienes, dienophiles, It may comprise one or more selected from alkenes, thiols, and tetrazines. The carboxylic reactive functional group can be selected from an azide or a strained alkyne. The click-reactive functional group can be selected from azide or dibenzocyclooctyne-amine. In addition, the click-reactive functional group can be selected from dienes or dienophiles. Furthermore, the click-reactive functional group can be selected from tetrazine or norbornene. Alternatively, the click-reactive functional group can be a tetrazine or a trans-cyclopentadiene. In addition, R1' may contain two or more click-reactive functional groups. It can be done.

[0350] In other specific embodiments, R1' is a carrier moiety, a fluorescent moiety, a drug moiety, or a radioactive moiety. Furthermore, R1' may include a drug moiety. In addition, R1' may include a VC linker. In another specific embodiment, R1' may comprise an antibody or analog thereof comprising a paratope. do.

[0351] In specific embodiments, D1 is a covalent bond, C 1~4 Alkylene, C 2~4 Alkenylene, C 2~ 4 alkynylene, and C 3~8 cycloalkylene. where D1 can be -CH2OCH2-. Additionally, D1 can be a covalent bond.

[0352] In specific embodiments, the antibody may be a human antibody. The antibody may be a non-human animal antibody. In a specific embodiment, the antibody is an immunoglobulin G (Ig In specific embodiments, the antibody may be a whole antibody. In embodiments, the antibody may be a fragment of an antibody.

[0353] In a specific embodiment, the antibody contains the amino acid sequence of formula 7-3 in only one of its two Fc domains. In another specific embodiment, the antibody may comprise both of its two Fc domains. The amino acid sequence may include the amino acid sequence of formula 7-3.

[0354] The present invention provides an antibody comprising one or more amino acid sequences selected from formulas 7-1, 7-2, and 7-3. In this case, the sequences of formulae 7-1 to 7-3 are as described above.

[0355] In specific embodiments, D1 can be a covalent bond.

[0356] In a specific embodiment, the antibody or fragment thereof comprises only the amino acid sequence of formula 7-1 and not formula 7-2. and 7-3. Furthermore, the antibody or fragment thereof does not contain the amino acid sequence of two of its Fc domains. In addition, an antibody may contain the amino acid sequence of Formula 7-1 in only one of its Fc domains. Both the amino acid sequence of Formula 7-1 may be included.

[0357] In another specific embodiment, the antibody or fragment thereof comprises only the amino acid sequence of Formula 7-2: The antibody or fragment thereof does not include the amino acid sequences of formulas 7-1 and 7-3. Furthermore, the antibody or fragment thereof does not include the amino acid sequences of formulas 7-1 and 7-3. In addition, the antibody may contain the amino acid sequence of Formula 7-2 in only one of its F Both of the c domains may contain the amino acid sequence of formula 7-2.

[0358] In a specific embodiment, the antibody or fragment thereof comprises only the amino acid sequence of formula 7-3 and not the amino acid sequence of formula 7-1 and 7-2. Furthermore, the antibody or fragment thereof does not contain the amino acid sequence of two of its Fc domains. In addition, an antibody may contain the amino acid sequence of formula 7-3 in only one of its Fc domains. Both the amino acid sequence of Formula 7-3 may be included.

[0359] 5.1. Antibodies Containing a First Click-Reactive Functional Group According to the present invention, an antibody containing a first click-reactive functional group is disclosed. Such compounds are designated herein by the symbol "H1-Ab."

[0360] The present application relates to a compound of formula 8:

[0361] [ka]

[0362] wherein H1 is a first click-reactive functional group; D1 is any alkylene, alkenylene, or alkynylene; X4 is NH, O, or S; Ab is antibody) The present invention provides an H1-Ab represented by the formula:

[0363] In specific embodiments, H1 is an alkyne, an azide, a strained alkyne, a diene, a dienophenoxy group, or a hydroxy group. The compound may include any one selected from the group consisting of olefins, alkenes, thiols, and tetrazines. , H1 can be an azide or a strained alkyne. Additionally, H1 can be a diene or a dienophile. Furthermore, H1 can be tetrazine or norbornene. Alternatively, H1 can be tetrazine, Or it can be trans-cyclooctene.

[0364] In specific embodiments, D1 is a covalent bond, C 1~4 Alkylene, C 2~4 Alkenylene, C 2~ 4 alkynylene, and C 3~8 cycloalkylene. where D1 can be -CH2OCH2-. Additionally, D1 can be a covalent bond.

[0365] In one specific embodiment, X4 can be NH.

[0366] In specific embodiments, the Ab can be a human antibody. In other specific embodiments, the Ab can be In a specific embodiment, the Ab is an immunoglobulin G (IgG). In a specific embodiment, the Ab may be a complete antibody. In a specific embodiment, the Ab can be a fragment of an antibody. In a specific embodiment, the Ab can be a wild-type antibody. In other specific embodiments, the Ab may be an engineered antibody.

[0367] In a specific embodiment, X4 and Ab can be connected via the Fab domain of the Ab. In another specific embodiment, X4 and the Ab can be connected via the Fc domain of the Ab. Furthermore, X4 and Ab can be connected via lysine 246 or 248 in the Fc domain of Ab. Additionally, X4 and Ab can be connected via lysine 246 in the Fc domain of the Ab. Additionally, X4 and Ab can be connected via lysine 248 of the Fc domain of the Ab. X4 and Ab can be connected via lysines 246 and 248 of the Fc domain of the Ab. In a typical embodiment, X4 and the Ab may be connected via only one of the two Fc domains of the Ab. In another specific embodiment, X4 and Ab bind to each other via both of the two Fc domains of the Ab. can be connected.

[0368] The present invention relates to a compound of the following formula 8-1: [Formula 8-1] GPSVFLFPP-(K)'-PKDTLMI (SEQ ID NO: 20) wherein G is glycine, P is proline, S is serine, and V is valine. where F is phenylalanine, L is leucine, K is lysine, and D is , is aspartic acid, T is threonine, M is methionine, and I is isoform is leucine, (K)' is

[0369] [ka]

[0370] wherein H1 is a first click-reactive functional group; D1 is any alkylene, alkenylene, or alkynylene. The antibody or fragment thereof comprises the amino acid sequence of the Fc domain. It has H1 connected via lysine 246 of the amino acid sequence, or a site corresponding to lysine 246.

[0371] In specific embodiments, H1 is an alkyne, an azide, a strained alkyne, a diene, a dienophenoxy group, or a hydroxy group. The compound may include any one selected from the group consisting of olefins, alkenes, thiols, and tetrazines. , H1 can be an azide or a strained alkyne. Additionally, H1 can be a diene or a dienophile. Furthermore, H1 can be tetrazine or norbornene. Alternatively, H1 can be tetrazine, Or it can be trans-cyclooctene.

[0372] In specific embodiments, D1 is a covalent bond, C 1~4 Alkylene, C 2~4 Alkenylene, C 2~ 4 alkynylene, and C 3~8 cycloalkylene. where D1 can be -CH2OCH2-. Additionally, D1 can be a covalent bond.

[0373] In specific embodiments, the antibody may be a human antibody. The antibody may be a non-human animal antibody. In a specific embodiment, the antibody is an immunoglobulin G (Ig In specific embodiments, the antibody may be a whole antibody. In embodiments, the antibody may be a fragment of an antibody. In specific embodiments, the antibody is a wild-type antibody. In other specific embodiments, the antibody may be an engineered antibody.

[0374] In a specific embodiment, the antibody contains the amino acid sequence of formula 8-1 in only one of its two Fc domains. In another specific embodiment, the antibody may comprise both of its two Fc domains. The amino acid sequence of formula 8-1 may be included.

[0375] The present invention relates to a compound of the following formula 8-2: [Formula 8-2] GPSVFLFPPKP-(K)'-DTLMI (SEQ ID NO: 21) wherein G is glycine, P is proline, S is serine, and V is valine. where F is phenylalanine, L is leucine, K is lysine, and D is , is aspartic acid, T is threonine, M is methionine, and I is isoform is leucine, (K)' is

[0376] [ka]

[0377] wherein H1 is a first click-reactive functional group; D1 is any alkylene, alkenylene, or alkynylene. The antibody or fragment thereof comprises the amino acid sequence of the Fc domain. It has H1 connected via lysine 248 of the amino acid sequence, or a site corresponding to lysine 248.

[0378] In specific embodiments, H1 is an alkyne, an azide, a strained alkyne, a diene, a dienophenoxy group, or a hydroxy group. The compound may include any one selected from the group consisting of olefins, alkenes, thiols, and tetrazines. , H1 can be an azide or a strained alkyne. Additionally, H1 can be a diene or a dienophile. Furthermore, H1 can be tetrazine or norbornene. Alternatively, H1 can be tetrazine, Or it can be trans-cyclooctene.

[0379] In specific embodiments, D1 is a covalent bond, C 1~4 Alkylene, C 2~4 Alkenylene, C 2~ 4 alkynylene, and C 3~8 cycloalkylene. where D1 can be -CH2OCH2-. Additionally, D1 can be a covalent bond.

[0380] In specific embodiments, the antibody may be a human antibody. The antibody may be a non-human animal antibody. In a specific embodiment, the antibody is an immunoglobulin G (Ig In specific embodiments, the antibody may be a whole antibody. In embodiments, the antibody may be a fragment of an antibody. In specific embodiments, the antibody is a wild-type antibody. In other specific embodiments, the antibody may be an engineered antibody.

[0381] In a specific embodiment, the antibody contains the amino acid sequence of formula 8-2 in only one of its two Fc domains. In another specific embodiment, the antibody may comprise both of its two Fc domains. The amino acid sequence may include the amino acid sequence of formula 8-2.

[0382] The present invention relates to a compound of the following formula 8-3: [Formula 8-3] GPSVFLFPP-(K)'-P-(K)'-DTLMI (SEQ ID NO: 22) wherein G is glycine, P is proline, S is serine, and V is valine. where F is phenylalanine, L is leucine, K is lysine, and D is , is aspartic acid, T is threonine, M is methionine, and I is isoform is leucine, (K)' is

[0383] [ka]

[0384] wherein H1 is a first click-reactive functional group; D1 is any alkylene, alkenylene, or alkynylene. The antibody or fragment thereof comprises the amino acid sequence of the Fc domain. H1 is connected via lysines 246 and 248 of the nucleotide sequence, or the region corresponding to lysines 246 and 248 of the nucleotide sequence. do.

[0385] In specific embodiments, H1 is an alkyne, an azide, a strained alkyne, a diene, a dienophenoxy group, or a hydroxy group. The compound may include any one selected from the group consisting of olefins, alkenes, thiols, and tetrazines. , H1 can be an azide or a strained alkyne. Additionally, H1 can be a diene or a dienophile. Furthermore, H1 can be tetrazine or norbornene. Alternatively, H1 can be tetrazine, Or it can be trans-cyclooctene.

[0386] In specific embodiments, D1 is a covalent bond, C1~4 Alkylene, C 2~4 Alkenylene, C 2~ 4 alkynylene, and C 3~8 cycloalkylene. where D1 can be -CH2OCH2-. Additionally, D1 can be a covalent bond.

[0387] In specific embodiments, the antibody may be a human antibody. The antibody may be a non-human animal antibody. In a specific embodiment, the antibody is an immunoglobulin G (Ig In specific embodiments, the antibody may be a whole antibody. In embodiments, the antibody may be a fragment of an antibody. In specific embodiments, the antibody is a wild-type antibody. In other specific embodiments, the antibody may be an engineered antibody.

[0388] In a specific embodiment, the antibody contains the amino acid sequence of formula 8-3 in only one of its two Fc domains. In another specific embodiment, the antibody may comprise both of its two Fc domains. The amino acid sequence may include the amino acid sequence of formula 8-3.

[0389] The present invention relates to an antibody comprising one or more amino acid sequences selected from formulas 8-1, 8-2, and 8-3. In this case, the sequences of formulae 8-1 to 8-3 are as described above.

[0390] In specific embodiments, D1 can be a covalent bond.

[0391] In a specific embodiment, the antibody or fragment thereof comprises only the amino acid sequence of formula 8-1 and not formula 8-2. and 8-3. Furthermore, the antibody or fragment thereof does not contain the amino acid sequence of two of its Fc domains. In addition, an antibody may contain the amino acid sequence of Formula 8-1 in only one of its Fc domains. Both the amino acid sequence of Formula 8-1 may be included.

[0392] In another specific embodiment, the antibody or fragment thereof comprises only the amino acid sequence of Formula 8-2: The antibody or fragment thereof does not include the amino acid sequences of formulas 8-1 and 8-3. Furthermore, the antibody or fragment thereof does not include the amino acid sequences of formulas 8-1 and 8-3. In addition, the antibody may contain the amino acid sequence of Formula 8-2 in only one of its F Both of the c domains may contain the amino acid sequence of formula 8-2.

[0393] In a specific embodiment, the antibody or fragment thereof comprises only the amino acid sequence of formula 8-3 and not the amino acid sequence of formula 8-1 and 8-2. Furthermore, the antibody or fragment thereof does not contain the amino acid sequence of two of its Fc domains. In addition, an antibody may contain the amino acid sequence of formula 8-3 in only one of its Fc domains. Both the amino acid sequence of Formula 8-3 may be included.

[0394] In specific embodiments, the antibody may be a human antibody. The antibody may be a non-human animal antibody. In a specific embodiment, the antibody is an immunoglobulin G (Ig In specific embodiments, the antibody may be a whole antibody. In embodiments, the antibody may be a fragment of an antibody. In specific embodiments, the antibody is a wild-type antibody. In other specific embodiments, the antibody may be an engineered antibody.

[0395] 5.2. Methods for Preparing Antibodies Containing a First Chemical Functionality Group According to the present invention, a method for preparing R1'-Ab and H1-Ab (hereinafter referred to as "R1'-Ab") is disclosed. It should be noted that the following description of the preparation method will be helpful in understanding the present invention. sea ​​bream.

[0396] For example, methods for preparing compounds of formula 7 are described. In specific embodiments, the compounds of formula 7 are The compound can be prepared through the reaction of Scheme 4 below.

[0397] [ka]

[0398] Since the first carbonyl group of the linker according to the present invention is mildly reactive, Cross-linking occurs when the first carbonyl group is in close proximity to an amine group on the antibody. First, R1'-L2-SSAI represented by formula 5 adheres to a specific site of the antibody and reacts. The R1'-L2-SSAI according to the present invention creates an environment in which the first carbonyl of the linker can Since it has an activated carbonyl group (i.e., a carbonyl group connected to X1) corresponding to the group The R1'-L2-SSAI of the present invention can undergo a nucleophilic substitution reaction, in which case the compound of formula 7 is The atom (X4) with a free electron pair in the antibody acts as a nucleophile that attacks the carbonyl group. In this case, site-specific antibodies can be prepared by designing a linker. The interactome (SSAI) is released while included in the leaving group, and the SSAI is removed from the final product. These characteristics favorably affect the physical properties of the antibody product, as described in Section 5.8 below. It has an effect.

[0399] In a specific embodiment, X4 can be NH2. Additionally, X4 can be the NH2 of a lysine residue. In other specific embodiments, X4 can be SH. Additionally, X4 can be a cysteine ​​residue. In other specific embodiments, X4 can be OH.

[0400] As a more specific example, a method for preparing an antibody or a fragment thereof comprising the amino acid sequence of formula 8-3 In a specific embodiment, the compound comprising the amino acid sequence of formula 8-3 is It can be prepared through the reaction of Scheme 5.

[0401] [ka]

[0402] The compound having the structure of formula 6-3 (the structure in which two cysteine ​​residues are optionally connected) is It has the sequence SSFI and is therefore directed to the Fc domain of the antibody (see section 3.2). The antibody containing the amino acid sequence of No. 1 contains a lysine residue in its Fc domain, and such a lysine An exemplary embodiment is lysine 246 of the Fc domain. or a residue corresponding to lysine 246 (hereinafter referred to as "lysine 246") serves as a nucleophile. In this case, the amine group of lysine 246 attacks the first carbonyl group of formula 6-3 to form Thus, H1 or R1 binds to a lysine residue in the Fc domain of the antibody to produce a compound of formula 8-1. In this case, a chemical functional group is transferred to any specific lysine residue. The design of the linker, SSAI, and R1'-L2-SSAI, as described in Sections 5.3 and 5.4 below, determines whether the R1'-L2-SSAI is a nucleotide sequence or not. may depend on

[0403] In accordance with the present invention, a method for preparing an antibody containing a first chemical functionality is disclosed.

[0404] The present invention provides a method for preparing an R1'-Ab, comprising the steps of: The agent for transferring the first chemical functional group to the antibody according to the present invention is combined with the antibody or fragment thereof. Reaction process The present invention provides a method comprising:

[0405] In a specific embodiment, the agent for transferring a first chemical functionality to an antibody is represented by Formula 5, 5-1 It may be any one selected from 5-1 to 5-3, and 6-1 to 6-3.

[0406] In specific embodiments, the antibody or fragment thereof may be a human antibody. In some embodiments, the antibody or fragment thereof may be a non-human animal antibody. The antibody or fragment thereof may be an immunoglobulin G (IgG). In a specific embodiment, an antibody or The fragment may be a complete antibody. In other specific embodiments, the antibody or fragment thereof is In a specific embodiment, the antibody or fragment thereof is a wild-type antibody. In other specific embodiments, the antibody or fragment thereof may be an engineered antibody.

[0407] In a specific embodiment, the present invention provides a method for the production of a lysine-containing antibody having a lysine residue in the Fc domain of the antibody. In this case, a method for preparing an antibody having a chemical functional group as described in Section 5.4 below is provided. This embodiment can be used as an agent for transferring a first chemical functional group to an antibody. can.

[0408] According to the present invention, a method for preparing an antibody containing a first click-reactive functional group is disclosed. can be.

[0409] The present invention provides a method for preparing H1-Ab, comprising the steps of: The agent for transferring the first click-reactive functional group to the antibody according to the invention is reacting with the fragment The present invention provides a method comprising:

[0410] In a specific embodiment, the agent for transferring the first click-reactive functional group to the antibody is , may be any one selected from formulas 6-1 to 6-3. An agent for transferring to an antibody may have the structure of formula 6-3.

