Method for synthesizing antibody-drug conjugate labeled by using position-selective antibody based on proximity effect

Abstract

The invention relates to a synthesis method of an antibody-drug conjugate labeled by a position-selective antibody based on proximity effect. According to the present application, it is possible to provide a method for synthesizing an antibody-drug conjugate by introducing a drug position-selectively into an antibody using a pyridine oxime derivative (labeled mediating protein) introduced into a binding protein comprising a non-standard amino acid.

Description

Technical Field

[0001] The present application relates to a method for synthesizing an antibody-drug conjugate using position-selective antibody labeling based on proximity effect. Background Art

[0002] Antibody-drug conjugates (ADCs), one of the fastest-growing classes of new anticancer drugs, consist of an antibody linked to a cytotoxic drug (payload). The antibody targets a protein specifically expressed in cancer cells, while the cytotoxic drug exhibits toxicity within the targeted cancer cells, minimizing the toxicity and side effects of low-molecular-weight anticancer agents on normal cells.

[0003] To develop an ADC for practical therapeutic use, multiple factors must be considered. Specifically, the appropriate antigen expressed on the target cancer cells, an antibody that selectively recognizes the antigen, and a toxic drug are all important factors. Furthermore, to conjugate the antibody and drug, appropriate conjugation technology and a suitable linker to connect the two substances are also important considerations.

[0004] Furthermore, the properties of ADCs are significantly influenced not only by the antibody, drug, and linker, but also by the location, quantity, and uniformity of the drug introduced. Consequently, this depends on the antibody-drug conjugate technology. The development of new antibody-drug conjugate technologies offers a broad platform technology applicable to a variety of ADC applications, regardless of the nature of the cancer cell.

[0005] The conjugation techniques currently used to introduce drugs into antibodies include (1) a chemical introduction method using lysine or cysteine and (2) a site-specific introduction method using an enzyme. First, the method using lysine on the surface of an antibody in the chemical method has the greatest advantage in that the produced antibody can be directly used, but has disadvantages of lacking position selectivity and low homogeneity of ADCs. The lack of position selectivity and the non-homogeneity of ADCs as described above can cause a decrease in the binding force of the antibody and difficulty in quality management during production. In contrast, the method using cysteine, although uniform ADCs can be synthesized, still exhibits low homogeneity and has problems of requiring introduction of cysteine by genetic modification or reduction of disulfide bonds of the antibody. Finally, for the method using an enzyme, although a highly efficient conjugation reaction can be performed with position selectivity, in most cases, there is a problem of requiring genetic modification to introduce an amino acid sequence recognizable by the enzyme into the antibody at the same time.

[0006] Therefore, there is a need to develop a method that can directly use an existing antibody production system without performing genetic modification and also synthesize uniform ADCs with position selectivity.

[0007] Prior Art Documents

[0008] Patent Documents

[0009] (Patent Document) Korean Patent Laid-Open Publication No. 10-2014-0046302 SUMMARY

[0010] The present application provides a conjugation-mediating protein conjugated by introducing a non-canonical amino acid into a binding protein and a pyridinium oxime derivative, an antibody labeling method using the conjugation-mediating protein, and a site-specific synthesis method of an antibody-drug conjugate.

[0011] However, the problems to be solved by the present application are not limited to those mentioned above, and other problems not mentioned will be further clearly understood by those skilled in the art through the following description.

[0012] The first aspect of the present application provides a labeled protein produced by conjugating a binding protein into which a non-standard amino acid is introduced and a pyridyloxime derivative.

[0013] The second aspect of the present application provides a labeling method using an antibody against the labeled protein.

[0014] The third aspect of the present application provides a position-selective synthesis method of an antibody-drug conjugate using an antibody against the labeled protein.

[0015] According to the present application, a method of position-selectively introducing a drug into an antibody using a pyridyloxime derivative (labeled protein) introduced into a binding protein containing a non-standard amino acid, thereby synthesizing an antibody-drug conjugate, can be provided.

[0016] According to the antibody-drug conjugate synthesis method of the present application, a natural antibody can be applied, and no additional change to an existing antibody production system is required, and thus a high utilization rate is expected.

[0017] In addition, the antibody-drug conjugate synthesis method according to the present application can be performed at a very high yield, and uniformity thereof can be ensured, and thus it is expected to be applicable to antibody new drugs as well as various protein new drugs. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 is a mode diagram of a position-selective synthesis method of an antibody-drug conjugate according to one embodiment of the present application.

[0019] Figure 2a and Figure 2b is a mode diagram of an antibody labeling method according to one embodiment of the present application.

[0020] Figure 3 is a mode diagram illustrating a protein composed of a Z-domain protein (Z-DM) and a Z-domain-specific affibody (Z-AFB) in one embodiment of the present application.

[0021] Figure 4are chemical formulas of 4-azido-L-phenylalanine (4-azido-L-phenylalanine; AzF), N6-[(2-azidoethoxy)carbonyl]-L-lysine (AzK), pyridinium oxime-alkyne (PyOx-Alkyne), PyOx_C6-Alkyne (Alkyne), N-alkylsulfonamide-fluorescein (NASA-FL), NASA-biotin (NASA-Bt), and NASA-N3 used in an embodiment of the present application.

[0022] Figure 5 is a schematic diagram illustrating a method of labeling Z-DM using genetic code expansion and Cu-catalyzed azide-alkyne cycloaddition (CuAAC) according to an embodiment of the present application.

[0023] Figure 6 is a schematic diagram illustrating positions of Z-AFB into which non-canonical amino acids (ncAA) are introduced and distances from the Z-AFB to labeling positions of Z-DM in an embodiment of the present application.

[0024] Figure 7 are matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) analysis spectra performed using Z-DM, Z-AFB_K4AzF-PyOx, Z-AFB_K28AzF-PyOx, Z-AFB_F32AzF-PyOx, and Z-AFB_K28AzK-PyOx in an embodiment of the present application.

[0025] Figure 8 is a chart illustrating labeling efficiency according to Z-AFB_K4AzF-PyOx, Z-AFB_K28AzF-PyOx, Z-AFB_F32AzF-PyOx, and Z-AFB_K28AzK-PyOx and distances to target lysines (Lys) of affimers K4, K28, and F32 in an embodiment of the present application.

[0026] Figure 9 are protease (Glu-C) degradation analysis spectra of a native Z-DM peptide fragment (peptide fragment 1) and a FL-labeled Z-DM fragment (peptide fragment 2) in an embodiment of the present application.

[0027] Figure 10 is a matrix-assisted laser desorption ionization-time of flight / time-of-flight (MALDI-TOF / TOF) tandem mass spectrometry (MS) analysis spectrum of peptide fragment 1 (KGSVDNKFNKE, SEQ ID NO: 4) in an embodiment of the present application.

[0028] Figure 11 is a MALDI-TOF / TOF tandem MS analysis spectrum of peptide fragment 2, which is a labeled peptide fragment confirming FL in K18, in an embodiment of the present application.

[0029] Figure 12a is a plot of labeling efficiency of Z-DM according to time in an embodiment of the present application, Figure 12b is a MALDI-TOF MS analysis spectrum for labeling Z-DM according to labeling times of 1 hour, 2 hours, 3 hours, and 4 hours in an embodiment of the present application.

[0030] Figure 13a and Figure 13b are mode diagrams illustrating labeling evaluation of non-binding proteins in an embodiment of the present application, respectively, and labeling efficiency plots of a strep-Z domain, an Fc fragment, lysozyme, and a tryptophan repressor (TrpR).

[0031] Figure 14 is a summary diagram illustrating a labeling method of an Fc fragment of human immunoglobulin G1 (IgG1) in an embodiment of the present application.