[0411] In specific embodiments, the antibody or fragment thereof may be a human antibody. In some embodiments, the antibody or fragment thereof may be a non-human animal antibody. The antibody or fragment thereof may be an immunoglobulin G (IgG). In a specific embodiment, an antibody or The fragment may be a complete antibody. In other specific embodiments, the antibody or fragment thereof is In a specific embodiment, the antibody or fragment thereof is a wild-type antibody. In other specific embodiments, the antibody or fragment thereof may be an engineered antibody.

[0412] In a specific embodiment, the present invention provides a method for the production of a lysine-containing antibody having a lysine residue in the Fc domain of the antibody.

[0013] A method for preparing an antibody having a click-reactive functional group is provided, which comprises the steps of: The embodiment described in paragraph 4 further includes an agent for transferring the first click-reactive functional group to the antibody. It can be used as.

[0413] According to the present invention, a kit for preparing an antibody or fragment thereof containing a first chemical functionality is provided. The following information will be disclosed.

[0414] The present invention is a kit for preparing an antibody or fragment thereof containing a first chemical functionality. Thus, an agent for transferring a first chemical functional group to an antibody according to the present invention, and a method for transferring the first chemical functional group to an antibody or its A kit containing the fragments is provided.

[0415] In a specific embodiment, the agent for transferring a first chemical functionality to an antibody is represented by Formula 5, 5-1 It may be any one selected from 5-1 to 5-3, and 6-1 to 6-3.

[0416] In specific embodiments, the antibody or fragment thereof may be a human antibody. In some embodiments, the antibody or fragment thereof may be a non-human animal antibody. The antibody or fragment thereof may be an immunoglobulin G (IgG). In a specific embodiment, an antibody or The fragment may be a complete antibody. In other specific embodiments, the antibody or fragment thereof is In a specific embodiment, the antibody or fragment thereof is a wild-type antibody. In other specific embodiments, the antibody or fragment thereof may be an engineered antibody.

[0417] Additionally, the present invention provides a method for preparing an antibody or fragment thereof containing a first chemical functionality. It is a a linker (R1'-L1) according to the present invention; a site-specific antibody interactome according to the present invention; and Antibodies or fragments thereof A kit comprising:

[0418] In specific embodiments, the linker is any one selected from formulas 1, 2, and 2-1 through 2-3. It is possible.

[0419] In specific embodiments, the site-specific antibody interactome is represented by Formulas 3, 3-1, and 4-1 through 4-6: It can be any one selected from:

[0420] In specific embodiments, the antibody or fragment thereof may be a human antibody. In some embodiments, the antibody or fragment thereof may be a non-human animal antibody. The antibody or fragment thereof may be an immunoglobulin G (IgG). In a specific embodiment, an antibody or The fragment may be a complete antibody. In other specific embodiments, the antibody or fragment thereof is In a specific embodiment, the antibody or fragment thereof is a wild-type antibody. In other specific embodiments, the antibody or fragment thereof may be an engineered antibody.

[0421] The present invention provides a method for the preparation of a medicament for the treatment of malaria, comprising administering to a patient a first chemical functional group transferred to a specific lysine residue in the Fc domain of the medicament. Kits for preparing antibodies are provided, in which case the antibodies are of the embodiments described in Section 5.4 below. The present invention provides a method for the preparation of a first functional group of an antibody, a linker, and a site-specific antibody intercalation site. It can be used as an interactome.

[0422] According to the present invention, an antibody or fragment thereof containing a first click-reactive functional group is prepared. A kit for the treatment is disclosed.

[0423] The present invention provides a method for preparing an antibody or a fragment thereof containing a first click-reactive functional group. A kit comprising a reagent for transferring a first click-reactive functional group to an antibody according to the invention. The present invention provides a kit comprising the agent, and the antibody or fragment thereof.

[0424] In a specific embodiment, the agent for transferring the first click-reactive functional group to the antibody is , may be any one selected from formulas 6-1 to 6-3. An agent for transferring to an antibody may have the structure of formula 6-3.

[0425] In specific embodiments, the antibody or fragment thereof may be a human antibody. In some embodiments, the antibody or fragment thereof may be a non-human animal antibody. The antibody or fragment thereof may be an immunoglobulin G (IgG). In a specific embodiment, an antibody or The fragment may be a complete antibody. In other specific embodiments, the antibody or fragment thereof is In a specific embodiment, the antibody or fragment thereof is a wild-type antibody. In other specific embodiments, the antibody or fragment thereof may be an engineered antibody.

[0426] Moreover, the present invention provides a method for preparing an antibody or fragment thereof containing a first click-reactive functional group. A kit for a linker according to the present invention (H1-L1); a site-specific antibody interactome according to the present invention; and Antibodies or fragments thereof A kit comprising:

[0427] In a specific embodiment, the linker is any one selected from formulas 2 and 2-1 to 2-3: Possible.

[0428] In specific embodiments, the site-specific antibody interactome is represented by Formulas 3, 3-1, and 4-1 through 4-6: It can be any one selected from:

[0429] In specific embodiments, the antibody or fragment thereof may be a human antibody. In some embodiments, the antibody or fragment thereof may be a non-human animal antibody. The antibody or fragment thereof may be an immunoglobulin G (IgG). In a specific embodiment, an antibody or The fragment may be a complete antibody. In other specific embodiments, the antibody or fragment thereof is In a specific embodiment, the antibody or fragment thereof is a wild-type antibody. In other specific embodiments, the antibody or fragment thereof may be an engineered antibody.

[0430] The present invention provides a first click-reactive functional group transferred to specific lysine residues in the Fc domain. Kits are provided for preparing antibodies having a group, in which case the group described in Section 5.4 below is used. Embodiments include a compound comprising an agent for transferring a first click-reactive functional group to an antibody, a linker, and can be used as a site-specific antibody interactome.

[0431] 5.3. Function of (Xa1)' and design principle of (Xa1)' position in R1'-L2-SSFI In this context, the compound of formula 6-3 is provided as an example to aid in understanding the invention. However, the scope of the present invention is not limited thereto. The following description also applies to Formulas 5, 5-1 to 5-3, and 6-1. The present invention applies to compounds of formula 6-3, which are provided merely as an example for convenience. Please note.

[0432] As discussed in Section 5.2, (Xa1)' of R1'-L2-SSFI can be nucleophilically substituted to attach R1' to the antibody. According to the present invention, the conditions for promoting the nucleophilic substitution reaction are as follows: Assume that the following requirements are met: (1) (Xa1)' is adjacent to a lysine residue in the Fc domain, and ( 2) The side chain to which R1' is attached is directed to a lysine residue. The yield and uniformity of the process are ( The position and direction of the Xa1)' side chain decreased as it moved away from the lysine residue of the Fc domain. Moreover, the substitution position of (3)(Xa1)' plays a significant role in the interaction between SSFI and the Fc domain. Preferably, this should not have a significant effect (see Section 3.2).

[0433] As can be seen indirectly in Figures 3 and 5, the Fc domain is composed of lysines 246 and 248. Confirm that the positions of SSFI that satisfy the conditions are 5, 6, 7, and 8 based on the following formula 6-3. Ta. [Formula 6-3] DCAWH-(Xa1)'-GELVWCT

[0434] In this case, the histidine at position 5 forms a salt bond with glutamic acid 380 in the Fc domain. Therefore, the histidine substitution may affect the interaction between SSFI and the Fc domain. Therefore, this does not satisfy the requirement of (3). Furthermore, the orientation of this side chain is different from that of lysine 246 and Since the glycine corresponding to position 7 is not close to 248, it does not satisfy the requirement of (2). However, the glycine residue was placed with the large (Xa1)' residue, which helps to form the bent structure of SSFI. It is not desirable to replace the glutamic acid at position 8 with arginine 255 of the Fc domain. The substitution of glutamic acid reduces the interaction between SSFI and the Fc domain by forming a salt bond with the Fc domain. Therefore, this does not satisfy the requirement of (3) (see Section 3.2). Since it satisfies all of the requirements 1), (2), and (3), it was determined that the 6-position is the most suitable for the (Xa1)' position. Therefore, the R1'-L2-SSFI of the present invention was completed based on these facts.

[0435] 5.4. The position of R1' transferred to the antibody may vary depending on the length of D2 of R1'-L1 and D3 of SSFI. do. The present disclosure is intended to explain the preferred design principles of the R1'-L2-SSFI according to the present invention. The design of 1'-L2-SSFI specifically transfers R1' to a specific lysine residue in the Fc domain. Furthermore, the present invention provides a method for preparing a preferred R1'-L2-SSFI by combining R1'-L1 and SSF This is intended to explain the design principles of I.

[0436] Those skilled in the art, having read Section 5.2 above, will recognize that the first carbonyl group of (Xa1)' of R1'-L2-SSFI is an Fc dopant. When the amine group of the main lysine is located adjacent to the amine group, a nucleophilic substitution reaction can occur. Based on the description in Section 3.2 above, one skilled in the art can also recognize that R1'-L2-SSFI , arranged with the Fc domain in a specific topology, and lysines 246 and 248 of the Fc domain It can be seen that the amine group is spaced a certain distance from the β carbon of (Xa1)'. By using this combination to design R1'-L2-SSFI, the β-carbon of (1)(Xa1)' can be The distance between the atom and the first carbonyl carbon (hereinafter referred to as "L c (2) specific topoisomers In the chemistry, the distance between the β carbon of (Xa1)' and the amine groups of lysines 246 and 248 of the Fc domain If the distance is the same as or similar to that of the desired lysine residue, it becomes possible to specifically label the desired lysine residue. This was expected.

[0437] The structure of R1'-L2-SSFI according to the present invention and L c As shown in Figure 8, D3, X3, carbon Atoms D2 and X1 are located between the β carbon of (Xa1)' and the first carbonyl group. Of these, D3 and X3 relate to the design of SSFI, and D2 and X1 relate to the design of L1-R1'.

[0438] For convenience, the present invention provides that D3 is C x alkylene, X3 is N, and D2 is C y Alkylene , C y Alkenylene, or C y alkynylene, X1 is S, and y is an integer of 1 or more. In this case, alkylene, alkenylene, and alkynylene are The linker structure shown in Figure 8 is c The software calculates the value The model was created using the software Discovery Studio. As a result, the x+y values ​​were determined. L c The values ​​are listed in Table 3. During the calculations, the chains are modeled at their maximum length. did.

[0439] [Table 3]

[0440] The following is provided to show the topology between R1'-L2-SSFI and the lysine residues of the Fc domain. The accompanying drawing is provided based on Figure 9. Figure 9 shows that the direction of the side chain of (Xa1)' (dotted line) is The topology between the R1'-L2-SSFI and Fc domains is shown parallel to the x-axis. As can be seen, the side chain of (Xa1)' is directed towards the amine groups of lysines 246 and 248 ( (See Section 5.3). Furthermore, the side chain of (Xa1)' is c Depending on the length of the It can be seen from the figure that the reaction is c The y-axis in the diagram along the exemplary length of 10 shows the view of FIG. 9 in a direction parallel to the arrow (thick solid arrow).

[0441] The distance between the β carbon of (Xa1)' and the amine groups of lysines 246 and 248 of the Fc domain (D 246,最小 , D 246,最大 , D 248,最小 , and D 248,最大 ) is described in Section 3.2. The β carbon of (Xa1)' is 246 The lysine residue at position L is closer to lysine 248 in the Fc domain. c Value is D 246,最小 Yo When the length is shorter than 100, the first carbonyl carbon reacts well with lysine 248 (Fig. 10), and L c Value is D 248,最大 In the longer case, the first carbonyl carbon reacts well with lysine 246 (Figure 11), and L c Value is D 246,最 小 That's all, D 248,最大 The first carbonyl carbon is selectively coupled to lysines 246 and 248 when: It was expected that the reaction would be responsive (Figure 12).

[0442] According to the present invention, a first chemical functional group is specifically transferred to lysine 248 of the Fc domain of an antibody. R1'-L2-SSFI for this purpose is disclosed (see Figure 11).

[0443] The present invention provides an agent for transferring a first chemical functional group to an antibody, the agent comprising: and the distance between the first carbonyl carbon (L c ) but D 246,最小 (approximately 11.668 Å) In this case, L c is approximately 6.5 Å, approximately 7 Å, approximately 8 Å, It may have a value of approximately 9 Å, approximately 10 Å, approximately 11 Å, or approximately 11.5 Å.

[0444] In a specific embodiment, R1'-L2-SSFI is selected from formulas 5, 5-1 to 5-3, and 6-1 to 6-3 If any one of the structures is present, D3 is C x alkylene, X3 is N, and D2 is C y a Lukiren, C y Alkenylene, or C y alkynylene, X1 is S, and y is 1 or more. x and y are integers, and the sum of x and y can be 1≦x+y≦5. Furthermore, the sum of x and y can be 1, 2, 3 , 4, or 5. For example, x can be 0 and y can be 1≦y≦5. In one specific embodiment, x can be 1 and y can be 1≦y≦4. In another specific embodiment, x can be 2 and y can be 1≦y≦3. , 3, and y can be 1≦y≦2. The corresponding numerical ranges are listed in Table 3. It is determined based on the value.

[0445] According to the present invention, a first chemical functional group is specifically transferred to lysine 246 of the Fc domain of an antibody. R1'-L2-SSFI for the purpose of

[0446] According to one aspect of the invention, the present invention provides an agent for transferring a first chemical functional group to an antibody. and the distance between the β carbon of (Xa1)′ and the first carbonyl carbon (L c ) but D 248,最大 (Oh For example, L c is approximately 16.5 Å , approximately 17 Å, approximately 18 Å, approximately 19 Å, approximately 20 Å, or approximately 20.5 Å .

[0447] In a specific embodiment, R1'-L2-SSFI is selected from formulas 5, 5-1 to 5-3, and 6-1 to 6-3 If any one of the structures is present, D3 is C x alkylene, X3 is N, and D2 is C y a Lukiren, C y Alkenylene, or C y alkynylene, X1 is S, and y is 1 or more. The sum of x and y is an integer, and may be 9 or greater. Furthermore, the sum of x and y may be 9, 10, 11, For example, x can be 0 and y can be 9≦y≦12. In an embodiment, x can be 1 and y can be 8≦y≦11. In another specific embodiment, x can be 2 and y can be 7≦y≦10. , 3, and y can be 6≦y≦9. Optionally, D2 can be alkynylene. When the side chain of (Xa1)' is an alkynylene, the side chain of (Xa1)' is sterically rigid, preventing bending of the side chain. It is possible.

[0448] According to the present invention, a first chemical functional group is selectively attached to lysine 246 or 248 of the Fc domain of an antibody. R1'-L2-SSFI for transfer is disclosed.

[0449] According to one aspect of the invention, the present invention provides an agent for transferring a first chemical functional group to an antibody. and the distance between the β carbon of (Xa1)′ and the first carbonyl carbon (L c ) but D 246,最小 (Oh (11.668Å) or more, D 248,最大 (approximately 16.208 Å) or less, In this case, L c is approximately 11.668 Å, approximately 12 Å, approximately 13 Å, approximately 14 Å, approximately 15 Å, approximately 15.5 Å, approximately 16 Å, or approximately 16.208 Å.

[0450] In a specific embodiment, R1'-L2-SSFI is selected from formulas 5, 5-1 to 5-3, and 6-1 to 6-3 If any one of the structures is present, D3 is C x alkylene, X3 is N, and D2 is C y a Lukiren, C y Alkenylene, or C y alkynylene, X1 is S, and y is 1 or more. The sum of x and y is an integer, and 6≦x+y≦8. In this case, the sum of x and y is 6, 7, or , or 8. For example, x can be 0 and y can be 6≦y≦8. In an embodiment, x can be 1 and y can be 5≦y≦7. In another specific embodiment, x can be 2 and y can be 4≦y≦6. and y may be 3≦y≦5.

[0451] According to the present invention, a first chemical functional group is specifically transferred to lysine 248 of the Fc domain of an antibody. A method for preparing R1'-L2-SSFI for the synthesis of SSFI is disclosed.

[0452] An example of a method for preparing an agent for transferring a first chemical functional group to an antibody, as described in Section 4.2. The present invention provides a method for preparing an R1'-L2-SSAI, comprising: Reacting a linker according to the present invention with a site-specific antibody interactome according to the present invention process Including, D2 of the linker is C y Alkylene, C y Alkenylene, or C y is alkynylene, D3 of the site-specific antibody interactome, C x is alkylene, y is an integer of 1 or more, and the sum of x and y is 1≦x+y≦5 Furthermore, the R1'-L2-SSAI prepared by the method is an anti- reacting with the antibody to specifically transfer a first chemical functional group to lysine 248 of the Fc domain of the antibody. This can be done.

[0453] According to the present invention, a first chemical functional group is specifically transferred to lysine 248 of the Fc domain of an antibody. A kit for preparing R1'-L2-SSFI for use in the synthesis of SSFI is disclosed.

[0454] An example of a method for preparing an agent 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, comprising: a linker according to the present invention; and Site-specific antibody interactome according to the present invention Including, D2 of the linker is C y Alkylene, C y Alkenylene, or C y is alkynylene, D3 of the site-specific antibody interactome, C x is alkylene, y is an integer of 1 or more, and the sum of x and y is 1≦x+y≦5 A kit is provided, which comprises:

[0455] According to the present invention, a first chemical functional group is specifically transferred to lysine 246 of the Fc domain of an antibody. A method for preparing R1'-L2-SSFI for the synthesis of SSFI is disclosed.

[0456] An example of a method for preparing an agent for transferring a first chemical functional group to an antibody, as described in Section 4.2. The present invention provides a method for preparing an R1'-L2-SSAI, comprising: Reacting a linker according to the present invention with a site-specific antibody interactome according to the present invention process Including, D2 of the linker is C y Alkylene, C y Alkenylene, or C y is alkynylene, D3 of the site-specific antibody interactome, C x is alkylene, y is an integer of 1 or more, and the sum of x and y is 9≦x+y≦12 Furthermore, the R1'-L2-SSAI prepared by the method is an anti- reacting with the antibody to specifically transfer a first chemical functional group to lysine 246 of the Fc domain of the antibody. This can be done.

[0457] According to the present invention, a first chemical functional group is specifically transferred to lysine 246 of the Fc domain of an antibody. A kit for preparing R1'-L2-SSFI for use in the synthesis of SSFI is disclosed.

[0458] An example of a method for preparing an agent 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, comprising: a linker according to the present invention; and Site-specific antibody interactome according to the present invention Including, D2 of the linker is C y Alkylene, C y Alkenylene, or C y is alkynylene, D3 of the site-specific antibody interactome, C x is alkylene, y is an integer of 1 or more, and the sum of x and y is 9≦x+y≦12 A kit is provided, which comprises:

[0459] According to the present invention, a first chemical functional group is selectively attached to lysine 246 or 248 of the Fc domain of an antibody. A method for preparing R1'-L2-SSFI for transfer is disclosed.

[0460] An example of a method for preparing an agent for transferring a first chemical functional group to an antibody, as described in Section 4.2. The present invention provides a method for preparing an R1'-L2-SSAI, comprising: Reacting a linker according to the present invention with a site-specific antibody interactome according to the present invention process Including, D2 of the linker is C y Alkylene, C y Alkenylene, or C y is alkynylene, D3 of the site-specific antibody interactome, C x is alkylene, y is an integer of 1 or more, and the sum of x and y is 6≦x+y≦8 Furthermore, the R1'-L2-SSAI prepared by the method is an anti- reacting with the antibody to selectively transfer a first chemical functional group to lysine 246 or 248 of the Fc domain of the antibody; It can be done.

[0461] According to the present invention, a first chemical functional group is selectively attached to lysine 246 or 248 of the Fc domain of an antibody. A kit for preparing R1'-L2-SSFI for transfer is disclosed.

[0462] An example of a method for preparing an agent 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, comprising: a linker according to the present invention; and Site-specific antibody interactome according to the present invention Including, D2 of the linker is C y Alkylene, C y Alkenylene, or C y is alkynylene, D3 of the site-specific antibody interactome, C x is alkylene, y is an integer of 1 or more, and the sum of x and y is 6≦x+y≦8 A kit is provided, which comprises:

[0463] According to the present invention, a first chemical functional group is specifically transferred to lysine 248 of the Fc domain. Disclosed are methods for preparing antibodies having the

[0464] As an example of a method for preparing an antibody containing a first chemical functionality as described in Section 5.2, A method for preparing an R1'-Ab, comprising: The agent for transferring the first chemical functional group to the antibody according to the present invention is combined with the antibody or fragment thereof. Reaction process Including, The agent for transferring the first chemical functional group to the antibody is a compound that binds the β carbon of (Xa1)' to the first carbonyl group. The distance between the carbon atoms (L c ) but shorter than approximately 11.668 Å The present invention provides a method characterized by:

[0465] In a specific embodiment, the agent for transferring a first chemical functionality to an antibody comprises D3 is C x alkylene, and D2 is C y Alkylene, C y Alkenylene, or C y Alkynylene can be, y can be an integer greater than or equal to 1, and the sum of x and y can be 1≦x+y≦5.

[0466] According to the present invention, a first chemical functional group is specifically transferred to lysine 248 of the Fc domain. A kit for preparing an antibody having the same is disclosed.

[0467] As an example of a method for preparing an antibody containing a first chemical functionality as described in Section 5.2, A kit for preparing R1'-Ab, comprising: an agent for transferring a first chemical functionality to an antibody according to the invention; and Antibodies or fragments thereof Including, The agent for transferring the first chemical functional group to the antibody is a compound that binds the β carbon of (Xa1)' to the first carbonyl group. The distance between the carbon atoms (L c ) but shorter than approximately 11.668 Å A kit is provided, which comprises:

[0468] In a specific embodiment, the agent for transferring a first chemical functionality to an antibody comprises D3 is C x alkylene, and D2 is C y Alkylene, C y Alkenylene, or C y Alkynylene can be, y can be an integer greater than or equal to 1, and the sum of x and y can be 1≦x+y≦5.

[0469] Optionally, as an example of a method for preparing an antibody containing a first chemical functionality as described in Section 5.2, The present invention provides a kit for preparing R1'-Ab, comprising: a linker according to the present invention; a site-specific antibody interactome according to the present invention; and Antibodies or fragments thereof Including, D2 of the linker is C y Alkylene, C y Alkenylene, or C y is alkynylene, D3 of the site-specific antibody interactome, C x is alkylene, y is an integer of 1 or more, and the sum of x and y is 1≦x+y≦5 A kit is provided, which comprises:

[0470] According to the present invention, a first chemical functional group is specifically transferred to lysine 246 of the Fc domain. Disclosed are methods for preparing antibodies having the

[0471] As an example of a method for preparing an antibody containing a first chemical functionality as described in Section 5.2, A method for preparing an R1'-Ab, comprising: The antibody or fragment thereof is combined with an agent for transferring a first chemical functional group to the antibody according to the invention. A process for enabling the reaction Including, The agent for transferring the first chemical functional group to the antibody is a compound that binds the β carbon of (Xa1)' to the first carbonyl group. The distance between the carbon atoms (L c ) but longer than approximately 16.208 Å The present invention provides a method characterized by:

[0472] In a specific embodiment, the agent for transferring a first chemical functionality to an antibody comprises D3 is C x alkylene, and D2 is C y Alkylene, C y Alkenylene, or C y Alkynylene can be, y may be an integer greater than or equal to 1, and the sum of x and y may be 9≦x+y≦12.

[0473] According to the present invention, a first chemical functional group is specifically transferred to lysine 246 of the Fc domain. A kit for preparing an antibody having the same is disclosed.

[0474] As an example of a method for preparing an antibody containing a first chemical functionality as described in Section 5.2, A kit for preparing R1'-Ab, comprising: an agent for transferring a first chemical functionality to an antibody according to the invention; and Antibodies or fragments thereof Including, The agent for transferring the first chemical functional group to the antibody is a compound that binds the β carbon of (Xa1)' to the first carbonyl group. The distance between the carbon atoms (L c ) but longer than approximately 16.208 Å A kit is provided, which comprises:

[0475] In a specific embodiment, the agent for transferring a first chemical functionality to an antibody comprises D3 is C x alkylene, and D2 is C y Alkylene, C y Alkenylene, or C y Alkynylene can be, y may be an integer greater than or equal to 1, and the sum of x and y may be 9≦x+y≦12.

[0476] Optionally, as an example of a method for preparing an antibody containing a first chemical functionality as described in Section 5.2, The present invention provides a kit for preparing R1'-Ab, comprising: a linker according to the present invention; a site-specific antibody interactome according to the present invention; and Antibodies or fragments thereof Including, D2 of the linker is C y Alkylene, C y Alkenylene, or C y is alkynylene, D3 of the site-specific antibody interactome, C x is alkylene, y is an integer of 1 or more, and the sum of x and y is 9≦x+y≦12 A kit is provided, which comprises:

[0477] According to the present invention, a first chemical bond selectively transferred to lysine 246 or 248 of the Fc domain A method for preparing an antibody having a functional group is disclosed.

[0478] As an example of a method for preparing an antibody containing a first chemical functionality as described in Section 5.2, A method for preparing an R1'-Ab, comprising: The agent for transferring the first chemical functional group to the antibody according to the present invention is combined with the antibody or fragment thereof. Reaction process Including, The agent for transferring the first chemical functional group to the antibody is a compound that binds the β carbon of (Xa1)' to the first carbonyl group. The distance between the carbon atoms (L c ) is greater than or equal to approximately 11.668 Å and less than or equal to approximately 16.208 Å The present invention provides a method characterized by:

[0479] In a specific embodiment, the agent for transferring a first chemical functionality to an antibody comprises D3 is C x alkylene, and D2 is C y Alkylene, C y Alkenylene, or C y Alkynylene can be, y may be an integer greater than or equal to 1, and the sum of x and y may be 6≦x+y≦8.

[0480] According to the present invention, a first chemical bond selectively transferred to lysine 246 or 248 of the Fc domain A kit for preparing functionalized antibodies is disclosed.

[0481] As an example of a method for preparing an antibody containing a first chemical functionality as described in Section 5.2, A kit for preparing R1'-Ab, comprising: an agent for transferring a first chemical functionality to an antibody according to the invention; and Antibodies or fragments thereof Including, The agent for transferring the first chemical functional group to the antibody is a compound that binds the β carbon of (Xa1)' to the first carbonyl group. The distance between the carbon atoms (L c ) is greater than or equal to approximately 11.668 Å and less than or equal to approximately 16.208 Å A kit is provided, which comprises:

[0482] In a specific embodiment, the agent for transferring a first chemical functionality to an antibody comprises D3 is C x alkylene, and D2 is C y Alkylene, C y Alkenylene, or C y Alkynylene can be, y may be an integer greater than or equal to 1, and the sum of x and y may be 6≦x+y≦8.

[0483] Optionally, as an example of a method for preparing an antibody containing a first chemical functionality as described in Section 5.2, The present invention provides a kit for preparing R1'-Ab, comprising: a linker according to the present invention; a site-specific antibody interactome according to the present invention; and Antibodies or fragments thereof Including, D2 of the linker is C y Alkylene, C y Alkenylene, or C y is alkynylene, D3 of the site-specific antibody interactome, C x is alkylene, y is an integer of 1 or more, and the sum of x and y is 6≦x+y≦8 A kit is provided, which comprises:

[0484] According to the present invention, a first chemical moiety is transferred to both lysines 246 and 248 of the Fc domain. Methods for preparing functionalized antibodies are disclosed.

[0485] The present invention provides a method for preparing an R1'-Ab, comprising the steps of: A first agent for transferring a first chemical functional group to the antibody is reacted with the antibody or fragment thereof. and A second agent for transferring a first chemical functional group to the antibody is reacted with the antibody or fragment thereof. Process The present invention provides a method comprising:

[0486] In a specific embodiment, the first agent for transferring the first chemical functionality to the antibody is a compound as described above. R1'-L2-SSFI for specifically transferring the first chemical functional group to lysine 248 of the Fc domain. Possibly, the second agent for transferring the first chemical functional group to the antibody is a compound similar to the first chemical functional group described above. It can be R1'-L2-SSFI to specifically transfer the group to lysine 246 of the Fc domain.

[0487] In a specific embodiment, the first agent for transferring the first chemical functionality to the antibody is a compound as described above. R1'-L2-SSFI for specifically transferring the first chemical functional group to lysine 246 of the Fc domain. Possibly, the second agent for transferring the first chemical functional group to the antibody is a compound similar to the first chemical functional group described above. It can be R1'-L2-SSFI to specifically transfer the group to lysine 248 of the Fc domain.

[0488] In a specific embodiment, the first agent for transferring the first chemical functional group to the antibody is or a fragment thereof; and reacting a second chemical functional group with the antibody to transfer the first chemical functional group to the antibody. The steps of reacting the agent with the antibody or fragment thereof may be performed sequentially.

[0489] According to the present invention, a first chemical moiety is transferred to both lysines 246 and 248 of the Fc domain. A kit for preparing functionalized antibodies is disclosed.

[0490] The present invention provides a kit for preparing R1'-Ab, comprising: a first agent for transferring a first chemical functional group to the antibody; a second agent for transferring the first chemical functional group to the antibody; and Antibodies or fragments thereof A kit comprising:

[0491] In a specific embodiment, the first agent for transferring the first chemical functionality to the antibody is a compound as described above. R1'-L2-SSFI for specifically transferring the first chemical functional group to lysine 248 of the Fc domain. Possibly, the second agent for transferring the first chemical functional group to the antibody is a compound similar to the first chemical functional group described above. It can be R1'-L2-SSFI to specifically transfer the group to lysine 246 of the Fc domain.

[0492] In a specific embodiment, the first agent for transferring the first chemical functionality to the antibody is a compound as described above. R1'-L2-SSFI for specifically transferring the first chemical functional group to lysine 246 of the Fc domain. Possibly, the second agent for transferring the first chemical functional group to the antibody is a compound similar to the first chemical functional group described above. It can be R1'-L2-SSFI to specifically transfer the group to lysine 248 of the Fc domain.

[0493] 5.5. Modifying the reactivity of the first carbonyl group makes the first chemical functional group more site-specific It becomes possible to transfer to As described above in Section 5.2, antibodies containing a first chemical functionality may be prepared by first chemistry of R1'-L2-SSFI. The first carbonyl group can be prepared by the nucleophile of the antibody attacking the functional group. Characterized by being less reactive than the second carbonyl group of the linker (see Section 2.2) The reactivity of the first carbonyl group is such that certain amine groups on the antibody react with certain amine groups on the antibody. The group can react with the first carbonyl group only if it is in close proximity to the first carbonyl group. Thus, the present invention provides a method for determining the first color by the neighborhood conditions for the reaction. This allows the carbonyl group to react poorly with any amine group, resulting in high regiospecificity of the reaction. This makes it possible.

[0494] The prior art disclosed in application numbers US 2018 / 0141976 A1 and WO 2018 / 199337 A1 does not disclose Fc-III The aim was to site-selectively modify lysine 246 or 248 of the Fc domain using analogs of Since the prior art uses a disuccinimidyl crosslinker, the first carbonyl group and The first and second carbonyl groups are equally highly reactive (see Section 2.2). Since the lysine group is highly reactive and has no proximity requirements for reaction, Residues are also likely to be labeled.

[0495] 5.6. Antibodies containing a first chemical functionality prepared in accordance with the present invention are highly homogeneous and highly productive. It has. (1) A desired number of first chemical functional groups are specifically transferred to (2) a specific site on the antibody. The technical challenges and technical solutions to achieve this are detailed in Sections 5.3 and 5.4. As can be seen from the following examples, the first chemical functional group-containing polymer prepared by the technical solution The chemical functional groups of the antibody that correspond to the Fc domain are transferred to specific lysine residues in high homogeneity and yield. Therefore, the present invention was completed based on these facts. did.

[0496] As described in the background art, highly homogeneous conjugate products obtained by antibody labeling can be obtained. (1) The function of the antibody conjugate is uniformly guaranteed. (2) The antibody conjugate is (3) It is safe because the effect is predictable, and (4) it labels antibodies while avoiding the functional regions of the antibodies. This has the advantage that it is possible to avoid a decrease in antibody function.

[0497] The prior art disclosed in application numbers US 2018 / 0141976 A1 and WO 2018 / 199337 A1 does not disclose Fc-III The aim was to site-selectively modify lysine 246 or 248 of the Fc domain using analogs of However, according to the prior art, the homogeneity and yield of antibodies are essential for two reasons. According to the prior art, first, the distance between the β carbon of (Xa1)' and the first carbonyl carbon is away(L c ) is approximately 15 Å, so lysine can be selectively labeled at either position 246 or 248. However, it is possible to specifically select and label one of lysines 246 and 248. Second, the first carbonyl group of the prior art crosslinker is highly reactive. Therefore, there is no proximity requirement for the reaction (see Section 5.5). As a result, the first carbonyl group Therefore, the prior art has not been able to produce antibody labels. However, the method has the drawback that the uniformity and yield of the product are inferior to those of the present invention.

[0498] 5.7. Antibodies containing a first chemical functionality according to the present invention have their FcRn binding site blocked Therefore, the function of the antibody is not reduced. (1) distant from the paratope; and (2) distant from the recognition site of the FcR, including FcRn. The design principles for the labeling site are described above in Section 1.1. Lysines 246 and 248 are located in the Fc domain. Because they are included in the main domain, lysines 246 and 248 are spaced from the paratope but are located at the corresponding The site may overlap with the recognition site of FcRn, which may be problematic.

[0499] FcRn has various functions, particularly in relation to IgG recycling, which reduces the half-life of antibodies. It is known that it plays an important role in prolonging the life of antibodies. That is, when an antibody is used as a therapeutic agent, an imaging agent, etc., the interaction between FcRn and the antibody is circular. This does not occur smoothly, and as a result, the antibody's short half-life makes it difficult for the antibody to function normally. It becomes possible.

[0500] 5.7.1. Lysines 246 and 248 are Distant from the FcRn Binding Site of the Antibody The distance between the lysine residue of the Fc domain and the FcRn-binding site of Fc is Figure 13 shows the binding structure between the Fc domain and FcRn. The positions of the FcRn binding site and lysines 246 and 248 in the Fc domain are also shown. Computer modeling was used to show the binding structure between the Fc domain and FcRn (Ying et al. T, Ju TW, Wang Y, Prabakaran P, Dimitrov DS., Interactions of IgG1 CH2 and CH3 D omains with FcRn; Front Immunol. 2014; 5:146, Monnet C, Jorieux S, Urbain R etc., Selection of IgG Variants with Increased FcRn Binding Using Random and Direc ted Mutagenesis: Impact on Effector Functions. Front Immunol. 2015; 6:39) (Figure (See the left figure of 13). The FcRn binding site and lysines 246 and 248 are also shown in the same paper. is shown in the Fc domain (see the right diagram of Figure 13). It can be seen that the side chain of 8 faces away from the FcRn binding site.

[0501] From these facts, when selecting antibody labeling sites, the antibody labeling site should be selected between FcRn and Fc domains. In fact, the antibody labeling site was designed to The half-life of the prepared antibody must not be shortened so as not to affect FcRn binding as expected. was confirmed (see Example 5).

[0502] 5.8. A process for preparing an antibody containing a first chemical functionality according to the present invention is characterized by SSAI elimination. It is a sign. As can be seen from Schemes 4 and 5 in Section 5.2, the process for preparing R1'-Ab according to the present invention is Since SSAI is included in the leaving group of the nucleophilic substitution reaction, SSAI is eliminated from the final product R1'-Ab. The binding structure between the Fc domain and the SSFI according to the present invention (see Section 3.2) is characterized by the following: The binding site between FcRn and FcRn was analyzed by comparing it with that between FcRn and FcRn. It was confirmed that the SSFI covers the R1'-Ab (Fig. 14). If not released during this process, SSFIs can adversely affect the physical properties of antibodies (e.g., half-life, etc.). It can be expected to have an impact.

[0503] The prior art disclosed in application number US 2018 / 0141976 A1 uses an analog of Fc-III to inhibit Fc The aim is to site-selectively modify lysine 246 or 248 of the domain. In the prior art, SSFI is not released during the preparation process, so SSFI is not released into the final antibody-labeled product. The present invention differs from the present invention in that

[0504] The prior art disclosed in application WO 2018 / 199337 A1 uses analogs of Fc-III, where III The purpose of this method is to site-selectively modify lysine 246 or 248 of the Fc domain. However, the corresponding prior art does not include SS in the preparation process. The method further includes a step of cleaving the "divalent cleavable linker" to remove the FI, The present invention provides a method for eliminating SSFI during the conjugation reaction without any additional process. Moreover, the cleavable linker, which is a divalent group according to the corresponding invention, Cleavage has the disadvantage that the disulfide bonds that form the structure of the antibody may be broken. This problem is solved because the invention uses a nucleophilic substitution reaction that occurs easily without any special conditions. can be dramatically improved.