[0032] Figure 15 is an image of superimposing a crystal structure of a Z-domain variant (Z-M) protein composed of an AlphaFold2 program and a 3-helix Fc binding protein derived from PDB ID 5U4Y in an embodiment of the present application.

[0033] Figure 16 is a mode diagram illustrating a distance of a Z-M protein and an Fc fragment for selecting an optimal position for introducing an ncAA and a PyOx in a Z-M protein in an embodiment of the present application.

[0034] Figure 17is a high performance liquid chromatography (HPLC) analysis chart of a labeling experiment of Fc fragments of Z-M variants (Z-M_M8AzK-PyOx, Z-M_M8AzK-PyOx_C6, Z-M_D37AzK-PyOx, Z-M_K33AzK-PyOx, and Z-M_H19AzK-PyOx) and NASA probes (NASA-FL and NASA-Bt) in an embodiment of the present application.

[0035] Figure 18 is a model diagram illustrating structural hindrance of target lysines of H19, K33, and D37 of Z-M proteins in an embodiment of the present application.

[0036] Figure 19 is a model diagram illustrating a distance between M8 of Z-M proteins and K248 and K246 of Fc fragments in an embodiment of the present application.

[0037] Figure 20 is a protease (trypsin) degradation analysis spectrum (MALDI-TOF / TOF tandem MS) of a native Fc fragment (peptide fragment 5), an FL-labeled Fc fragment (peptide fragment 6), and a Bt-labeled Fc fragment (peptide fragment 7) in an embodiment of the present application.

[0038] Figure 21 is a MALDI-TOF / TOF tandem MS analysis spectrum of peptide fragment 5 (ASDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISR, SEQ ID NO: 6) in an embodiment of the present application.

[0039] Figure 22 is a MALDI-TOF / TOF tandem MS analysis spectrum of labeled peptide fragment 6 confirming biotin in K248 in an embodiment of the present application.

[0040] Figure 23 is a MALDI-TOF / TOF tandem MS analysis spectrum of labeled peptide fragment 7 confirming FL in K248 in an embodiment of the present application.

[0041] Figure 24 is a HPLC analysis chart of trastuzumab wild type (WT) and Bt-labeled trastuzumab in an embodiment of the present application.

[0042] Figure 25Protease (Trypsin) degradation analysis spectrum (MALDI-TOF / TOF tandem MS) of native trastuzumab fragments (peptide fragment 8) and Bt-labeled trastuzumab fragments (peptide fragment 9) in one embodiment of the present application.

[0043] Figure 26 MALDI-TOF / TOF tandem MS analysis spectrum of peptide fragment 8 (GPSVFLFPPKPKDTLMISR, SEQ ID NO: 8) in one embodiment of the present application.

[0044] Figure 27 MALDI-TOF / TOF tandem MS analysis spectrum of peptide fragment 9 in one embodiment of the present application.

[0045] Figure 28 Overview of labeling method using NASA-N3 to introduce BCN-VC-PABC-MMAE into trastuzumab in one embodiment of the present application.

[0046] Figure 29 HPLC analysis chart of trastuzumab WT, trastuzumab-N3 and trastuzumab-(VC-PABC-MMAE)2 in one embodiment of the present application.

[0047] Figure 30 HPLC analysis chart of trastuzumab WT and trastuzumab-N3 in one embodiment of the present application.

[0048] Figure 31 Orbitrap-MS analysis chart of trastuzumab-WT degraded by immunoglobulin G degrading enzyme (IdeS) in one embodiment of the present application.

[0049] Figure 32 Orbitrap-MS analysis chart of trastuzumab-N3 degraded by IdeS in one embodiment of the present application.

[0050] Figure 33 Protease (Trypsin) degradation analysis spectrum (MALDI-TOF / TOF tandem MS) of native trastuzumab fragments (peptide fragment 10) and trastuzumab-N3 fragments (peptide fragment 11) in one embodiment of the present application.

[0051] Figure 34 MALDI-TOF / TOF tandem MS analysis spectrum of peptide fragment 10 (GPSVFLFPPKPKDTLMISR, SEQ ID NO: 8) in one embodiment of the present application.

[0052] Figure 35MALDI-TOF / TOF tandem MS analysis spectrum of the peptide fragment 11 in one embodiment of the present application.

[0053] Figure 36 In vitro (in vitro) anti-tumor activity chart according to the concentration of emicertumab (Kadcyla), trastuzumab WT and trastuzumab-(VC-PABC-MMAE)2 against MDA-MB-231, MDA-MB-453 and SK-BR-3 in one embodiment of the present application. DETAILED DESCRIPTION

[0054] Next, the embodiments and examples of the present application will be described in detail with reference to the accompanying drawings, so that those skilled in the art to which the present application pertains can easily implement the present application. However, the present application can be implemented in various different forms, and is not limited to the embodiments and examples described herein. In addition, in the accompanying drawings, portions unrelated to the description are omitted in order to clearly describe the present application, and in the entire specification, like reference numerals are assigned to like parts.

[0055] In the entire specification of the present application, when it is described that a certain part is "connected" to another part, it includes not only the case of "directly connected" but also the case of "electrically connected" with another element interposed therebetween.

[0056] In the entire specification of the present application, when it is described that a certain part is "on" another part, it includes not only the case where the certain part is in contact with the other part but also the case where another part is interposed between the two parts.

[0057] In addition, in the entire specification of the present application, when it is described that a certain part "includes" a certain component, it does not mean that other components are excluded unless explicitly stated otherwise, but means that other components can also be included.

[0058] The terms "about" and "substantially" and the like used in the present specification give inherent manufacturing and material tolerances in the meaning of the mentioned values or close thereto, and are used to prevent dishonest infringers from improperly using the disclosure of the precise or absolute values mentioned to help understand the present application.

[0059] The term "step for ~" used in the entire specification of the present application does not mean "step for ~".

[0060] The term "the combination(s)" included in the description of the present application in the form of a Markush group means a mixture or combination of one or more selected from the group consisting of the components recited in the description of the present application in the form of a Markush group, and means one or more selected from the group consisting of the components recited in the description of the present application.

[0061] In the description of the present application, the recitation of "A and / or B" means "A or B or A and B".

[0062] In the description of the present application, in the case where a functional group is substituted, at least one selected from the group consisting of acyl, amino (including simple amino, monoalkylamino and dialkylamino, monoarylamino and diarylamino, and alkylarylamino), acylamino (including carbamoyl and ureido), alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, alkoxycarbonyl, carboxyl, carboxylate, aminocarbonyl, monoalkylaminocarbonyl and dialkylaminocarbonyl, cyano, azido, halogen, hydroxyl, nitro, trifluoromethyl, mercapto, alkylmercapto, arylmercapto, alkylthiocarbonyl, thiocarboxylate, alkyl (as a non-limiting example, the number of carbon atoms is 1 to 20), alkenyl (as a non-limiting example, the number of carbon atoms is 2 to 20), alkynyl (as a non-limiting example, the number of carbon atoms is 2 to 20), cycloalkyl (as a non-limiting example, the number of carbon atoms is 3 to 20), heterocycloalkyl (as a non-limiting example, the number of carbon atoms is 3 to 20), aryl (as a non-limiting example, the number of carbon atoms is 4 to 20), heteroaryl (as a non-limiting example, the number of carbon atoms is 3 to 20), lower alkoxy, aryloxy, aryloxycarbonyloxy, benzyloxy, benzyl, sulfinyl, alkylsulfinyl, sulfonyl, sulfate, sulfonate, sulfamidate, phosphate, phosphonate, phosphinidate, oxo, guanidine, imino, and formyl can be included, but is not limited thereto.

[0063] Next, the implementation examples of the present application are described in detail, but the present application is not limited thereto.

[0064] The first aspect of the present application provides a conjugation-mediating protein formed by conjugating a binding protein into which a non-canonical amino acid is introduced and a pyridinium oxime derivative.