[0505] 5.9. Antibodies containing a first click-reactive functional group according to the present invention can be synthesized into highly bioreactive chemistries. Since it does not contain any functional groups, no side reactions occur other than the conjugate formation reaction. The antibody prepared by the method for preparing R1'-Ab according to the present invention has the formula 7:

[0506] [ka]

[0507] wherein R1' is transferred to lysine 246 or 248, X4 can be NH, and D1 is any alkylene (which may be aryl, alkenylene, or alkynylene) In this case, the -NH-(CO)- bonded to the antibody is generally stable in vivo. Moreover, D1 generally has a structure that is not highly bioreactive. Therefore, the highly bioreactive structure is not added to any molecules other than the labeled molecule (R1'). It is expected that the R1'-Ab prepared according to the invention will have a high safety profile.

[0508] In particular, when the R1'-Ab is an H1-Ab with a click-reactive residue transferred to it, Therefore, the yield of the click chemistry reaction is This can be improved because secondary reactions other than the black chemical reaction do not occur.

[0509] The prior art disclosed in application WO 2018 / 199337 A1 uses an analog of Fc-III to inhibit Fc Lysine 246 or 248 of the domain was site-selectively modified to introduce a bioorthogonal functional group. However, the corresponding prior art is intended to use antibodies according to the corresponding prior art. The product is formed by cleaving the "divalent group cleavable linker" while cleaving the thiol, hydroxyl, , carboxylic acid, phosphoric acid, amine, etc. This is different from the present invention. Such additional chemical functional groups are bioreactive functional groups, and therefore, This may affect the safety of the product.

[0510] 6. Payload (C m -H2) In accordance with the present invention, payloads are disclosed. Such compounds are referred to herein by the symbol "C m -H2".

[0511] The present application provides a compound of formula 9: [Formula 9] C m -H2 (In the formula, C m is the cargo part, H2 is the second click-reactive functional group C, represented by m -Provide H2.

[0512] In a specific embodiment, C m comprises a carrier moiety, a fluorescent moiety, a drug moiety, or a radioactive moiety Moreover, R1' may include a drug moiety. In addition, R1' may include a VC linker. In a specific embodiment, R1' may comprise an antibody or analog thereof comprising a paratope.

[0513] In a specific embodiment, C m contains a drug moiety, the drug moiety may be an anti-cancer drug In addition, anticancer drugs include DM1, DM3, DM4, abrin, ricin A, Pseudomonas exotoxin, and Relatin, diphtheria toxin, tumor necrosis factor, α-interferon, β-interferon , nerve growth factor, platelet-derived growth factor, tissue plasminogen activator, cytokines, Apoptosis inducers, antiangiogenic agents, lymphokines, taxanes, DNA-alkylating agents, Tracycline, tubulysin analogues, duocarmycin analogues, auristatin E, Uristatin F, a maytansinoid, contains a reactive polyethylene glycol residue and is a cytotoxic Agents, Taxon, Cytochalasin B, Gramicidin D, Ethidium Bromide, Emetine, Mitoma Isin, etoposide, tenoposide, vincristine, vinblastine, colchicine, Xorubicin, daunorubicin, dihydroxyanthracin dione, mitoxantrone, Mithramycin, actinomycin D, 1-dihydrotestosterone, glucocorticoids , procaine, tetracaine, lidocaine, propranolol, puromycin, mesothelioma Trexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil Decarbazine, mechlorethamine, thiotepa, chlorambucil, melphalan, carmustine fluticasone, lomustine, cyclophosphamide, busulfan, dibromomannitol, streptavidin Tozotocin, mitomycin C, cisplatin, dactinomycin, bleomycin, thramycin, calicheamicin, abiraterone, bendamustine, bortezomib, calcitriol, Boplatin, cabazitaxel, dasatinib, docetaxel, epirubicin, erlotinib , everolimus, gemcitabine, gefitinib, idarubicin, imatinib, hydroxybenzoates Urea, lapatinib, leuprorelin, melphalan, nedaplatin, nilotinib, oxaliplatin Saliplatin, pazopanib, pemetrexed, picoplatin, romidepsin, satrap tinib, sorafenib, vemurafenib, sunitinib, teniposide, triplatin, and bilephrine Norelbine.

[0514] In a specific embodiment, C m is a plurality of carrier moieties, fluorescent moieties, drug moieties, or radioactive moieties Furthermore, C m may contain two or more drug moieties. m is the VC linker may include:

[0515] In specific embodiments, H2 is an alkyne, an azide, a strained alkyne, a diene, a dienophenoxy group, or a hydroxy group. The compound may include any one selected from the group consisting of olefins, alkenes, thiols, and tetrazines. , H2 can be an azide or a strained alkyne. Additionally, H2 can be a diene or dienophile. Furthermore, H2 can be tetrazine or norbornene. Or it can be trans-cyclooctene.

[0516] In a specific embodiment, when H2 reacts with an H1-Ab according to the present invention, H2 reacts with the first click. The reactive functional group (H1) may be complementary to the reactive functional group (H1).

[0517] 7. Antibody-payload conjugates (C m -Ab) According to the present invention, a conjugate of an antibody and a payload (i.e., an antibody-payload conjugate) is Such compounds are referred to herein by the symbol "C m -Ab" .

[0518] The present application provides a compound of formula 10:

[0519] [ka]

[0520] (In the formula, C m is the cargo part, B is a group that initiates a click chemistry reaction between the first click reactive functional group and the second click reactive functional group. It is a structure formed by reaction, D1 is any alkylene, alkenylene, or alkynylene; X4 is NH, O, or S; Ab is antibody) C, represented by m -Provide Ab.

[0521] The contents of the cargo portion are as disclosed in Section "6. Payload."

[0522] In specific embodiments, B is an alkyne, an azide, a strained alkyne, a diene, a dienophenoxy group, or a hydroxy group. Any one selected from the group consisting of olefins, alkenes, thiols, and tetrazines, and its partner - a structure formed by a click chemistry reaction between a "click reactive functional group" Moreover, B

[0523] [ka]

[0524] where both A1 and A2 are connected to a cargo moiety or D1 such that A1 and A2 are not connected to the same moiety. Connect with R x is H, halogen, and C 1~3 alkyl) Furthermore, B can be any one selected from

[0525] [ka]

[0526] In addition, B can be

[0527] [ka]

[0528] It is possible.

[0529] In specific embodiments, D1 is a covalent bond, C 1~4 Alkylene, C 2~4 Alkenylene, C 2~ 4 alkynylene, and C 3~8 cycloalkylene. where D1 can be -CH2OCH2-. Additionally, D1 can be a covalent bond.

[0530] In one specific embodiment, X4 can be NH.

[0531] In specific embodiments, the Ab can be a human antibody. In other specific embodiments, the Ab can be In a specific embodiment, the Ab is an immunoglobulin G (IgG). In a specific embodiment, the Ab may be a complete antibody. Alternatively, the Ab may be a fragment of an antibody.

[0532] In a specific embodiment, X4 and Ab can be connected via the Fab domain of the Ab. In another specific embodiment, X4 and the Ab can be connected via the Fc domain of the Ab. Furthermore, X4 and Ab can be connected via lysine 246 or 248 in the Fc domain of Ab. Additionally, X4 and Ab can be connected via lysine 246 in the Fc domain of the Ab. Additionally, X4 and Ab can be connected via lysine 248 of the Fc domain of the Ab. X4 and Ab can be connected via lysines 246 and 248 of the Fc domain of the Ab. In a typical embodiment, X4 and the Ab may be connected via only one of the two Fc domains of the Ab. In another specific embodiment, X4 and Ab bind to each other via both of the two Fc domains of the Ab. can be connected.

[0533] The present invention relates to a compound of the following formula 10-1: [Formula 10-1] GPSVFLFPP-(K)"-PKDTLMI (SEQ ID NO: 23) wherein G is glycine, P is proline, S is serine, and V is valine. where F is phenylalanine, L is leucine, K is lysine, and D is , is aspartic acid, T is threonine, M is methionine, and I is isoform is leucine, (K)" is

[0534] [ka]

[0535] (In the formula, C m is the cargo part, B is a group that initiates a click chemistry reaction between the first click reactive functional group and the second click reactive functional group. It is a structure formed by reaction, D1 is any alkylene, alkenylene, or alkynylene. The antibody or fragment thereof comprises the amino acid sequence of the Fc domain. a cargo moiety that conjugates to lysine 246 of the ribozyme or a moiety corresponding to lysine 246 .

[0536] The contents of the cargo portion are as disclosed in Section "6. Payload."

[0537] In specific embodiments, B is an alkyne, an azide, a strained alkyne, a diene, a dienophenoxy group, or a hydroxy group. Any one selected from the group consisting of olefins, alkenes, thiols, and tetrazines, and its partner - a structure formed by a click chemistry reaction between a "click reactive functional group" Moreover, B

[0538] [ka]

[0539] where both A1 and A2 are connected to a cargo moiety or D1 such that A1 and A2 are not connected to the same moiety. Connect with R x is H, halogen, and C 1~3 alkyl) Furthermore, B can be any one selected from

[0540] [ka]

[0541] In addition, B can be

[0542] [ka]

[0543] It is possible.

[0544] In specific embodiments, D1 is a covalent bond, C 1~4 Alkylene, C 2~4 Alkenylene, C 2~ 4 alkynylene, and C 3~8 cycloalkylene. where D1 can be -CH2OCH2-. Additionally, D1 can be a covalent bond.

[0545] In specific embodiments, the antibody may be a human antibody. The antibody may be a non-human animal antibody. In a specific embodiment, the antibody is an immunoglobulin G (Ig In specific embodiments, the antibody may be a whole antibody. In embodiments, the antibody may be a fragment of an antibody.

[0546] In a specific embodiment, the antibody contains the amino acid sequence of formula 10-1 in only one of its two Fc domains. In another specific embodiment, the antibody may comprise both of its two Fc domains. The amino acid sequence may include the amino acid sequence of formula 10-1.

[0547] The present invention relates to a compound of the following formula 10-2: [Formula 10-2] GPSVFLFPPKP-(K)"-DTLMI (SEQ ID NO: 24) wherein G is glycine, P is proline, S is serine, and V is valine. where F is phenylalanine, L is leucine, K is lysine, and D is , is aspartic acid, T is threonine, M is methionine, and I is isoform is leucine, (K)" is

[0548] [ka]

[0549] (In the formula, C m is the cargo part, B is a group that initiates a click chemistry reaction between the first click reactive functional group and the second click reactive functional group. It is a structure formed by reaction, D1 is any alkylene, alkenylene, or alkynylene. The antibody or fragment thereof comprises the amino acid sequence of the Fc domain. a cargo moiety that conjugates to lysine 246 of the ribozyme or a moiety corresponding to lysine 246 .

[0550] The contents of the cargo portion are as disclosed in Section "6. Payload."

[0551] In specific embodiments, B is an alkyne, an azide, a strained alkyne, a diene, a dienophenoxy group, or a hydroxy group. Any one selected from the group consisting of olefins, alkenes, thiols, and tetrazines, and its partner - a structure formed by a click chemistry reaction between a "click reactive functional group" Moreover, B

[0552] [ka]

[0553] where both A1 and A2 are connected to a cargo moiety or D1 such that A1 and A2 are not connected to the same moiety. Connect with R x is H, halogen, and C 1~3 alkyl) Furthermore, B can be any one selected from

[0554] [ka]

[0555] In addition, B can be

[0556] [ka]

[0557] It is possible.

[0558] In specific embodiments, D1 is a covalent bond, C 1~4 Alkylene, C 2~4 Alkenylene, C 2~ 4 alkynylene, and C3~8 cycloalkylene. where D1 can be -CH2OCH2-. Additionally, D1 can be a covalent bond.

[0559] In specific embodiments, the antibody may be a human antibody. The antibody may be a non-human animal antibody. In a specific embodiment, the antibody is an immunoglobulin G (Ig In specific embodiments, the antibody may be a whole antibody. In embodiments, the antibody may be a fragment of an antibody.

[0560] In a specific embodiment, the antibody contains the amino acid sequence of formula 10-2 in only one of its two Fc domains. In another specific embodiment, the antibody may comprise both of its two Fc domains. The amino acid sequence may include the amino acid sequence of formula 10-2.

[0561] The present invention relates to a compound of formula 10-3: [Formula 10-3] GPSVFLFPP-(K)"-P-(K)"-DTLMI (SEQ ID NO: 25) wherein G is glycine, P is proline, S is serine, and V is valine. where F is phenylalanine, L is leucine, K is lysine, and D is , is aspartic acid, T is threonine, M is methionine, and I is isoform is leucine, (K)" are each independently

[0562] [ka]

[0563] (In the formula, C m is the cargo part, B is a group that initiates a click chemistry reaction between the first click reactive functional group and the second click reactive functional group. It is a structure formed by reaction, D1 is any alkylene, alkenylene, or alkynylene. The antibody or fragment thereof comprises the amino acid sequence of the Fc domain. a cargo moiety that conjugates to lysine 246 of the ribozyme or a moiety corresponding to lysine 246 .

[0564] The contents of the cargo portion are as disclosed in Section "6. Payload."

[0565] In specific embodiments, B is an alkyne, an azide, a strained alkyne, a diene, a dienophenoxy group, or a hydroxy group. Any one selected from the group consisting of olefins, alkenes, thiols, and tetrazines, and its partner - a structure formed by a click chemistry reaction between a "click reactive functional group" Moreover, B

[0566] [ka]

[0567] where both A1 and A2 are connected to a cargo moiety or D1 such that A1 and A2 are not connected to the same moiety. Connect with R x is H, halogen, and C 1~3 alkyl) Furthermore, B can be any one selected from

[0568] [ka]

[0569] In addition, B can be

[0570] [ka]

[0571] It is possible.

[0572] In specific embodiments, D1 is a covalent bond, C 1~4 Alkylene, C 2~4 Alkenylene, C 2~ 4 alkynylene, and C 3~8 cycloalkylene. where D1 can be -CH2OCH2-. Additionally, D1 can be a covalent bond.

[0573] In specific embodiments, the antibody may be a human antibody. The antibody may be a non-human animal antibody. In a specific embodiment, the antibody is an immunoglobulin G (Ig In specific embodiments, the antibody may be a whole antibody. In embodiments, the antibody may be a fragment of an antibody.

[0574] In a specific embodiment, the antibody contains the amino acid sequence of formula 10-3 in only one of its two Fc domains. In another specific embodiment, the antibody may comprise both of its two Fc domains. The amino acid sequence may include the amino acid sequence of formula 10-3.

[0575] The present invention comprises one or more amino acid sequences selected from formulas 10-1, 10-2, and 10-3. An antibody or a fragment thereof is provided, in which the contents of formulas 10-1 to 10-3 are as described above.

[0576] In specific embodiments, D1 can be a covalent bond.

[0577] In a specific embodiment, the antibody or fragment thereof comprises only the amino acid sequence of formula 10-1, and Furthermore, the antibody or fragment thereof does not contain the amino acid sequences of 10-2 and 10-3. In addition, an antibody may contain the amino acid sequence of Formula 10-1 in only one of its Fc domains. Both domains may contain the amino acid sequence of Formula 10-1.

[0578] In other specific embodiments, the antibody or fragment thereof comprises only the amino acid sequence of Formula 10-2: The antibody or fragment thereof does not include the amino acid sequences of formulas 10-1 and 10-3. In addition, the antibody may contain the amino acid sequence of Formula 10-2 in only one of the domains. The Fc domain of each of the antibodies may comprise the amino acid sequence of Formula 10-2.

[0579] In a specific embodiment, the antibody or fragment thereof comprises only the amino acid sequence of formula 10-3, and Furthermore, the antibody or fragment thereof does not contain the amino acid sequences of 10-1 and 10-2. In addition, an antibody may contain the amino acid sequence of Formula 10-3 in only one of its Fc domains. Both domains may contain the amino acid sequence of formula 10-3.

[0580] 7.1. Antibody Drug Conjugates (ADCs) According to the present invention, a novel antibody drug conjugate (ADC) is disclosed. means that the payload in the antibody-payload conjugate comprises a drug moiety.

[0581] The present invention relates to a C carboxylate according to the present invention, wherein the cargo moiety comprises a drug moiety. m -Provide Ab.

[0582] In specific embodiments, the cargo moiety may comprise two or more drug moieties.

[0583] In a specific embodiment, the drug moiety can be an anti-cancer drug. Additionally, the anti-cancer drug can be a DM 1, DM3, DM4, abrin, ricin A, Pseudomonas exotoxin, cholera toxin, diphtheria toxin , tumor necrosis factor, α-interferon, β-interferon, nerve growth factor, platelet-derived Growth factors, tissue plasminogen activators, cytokines, apoptosis inducers, anti-inflammatory drugs Angiogenic agents, lymphokines, taxanes, DNA-alkylating agents, anthracyclines, tubulin maytansin analogs, duocarmycin analogs, auristatin E, auristatin F, maytansin Cytotoxic agents containing reactive polyethylene glycol residues, taxa, cytochalasins methicillin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide Cid, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin dihydroxyanthracindione, mitoxantrone, mithramycin, actinoma Isin D, 1-dihydrotestosterone, glucocorticoids, procaine, tetracaine , lidocaine, propranolol, puromycin, methotrexate, 6-mercapto Purine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine, mechlorethamine thiazol-3, thiotepa, chlorambucil, melphalan, carmustine, lomustine, cyclophosphamide, Famid, busulfan, dibromomannitol, streptozotocin, mitomycin C, cisplatin, dactinomycin, bleomycin, anthramycin, calicheamicin Shin, abiraterone, bendamustine, bortezomib, carboplatin, cabazitaxel , dasatinib, docetaxel, epirubicin, erlotinib, everolimus, gemcitabine cerebrospinal fluid, gefitinib, idarubicin, imatinib, hydroxyurea, lapatinib, leuproreductase inhibitor, leuproreductase inhibitor Lorelin, melphalan, nedaplatin, nilotinib, oxaliplatin, pazopanib, Pemetrexed, picoplatin, romidepsin, satraplatin, sorafenib, Vemura one selected from phenib, sunitinib, teniposide, triplatin, and vinorelbine It may include one or more.

[0584] In specific embodiments, the cargo moiety may comprise a VC linker.

[0585] 7.2. Methods for Preparing Antibody-Payload Conjugates According to the present invention, a method for preparing an antibody-payload conjugate is disclosed.

[0586] The antibody-payload conjugates of the present invention are m -H2 to react with H1-Ab In this case, the H1-Ab content can be prepared by the method disclosed in Section 5.1. The contents of this document apply, and the contents disclosed in Section 6 are m -H2. First click of H1-Ab C reactive functional groups and m If the second click-reactive functional groups of -H2 are complementary to each other, The first click-reactive functional group of H1-Ab and C m The second click-reactive functional group, -H2, When the toner functions as a click-reactive functional group, a click chemistry reaction occurs, and the present invention The antibody-payload conjugate can be prepared by the click chemistry reaction. As explained in the "Definition" section, click chemistry is a bioorthogonal reaction, where the reaction is very It has the advantage that it occurs at a rapid reaction rate and is used to form strong bond structures. Click chemistry therefore has the same advantages as in Sections 7.3 and 7.4 below.

[0587] The present invention relates to a method for preparing an antibody-payload conjugate, comprising the steps of: reacting an antibody containing a click-reactive functional group of the formula (I) with a payload according to the present invention; wherein H2 of the payload comprises a second click-reactive functional group complementary to the first click-reactive functional group. The present invention provides a method for producing a hydroxylase-containing compound, characterized in that the hydroxylase-containing compound is a functional group.

[0588] In this case, the antibody provided in Section 5.1 is converted to an antibody containing a first click-reactive functional group. The payloads provided in Section 6 apply to the payload. Lysine 246 and / or All antibodies containing a first click-reactive functional group at lysine 248 are disclosed in Section 5.1. Therefore, antibody-payloads in which cargo moieties are conjugated to lysine 246 and / or lysine 248 The conjugates are preferably fully reproducibly disclosed (see Equation 10 in Section 7 and (See 10-1 to 10-3).

[0589] The present invention provides a kit for preparing an antibody-payload conjugate of the present invention, an antibody containing a first click-reactive functional group according to the present invention; and a payload according to the present invention. , H2 of the payload is a second click-reactive functional group complementary to the first click-reactive functional group. The kit is characterized by:

[0590] In this case, the antibody provided in Section 5.1 is converted to an antibody containing a first click-reactive functional group. The payloads provided in Section 6 apply to the payload.

[0591] Optionally, the present invention provides a kit for preparing an antibody-payload conjugate, , antibody; an agent for transferring a first click-reactive functional group to an antibody according to the invention; and Payload according to the present invention A kit comprising:

[0592] In a specific embodiment, the agent for transferring the first click-reactive functional group to the antibody is The first click-reactive functional group is attached specifically to lysine 246 or 248 of the Fc domain of the antibody according to the present invention. In another specific embodiment, the first clone may be an H1-L2-SSFI for targeting the clone to metastasize. The agent for transferring the click-reactive functional group to the antibody is a compound that transfers the first click-reactive functional group to the antibody of the present invention. The H1-L2-SSFI can be used to specifically transfer the antibody to lysine 246 in the Fc domain of the antibody. In yet another specific embodiment, a first click-reactive functional group is transferred to the antibody. The agent for this purpose is a compound in which the first click-reactive functional group is attached to lysine 248 of the Fc domain of the antibody according to the present invention. It may be H1-L2-SSFI for specific transfer.

[0593] Optionally, the present invention provides a kit for preparing an antibody-payload conjugate, , antibody; a linker according to the present invention; a site-specific antibody interactome according to the present invention; and Payload according to the present invention A kit comprising:

[0594] In a specific embodiment, D2 of the linker is C y Alkylene, C y Alkenylene, or C y Al D3 of the site-specific antibody interactome is Cx alkylene, and y is 1 Furthermore, the sum of x and y may be 1≦x+y≦5. The sum of y may be 6≦x+y≦8. Furthermore, the sum of x and y may be 9≦x+y≦12.

[0595] 7.3. Antibody-payload conjugates according to the present invention can be stably synthesized using bioorthogonal reactions form a bond. The antibody-payload conjugates according to the present invention are formed through click chemistry reactions. Click chemistry is a biomimetic reaction that does not affect naturally occurring biochemical events in vivo. Furthermore, the bond formed by the bioorthogonal reaction is similar to that formed by the in vivo reaction. Therefore, the antibody-payload conjugate according to the present invention is not recognized by the antibody and cargo moieties have the advantage that they form very stable bonds in vivo.

[0596] 7.4. Antibody-payload conjugates prepared according to the present invention are highly homogeneous and yield It has. As described above in Section 5.6, an antibody containing a first Click-reactive functional group according to the present invention can be prepared by: The antibody-payload prepared from the antibody of the present invention has high homogeneity and yield. The Cm-Ab conjugate also has the advantage of high homogeneity and yield. The homogeneity needs to be high so that the antibody conjugate has uniform performance.

[0597] Prepared according to the prior art disclosed in application numbers US 2018 / 0141976 A1 and WO 2018 / 199337 A1 In the case of antibody conjugates that are to be purified, the antibody conjugates may be purified by the methods described above in Section 5.6. Since the efficiency and uniformity are poor, there can be a negative impact on the uniformity of performance.

[0598] 7.5. Antibody-payload conjugates according to the present invention have their FcRn binding site blocked Therefore, the function of the antibody is not reduced. C according to the present invention m -Ab has the same advantages as described in Sections 5.7 and 5.8. In particular, The pharmacokinetic (PK) properties of ADCs used in this study have been significantly improved because ADCs have extended half-lives. It can be improved.

[0599] FcRn-binding portion of ADCs prepared by the prior art disclosed in Application No. US 2018 / 0141976 A1 The site is blocked because SSFI is included in the final antibody labeling product. The resulting ADCs have superior PK properties compared to corresponding prior art ADCs.

[0600] 7.6. The antibody-drug conjugates of the present invention do not contain highly bioreactive chemical functional groups. The antibody-drug conjugates produced by Akari are stable. C according to the present invention m -Ab has the same advantages as described in Section 5.9. In particular, A The pharmacodynamic (PD) properties of ADCs are significantly improved as ADCs enable smooth antibody-drug interaction. It is possible.

[0601] In the case of ADCs prepared by the prior art disclosed in application number WO 2018 / 199337 A1, thiol Additional chemical functional groups such as aryl, hydroxy, carboxylic acid, phosphate, amine, etc. may be added to the final The ADCs according to the present invention are therefore comparable to the corresponding prior art ADCs. It has superior PD characteristics compared to

[0602] 8. Composition According to the present invention, a composition comprising an antibody is disclosed, wherein the antibody is a first antibody according to the present invention. Alternatively, the antibody may be an antibody-payload according to the present invention. Moreover, the antibody may be an antibody-drug conjugate according to the invention. It is possible.

[0603] The composition of the present invention can be used for various purposes depending on the function of the antibody contained in the composition. For example, if the first chemical functionality or cargo moiety contains a radioactive moiety, The resulting composition can be used as a radioactive imaging agent, etc. Alternatively, the first chemical functional group When the group or cargo moiety comprises a fluorescent moiety, the corresponding composition can be used in an enzyme-linked immunosorbent assay. Alternatively, the first chemical functional group can be used as a label for use in ELISA, etc. Alternatively, if the cargo moiety comprises a drug moiety, the corresponding composition can be used as a pharmaceutical composition. In this case, the components of the composition generally used in the related technical field may be the same as those of the present invention. Moreover, the composition ratio of the corresponding composition commonly used in the related art is within the clear range. are also within the scope of the present invention.

[0604] The present invention provides a composition comprising an antibody containing a first chemical functionality according to the present invention. In specific embodiments, the antibody containing the first chemical functionality is represented by Formulas 7, 7-1 to 7-3, 8, and 8-1 It may be at least one selected from the following:

[0605] Furthermore, the present invention includes antibodies containing a first click-reactive functional group according to the present invention, Compositions are provided, in specific embodiments, comprising an antibody containing a first click-reactive functional group. can be at least one selected from formulas 8 and 8-1 to 8-3.

[0606] Additionally, the present invention provides a composition comprising an antibody-payload conjugate according to the present invention. In this case, the contents of Section 7 apply to the contents of the antibody-payload conjugate. do.

[0607] Optionally, the present invention provides a composition comprising an antibody-drug conjugate according to the present invention. In this case, the contents of Section 7.1 apply to the contents of the antibody-drug conjugate.

[0608] Pharmaceutical Composition The following is a description of the composition, when it is a pharmaceutical composition used for diagnostic, prophylactic, and / or therapeutic purposes. In the context of the present invention, the term "pharmaceutical composition" refers to any R1'-Ab , H1-Ab, C m -Ab and ADC are used interchangeably with the term "antibody or fragment thereof."

[0609] The present invention provides a pharmaceutical composition comprising an antibody or a fragment thereof according to the present invention. The composition may be a composition for treating cancer. Further, the cancer may be 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 cancer The cancer may include any one selected from germ cell cancer, thymoma, and thymic carcinoma. , it could be breast cancer.

[0610] The present invention relates to a method for treating a cancer, comprising administering to a subject a pharmaceutical composition comprising an antibody or a fragment thereof according to the present invention. Furthermore, the therapeutic method can be a method for treating cancer. Cancers include bladder cancer, bone cancer, brain cancer, breast cancer, heart cancer, cervical cancer, colon cancer, and rectal cancer. Cancer, esophageal cancer, fibrosarcoma, gastric cancer, stomach cancer, head and neck cancer, Kaposi's disease tumor, kidney cancer, leukemia, liver cancer, lung cancer, lymphoma, melanoma, myeloma, ovarian cancer, pancreatic cancer, Any one selected from penile cancer, prostate cancer, testicular germ cell cancer, thymoma, and thymic carcinoma Further, the cancer may be breast cancer.

[0611] To prepare a pharmaceutical or sterile composition comprising an antibody or a fragment thereof, a method according to the present invention is The antibody or fragment thereof can be mixed with a pharmaceutically acceptable carrier or excipient. The substance is used for cancer (e.g., breast cancer, colorectal cancer, lung cancer, multiple myeloma, ovarian cancer, liver cancer) , gastric cancer, pancreatic cancer, acute myeloid leukemia, chronic myeloid leukemia, osteosarcoma, squamous cell carcinoma, Peripheral nerve sheath tumor, Schwannoma, head and neck cancer, bladder cancer, esophageal cancer, Barrett's esophagus cancer, Glioblastoma, clear cell sarcoma of soft tissue, malignant mesothelioma, neurofibromatosis, renal cancer, melanoma, pre- Treats or prevents the development of certain conditions (prostate cancer, benign prostatic hyperplasia (BPH), gynecomastia, rhabdomyosarcoma, and endometriosis) may further comprise one or more other therapeutic agents suitable for prophylaxis.

[0612] Therapeutic and diagnostic agents can be formulated into formulations such as, for example, lyophilized powders, slurries, aqueous solutions, lotions, etc. In the form of a liquid or suspension, the liquid may be mixed with a physiologically acceptable carrier, additive, or stabilizer. can be prepared by combining (e.g., Hardman et al., Goodman and Gilman's The Pharmacological Basis of Therapeutics, McGraw-Hill, New York, NY, 2001; G ennaro, Remington: The Science and Practice of Pharmacy, Lippincott, Williams, a. nd Wilkins, New York, NY, 2000; Avis et al. (eds.), Pharmaceutical Dosage Forms: P arenteral Medications, Marcel Dekker, NY, 1993; Lieberman et al. (eds.), Pharmaceuti cal Dosage Forms: Tablets, Marcel Dekker, NY, 1990; Lieberman et al. (eds.) Pharmace utical Dosage Forms: Disperse Systems, Marcel Dekker, NY, 1990; Weiner and Ko tkoskie, Excipient Toxicity and Safety, Marcel Dekker, Inc., New York, NY, 2. (See Year 000).

[0613] In a specific embodiment, the clinical supply form (CSF) of an antibody-drug conjugate according to the invention comprises The frozen solution present in the vial containing the ADC, sodium succinate, and polysorbate 20 The lyophilisate can be reconstituted with water for injection and the solution will have a pH of approximately 5.0, containing ADC, sodium succinate, sucrose, and polysorbate 20. Sequential i.v. For internal administration, the resulting solution is usually further diluted in a carrier solution.

[0614] The choice of therapeutic dosing regimen depends on the serum or tissue replacement rate of the substance, the level of symptoms, the effectiveness of the substance, Various factors, including immunogenicity and target cell accessibility in the biological matrix, In a specific embodiment, the dosing regimen comprises the amount of therapeutic agent delivered to the patient: To maximize the effectiveness of the biologic agent administered, the acceptable level of side effects is met. The amount of will depend in part on the particular agent and the severity of the condition being treated. can be used to select appropriate doses of antibodies, cytokines, and small molecules (e.g., Waw rzynczak, Antibody Therapy, Bios Scientific Pub. Ltd, Oxfordshire, UK, 1996; Kresina (ed.), Monoclonal Antibodies, Cytokines and Arthritis, Marcel Dekker, N. New York, NY, 1991; Bach (ed.), Monoclonal Antibodies and Peptide Therapy in A 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, 1 999; 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, 2 003; see Lipsky et al., New Engl. J. Med. 343:1594-1602, 2000).

[0615] Suitable dosages may be determined, for example, by methods known in the relevant art or believed to affect treatment. parameters or factors that affect the treatment or that are expected to affect the treatment. The dose is determined by the clinician using a number of different parameters or factors. Start with a low dose and then increase it slightly until the desired or appropriate effect is achieved for any side effects. Important diagnostic measurements include, for example, the symptoms of inflammation or the amount of inflammatory cytokines produced. Includes the input level.

[0616] The actual dosage level of the active ingredient of the pharmaceutical composition according to the present invention is determined so as not to cause any toxicity to the patient. activity that achieves the desired therapeutic response in a given patient, composition, and mode of administration without The dosage level selected may vary to achieve an effective amount of the component. The activity, route of administration, time of administration, use of a given composition, or its ester, salt, or amide The secretion rate of a particular compound used, the duration of treatment, the combination of a particular compound used, other drugs, compounds, and / or substances used in combination with the A variety of factors, including the patient's condition, general health, and past medical history, as well as other factors known in the medical arts, may be considered. This can be determined depending on pharmacokinetic factors.

[0617] The composition comprising the antibody or fragment thereof according to the present invention can be administered, for example, daily, weekly, 1 to 7 times a week, Every other week, once every 3 weeks, once every 4 weeks, once every 5 weeks, once every 6 weeks, once every 7 weeks, or once every 8 weeks Doses can be given intravenously, subcutaneously, by continuous infusion, or as a single dose. and may be given topically, orally, intranasally, rectally, intramuscularly, intracerebrally, or by inhalation. Certain administration protocols may be used to determine the maximum dose or doses that avoid significant unwanted side effects. Includes frequency of administration.

[0618] The dose administered to a patient for the antibody or fragment thereof according to the present invention is 0.0001 mg / kg to 100 The dose can range from 0.0001 mg / kg to 20 mg / kg, 0.0001 mg / kg to 10 mg / kg, mg / kg, 0.0001mg / kg~5mg / kg, 0.0001~2mg / kg, 0.0001~1mg / kg, 0.0001mg / kg~0.75mg / kg, 0.0001mg / kg~0.5mg / kg, 0.0001mg / kg~0.25mg / kg, 0.0001~0.15mg / kg, 0.0001~0. 10 mg / kg, 0.001 to 0.5 mg / kg, 0.01 to 0.25 mg / kg, or 0.01 to 0.10 mg / kg (patient's weight) The dose of the antibody or fragment thereof according to the present invention may be calculated based on the patient's mass (kilograms (kg)): It can be calculated by multiplying the administered dose (mg / kg).

[0619] The antibody or fragment thereof according to the present invention can be administered repeatedly, and the administration is carried out in at least one days, 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 at monthly intervals. The strips can be administered repeatedly every three weeks.

[0620] The effective amount to be administered to a particular patient will depend on the condition being treated, the patient's general health, the manner of administration, and the amount of These may vary depending on factors such as the formula, route, and dosage, as well as the severity of side effects (e.g., See Maynard et al., A Handbook of SOPs for Good Clinical Practice, Interpharm Press. , Boca Raton, Fla., 1996; Dent, Good Laboratory and Good Clinical Practice, Ur. ch Publ., London, UK, 2001).

[0621] The route of administration may be, for example, topical or cutaneous application, intravenous, intraperitoneal, intracerebral, or intramuscular. by intraocular, intra-arterial, intramedullary, intralesional injection or infusion, or by sustained release systems or may be due to grafting (e.g., Sidman et al., Biopolymers 22:547-556, 1983; Langer et al., J. Biomed. Mater. Res. 15:167-277, 1981; Langer, Chem. Tech. 12:9 8-105, 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; US Patent No. 6,350,46 (See U.S. Patent No. 6,316,024.) If desired, the composition may also contain a solubilizing agent, and A local anesthetic (e.g., lidocaine) may be included to ease pain at the site of the injection. The product may also be formulated for use in an inhaler or nebulizer, or may be administered by aerosolization. It can be used for pulmonary administration by formulation using, for example, U.S. Patent No. 6,019,966. No. 8, U.S. Patent No. 5,985,320, U.S. Patent No. 5,985,309, U.S. Patent No. 5,934,272, U.S. Patent No. No. 5,874,064, U.S. Pat. No. 5,855,913, U.S. Pat. No. 5,290,540, and U.S. Pat. No. 80,078; and PCT Application Nos. WO 92 / 19244, WO 97 / 32572, WO 97 / 44013, WO 98 / 31346 and WO 99 / 66903, the entire contents of which are incorporated herein by reference.

[0622] Moreover, the compositions of the present invention can be prepared using one or more of a variety of methods known in the relevant art. As will be appreciated by those skilled in the art, the compounds may be administered via one or more of the following routes of administration: The route and / or mode of administration will depend on the desired results. The route of administration chosen for the compound is intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, intrathecal, or intravenous. Parenteral administration includes other parenteral routes, such as administration by injection or infusion. Generally, it refers to a mode of administration by injection other than enteral and local administration, and includes intravenous, intramuscular, intraarterial, Intrathecal, intracapsular, intraorbital, intracardial, intradermal, intraperitoneal, transtracheal, subcutaneous, subcutaneous, intraarticular, subcapsular, These include, but are not limited to, intrathecal, intrathecal, epidural, and intrasternal injection and infusion. Alternatively, the compositions of the present invention may be administered parenterally, e.g., topically, intradermally, or intradermally. or via intramucosal routes, for example, intranasally, orally, vaginally, rectally, It can be administered sublingually or topically. In a specific embodiment, the antibody or In another specific embodiment, the fragment can be administered by injection. The antibody or fragment thereof can be administered subcutaneously.