[0065] In the present application, "binding protein" refers to all types of proteins into which non-canonical amino acids are genetically introduced as will be described later. In this case, "genetic introduction" refers to the substitution of a nucleic acid sequence corresponding to at least one amino acid contained in the binding protein with another nucleic acid sequence regardless of the type of the binding protein, thereby substituting the amino acid contained in the binding protein with another amino acid.

[0066] For example, the binding protein can be a protein derived from a protein known to bind to an antibody, i.e., Protein A, Protein G, Protein L, or a similar protein that functions identically thereto, but is not limited thereto.

[0067] Specifically, the binding protein can be a B-domain variant of Staphylococcal Protein A present in the cell of Staphylococcus.

[0068] According to one embodiment of the present application, the B-domain variant is composed of an amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2 (hereinafter, referred to as "Z-M").

[0069] In the present application, "non-canonical amino acids" refer to all amino acids other than one of 20 common amino acids, selenocysteine, and pyrrolysine, and as other terms that can be used in a similar meaning to the term "non-canonical amino acids", include, for example, "unnatural amino acids", "non-naturally encoded amino acids", and "non-naturally occurring amino acids", but can have slightly different scientific definitions. The naturally encoded amino acids include 20 common amino acids, selenocysteine, or pyrrolysine, but are not limited thereto.

[0070] For example, the non-standard amino acid can be selected from the group consisting of 4-azido-L-phenylalanine, N6-((2-azidoethoxy)carbonyl)-L-lysine, 4-acetyl-phenylalanine, O-propargyl-tyrosine, N6-(propargyloxy)carbonyl-lysine, N6-(bicyclononyloxy)carbonyl-lysine, N6-(((2-methylcyclopropenyl)methoxy)carbonyl)-lysine, N6-(trans-cyclooctenyloxy)carbonyl-lysine, and 4-(6-methyltetrazinyl)phenylalanine represented by Chemical Formula 1 to Chemical Formula 9 below, but is not limited thereto.

[0071] [Chemical Formula 1]

[0072]

[0073] [Chemical Formula 2]

[0074]

[0075] [Chemical Formula 3]

[0076]

[0077] [Chemical Formula 4]

[0078]

[0079] [Chemical Formula 5]

[0080]

[0081] [Chemical Formula 6]

[0082]

[0083] [Chemical Formula 7]

[0084]

[0085] [Chemical Formula 8]

[0086]

[0087] [Chemical Formula 9]

[0088]

[0089] Specifically, the non-standard amino acid can be 4-azido-L-phenylalanine, N6-((2-azidoethoxy)carbonyl)-L-lysine, O-propargyl-tyrosine, or N6-(propargyloxy)carbonyl-lysine. ε -((2-azidoethoxy)carbonyl)-L-lysine, O-propargyl-tyrosine, or N6-(propargyloxy)carbonyl-lysine.

[0090] According to one embodiment of the present application, the non-standard amino acid can be introduced by substitution with at least one or more amino acids included in the binding protein.

[0091] Amino acid positions of the binding protein that can be substituted with the non-standard amino acid include positions excluded from sites that bind to one or more binding partners, i.e., potential receptor binding sites, positions that can be completely or partially exposed to a solvent, positions that have little or no hydrogen bonding interaction with adjacent residues, positions that are least exposed to adjacent reactive residues, positions that are predicted to be highly flexible or structurally rigid according to the three-dimensional structure of the protein, positions that are bound or unbound to a relevant receptor, ligand, or binding protein, positions that are located at sites that are bound or unbound to other proteins or other biologically active molecules, or positions that can adjust the conformation of the protein itself or a dimer or multimer comprising one or more proteins by changing the flexibility or rigidity of the overall structure as needed, but are not limited thereto.

[0092] According to one embodiment of the present application, positions that can not affect binding to each other and are located around the binding site were identified based on the X-ray crystal structure of the Z-domain and affibody, and the positions were selected as introduction positions of the non-standard amino acid (e.g., K7 or D36).

[0093] In addition, according to another embodiment of the present application, a position at which interference with Fc binding of a generated 3-helix Fc binding protein (Z-M) can be minimized and which is adjacent to an Fc fragment is identified based on a crystal structure of a complex formed by a 2-helix Fc binding protein and an Fc fragment, and the position is selected as a position into which a non-standard amino acid is introduced (e.g., M8, H19, K33, or D37).

[0094] The binding protein into which the non-standard amino acid is introduced, which is manufactured according to the embodiment, is composed of the amino acid sequence represented by SEQ ID NO: 3.

[0095] Specifically, the amino acid sequence represented by SEQ ID NO: 3 is a binding protein, i.e., a Z-domain variant (Z-M), in which an eighth amino acid, i.e., methionine (M), is substituted with a non-standard amino acid, i.e., N6-[(2-azidoethoxy)carbonyl]-L-lysine (AzK).

[0096] In the present application, "pyridinium oxime derivative" can be represented by Formula 1 below, but is not limited thereto.

[0097] [Formula 1]

[0098]

[0099] In Formula 1,

[0100] A is nothing or an alkylene group of substituted or unsubstituted C 1-10 , a cycloalkylene group of substituted or unsubstituted C 5-10 , a phenylene group of substituted or unsubstituted C 5-10 , a polyethylene glycol group of substituted or unsubstituted C 2-10 , an alkylene group of substituted or unsubstituted C 1-10 , a cycloalkylene group of substituted or unsubstituted C 5-10 , a phenylene group of substituted or unsubstituted C 5-20 , a polyethylene glycol group of substituted or unsubstituted C , and a combination thereof,

[0101] R a , and R b are each independently nothing, an alkylene group of substituted or unsubstituted C 1-10 , or a polyethylene glycol group of substituted or unsubstituted C 2-10 , a cycloalkylene group of substituted or unsubstituted C

[0102] B is selected from , and a combination thereof,

[0103] W is -F, -Cl, -Br, -NO2, or -CF3,

[0104] n is 0 to 4.

[0105] wherein the C 1-10 alkylene group can be selected from methylene, ethylene, propylene, butylene, pentylene, hexylene, heptylene, nonylene, decylene, and isomers thereof.

[0106] wherein the C 2-10 polyethylene glycol group can be represented by [- (C2H4) -O-] m m can be an integer of 1 to 5.

[0107] wherein according to the combination of the linking groups listed as A, there can be a linking site in the form of -(CH2) o -O-(CH2) p -, -(CH2) o -(C6H4)-(CH2) p -, -(CH2) o -NH-(CH2) p -, -(CH2) o -NH-(C6H4)-(CH2) p -, -(CH2) o -O-(C6H4)-(CH2) p -, -(CH2) o -(C6H4)-NH-(CH2) p -, and -(CH2) o -(C6H4)-O-(CH2) p - (wherein o and p are each independently 0 to 5).

[0108] The hydroxyl group present in the pyridine oxime can have a pK a value of 8 or less and can exist in an anionic state under general in vivo conditions, so that it can react with a labeling substance (a binding protein into which a non-standard amino acid is introduced) with high efficiency and selectivity.

[0109] According to one embodiment of the present application, the non-standard amino acid introduced into the binding protein and the pyridyloxime derivative can be reacted by bioorthogonal reactions such as copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) or strain-promoted azide-alkyne cycloaddition (SPAAC), but are not limited thereto.

[0110] Specifically, the bioorthogonal reaction can be a reaction of B of the pyridyloxime derivative of Formula 1 with a terminal functional group of the non-standard amino acid described above.

[0111] For example, a structure formed by a binding reaction of B of the pyridyloxime derivative of Formula 1 with a terminal functional group of the non-standard amino acid described above can be as follows.

[0112]

[0113] The CuAAC can be effectively performed in the presence of copper ions (Cu 2+ ), a reducing agent (ascorbic acid, which converts Cu 2+ into Cu + ), and a ligand (stabilization of copper ions).