[0623] When the antibody or fragment thereof according to the present invention is administered using a controlled or delayed release system, In some cases, a pump can be used to achieve controlled or delayed release (Langer, supra; Sefto n, CRC Crit. Ref Biomed. Eng. 14:20, 1987; Buchwald et al., Surgery 88:507, 1980; (See Saudek et al., N. Engl. J. Med. 321:574, 1989). Controlled or delayed release can be achieved in various therapies (e.g., Medical Applications). Ons of Controlled Release, Langer and Wise (eds.), CRC Press, Boca Raton, Fla., 19 74; Controlled Drug Bioavailability, Drug Product Design and Performance, Smol En and Ball (eds.), Wiley, New York, 1984; Ranger and Peppas, J. Macromol. Sci. R See ev. Macromol. Chem. 23:61, 1983; also 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 Application No. WO 99 / 15154; and PCT Application No. WO 99 Examples of polymers used in sustained release formulations include poly(2-hydroxyethyl) methacrylate), poly(methyl methacrylate), poly(acrylic acid), poly(ethylene- co-vinyl acetate), poly(methacrylic acid), polyglycolide (PLG), polyanhydride ide, poly(N-vinylpyrrolidone), poly(vinyl alcohol), polyacrylamide, poly (ethylene glycol), polylactide (PLA), poly(lactide-co-glycolide) (PLGA), and Examples of the polyol include, but are not limited to, polyols and polyorthoesters. In embodiments, the polymers used in the sustained release formulation are inert and free of filterable impurities. Controlled or delayed release systems are suitable for prophylactic purposes and are stable in storage, sterilizable, and biodegradable. or can be located adjacent to the therapeutic target, thus requiring a fraction of the systemic dose (e.g., Goodson, Medical Applications of Controlled Release, supra, Vol. 2, pp. 115-138 , 1984).

[0624] Controlled release systems are discussed in a review article by Langer, Science 249:1527-1533, 1990. Any prior art known to those skilled in the art can be used to produce one or more Sustained release formulations containing antibodies or fragments thereof can be produced. See, e.g., U.S. Pat. No. 526,938, PCT Application No. WO 91 / 05548, PCT Application 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. Bio act. Mater. 24:853-854, 1997; and Lam et al., Proc. Int'l. Symp. Control Rel. See Bioact. Mater. 24:759-760, 1997, the entire contents of which are incorporated herein by reference. be incorporated into the book.

[0625] When the antibody or fragment thereof according to the present invention is administered topically, the antibody or fragment thereof may be applied in an ointment. , creams, transdermal patches, lotions, gels, shampoos, sprays, aerosols The pharmaceutical composition may be formulated in the form of a pill, solution, or emulsion, or in other forms known to those skilled in the art. For example, Remington's Pharmaceutical Sciences and Introduction to Pharma See Medical Dosage Forms, 19th ed., Mack Pub. Co., Easton, Pa. (1995). Non-spray topical dosage forms include a carrier or one or more additives suitable for topical application. In some cases, viscous, semi-solid, or solid forms with kinematic viscosities higher than water are commonly used. It can be used as a topical dosage form. Suitable preparations include solutions, suspensions, emulsions, and cereals. These include creams, ointments, powders, liniments, ointments, etc., all of which must be sterilized. , or, if necessary, auxiliary substances that affect various properties, such as osmolality (e.g. preservatives, stabilizers, wetting agents, buffers, or salts), but in the present invention, these In some cases, other suitable topical dosage forms include those containing the active ingredient in solid or liquid form. A mixture of volatile substances (e.g., gases such as Freon) that is compressed after being combined with an inert carrier. This includes spray-type aerosol preparations in a squeeze bottle or spray-type aerosol preparations. In such cases, humectants or humectants can also be added to pharmaceutical compositions and dosage forms. Examples of such additional ingredients are known in the relevant art.

[0626] When a composition comprising an antibody or fragment thereof is administered intranasally, the composition may be administered as an aerosol, It can be formulated in the form of a spray, mist, or drops. The prophylactic or therapeutic agent to be administered may be sprayed in a suitable propellant (e.g., dichlorodifluoromethane, trichlorodifluoromethane, etc.). dichlorotetrafluoromethane, dichlorotetrafluoroethane, carbon dioxide, or other suitable gas) The compound can be conveniently delivered in an aerosol spray presentation from pressurized packs or from a nebulizer using In the case of a pressurized aerosol, the dosage unit may comprise a valve to deliver a metered amount. This can be determined by the following: Capsules and cartridges containing a powder mix of the compound and a suitable powder base, For example, it can be formulated with lactose or starch.

[0627] a second therapeutic agent, such as a cytokine, steroid, chemotherapeutic agent, antibiotic, or radiation Methods for co-administration or treatment with steroids are known in the art (see, for example, Hardman et al. (eds.) (200 1) Goodman and Gilman's The Pharmacological Basis of Therapeutics, 10th edition, McGrath W-Hill, New York, NY; Poole and Peterson (eds.) (2001) Pharmacotherapeutics for A Advanced Practice: A Practical Approach, Lippincott, Williams & Wilkins, Phila. , Pa.; Chabner and Longo (eds.) (2001) Cancer Chemotherapy and Biotherapy, Lippincott (See, Williams & Wilkins, Inc., Philadelphia, Pa.) An effective amount of a therapeutic agent is at least 10%; reducing symptoms by at least 20%; at least approximately 30%; at least 40%; or at least 50% This can be done.

[0628] Additional therapies (e.g., , prophylactic or therapeutic agent) may be administered at intervals of less than 5 minutes, less than 30 minutes, one hour, or approximately one hour. intervals of approximately 1 hour to approximately 2 hours, intervals of approximately 2 hours to approximately 3 hours, and intervals of approximately 3 hours. 10:00 - 4 hour intervals, 4 hour - 5 hour intervals, 5 hour - 6 hour intervals intervals between, intervals of approximately 6 hours to approximately 7 hours, intervals of approximately 7 hours to approximately 8 hours, 8 hours to approximately 9 hours intervals, approximately 9 hours to approximately 10 hours intervals, approximately 10 hours to approximately 1 Every hour, every 11 to 12 hours, every 12 to 18 hours, 6:00 PM Between 24 hours, between 24 hours and 36 hours, between 36 hours and 48 hours, between 48 hours and 52 hours Intervals: 52-60 hours, 60-72 hours, 72-84 hours, 84-96 hours or 96 to 120 hours apart, together with the antibody or fragment thereof according to the present invention. Two or more therapeutic agents may be administered during the same patient visit. .

[0629] In a specific embodiment, the antibodies or fragments thereof according to the invention have an appropriate distribution in vivo. For example, the blood-brain barrier (BBB) ​​is a barrier that allows some high Exclude hydrophilic compounds. To ensure that the therapeutic compounds of the present invention cross the BBB (as required), In some cases, these may be formulated, for example, in liposomes. For methods of preparation, see, e.g., U.S. Pat. No. 4,522,811; U.S. Pat. No. 5,374,548; and See U.S. Patent No. 5,399,331. Liposomes can be selectively delivered to specific cells or organs. The drug may contain one or more transportable moieties, thereby improving targeted drug delivery (e.g. (See, e.g., Ranade, (1989) J. Clin. Pharmacol. 29:685). Exemplary targeting moieties include folic acid or biotin (see, e.g., U.S. Pat. No. 5,416,016 to Low et al.); mannosides (Umez antibodies (see Bloeman et al. (1995) Biochem. Biophys. Res. Commun. 153:1038); ) FEBS Lett. 357:140; see 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). nen; ML Laukkanen (1994) FEBS Lett. 346:123 pages; JJ Killion; IJ Fidler (1 994) See Immunomethods 4:273.

[0630] The present invention provides a pharmaceutical composition comprising an antibody or a fragment thereof according to the present invention for use in a patient in need thereof. The present invention provides protocols for administering the compound alone or in combination with other therapies to mammals. Therapeutic agents (e.g., prophylactic or therapeutic agents) for the specified combination therapy may be administered simultaneously or sequentially to a subject. Therapeutic agents (e.g., prophylactic or therapeutic agents) for the combination therapy of the present invention may also be administered cyclically. Cycling therapy can involve administering a first therapeutic agent (e.g., a first prophylactic agent) for a predetermined period of time. or therapeutic agent), for a predetermined period of time, administering a second therapeutic agent (e.g., a second prophylactic or therapeutic agent), a therapeutic agent (e.g., an anti-inflammatory drug), followed by sequentially repeating the steps of administering the therapeutic agent (e.g., an anti-inflammatory drug), to reduce the development of resistance to one of the therapeutic agents (e.g., agonists) and / or to prevent or reduce the side effects of one of the It includes a predetermined cycle for improving the

[0631] The therapeutic agents (e.g., prophylactic or therapeutic agents) for the combination therapies of the invention can be administered simultaneously to a subject. can.

[0632] The term "concurrently" refers to, but is not limited to, when the therapies (e.g., prophylactic or therapeutic agents) are administered at exactly the same time. It is not necessary to administer the antibody or fragment thereof to the patient at once, but rather to administer the pharmaceutical composition comprising the antibody or fragment thereof according to the present invention sequentially. compared to when the antibody of the invention is administered to a subject at a different time than other therapies. This means that the doses are administered at intervals that may serve to provide greater benefit. Each therapeutic agent may be administered to a subject simultaneously in any order or sequentially at different time points. However, if they are not administered simultaneously, the amount of steroids present may be insufficient to produce the desired therapeutic or prophylactic effect. Each therapeutic agent should be administered in any suitable manner, with the administration time being as short as possible. The drug may be administered to a subject via any suitable route. In this case, the therapeutic agent (e.g., prophylactic or therapeutic agent) may be administered at intervals of less than 15 minutes, less than 30 minutes, or 1 hour. Intervals of less than 1 hour, intervals of approximately 1 hour, intervals of approximately 1 hour to approximately 2 hours, intervals of approximately 2 hours to approximately Every 3 hours, Every 3 to 4 hours, Every 4 to 5 hours , approximately 5 to 6 hour intervals, approximately 6 to 7 hour intervals, approximately 7 hours Approximately every 8 hours, between approximately 8 and 9 hours, between approximately 9 and 10 hours Every 10 to 11 hours, Every 11 to 12 hours, 24 hours, The subject may be administered the drug at 48 hour, 72 hour, or weekly intervals. In certain embodiments, two or more therapies (e.g., prophylactic or therapeutic agents) are administered by a patient during the same visit. can be administered to

[0633] The prophylactic or therapeutic agents of the combination therapy can be administered to a subject in the same pharmaceutical composition. Alternatively, in separate pharmaceutical compositions, the prophylactic or therapeutic agents for the combination therapy may be administered simultaneously to a subject. The prophylactic or therapeutic agents may be administered to a subject via the same or different routes of administration. This can be done. [Example]

[0634] 1. To prepare an agent for site-specific transfer of a first click-reactive functional group to an antibody Preparation example of compound for 1.1. Synthesis of linker (H1-L1) and confirmation of its structure 1.1.1.: Compound I (SO1 linker: NHS and norbornene)

[0635] [ka]

[0636] Synthesis of Compound I (Scheme 6, Figure 15) Synthesis of Compound 1 2 g (10.98 mmol, 1.0 equiv.) of 2-(bicyclo[2.2.1]hept-5-en-2-ylmethoxy)acetic acid , dissolved in 50 mL of DCM, and then added 0.085 mL (1.098 mmol, 0.1 equiv.) of DMF and 1.91 mL (21.96 mmol) of 2.0 equivalents) of oxalyl chloride was added dropwise at room temperature with stirring. The reaction solution was stirred for 3 hours. After that, it was concentrated under reduced pressure to give 1.97 g of the target compound (yield: 90%).

[0637] Synthesis of compound 2 1.97 g (9.88 mmol, 1.0 equivalent) of compound 1 was dissolved in 20 mL of acetonitrile (ACN) and then 0 0.68 mL (9.88 mmol, 1.0 equiv.) thioglycolic acid and 2.06 mL (14.82 mmol, 1.5 equiv.) triglyceride Ethylamine was added dropwise with stirring at room temperature. The reaction solution was stirred for 18 hours, and then heated 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. The residue was purified by distillation with DCM:MeOH=10:1 to give 2.05 g (yield: 79%) of the target compound.

[0638] 1 H NMR (500 MHz, CDCl3) δ 1 H NMR (500 MHz, CDCl3) δ 6.15 - 6.01 (m, 2H), 4.2 0 (t, J = 5.0 Hz, 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, 8.6 Hz, 4H), 1.15 (ddd, J = 16.0, 7.6, 3.3 Hz, 1H).

[0639] Synthesis of Compound I 2.05 g (7.81 mmol, 1.0 equiv.) of compound 2 was dissolved in 50 mL of ACN, followed by addition of 2.76 g (14.44 mmol, 1 equiv.) of the compound. 0.8 equivalents) of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCi), and 2.21 g (19 N-hydroxysuccinimide (NHS) (0.25 mmol, 2.46 equiv.) was added with stirring at room temperature. The reaction solution was stirred for 18 hours and then concentrated under reduced pressure. Then, water was added to the reaction solution. The reaction solution was extracted three times with EA. The organic layer was collected, dried over magnesium sulfate, and then The mixture was concentrated under reduced pressure, and the residue was then purified by chromatography (EA:Hex=2:1) ​​on silica gel. The product was purified by column chromatography to give 2.7 g (yield: 98%) of the target compound.

[0640] 1 H NMR (500 MHz, CDCl3) δ 6.15 - 6.03 (m, 2H), 4.45 (s, 1H), 4.21 (d, J = 5.0 Hz, 1H), 3.97 (s, 1H), 3.65 (dd, J = 12.0, 7.3 Hz, 1H), 3.52 (t, J = 8.9 Hz, 1H ), 2.83 (dd, J = 24.4, 11.8 Hz, 6H), 1.81 - 1.67 (m, 1H), 1.30 (dq, J = 27.0, 9. 5 Hz, 4H), 1.16 (ddd, J = 15.3, 7.4, 3.8 Hz, 1H).

[0641] Confirmation of the structure of compound I 1 H NMR (500 MHz, CDCl3) δ 6.15 - 6.03 (m, 2H), 4.45 (s, 1H), 4.21 (d, J = 5.0 Hz, 1H), 3.97 (s, 1H), 3.65 (dd, J = 12.0, 7.3 Hz, 1H), 3.52 (t, J = 8.9 Hz, 1H ), 2.83 (dd, J = 24.4, 11.8 Hz, 6H), 1.81 - 1.67 (m, 1H), 1.30 (dq, J = 27.0, 9. 5 Hz, 4H), 1.16 (ddd, J = 15.3, 7.4, 3.8 Hz, 1H). LRMS (ESI): m / z 371.2 [M+ NH4 + ]

[0642] The structure of Compound I was confirmed by mass spectrometry, and the results are shown in FIG.

[0643] 1.1.2. Compound II (SO2 Linker: NHS and Norbornene)

[0644] [ka]

[0645] Synthesis of Compound II (Scheme 7, Figure 17) Synthesis of compound 3 0.55 g (2.33 mmol, 1.0 equivalent) of compound 1 was dissolved in 5 mL of acetonitrile (ACN) and then 0.2 mL (2.33 mmol, 1.0 equiv.) of 3-mercaptopropionic acid and 0.49 mL (3.49 mmol, 1.5 equiv.) of Triethylamine was added dropwise at room temperature while stirring. The reaction solution was stirred for 11 hours, then the The reaction mixture was concentrated under reduced pressure. Then, water was added to the reaction mixture, and the reaction mixture was extracted three times with ethyl acetate (EA). The organic layer was dried over magnesium sulfate, concentrated under reduced pressure, and then passed through a silica gel column. Purification by chromatography (DCM:MeOH=10:1) gave 0.56 g (yield: 89%) of the target compound.

[0646] Synthesis of Compound II 0.56 g (2.07 mmol, 1.0 equivalent) of compound 3 was dissolved in 10 mL of acetonitrile (ACN) and 0.81 g ( 4.21 mmol, 2.0 equiv) of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCi), and 0.65 g (5.61 mmol, 2.7 equivalents) of N-hydroxysuccinimide (NHS) were added at room temperature without stirring. The reaction solution was stirred for 12 hours and then concentrated under reduced pressure. The reaction solution was extracted three times with EA. The organic layer was dried over magnesium sulfate. The mixture was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (EA:Hex=2: 1) to obtain 0.63 g (yield: 83%) of the target compound.

[0647] Confirmation of the structure of compound II 1 H NMR (500 MHz, CDCl3) δ 6.16-6.14 (m, 2H), 4.45 (s, 1H), 4.21 - 4.08 (m, 1H ), 3.69 - 3.58 (m, 1H), 3.51 (dt, J = 14.5, 8.9 Hz, 1H), 3.22 (t, J = 7.1 Hz, 2H ), 2.96 (t, J = 7.1 Hz, 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). LRMS (ESI): m / z 385.1 [M+ NH4 + ]

[0648] The structure of Compound II was confirmed by mass spectrometry, and the results are shown in Figure 18.

[0649] 1.1.3. Compound III (SO3 Linker: NHS and Norbornene)

[0650] [ka]

[0651] Synthesis of Compound III (Scheme 8, Figure 19) Synthesis of compound 4 0.5 g (3.62 mmol, 1.0 equiv.) of exo-5-norbornenecarboxylic acid was dissolved in 20 mL of DCM. After this, 0.028 mL (0.37 mmol, 0.94 equiv.) of DMF and 0.63 mL (7.24 mmol, 2.0 equiv.) of oxychloride were added. Salil was added dropwise with stirring at room temperature. The reaction solution was stirred for 3 hours and then concentrated under reduced pressure. As a result, 0.47 g (yield: 83%) of the target compound was obtained.

[0652] Synthesis of compound 5 0.47 g (3.0 mmol, 1.0 equiv.) of compound 4 was dissolved in 15 mL of acetonitrile (ACN) and then 0.2 1 mL (3.0 mmol, 1.0 equiv.) of thioglycolic acid and 0.63 mL (4.5 mmol, 1.5 equiv.) of triethyl 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. Purification with (DCM:MeOH=10:1) gave 0.3 g (yield: 47%) of the target compound.

[0653] 1H NMR (500 MHz, CDCl3) δ 9.82 (brs, 1H), 6.24 - 6.07 (m, 2H), 3.76 (s, 2H), 3.12 (s, 1H), 2.97 (s, 1H), 2.54 (dd, J = 9.0, 4.7 Hz, 1H), 2.04 - 1.87 (m, 1H), 1.57 (d, J = 8.5 Hz, 1H), 1.48 - 1.35 (m, 2H).

[0654] Synthesis of Compound III 0.26 g (1.22 mmol, 1.0 equiv.) of compound 5 was dissolved in 15 mL of acetonitrile (ACN) and 0.35 g ( 1.83 mmol, 1.5 equiv) of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCi), and 0.28 g (2.44 mmol, 2.0 equivalents) of N-hydroxysuccinimide (NHS) were added at room temperature without stirring. The reaction solution was stirred for 3 hours and then concentrated under reduced pressure. The reaction solution was extracted with EA three times, and the organic layer was collected and dried over magnesium sulfate. The residue was then purified by chromatography (EA:Hex=2:1). The residue was purified on a silica gel column to give 0.32 g (yield: 85%) of the target compound.

[0655] Confirmation of the structure of compound III 1 H NMR (500 MHz, CDCl3) δ 6.21 (dd, J = 5.5, 3.0 Hz, 1H), 6.15 (dd, J = 5.5, 3.1 Hz, 1H), 4.01 (s, 2H), 3.15 (s, 1H), 2.98 (s, 1H), 2.86 (s, 4H), 2.54 (dd, J = 9.2, 4.6 Hz, 1H), 2.02 (dt, J = 11.9, 4.0 Hz, 1H), 1.59 (d, J = 8.6 Hz, 1H), 1.46 - 1.36 (m, 2H). LCMS (ESI): m / z 332.16 [M+Na + ]

[0656] The structure of compound III was confirmed by mass spectrometry, and the results are shown in Figure 20.

[0657] 1.1.4. Compound IV (SO4 Linker: NHS and Azide)

[0658] [ka]

[0659] Synthesis of Compound IV (Scheme 9, Figures 21 and 22) Synthesis of compound 6 2-(2-chloroethoxy)ethanol (2 mL, 18.94 mmol) was dissolved in distilled water (12 mL) and NaN3 (3 To this was added 1.08 g, 47.35 mmol, 2.5 eq. 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 approximately 10 minutes. The reaction mixture was extracted three times with diethyl ether, and the organic layer was extracted with magnesium sulfate. The filtrate was concentrated under reduced pressure to give 2.47 g (yield: 99%) of the target compound. Ta.

[0660] Synthesis of compound 7 2.47 g (18.84 mmol) of compound 6 was dissolved in acetone (50 mL) and then added to 1 M Jones's reagent (75.36 m L, 75.36 mmol, 4 eq) was added slowly at 0° C. The reaction mixture was stirred at 0° C. for 3 hours. After warming to room temperature and stirring for approximately 10 minutes, the reaction mixture was extracted three times with ethyl acetate (EA). The organic layer was dried over magnesium sulfate and filtered. The filtrate was concentrated under reduced pressure to give 2.69 ml of ethyl acetate. g (yield: 98%) of the target compound was obtained.

[0661] Synthesis of compound 8 2.69 g (18.57 mmol, 1.0 equiv.) of compound 7 was dissolved in 50 mL of DCM, followed by 0.1 mL (1.29 mmol, 0 0.07 equiv.) DMF and 2.43 mL (27.86 mmol, 1.5 equiv.) oxalyl chloride were added to the flask at room temperature without stirring. The reaction solution was stirred for 3 hours and then concentrated under reduced pressure to give 2.42 g (yield: 80%) of the target compound. The target compound was obtained.

[0662] Synthesis of compound 9 0.88 g (5.40 mmol, 1.0 equiv.) of compound 8 was dissolved in 30 mL of DCM, followed by addition of 0.8 g (5.40 mmol, 1.0 equiv.) of the compound 8. 1.41 mL (8.10 mmol, 1.5 equiv.) of tert-butyl 2-mercaptoacetate and 1.41 mL (8.10 mmol, 1.5 equiv.) of N,N-dimercaptoacetate Isopropylethylamine was added dropwise at room temperature with stirring. The reaction solution was stirred for 2 hours. Then, water was added to the reaction solution, and the reaction solution was diluted with dichloromethane The organic layer was dried over magnesium sulfate, concentrated under reduced pressure, and then extracted three times with dichloromethane (DCM). The product was purified by column chromatography (Hex:EA=5:1) to give 0.73 g (yield: 49%) of the target compound.

[0663] Synthesis of compound 10 0.73 g (2.66 mmol, 1 equiv.) of compound 9 was dissolved in 10 mL of DCM, followed by 10 mL (129.8 mmol, 48 equiv.) of DCM. Amount of trifluoroacetic acid was added dropwise at room temperature while stirring. After stirring for 8 hours, The mixture was concentrated under reduced pressure to give 0.40 g (yield: 70%) of the target compound.

[0664] Synthesis of Compound IV 1.97 g (9.0 mmol, 1.0 equiv.) of compound 10 was dissolved in 25 mL of acetonitrile (ACN) and 2.58 g ( 13.5 mmol, 1.5 equiv) of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCi), and 2.07 g (18.0 mmol, 2.0 equivalents) of N-hydroxysuccinimide (NHS) were added at room temperature without stirring. The reaction solution was stirred for 3 hours and then concentrated under reduced pressure. The reaction solution was extracted with EA three times, and the organic layer was collected and dried over magnesium sulfate. The residue was then purified by chromatography (EA:Hex=2:1). The residue was purified on a silica gel column to give 1.03 g (yield: 36%) of the target compound.

[0665] Confirmation of the structure of compound IV 1 H NMR (500 MHz, CDCl3) δ 4.30 (s, 2H), 4.00 (s, 2H), 3.87 - 3.75 (m, 2H), 3. 55 - 3.47 (m, 2H), 2.86 (s, 4H). LRMS (ESI): m / z 334.0 [M+ NH4 + ]

[0666] The structure of compound IV was confirmed by mass spectrometry, and the results are shown in Figure 23.

[0667] 1.2. Synthesis and structural confirmation of site-specific Fc interactome (SSFI) 1.2.1. Synthesis and structural confirmation of SSFI (Xa1 is a linker) Synthesis of SSFI(6Lys)

[0668] [ka]

[0669] List of Fmoc amino acids used and order of introduction 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.

[0670] Preparation method (a) Introduction of amino acids The amounts of reagents used in the following process were based on 0.25 mmol. A transparent amide resin (Peptide International Inc., USA) (100 ml / g) was placed in a synthesis reactor. One millimolar of each Fmoc-amino acid block was weighed and added to the peptides from the C-terminus to the N-terminus in the above order. The amino acid sequence of the tide was prepared.

[0671] For activating Fmoc-amino acids and coupling the activated residue to a clear amide resin The reactions were carried out sequentially starting from the C-terminal amino acid.

[0672] Fmoc removal was carried out with 20% piperidine in DMF, and the prepared amino acids corresponding to the sequence were The mixture was incubated with 2 mL of 0.5 M HOBt in DMF, 2 mL of 0.5 M HBTU in DMF, and 174 μL of DIPEA for 5 min. Mix the resulting mixture in the reactor containing the resin for 2 hours. The activation and introduction of residues were carried out by

[0673] The transfection reaction was confirmed using the Kaiser test. If this occurred, the incorporation reaction was repeated once more, or capping was performed using a 20% Ac2O solution in DMF. In each of the incorporation reaction and Fmoc removal, the resin was washed with 100 ml of HCl before proceeding to the next step. This process was repeated until the target peptide sequence was completed. The return was carried out.

[0674] (b) Introduction of H-PEG8-OH After the amino acid introduction is complete, add 1 mL of 0.5 M H-PEG8-OH to the N-terminus of the sequence. Solution of Fmoc-N-amide-dPEG8-acid in DMF, 1 mL of 0.5 M HBTU in DMF, 1 mL of 0.5 M HOBt in DMF The DMF solution and 87 μL of DIPEA were mixed for 5 min, and the resulting mixture was added to the reaction mixture containing the reactive resin. The reactor was mixed for 2 hours.

[0675] The progress of the reaction was monitored by the Kaiser test. If this occurs, the reaction time is extended for another 1 to 3 hours, or the reaction solution is discarded and the reaction is repeated as described above. The process was repeated again. The N-terminal Fmoc protecting group was removed using 20% ​​piperidine in DMF. After the run using the HPLC, the peptide-bound resin was dried and weighed.

[0676] (c) 250 mg of the peptide-bound resin prepared in step (b) was dissolved in 2 mL of TFA, TIS, water, and EDT. The peptide was cleaved from the resin by stirring with the mixture (94:1.0:2.5:2.5) at room temperature for 120 min. The cleavage mixture was filtered and the filtrate was concentrated to approximately half its volume using nitrogen gas. After that, ether was poured into the mixture to precipitate the peptide. The dried precipitate was washed three times with ethanol and dried under nitrogen gas. The dried precipitate was dissolved in 0.1% TFA-30% ACN in water. The mixture was stirred for 6 hours and then concentrated.

[0677] The concentrate was dissolved in 5% DMSO-20% ACN in 0.01M ammonium acetate buffer (pH 6.5). The solution was dissolved in 0.1 mg / mL of HCl and then stirred for 3 days while exposed to air. The progress of the bond formation reaction was monitored by HPLC. If so, the reaction solution was lyophilized to obtain the peptide precipitate.

[0678] (d) Purification The peptide precipitates obtained by freeze-drying in step (c) are listed in Table 4 below. The eluates were separated under the preparative LC primary purification conditions listed in Table 5 below. The resulting peptide was purified under the conditions described above and then lyophilized. The purity of the resulting peptide was 90% or more as determined by analytical HPLC. The molecular weight of the synthesized peptide was confirmed using LC / MS mass spectrometry. did.

[0679] H-PEG8-Asp-Cys*-Ala-Trp-His-Lys-Gly-Glu-Leu-Val-Trp-Cys*-Thr-NH2(Cys*:Disul fido binding site)

[0680] [Table 4]

[0681] [Table 5]

[0682] Confirmation of the structure of SSFI(6Lys) Synthesis of SSFI(6Lys) by matrix-assisted laser desorption / ionization (MALDI) mass spectrometry This was confirmed by molecular weight measurement. Measuring equipment: Ultraflextreme (Bruker) Measurement matrix: CHCA (α-cyano-4-hydroxycinnamic acid) and DHB (2,5-dihydroxy benzoic acid) Calculated molecular weight: 2010.29 g / mol Measured molecular weight (M+H) + :2011.89g / mol

[0683] The mass spectrometry results of SSFI(6Lys) are shown in FIG.

[0684] 1.2.2. Synthesis and structural confirmation of SSFI (Xa1 is ornithine)

[0685] [ka]

[0686] Synthesis of SSFI(6Orn) List of Fmoc amino acids used and order of introduction 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-Orn(Boc)-OH, Fmoc-L-His(Trt)-OH , Fmoc-L-Trp(Boc)-OH, Fmoc-Ala-OH, Fmoc-Cys(Trt)-OH, Fmoc-Asp(tBu)-OH.

[0687] Preparation method (a) Introduction of amino acids The amounts of reagents used in the following process were based on 0.25 mmol. A transparent amide resin (Peptide International Inc., USA) (100 ml / g) was placed in a synthesis reactor. One millimolar of each Fmoc-amino acid block was weighed and added to the C-terminal to N-terminal peptides described above. The amino acid sequence was prepared.

[0688] For activating Fmoc-amino acids and coupling the activated residue to a clear amide resin The reactions were carried out sequentially starting from the C-terminal amino acid.

[0689] Fmoc removal was carried out with 20% piperidine in DMF, and the prepared amino acids corresponding to the sequence were The mixture was incubated with 2 mL of 0.5 M HOBt in DMF, 2 mL of 0.5 M HBTU in DMF, and 174 μL of DIPEA for 5 min. Mix the resulting mixture in the reactor containing the resin for 2 hours. The activation and introduction of residues were carried out by

[0690] The transfection reaction was confirmed using the Kaiser test. If this occurred, the incorporation reaction was repeated once more, or capping was performed using a 20% Ac2O solution in DMF. In each of the incorporation reaction and Fmoc removal, the resin was washed with 100 ml of HCl before proceeding to the next step. This process was repeated until the target peptide sequence was completed. The return was carried out.

[0691] (b) Introduction of H-PEG8-OH After the amino acid introduction is complete, add 1 mL of 0.5 M H-PEG8-OH to the N-terminus of the sequence. Solution of Fmoc-N-amide-dPEG8-acid in DMF, 1 mL of 0.5 M HBTU in DMF, 1 mL of 0.5 M HOBt in DMF The DMF solution and 87 μL of DIPEA were mixed for 5 min, and the resulting mixture was added to the reaction mixture containing the reactive resin. The reactor was mixed for 2 hours.

[0692] The progress of the reaction was monitored by the Kaiser test. If this occurs, the reaction time is extended for another 1 to 3 hours, or the reaction solution is discarded and the reaction is repeated as described above. The process was repeated again. The N-terminal Fmoc protecting group was removed using 20% ​​piperidine in DMF. After the run using the HPLC, the peptide-bound resin was dried and weighed.

[0693] (c) 250 mg of the peptide-bound resin prepared in step (b) was dissolved in 2 mL of TFA, TIS, water, and EDT. The peptide was cleaved from the resin by stirring with the mixture (94:1.0:2.5:2.5) at room temperature for 120 min. The cleavage mixture was filtered and the filtrate was concentrated to approximately half its volume using nitrogen gas. After that, ether was poured into the mixture to precipitate the peptide. The dried precipitate was washed three times with ethanol and dried under nitrogen gas. The dried precipitate was dissolved in 0.1% TFA-30% ACN in water. The mixture was stirred for 6 hours and then concentrated.

[0694] The concentrate was dissolved in 5% DMSO-20% ACN in 0.01M ammonium acetate buffer (pH 6.5). The solution was dissolved in 0.1 mg / mL of HCl and then stirred for 3 days while exposed to air. The progress of the bond formation reaction was monitored by HPLC. If so, the reaction solution was lyophilized to obtain the peptide precipitate.

[0695] (d) Purification The peptide precipitates obtained by freeze-drying in step (c) are listed in Table 6 below. The eluates were separated under the preparative LC primary purification conditions listed in Table 7 below. The resulting peptide was purified under the conditions described above and then lyophilized. The purity of the resulting peptide was 90% or more as determined by analytical HPLC. The molecular weight of the synthesized peptide was confirmed using LC / MS mass spectrometry. did.

[0696] H-PEG8-Asp-Cys*-Ala-Trp-His-Orn-Gly-Glu-Leu-Val-Trp-Cys*-Thr-NH2(Cys*:Disul fido binding site)

[0697] [Table 6]

[0698] [Table 7]

[0699] Confirmation of the structure of SSFI(6Orn) The synthesis of SSFI (6-ornithine) was performed using matrix-assisted laser desorption / ionization (MALDI) mass spectrometry. This was confirmed by molecular weight measurement by HPLC. Measuring equipment: Ultraflextreme (Bruker) Measurement matrix: CHCA (α-cyano-4-hydroxycinnamic acid) and DHB (2,5-dihydroxy benzoic acid) Calculated molecular weight: 1996.26 g / mol Measured molecular weight (M+2H) 2+ :999.13g / mol

[0700] The mass spectrometry results of SSFI (6Orn) are shown in FIG.

[0701] 1.2.3. Synthesis and structural confirmation of SSFI (Xa1 is diaminobutyric acid (Dab))

[0702] [ka]

[0703] Synthesis of SSFI(6Dab) List of Fmoc amino acids used and order of introduction 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-Dab(Boc)-OH, Fmoc-L-His(Trt)-OH , Fmoc-L-Trp(Boc)-OH, Fmoc-Ala-OH, Fmoc-Cys(Trt)-OH, Fmoc-Asp(tBu)-OH.

[0704] Preparation method (a) Introduction of amino acids The amounts of reagents used in the following process were based on 0.25 mmol. A transparent amide resin (Peptide International Inc., USA) (100 ml / g) was placed in a synthesis reactor. One millimolar of each Fmoc-amino acid block was weighed and added to the peptides from the C-terminus to the N-terminus in the above order. The amino acid sequence of the tide was prepared.

[0705] For activating Fmoc-amino acids and coupling the activated residue to a clear amide resin The reactions were carried out sequentially starting from the C-terminal amino acid.

[0706] Fmoc removal was carried out with 20% piperidine in DMF, and the prepared amino acids corresponding to the sequence were The mixture was incubated with 2 mL of 0.5 M HOBt in DMF, 2 mL of 0.5 M HBTU in DMF, and 174 μL of DIPEA for 5 min. Mix the resulting mixture in the reactor containing the resin for 2 hours. The activation and introduction of residues were carried out by

[0707] The transfection reaction was confirmed using the Kaiser test. If this occurred, the incorporation reaction was repeated once more, or capping was performed using a 20% Ac2O solution in DMF. In each of the incorporation reaction and Fmoc removal, the resin was washed with 100 ml of HCl before proceeding to the next step. This process was repeated until the target peptide sequence was completed. The return was carried out.

[0708] (b) Introduction of H-PEG8-OH After the amino acid introduction is complete, add 1 mL of 0.5 M H-PEG8-OH to the N-terminus of the sequence. Solution of Fmoc-N-amide-dPEG8-acid in DMF, 1 mL of 0.5 M HBTU in DMF, 1 mL of 0.5 M HOBt in DMF The DMF solution and 87 μL of DIPEA were mixed for 5 min, and the resulting mixture was added to the reaction mixture containing the reactive resin. The reactor was mixed for 2 hours.

[0709] The progress of the reaction was monitored by the Kaiser test. If this occurs, the reaction time is extended for another 1 to 3 hours, or the reaction solution is discarded and the reaction is repeated as described above. The process was repeated again. The N-terminal Fmoc protecting group was removed using 20% ​​piperidine in DMF. After the run using the HPLC, the peptide-bound resin was dried and weighed.