[0114] The CuAAC is a reaction having high sensitivity and specificity, Figure 5 is a schematic diagram illustrating the process of the CuAAC. As Figure 5 shown, the azido group (N3) contained in a binding protein into which an azido group (N3)-containing non-standard amino acid is introduced by gene is coupled with a pyridyloxime derivative by CuAAC.

[0115] The pyridyloxime derivative and the CuAAC have a high rate constant (0.1 to 1.0 M -1 s -1 ), and a relatively small triazole is generated after the reaction, and thus are recognized as suitable for bioconjugation between biochemical molecules such as proteins. In order to apply CuAAC to protein conjugation, it is necessary to introduce an azido group (alkynyl group) into a binding protein. Since the azido group (alkynyl group) can be introduced into a binding protein by a genetic method, effective labeling of a binding protein can be achieved by applying CuAAC to an amino acid containing a gene-introduced azido group (alkynyl group).

[0116] The coupling reaction can be initiated by mixing the binding protein and the pyridine oxime derivative, but is not limited thereto.

[0117] In the present application, the nucleic acid sequence encoding the "non-standard amino acid-introduced binding protein" is not limited to a nucleic acid sequence that is modified based on the amino acid sequence of a wild type protein, and in which a specific amino acid included in the amino acid sequence is introduced, substituted, or deleted, but can be a nucleic acid sequence that is selectively or non-selectively modified according to a method known in the art. For example, it can include a method of substituting a codon encoding a specific amino acid in a protein gene with a TAG codon, a method of introducing a plasmid including a protein gene having the substituted codon as a stop codon, a plasmid including an appropriate tRNA gene, and an appropriate aminoacyl-tRNA synthetase gene into a cell such as E. coli, to produce a transformed cell, a method of culturing the transformed cell in a medium including a non-standard amino acid, but is not limited thereto.

[0118] In the process of producing a protein mutant by the transformed cell, the non-standard amino acid can be bound to tRNA by the aminoacyl-tRNA synthetase, and the tRNA bound to the non-standard amino acid can recognize the substituted nucleic acid sequence (TAG) and insert the non-standard amino acid into an appropriate site of the translated protein, but is not limited thereto.

[0119] The non-standard amino acid can be present at any position on a protein including any terminal position or any internal position. The non-standard amino acid-introduced protein can be produced by biosynthesis or solid-phase peptide synthesis, but is not limited thereto. The "biosynthesis" refers to a method including the use of at least one of a polynucleotide, a codon, tRNA, and a ribosome, and a cell translation system, but is not limited thereto.

[0120] In the present application, the "conjugation-mediating protein" refers to a protein that does not include a direct labeling material, but plays a mediating role in catalyzing labeling by binding to a target protein.

[0121] The second aspect of the present application provides a labeling method of an antibody including the following steps:

[0122] The first step of binding the conjugation-mediating protein to a native antibody

[0123] a second step in which the labeling mediator protein reacts with the N-alkyl sulfonamide derivative having the probe bound thereto, thereby forming an active ester intermediate; and

[0124] a third step in which a lysine included in an Fc domain of the native antibody reacts with the intermediate, thereby forming an amide bond.

[0125] According to an embodiment of the present application, the first step, the second step, and the third step of the labeling method of the antibody are sequentially described, but this is only a description for explaining the reaction, and the order of the steps is not limited. Specifically, the labeling method of the antibody can be a reaction that is substantially simultaneously or almost simultaneously performed.

[0126] According to an embodiment of the present application, the first step can be performed by mixing a labeling mediator protein labeled with a pyridine oxime derivative with a native antibody.

[0127] In the present application, the "native antibody" refers to all types of antibodies naturally produced as a result of the function of the human immune system without damage, and can be used in the same meaning as "native antibody" and "intact antibody" and the like. The native antibody is different from an antibody obtained by a recombinant library method (phage or transformed mouse) in some aspects, and has a unique characteristic that can be an ideal treatment method for human viral diseases.

[0128] A complete antibody is a structure having two full-length light chains and two full-length heavy chains, each of which is connected to a heavy chain by a disulfide bond. The heavy chain constant region includes gamma (γ), mu (μ), alpha (α), delta (δ), and epsilon (ε) types, and its subclasses include gamma 1 (γ1), gamma 2 (γ2), gamma 3 (γ3), gamma 4 (γ4), alpha 1 (α1), and alpha 2 (α2). The light chain constant region includes kappa (κ) and lambda (λ) types (Cellular and Molecular Immunology, Wonsiewicz, M. J., Ed., Chapter 45, pp. 41-50, W. B. Saunders Co. Philadelphia, PA (1991); Nisonoff, A., Introduction to Molecular Immunology, 2nd Ed., Chapter 4, pp. 45-65, Sinauer Associates, Inc., Sunderland, MA (1984)).

[0129] In the present application, the "heavy chain" refers to a full-length heavy chain including a variable region domain VH having an amino acid sequence of a sufficient variable region sequence for conferring specificity to an antigen, and three constant region domains, CH1, CH2, and CH3, and fragments thereof. In addition, the "light chain" in the present application refers to a full-length light chain including a variable region domain VL having an amino acid sequence of a sufficient variable region sequence for conferring specificity to an antigen, and a constant region domain, CL, and fragments thereof.

[0130] Specifically, the native antibody can be human immunoglobulin G1 (IgG1), but is not limited thereto.

[0131] According to one embodiment of the present application, the second step can be performed by mixing the N-alkylsulfonamide derivative having a probe with the native antibody having a label-mediated protein bound thereto.

[0132] In the present application, the "N-alkylsulfonamide (NASA) derivative having a probe" refers to a probe (P) in which N-alkylsulfonamide or a derivative thereof is substituted, and can be represented by Formula 2 below, but is not limited thereto.

[0133] [Formula 2]

[0134]

[0135] In Formula 2,

[0136] R' can be

[0137] R is absent or substituted or unsubstituted C 1-10 Alkylene, substituted or unsubstituted C 5-10 Cycloalkylene, substituted or unsubstituted C 5-10 Phenylene, substituted or unsubstituted C 2-10 A polyethylene glycol group, a C containing a heteroatom selected from N, O or S 1-10 an alkylene group, a C 5-10 Cycloalkylene, C containing heteroatoms selected from N, O or S 5-20 Phenylene, and the combination of the above,

[0138] R c and R d Each independently is absent, substituted or unsubstituted C 1-10 Alkylene or substituted or unsubstituted C 2-10 The polyethylene glycol group,

[0139] R1 is from CF3 or Select

[0140] R2 is from CF3, Select

[0141] R3 is H, linear or branched C 1-5 The alkyl group,

[0142] P is from Select

[0143] X1, X2 and X3 replace at least one hydrogen in the phenyl group, and are each independently F, Cl, Br, CF3 or NO2,

[0144] Y is O or NH,

[0145] Z is -NR e R f Substituted or unsubstituted linear or branched C 1-20 The alkyl group,

[0146] R e and R f Each independently is C 1-5 Alkyl groups or interconnected C 4-10 and may contain N, S or O heteroatoms.

[0147] Among them, the C1-10 The alkylene group may be selected from methylene, ethylene, propylene, butylene, pentylene, hexylene, heptylene, nonylene, decylene and isomers thereof.

[0148] Among them, the C 2-10 The polyethylene glycol group can be represented as [-(C2H4)-O-] m , m can be an integer from 1 to 5.

[0149] Among them, according to the C 1-10 The alkylene group and the C 2-10 The combination of polyethylene glycol groups may also have a linking portion in the form of -(CH2)-(CH2CH2)-O- or -(CH2CH2)-(CH2CH2)-O-.