[0710] (c) 250 mg of the peptide-bound resin prepared in step (b) was dissolved in 2 mL of TFA, TIS, water, and EDT. The peptide was cleaved from the resin by stirring with the mixture (94:1.0:2.5:2.5) at room temperature for 120 min. The cleavage mixture was filtered and the filtrate was concentrated to approximately half its volume using nitrogen gas. After that, ether was poured into the mixture to precipitate the peptide. The dried precipitate was washed three times with ethanol and dried under nitrogen gas. The dried precipitate was dissolved in 0.1% TFA-30% ACN in water. The mixture was stirred for 6 hours and then concentrated.

[0711] The concentrate was dissolved in 5% DMSO-20% ACN in 0.01M ammonium acetate buffer (pH 6.5). The solution was dissolved in 0.1 mg / mL of HCl and then stirred for 3 days while exposed to air. The progress of the bond formation reaction was monitored by HPLC. If so, the reaction solution was lyophilized to obtain the peptide precipitate.

[0712] (d) Purification The peptide precipitates obtained by freeze-drying in step (c) are listed in Table 8 below. The eluates were separated under the preparative LC primary purification conditions listed in Table 9 below. The resulting peptide was purified under the conditions described above and then lyophilized. The purity of the resulting peptide was 90% or more as determined by analytical HPLC. The molecular weight of the synthesized peptide was confirmed using LC / MS mass spectrometry. did.

[0713] H-PEG8-Asp-Cys*-Ala-Trp-His-Dab-Gly-Glu-Leu-Val-Trp-Cys*-Thr-NH2(Cys*:Disul fido binding site)

[0714] [Table 8]

[0715] [Table 9]

[0716] Confirmation of the structure of SSFI(6Dab) Synthesis of SSFI(6Dab) by matrix-assisted laser desorption / ionization (MALDI) mass spectrometry This was confirmed by molecular weight measurement. Measuring equipment: Ultraflextreme (Bruker) Measurement matrix: CHCA (α-cyano-4-hydroxycinnamic acid) and DHB (2,5-dihydroxy benzoic acid) Calculated molecular weight: 1982.24 g / mol Measured molecular weight (M+2H) 2+ :992.33g / mol

[0717] The mass spectrometry results of SSFI (6Dab) are shown in FIG.

[0718] 1.2.4. Synthesis and structural confirmation of SSFI (Xa1 is diaminopropionic acid (Dab))

[0719] [ka]

[0720] Synthesis of SSFI(6Dap) Sequence: Nor.-PEG8-DCAWHA(β-aminoalanine, Dap)GELVWCT-CONH2 List of Fmoc amino acids used and order of introduction 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-Dap(Boc)-OH, Fmoc-L-His(Trt)-OH , Fmoc-L-Trp(Boc)-OH, Fmoc-Ala-OH, Fmoc-Cys(Trt)-OH, Fmoc-Asp(tBu)-OH.

[0721] Preparation method (a) Introduction of amino acids The amounts of reagents used in the following process were based on 0.25 mmol. A transparent amide resin (Peptide International Inc., USA) (100 ml / g) was placed in a synthesis reactor. One millimolar of each Fmoc-amino acid block was weighed and added to the peptides from the C-terminus to the N-terminus in the above order. The amino acid sequence of the tide was prepared.

[0722] For activating Fmoc-amino acids and coupling the activated residue to a clear amide resin The reactions were carried out sequentially starting from the C-terminal amino acid.

[0723] Fmoc removal was carried out with 20% piperidine in DMF, and the prepared amino acids corresponding to the sequence were The mixture was incubated with 2 mL of 0.5 M HOBt in DMF, 2 mL of 0.5 M HBTU in DMF, and 174 μL of DIPEA for 5 min. Mix the resulting mixture in the reactor containing the resin for 2 hours. The activation and introduction of residues were carried out by

[0724] The transfection reaction was confirmed using the Kaiser test. If this occurred, the incorporation reaction was repeated once more, or capping was performed using a 20% Ac2O solution in DMF. In each of the incorporation reaction and Fmoc removal, the resin was washed with 100 ml of HCl before proceeding to the next step. This process was repeated until the target peptide sequence was completed. The return was carried out.

[0725] (b) Introduction of H-PEG8-OH After the amino acid introduction is complete, add 1 mL of 0.5 M H-PEG8-OH to the N-terminus of the sequence. Solution of Fmoc-N-amide-dPEG8-acid in DMF, 1 mL of 0.5 M HBTU in DMF, 1 mL of 0.5 M HOBt in DMF The DMF solution and 87 μL of DIPEA were mixed for 5 min, and the resulting mixture was added to the reaction mixture containing the reactive resin. The reactor was mixed for 2 hours.

[0726] The progress of the reaction was monitored by the Kaiser test. If this occurs, the reaction time is extended for another 1 to 3 hours, or the reaction solution is discarded and the reaction is repeated as described above. The process was repeated again. The N-terminal Fmoc protecting group was removed using 20% ​​piperidine in DMF. After the run using the HPLC, the peptide-bound resin was dried and weighed.

[0727] (c) 250 mg of the peptide-bound resin prepared in step (b) was dissolved in 2 mL of TFA, TIS, water, and EDT. The peptide was cleaved from the resin by stirring with the mixture (94:1.0:2.5:2.5) at room temperature for 120 min. The cleavage mixture was filtered and the filtrate was concentrated to approximately half its volume using nitrogen gas. After that, ether was poured into the mixture to precipitate the peptide. The dried precipitate was washed three times with ethanol and dried under nitrogen gas. The dried precipitate was dissolved in 0.1% TFA-30% ACN in water. The mixture was stirred for 6 hours and then concentrated.

[0728] The concentrate was dissolved in 5% DMSO-20% ACN in 0.01M ammonium acetate buffer (pH 6.5). The solution was dissolved in 0.1 mg / mL of HCl and then stirred for 3 days while exposed to air. The progress of the bond formation reaction was monitored by HPLC. If so, the reaction solution was lyophilized to obtain the peptide precipitate.

[0729] (d) Purification The peptide precipitates obtained by freeze-drying in step (c) are listed in Table 10 below. The compounds were separated under the preparative LC primary purification conditions listed above and further purified under the preparative LC secondary purification conditions listed in Table 11 below. The peptide was purified under the same conditions as in Example 1 and lyophilized. The purity and molecular weight of the synthesized peptides were confirmed using LC / MS mass spectrometry. Confirmed.

[0730] H-PEG8-Asp-Cys*-Ala-Trp-His-Dap-Gly-Glu-Leu-Val-Trp-Cys*-Thr-NH2(Cys*:Disul fido binding site)

[0731] [Table 10]

[0732] [Table 11]

[0733] Confirmation of the structure of SSFI(6Dap) Measurement equipment: Quattro Premier Xe (Waters) Calculated molecular weight: 1968.21 g / mol Measured molecular weight (M+2H) 2+:984.71g / mol

[0734] The mass spectrometry results of SSFI (6Dap) are shown in FIG.

[0735] 1.3. Synthesis of VC linker and confirmation of its structure 1.3.1. Synthesis of VC linker (DD2) and confirmation of its structure

[0736] [ka]

[0737] Synthesis of VC linker (DD2) Synthesis of compound 11 5 g (14.7 mmol, 1.0 equiv.) of Fmoc-Val-OH and 1.7 g (14.7 mmol, 1.0 equiv.) of N-hydroxybenzoxazole Succinimide (NHS) was dissolved in 140 mL of dimethoxyethane (DME) and stirred. mol, 1.1 equivalents) 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 suspended matter, and the filtrate was concentrated under reduced pressure. The solution was dissolved in acetone and stored in a refrigerator at low temperature for 4 hours. The reformed supernatant was removed by filtration under pressure and used in the next reaction without any purification ( Crude yield: 5.5g, 86%). TLC(EA:Hex=1:1);R f =0.5.

[0738] Synthesis of compound 12 2.0 g (11.5 mmol, 1.0 equivalent) of L-citrulline was dissolved in a 1:1 mixture of tetrahydrofuran (THF) and water. The mixture was dissolved in 100 mL of the solution and stirred. 988 mg of sodium bicarbonate was added to it and stirred. Then, 5.0 g (11.5 mmol, 1.0 equivalent) of compound 8 was dissolved in 80 mL of acetone to form a reaction solution. The resulting mixture was stirred for 21 hours and then concentrated under reduced pressure to give a The organic solvent was removed. The aqueous layer was washed with ethyl acetate (EA) and then 2N HCl was added dropwise. The pH was adjusted to 3 by adding EA to the solution, and the precipitate in the organic layer was extracted. was dried over saturated brine and sodium sulfate and used in the next reaction without any purification. (Crude yield: 5.6 g, 98%). TLC (DCM:MeOH=10:1, 1 drop of formic acid); R f =0.1.

[0739] Synthesis of compound 13 Dissolve 2.84 g (5.72 mmol, 1.0 eq) of 10% piperidine in 50 mL of N,N-dimethylformamide (DMF). Amount of Compound 12 was added dropwise and stirred. After 4 hours, the resulting mixture was concentrated under reduced pressure to give the reaction mixture. The solution was removed and the residue was dissolved in water and filtered under reduced pressure to remove any precipitate that formed. was concentrated to give the target compound, which was used in the next reaction without any purification. C (DCM:MeOH=10:1); R f =0.01.

[0740] Synthesis of compound 14 54 mg (0.22 mmol, 1.0 equiv.) of compound 10 was dissolved in 2 mL of DMF and 0.05 mL (0.264 mmol, 1.2 equiv.) of 10 was added. Amount of N,N-diisopropylethylamine (DIPEA) was added dropwise to it. Compound 13 was added to 2 mL of The solution was dissolved completely in water, and 0.05 mL (0.528 mmol, 2.4 equivalents) of acetic anhydride was added at room temperature. After 3 hours, the resulting mixture was concentrated under reduced pressure to remove the reaction solution. The product was purified by reverse phase column chromatography to give the target compound. TLC (DCM:MeOH=10:1) ;R f =0.05.

[0741] Synthesis of compound 15 1 g (3.16 mmol, 1.0 equiv.) of compound 14 was dissolved in 30 mL of a 2:1 mixture of DCM and methanol. , 868 mg (3.48 mmol, 1.1 equivalents) of N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) was added thereto and stirred. Alcohol was added thereto and stirred for 5 hours. The resulting mixture was concentrated under reduced pressure to give the reaction mixture. The solvent was removed and the concentrate was purified by column chromatography (10% MeOH in DCM) to give 419m g of the target compound was obtained (yield: 32%). TLC (DCM:MeOH=10:1); f =0.1.

[0742] Synthesis of compound DD2 109 mg (0.3 mmol, 1.2 equiv.) of phenol-activated SN38 and 0.06 mL (0.33 mmol, 1.3 equiv.) of DI PEA was prepared by dissolving 105 mg (0.25 mmol, 1.0 equiv.) 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 give the target compound. The product was obtained by TLC (DCM:MeOH=10:1); f =0.2.

[0743] Confirmation of the structure of the VC linker (DD2) LRMS (ESI): m / z 840.4 [M+H + ]

[0744] The results are shown in FIG.

[0745] 1.3.2. Synthesis of VC linker (DD3) and confirmation of its structure

[0746] [ka]

[0747] [ka]

[0748] Synthesis of VC linker (DD3) Synthesis of compound 16 5 g (14.7 mmol, 1.0 equiv.) of Fmoc-Val-OH and 1.7 g (14.7 mmol, 1.0 equiv.) of N-hydroxybenzoxazole Succinimide (NHS) was dissolved in 140 mL of dimethoxyethane (DME) and stirred. mol, 1.1 equivalents) 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 suspended matter, and the filtrate was concentrated under reduced pressure. The solution was dissolved in acetone and stored in a refrigerator at low temperature for 4 hours. The reformed supernatant was removed by filtration under pressure and used in the next reaction without any purification ( Crude yield: 5.5g, 86%). TLC(EA:Hex=1:1);R f =0.5.

[0749] Synthesis of compound 17 2.0 g (11.5 mmol, 1.0 equivalent) of L-citrulline was dissolved in a 1:1 mixture of tetrahydrofuran (THF) and water. The mixture was dissolved in 100 mL of the solution and stirred. 988 mg of sodium bicarbonate was added to it and stirred. Then, 5.0 g (11.5 mmol, 1.0 equivalent) of compound 16 was dissolved in 80 mL of acetone to form a r...

Claims

1. 1. A modified antibody or fragment thereof comprising a bioorthogonal click-reactive functional group, the modified antibody comprises one or more modified heavy chains; each of the modified heavy chains comprises a modified lysine residue at position 248 of the heavy chain amino acid sequence; The rank is determined by the EU numbering system, The modified lysine residue contains a bioorthogonal click-reactive functional group at the terminus of the modified antibody or Fragment of.

2. The modified lysine residue has the following structure: 【Chemistry 1】 (In the formula, H 1 is a group bearing a bioorthogonal click-reactive functional group, D 1 is a bond or C 1～4 Alkylene, C 2～4 Alkenylene, C 2～4 Alkynylene, and C 3～8 cycloalkylene) 2. The modified antibody of claim 1, having:

3. D 1 is a bond or an unsubstituted C 1～4 Alkylene, unsubstituted C 2～4 Alkenylene, unsubstituted C 2～4 Alkynylene and unsubstituted C 3～8 cycloalkylene according to claim 2 Modified antibodies.

4. D 1 The modified antibody of claim 2 or 3, wherein said amino acid sequence comprises one or more heteroatoms.

5. D 1 is -[CH 2 ] a -O-[CH 2 ] b - and Each of a and b is independently selected from 0 to 4, and each of a and b is an integer. 、 the sum of a and b is greater than or equal to 0 and less than or equal to 4; A modified antibody described in any one of claims 2 to 4.

6. D 1 is -CH 2 CH 2 OCH 2 -or-CH 2 OCH 2 The modification according to any one of claims 2 to 5, wherein antibody.

7. H 1 is a bioorthogonal click-reactive functional group. Decorative antibodies.

8. The bioorthogonal click-reactive functional groups can be synthesized by Huisgen 1,3-dipolar cycloaddition and Diels -Alder reaction, The modified antibody of any one of items 1 to 7.

9. The bioorthogonal click-reactive functional group can be an azide group, a tetrazine group, a dibenzocyclohexane group, or a cyclohexane group. The modified hydroxyl group according to any one of claims 1 to 7, wherein the modified hydroxyl group is selected from the group consisting of a cutin group and a norbornene group. Decorative antibodies.

10. The modified lysine residue has the following structure: 【Chemistry 2】 8. The modified antibody of any one of claims 1 to 7, having:

11. The modified lysine residue may have a terminal hydroxyl group, a terminal carboxyl group, a terminal thiol group, a terminal The present invention relates to a method for producing a polymerizable composition comprising the steps of: (a) preparing a polymerizable composition comprising a polymerizable compound having a terminal amino group and a terminal aldehyde group; 11. The modified antibody of any one of claims 1 to 10.

12. The modified heavy chain has the formula 8-2: [Formula 8-2] GPSVFLFPPKP-(K)'-DTLMI (wherein (K)' is the modified lysine residue) 12. The modified antibody of claim 1, comprising an amino acid sequence having the structure:

13. 13. The modified antibody of any one of claims 1 to 12, comprising two modified heavy chains.

14. 1. A method for preparing an antibody-payload conjugate, comprising: The modified antibody or fragment thereof comprising a first click-reactive functional group is combined with a cargo unit and a second with a payload comprising a click-reactive functional group; The antibody-payload conjugate is formed by reacting a first click-reactive functional group of the modified antibody with a formed by a click chemistry reaction between a second click-reactive functional group of the payload 、 the modified antibody comprises one or more modified heavy chains; each of the modified heavy chains comprises a modified lysine residue at position 248 of the heavy chain amino acid sequence; The 48th place is determined based on the EU numbering system, The modified lysine residue has a terminal bioorthogonal click-reactive functional group, which is the first click-reactive functional group. containing a block reactive functional group, The antibody-payload conjugate comprises an antibody unit derived from the modified antibody and a a payload unit derived from the payload, The payload unit of the antibody-payload conjugate comprises the antibody unit The method of claim 1, wherein the antibody unit is linked to the antibody unit via K248 of the

15. The modified lysine residue has the following structure: 【Transformation 3】 (In the formula, H 1 is a group bearing a bioorthogonal click-reactive functional group, D 1 is a bond or C 1～4 Alkylene, C 2～4 Alkenylene, C 2～4 Alkynylene, and C 3～8 cycloalkylene) 15. The method of claim 14, comprising:

16. D 1 is a bond or an unsubstituted C 1～4 Alkylene, unsubstituted C 2～4 Alkenylene, unsubstituted C 2～4 Alkynylene and unsubstituted C 3～8 16. The method of claim 15, wherein the alkyl group is selected from the group consisting of cycloalkylene. How to do it.

17. D 1 The method of claim 15 or 16, wherein comprises one or more heteroatoms.

18. D 1 is -[CH 2 ] a -O-[CH 2 ] b - and Each of a and b is independently selected from 0 to 4, and each of a and b is an integer. 、 the sum of a and b is greater than or equal to 0 and less than or equal to 4; 18. The method according to any one of claims 15 to 17.

19. D 1 is -CH 2 CH 2 OCH 2 -or-CH 2 OCH 2 The method according to any one of claims 15 to 18, wherein Law.

20. H 1 is a bioorthogonal click-reactive functional group according to any one of claims 15 to 19. method.

21. The bioorthogonal click-reactive functional groups can be synthesized by Huisgen 1,3-dipolar cycloaddition and Diels -Alder reaction, 21. The method according to any one of items 14 to 20.

22. The bioorthogonal click-reactive functional group can be an azide group, a tetrazine group, a dibenzocyclohexane group, or a cyclohexane group.

21. The copolymer of claim 14, wherein the copolymer is selected from the group consisting of a cutin group and a norbornene group. method.

23. The modified lysine residue has the following structure: 【Chemistry 4】 21. The method of any one of claims 14 to 20, comprising:

24. The modified lysine residue may have a terminal hydroxyl group, a terminal carboxyl group, a terminal thiol group, a terminal 3. The method of claim 1, wherein the compound is free of terminal bioreactive groups selected from terminal amino groups and terminal aldehyde groups.

24. The method of any one of claims 14 to 23.

25. the cargo unit comprises a carrier moiety, a fluorescent moiety, a drug moiety, or a radioactive moiety.

25. The method according to any one of items 14 to 24.

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