[0150] Wherein, according to the combination of the linking groups listed as R, there may be -(CH2) o -O-(CH2) p -、-(CH2) o -(C6H4)-(CH2) p -、-(CH2) o -NH-(CH2) p -、-(CH2) o -NH-(C6H4)-(CH2) p -、-(CH2) o -O-(C6H4)-(CH2) p -、-(CH2) o -(C6H4)-NH-(CH2) p -and-(CH2) o -(C6H4)-O-(CH2) p - (wherein o and p are each independently 0 to 5) is a linking site.

[0151] Specifically, the N-alkylsulfonamide derivative conjugated to the probe can be N-alkylsulfonamide-fluorescein (NASA-FL), N-alkylsulfonamide-biotin (NASA-Bt), N-alkylsulfonamide-N3, N-alkylsulfonamide-thiol, tetrazine, cyclopropene, trans-cyclooctene, bicyclo[6.1.0]non-4-yne (BCN) and / or dibenzocyclooctyne (DBCO).

[0152] More specifically, the probe-bound N-alkyl sulfonamide derivative can be N-alkyl sulfonamide-fluorescein (NASA-FL), N-alkyl sulfonamide-biotin (NASA-Bt), or N-alkyl sulfonamide-N3.

[0153] In the present application, the label-mediated protein and the probe-bound N-alkyl sulfonamide derivative can form an active ester intermediate through a mutual specific reaction.

[0154] In the present application, the "active ester intermediate" refers to an acyl-oxime intermediate.

[0155] In the present application, the active ester intermediate can be bound to lysine included in the Fc domain of the natural antibody.

[0156] Specifically, the active ester intermediate can be bound to lysine by forming an amide bond.

[0157] In the present application, the "Fc domain" refers to a single heavy chain Fc region of an antibody, and the Fc domain can be composed of one to four domains selected from the group consisting of CH1, CH2, CH3, and CH4 domains.

[0158] Specifically, the Fc domain can additionally include a hinge region.

[0159] Further, specifically, the Fc domain can be selected from the group consisting of IgG, IgA, IgD, IgE, IgM, a combination thereof, and a hybrid thereof.

[0160] According to an embodiment of the present application, the Fc domain protein can be composed of the amino acid sequence represented by SEQ ID NO: 5.

[0161] Detailed descriptions of parts repeated with the first aspect of the present application are omitted, but the descriptions made with respect to the first aspect of the present application can be equally applied even in the case where the descriptions thereof are omitted in the second aspect.

[0162] The third aspect of the present application provides a site-specific synthesis method of an antibody-drug conjugate including the following steps:

[0163] a first step of binding a labeling mediator protein to a natural antibody;

[0164] a second step of reacting the labeling mediator protein with an N-alkylsulfonamide derivative to which a probe is bound, thereby forming an active ester intermediate;

[0165] a third step of reacting a lysine included in an Fc domain of the natural antibody with the intermediate, thereby forming an amide bond; and

[0166] a fourth step of labeling a drug by reacting the probe bound to the lysine.

[0167] or

[0168] a first step of binding a labeling mediator protein to a natural antibody;

[0169] a second step of reacting the labeling mediator protein with an N-alkylsulfonamide derivative to which a drug is bound, thereby forming an active ester intermediate; and

[0170] a third step of reacting a lysine included in an Fc domain of the natural antibody with the intermediate, thereby forming an amide bond.

[0171] In the present application, the "N-alkylsulfonamide (NASA) derivative to which a probe is bound" refers to a probe (P) to which an N-alkylsulfonamide or a derivative thereof is substituted, and can be represented by Formula 2 below, but is not limited thereto.

[0172] [Formula 2]

[0173]

[0174] In the Formula 2,

[0175] R' can be

[0176] R is nothing or an alkylene group substituted or unsubstituted with C 1-10 , a cycloalkylene group substituted or unsubstituted with C 5-10 , a phenylene group substituted or unsubstituted with C 5-10 , a polyethylene glycol group, a C 2-10 alkylene group including a heteroatom selected from N, O, or S, a C 1-10 cycloalkylene group including a heteroatom selected from N, O, or S, a C 5-10 phenylene group including a heteroatom selected from N, O, or S, 5-20 and selected from the combinations thereof, ​

[0177] R c and R d Each independently is absent, substituted or unsubstituted C 1-10 Alkylene or substituted or unsubstituted C 2-10 The polyethylene glycol group,

[0178] R1 is from CF3 or Select

[0179] R2 is from CF3, Select

[0180] R3 is H, linear or branched C 1-5 The alkyl group,

[0181] P is from Select

[0182] X1, X2 and X3 replace at least one hydrogen in the phenyl group, and are each independently F, Cl, Br, CF3 or NO2,

[0183] Y is O or NH,

[0184] Z is -NR e R f Substituted or unsubstituted linear or branched C 1-20 The alkyl group,

[0185] R e and R f Each independently is C 1-5 Alkyl groups or interconnected C 4-10 and may contain N, S or O heteroatoms.

[0186] Among them, the C 1-10 The alkylene group may be selected from methylene, ethylene, propylene, butylene, pentylene, hexylene, heptylene, nonylene, decylene and isomers thereof.

[0187] Among them, the C 2-10 The polyethylene glycol group can be represented as [-(C2H4)-O-] m , m can be an integer from 1 to 5.

[0188] Among them, according to the C 1-10 The alkylene group and the C 2-10 The combination of polyethylene glycol groups may also have a linking portion in the form of -(CH2)-(CH2CH2)-O- or -(CH2CH2)-(CH2CH2)-O-.

[0189] wherein, according to the combination of the linking groups listed as R, there can be, for example, -(CH2) o -O-(CH2) p -, -(CH2) o -(C6H4)-(CH2) p -, -(CH2) o -NH-(CH2) p -, -(CH2) o -NH-(C6H4)-(CH2) p -, -(CH2) o -O-(C6H4)-(CH2) p -, -(CH2) o -(C6H4)-NH-(CH2) p - and -(CH2) o -(C6H4)-O-(CH2) p -(wherein o and p are each independently 0 to 5).

[0190] Specifically, the probe-bound N-alkylsulfonamide derivative can be N-alkylsulfonamide-fluorescein (NASA-FL), N-alkylsulfonamide-biotin (NASA-Bt), N-alkylsulfonamide-N3, N-alkylsulfonamide-thiol, tetrazine, cyclopropene, trans-cyclooctene, bicyclo[6.1.0]non-4-yne (BCN), and / or dibenzocyclooctyne (DBCO).

[0191] More specifically, the probe-bound N-alkylsulfonamide derivative can be N-alkylsulfonamide-fluorescein (NASA-FL), N-alkylsulfonamide-biotin (NASA-Bt), or N-alkylsulfonamide-N3.

[0192] In the present application, the label-mediated protein and the probe-bound N-alkylsulfonamide derivative can form an active ester intermediate through a mutual specific reaction.

[0193] In the present application, the "probe" bound to lysine can be a moiety represented as P in the probe-bound N-alkylsulfonamide derivative represented by the formula 2.

[0194] In an implementation example of the present application, in the case where the marker-mediated protein directly reacts with the N-alkyl sulfonamide derivative to which a drug is bound, the drug can perform a function as a probe. In the case as described above, the N-alkyl sulfonamide derivative to which a drug is bound can be represented by the following Formula 3.

[0195] [Formula 3]

[0196]

[0197] In the Formula 3,

[0198] R' can be

[0199] R is nothing or an alkylene group of C 1-10 , a substituted or unsubstituted C 5-10 , a cycloalkylene group of C 5-10 , a phenylene group of C 2-10 , a polyethylene glycol group of C 1-10 , an alkylene group of C 5-10 , a cycloalkylene group of C 5-20 , a phenylene group of C , and a combination thereof,

[0200] R c , and R d are each independently nothing, a substituted or unsubstituted C 1-10 , an alkylene group of C 2-10 , a polyethylene glycol group of C

[0201] R1 is selected from CF3 or ,

[0202] R2 is selected from CF3, ,

[0203] R3 is H, a linear or branched C 1-5 , an alkyl group of C

[0204] D is a drug or a drug including a cleavable linker,

[0205] X1, X2, and X3 substitute at least one hydrogen in a phenyl group, and are each independently F, Cl, Br, CF3, or NO2,

[0206] Y is O or NH,

[0207] Z is -NR eR f substituted or unsubstituted linear or branched C 1-20 alkyl,

[0208] R e and R f each independently is a C 1-5 alkyl or a C 4-10 cyclic alkyl, with or without heteroatoms of N, S or O.

[0209] wherein the C 1-10 alkylene can be selected from methylene, ethylene, propylene, butylene, pentylene, hexylene, heptylene, nonylene, decylene and isomers thereof.

[0210] wherein the C 2-10 polyethylene glycol group can be represented as [- (C2H4)-O-] m , and m can be an integer from 1 to 5.

[0211] wherein according to the combination of the C 1-10 alkylene and the C 2-10 polyethylene glycol group, there can be a connecting site in the form of, for example, -(CH2)-(CH2CH2)-O- or -(CH2CH2)-(CH2CH2)-O-.

[0212] wherein according to the combination of the connecting groups listed as R, there can be a connecting site in the form of, for example, -(CH2) o -O-(CH2) p -, -(CH2) o -(C6H4)-(CH2) p -, -(CH2) o -NH-(CH2) p -, -(CH2) o -NH-(C6H4)-(CH2) p -, -(CH2) o -O-(C6H4)-(CH2) p -, -(CH2) o -(C6H4)-NH-(CH2) p - and -(CH2) o -(C6H4)-O-(CH2) p - (wherein o and p are each independently 0 to 5).

[0213] In the present application, "payload" refers to a low-molecular synthetic compound that occupies a considerable portion of actual anticancer effects, and is one of the constituent elements of antibody-payload conjugates as important as antibodies. In the present application, the payload includes all types of compounds that exhibit cytotoxicity and anticancer effects.

[0214] According to one embodiment of the present application, the payload can be a drug or a drug comprising a cleavable linker.

[0215] The position-selective synthesis method of antibody-payload conjugates according to one embodiment of the present application can be applied without limitation to payloads that can be used in antibody-payload conjugates, as long as there are no restrictions applicable to the present application. For example, the payload can be selected from monomethyl auristatin E (MMAE), Maytansinoids (as non-limiting examples, DM1 and DM4), Calicheamicin, exatecan, SN-38, monomethyl auristatin F (MMAF), and derivatives thereof.

[0216] In one embodiment of the present application, the linker can be used without limitation to linkers that are commonly used in the technical field and are useful for antibody-payload conjugates. Among them, the linker is a chemical linking loop that links an antibody and a payload, should maintain stability when circulating or distributing in the body, and is selectively cleaved only when penetrating into a tumor and into a cell, thereby releasing the bound payload. In general, the linker is composed of an attachment-spacer-release, and a new linker can be developed by modifying and / or improving one or more constituent elements in the commercially available linker composition. In one embodiment of the present application, the linker can use not only linkers commonly used in the technical field, but also new linkers developed without limitation.

[0217] In one embodiment of the present application, the linker as a "cleavable linker" may include N-succinimidyl-4-(2-pyridyldithio) pentanoate [N-succinimidyl-4-(2-pyridyldithio) pentanoate; SPP], N-succinimidyl-4-(2-pyridyldithio) butanoate [N-succinimidyl-4-(2-pyridyldithio) butanoate; SPDB], sulfo-SPDB (sulfo-SPDB), MDS, DMDS, DSDM or NDMDS, etc. Disulfide linkers; enzymatically cleavable linkers such as phenylalanine-lysine-p-aminobenzyloxycarbonyl (Phe-Lys-PABC), valine-citrulline-p-aminobenzyloxycarbonyl (Val-Cit-PABC), valine-alanine-p-aminobenzyloxycarbonyl (Val-Ala-PABC), valine-citrulline (MHVCBC) or phenylalanine-lysine (MHFKBC); hydrazone linkers such as methylhydrazone hexanoate (MHH); or glucuronic acid (GBC) The invention also provides a glucosidase-sensitive linker such as a glucuronic acid), glucuronic acid (GBCDN) or a β-glucuronide linker, or a non-cleavable linker including maleimide-polyethylene glycol-N-hydroxysuccinimide ester (Mal-PEG-NHS), N-succinimidyl-4-(N-maleimidomethyl)cyclohexanecarboxylate [SMCC], maleimide-alkane linker (Mal-alkanelinker), bis-maleimidopolyethylene glycol (BMPEO), N-(β-maleimidopropyloxy)succinimide ester [BMPS], ε-maleimidocaproic acid N-hydroxysuccinimide ester (ε-maleimidocaproic acid N-hydroxysuccinimide ester (ε-maleimidocaproic acid N-hydroxysuccinimide ester (ε-maleimidocaproic acid N-hydroxysuccinimide ester (ε-maleimidocaproic acid N-hydroxysuccinimide ester (ε-maleimidocaproic acid N-hydroxysuccinimide ester (ε-maleimidocaproic acid N-hydroxysuccinimide ester (ε-maleimidocaproic acid N-hydroxysuccinimide ester (ε-maleimidocaproic acid N-hydroxysuccinimide ester (ε-maleimidocaproic acid N-hydroxysuccinimide ester ( N-hydroxysuccinimide ester, EMCS), γ-maleimidobutyric acid N-succinimidyl ester (GMBS), m-maleimidobenzoyl-N-hydroxysuccinimide ester (N-succinimidyl 3-(bromoacetamido)propionate [SBAP], N-succinimidyl iodoacetate (SIA), N-succinimidyl-4-(iodoacetyl)-aminobenzoate [SIAB], N-succinimidyl 4-(p-maleimidophenyl)butyrate [SMPB], or succinimidyl-6-(maleimidopropionamido)hexanoate [SMPH], but are not limited thereto.

[0218] Specifically, the "cleavable linker" can be valine-citrulline-p-aminobenzyloxycarbonyl (Val-Cit-PABC), but is not limited thereto. - Specifically, the "cleavable linker" can be valine-citrulline-p-aminobenzyloxycarbonyl (Val-Cit-PABC), but is not limited thereto.

[0219] The drug can be introduced in a form in which the drug is bound to the probe after the probe is bound to the native antibody, or in a form in which an N-alkyl sulfonamide derivative to which the drug is bound is used, and is introduced into the native antibody by mediating the formation of an amide bond between the protein and the antibody by means of the label.

[0220] For parts repeated in the first aspect and / or the second aspect of the present application, detailed explanations are omitted, but the explanations made for the first aspect and / or the second aspect of the present application can be equally applied even in the case where the explanations thereof are omitted in the second aspect.

[0221] Next, the present application will be more specifically explained using examples, but the following examples are only illustrative to help understanding of the present application, and the content of the present application is not limited to the following examples.

[0222]

EXAMPLES

[0223] 1. Test substance and analysis method

[0224] 1.1. Test substance

[0225] The materials required for protein labeling were used directly after purchase without additional purification steps.

[0226] When introducing non-standard amino acids (ncAAs), N6-[(2-azidoethoxy)carbonyl]-L-lysine (AzK) was purchased from Iris Biotech, and 4-azido-L-phenylalanine (AzF) was purchased from Santa Cruz Biotechnology. NaCl, Na2HPO4, KH2PO4, (NH4)2SO4, MgSO4, CuSO4, NaOH, ethylenediaminetetraacetic acid (EDTA), glycerol, sodium-L-ascorbate, aminoguanidine hydrochloride, and amino acids were purchased from TGI Chemicals. Lactose and sodium dodecyl sulfate (SDS) were purchased from Sigma-Aldrich. Arabinose and chloramphenicol were purchased from Alfa Aesar. Ampicillin, kanamycin, and dithiothreitol (DTT) were purchased from Fisher BioReagent, Wako, and BioBasic, respectively.

[0227] Ni-nitrilotriacetic acid (Ni-NTA) agarose was purchased from Qiagen. 4- Hydroxyethylpiperazineethanesulfonic acid (HEPES) and Luria-Bertani (LB) liquid medium were purchased from Dojindo Laboratories and Gibco, respectively.

[0228] Sinapic acid (SA), a-cyano-4-hydroxycinnamic acid (CHCA), and 2,5-dihydroxybenzoic acid (DHB) were purchased from Sigma-Aldrich when performing matrix-associated laser desorption ionization time-of-flight (MALDI-TOF) analysis.

[0229] HPLC-grade acetonitrile (ACN), formic acid, and trifluoroacetic acid (TFA) were purchased from Trag-Heal when performing high-performance liquid chromatography (HPLC) analysis. 4-Nitrobenzenesulfonamide and 4-nitrobenzyl bromide were purchased from Combi-Blocks. 5(6)-Carboxyfluorescein was purchased from Sigma-Aldrich, pyridine-4-carboxaldoxime, 4-bromo-1-butyne, and 6-chloro-1-hexyne were purchased from Alfa Aesar. 2-Azidoacetic acid and N-succinimidyl D-biotinate were purchased from TCI. Fluorenylmethoxycarbonyl-valine-citrulline-p- aminobenzyloxycarbonyl-p-nitrophenyl ester (Fmoc-Val-Cit-PABC-PNP), monomethyl auristatin E (MMAE), and bicyclononene-polyethylene glycol 4-acid (BCN-PEG4-acid) were purchased from Angene, Chemscene, and BLD Pharm, respectively.

[0230] In the antibody labeling experiment, trastuzumab was purchased from Roche, and IdeS was purchased from ACROBiosystems. Sequencing Grade Modified Trypsin was purchased from Promega.

[0231] In the deoxyribonucleic acid (DNA) manipulation, PrimeSTAR High-Fidelity DNA Polymerase and restriction enzymes were purchased from Takara Bio. DNA oligos were purchased from Macrogen, and PCR / Gel purification kits and plasmid miniprep kits were purchased from Nucleogen. For the preparation of proteins, Ultra centrifugal filter tubes, Pipette Tips were purchased from Merck Millipore. Pierce TM C18 spin columns and Slide-A-Lyzer TM Dialysis cassettes were purchased from Thermofisher Scientific, and PD SpinTrap G-25 was purchased from Cytiva.

[0232] 1.2. Analytical methods

[0233] 1) analytical-high performance liquid chromatography (HPLC)

[0234] In reversed-phase liquid chromatography (RPLC) analysis, each sample (3 pg) was analyzed using an Agilent 1260 Infinity I with Poroshell 300SB-C8 (Agilent, 1.0 x 75 mm, 5 pm) at a flow rate of 1 mL / min. The initial mobile phase A (0.1% TFA in water) was kept at 80% for 2 min. The mobile phase B (0.1% TFA in ACN) was ramped from 20% to 60% over 2-8 min. Additionally, over the next 2 min (8-10 min), all samples in the column were eluted by ramping the mobile phase B from 60% to 98% and detected at 280 nm using a variable wavelength detector (VWD).

[0235] In hydrophobic interaction chromatography (HIC) analysis, each sample (10 pg) was analyzed using a high-performance liquid chromatograph equivalent to a MAbPac HIC-Butyl HPLC (Thermo Scientific TM , 4.6 x 100 mm, 5 pm) at a flow rate of 1 mL / min. A gradient change from 0% to 100% was applied over 1-15 min. Buffer A: (1.5 M ammonium sulfate and 50 mM sodium phosphate, pH 7.0); Buffer B: (50 mM sodium phosphate, pH 7.0); Flow rate = 1 mL / min. Samples were detected at 280 nm using a VWD.

[0236] 2) Mass analysis of Fc fragments and trastuzumab

[0237] Each sample (30 pg) was washed using a PD SpinTrap G-25 containing ammonium bicarbonate buffer (50 mM) and treated with 30 units of IdeS enzyme prior to analysis. Samples were analyzed using an ACQUITY UPLC Protein BEH C4 column (Waters, 2.1 x 50 mm, 1.7 pm) and linear ion trap Orbitrap mass analyzer coupled with ultra-high performance liquid chromatograph (Ultimate) 3000 (UHPLC, Thermo Fisher Scientific) were used for analysis. In the UHPLC method, mobile phase A (0.1% TFA in water) was kept at 80% for the first 2 min. Mobile phase B (0.1% TFA in ACN) was ramped from 20% to 90% from 1 min to 10 min. Orbitrap-MS was detected in the extended mass range of 500 m / z to 4,000 m / z. Peak deconvolution was performed using UniDec software.

[0238] 3) Antibody drug conjugate (ADC) purification with HIC

[0239] For purification of unpurified trastuzumab-MMAE, a HIC column (0.8 x 10 cm, 5 mL) pre-packed with Skillpak Phenyl-650S HIC resin (Tosoh Bioscience) was connected to an AKTA pure (Cytiva). The whole process was performed at room temperature. Prior to loading, the column was equilibrated with 20 column volumes of buffer A (50 mM sodium phosphate, pH 7.0, 2 M sodium chloride). For loading to the column, 0.25 mL of unpurified ADC (1 mg / mL in formulation buffer) was mixed with 0.25 mL of buffer A. The resulting mixture (total volume 0.5 mL) was then loaded to the column and eluted using a linear gradient from 100% buffer A to 100% buffer B (50 mM sodium phosphate, pH 7.0, 20% isopropanol (IPA, v / v)).

[0240] 4) Nuclear magnetic resonance (NMR)

[0241] 1 H NMR spectra were recorded at room temperature using a Bruker 400 MHz spectrometer and processed by MestReNova 6.0.2 using automatic phasing and polynomial baseline correction functions, or using a Varian 400 MHz spectrometer. In general 13 CNMR spectra were recorded on a Bruker 400 MHz or Bruker 700 MHz spectrometer with complete proton decoupling. 13 C resonances are reported in ppm units with respect to the solvent residual peak in CDC13 (77.16 ppm).

[0242] 5) Fourier-transform infrared spectroscopy (FTIR)

[0243] Infrared spectra were recorded on a JASCO FT / IR-4600 spectrometer, v max recorded in cm -1 High resolution mass spectra were obtained on a JEOL JMS-700 instrument equipped with fast atom bombardment (FAB) mode. Analytical thin-layer chromatography was performed using pre-coated Silica Gel F254 plates (0.25 mm thickness) while thin-layer chromatography (TLC) was visualized by irradiation of an ultraviolet (UV) lamp. Normal-phase column chromatography was performed using Silica Gel (32 to 62 pm) with an Agilent 1260 series instrument fitted with a COSMOSIL diode array detector and a chromatographic column under appropriate mobile phase composition and gradient conditions. High resolution mass spectra were obtained on a JEOL JMS-700 instrument equipped with fast atom bombardment (FAB) mode. Analytical thin-layer chromatography was performed using pre-coated Silica Gel F254 plates (0.25 mm thickness) while thin-layer chromatography (TLC) was visualized by irradiation of an ultraviolet (UV) lamp. Normal-phase column chromatography was performed using Silica Gel (32 to 62 pm) with an Agilent 1260 series instrument fitted with a COSMOSIL diode array detector and a chromatographic column under appropriate mobile phase composition and gradient conditions. High resolution mass spectra were obtained on a JEOL JMS-700 instrument equipped with fast atom bombardment (FAB) mode. Analytical thin-layer chromatography was performed using pre-coated Silica Gel F254 plates (0.25 mm thickness) while thin-layer chromatography (TLC) was visualized by irradiation of an ultraviolet (UV) lamp. Normal-phase column chromatography was performed using Silica Gel (32 to 62 pm) with an Agilent 1260 series instrument fitted with a COSMOSIL diode array detector and a chromatographic column under appropriate mobile phase composition and gradient conditions.

[0244] 2. Test procedure

[0245] 2.1. Protein production

[0246] 1) Protein expression, ncAA incorporation and purification in E. coli

[0247] To obtain strep-tagged Z-domain protein (STZ), the Z-domain WT gene was amplified by gene synthesis and overlapped with a strep-tag sequence (GWSHPQFEK) at the N-terminus and a hexa-histidine tag sequence at the C-terminus to generate the STZ gene. pET20b-strep Z-domain was generated using Ndel and EcoRI restriction sites and the pET20b vector. Next, E. coli BL21(DE3) cells were transformed with pET20b-StrepZ-domain and 6 mL of LB broth containing ampicillin (100 pg / mL) was used for overnight amplification at 37°C. The amplified cells (1 mL) were inoculated into a defined medium (50 mM Na2HP04, 50 mM KH2P04, 25 mM (NH4)2S04, 2 mM MgS04, 0.1% trace metals, 0.5% glycerol, 0.05% glucose, 0.2% soil, and 5% amino acids). An equal amount of ampicillin was added and the cells were incubated overnight at 37°C. The cells were obtained by centrifugal force (10,000 rpm, 5 min) and the cell pellet was frozen at -20°C for additional purification. The pellet was thawed according to the manufacturer's protocol and purified using Ni-NTA agarose resin. The concentration of the purified protein was determined by UV absorbance at 280 nm and the purity was checked using 8-12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The molar extinction coefficient of the protein was calculated using the Biomol Protein Extinction Coefficient Calculator (http: / / www.biomol.net / en / tools / proteinextinction.htm).

[0248] Affibodies were obtained by gene synthesis and a hexa-histidine tag was attached using overlap PCR. The gene of Z-M variant was generated from the minimized Z-domain of Protein A and the sequence of the third helix of Z-domain WT was added. In addition, a hexa-histidine tag was added at the C-terminus of the gene. Affibodies were inserted into the NcoI / KpnI restriction enzyme sites of pBAD- affibody and pET20b-Z-M variant of the gene of Z-M variant and pBAD between pET20b and Ndel / EcoRI, respectively, to generate pBAD-affibody and pET20b-Z-M variant of the gene of Z-M variant. pBAD-affibody mutants (K4, K28, F32TAG) and pET20b-Z-M variant (M8, H19, K33, D37TAG) were generated by site-directed mutagenesis. For pBAD-affibody F32TAG, pET20b-Z-M M8 and D37TAG, to avoid self-labeling of the probe, K28CGT_F32TAG, K5CGT_M8TAG and K33CGT_D37TAG were generated by replacing the nearest Lys with Arg (CGT codon), respectively. Next, the plasmids were co-transformed with pEvol-AzFRS or pEvol-AzKRS in DH10B or BL21 cells. The transformed cells were expressed and purified as described above using unnatural amino acids (AzF 100 μM or AzK 1 mM) and chloramphenicol (35 μg / mL). Concentration and purity were also measured by the same method.

[0249] 2) Expression and purification of IgG1 Fc domain

[0250] pCDNA3.4-Fc domain was generated by inserting into the Xho1 and BamH1 sites of pCDNA3.4 (Invitrogen, USA) after amplification of Fc domain gene by PCR. The plasmids containing Fc domain were transiently transfected into FreeStyle 293 cells (Invitrogen, USA) using PEI-transfection reagent according to the manufacturer's protocol (Polysciences, USA). For expression, FreeStyle 293 cells transiently transfected with each Fc domain construct were incubated in 10 mL of FreeStyle 293 expression medium (Invitrogen, USA) at 37°C for 72 hours. The culture medium was centrifuged at 3000 rpm for 10 minutes and the supernatant was filtered through a 0.22 μm filter. The filtered medium was purified by protein A affinity chromatography according to the manufacturer's protocol (GE Healthcare, USA). The eluate was dialyzed against PBS and the dialyzed sample was concentrated and purified by size exclusion chromatography using a Superdex 200 column (GE Healthcare, USA). TM 293 cells (Invitrogen, USA) using PEI-transfection reagent according to the manufacturer's protocol (Polysciences, USA). For expression, FreeStyle 293 cells transiently transfected with each Fc domain construct were incubated in 10 mL of FreeStyle 293 expression medium (Invitrogen, USA) at 37°C for 72 hours. The culture medium was centrifuged at 3000 rpm for 10 minutes and the supernatant was filtered through a 0.22 μm filter. The filtered medium was purified by protein A affinity chromatography according to the manufacturer's protocol (GE Healthcare, USA). The eluate was dialyzed against PBS and the dialyzed sample was concentrated and purified by size exclusion chromatography using a Superdex 200 column (GE Healthcare, USA). TMThe supernatant of 293 cells was collected by centrifugation at 4 ° C and 8,000 rpm for 20 minutes. The expressed Fc domain was purified using a HitrapProtein A HP column according to the manufacturer's protocol (Cytiva, Switzerland), and the column was balanced with A buffer (1.8 mM potassium dihydrogen phosphate, 10 mM potassium dihydrogen phosphate, 137 mM sodium chloride, 2.7 mM potassium chloride, pH 7.4). After the supernatant containing the Fc domain was loaded onto the column and unbound impurities were removed by washing, the purified Fc domain was eluted using B buffer (100 mM citrate, 100 mM NaCl, pH 3.0). The residual purification buffer was removed by dialyzing the purified Fc domain against A buffer three times, and the purified Fc domain was purified by using the molar absorptivity of the Fc domain (7.157 × 10 4 cm -1 M -1 ) The concentration of the Fc domain was determined by measuring the absorbance at 280 nm.

[0251] 3) Copper(I)-catalyzed azide-alkynecycloaddition (CuAAC)

[0252] The purified affimers and ZM protein variants were lysed using Slide-A-Lyzer TM The reaction mixture was dialyzed against 20 mM potassium phosphate buffer (100 mM NaCl, pH 7.0) using a dialysis kit and 3.5 KMWCO. A mixture of 20 μM of each protein, PyOx (4 mM), CuSO4:BTTAA premix (300 μM:1500 μM), sodium-L-ascorbate (5 mM) and aminoguanidine hydrochloride (5 mM) was stirred at 25 ° C and 250 rpm for 1 hour. The reaction mixture was dialyzed against 50 mM HEPES buffer (pH 7.2 or 8.0) and used for the additional reaction. Ultra centrifugal filter (3KMWCO) was used for concentration.

[0253] Protein labeling

[0254] 1) Using STZ to label NASA-FL

[0255] Affinity mutant containing PyOx (30 μΜ) was mixed with STZ (15 μΜ). NASA-FL (75 μΜ) was added to the mixture and incubated in HEPES buffer (50 mM, pH 7.2) at 37 °C for 4 hours. The reaction was quenched using a mixture of L-Lys (100 mM) at a final concentration of 5 mM and But-PyOx. The labeled NASA-FL was desalted using ZipTip containing 0.6 μL of C18 resin according to the manufacturer's protocol, and the labeling yield was determined by MALDI-TOF MS analysis (matrix: sinapic acid).

[0256] 2) Fc fragment labeling of NASA-FL for PyOx position selection of Z-M protein variants

[0257] Z-M variant containing PyOx (30 μΜ) was mixed with Fc fragment (7.5 μΜ). NASA-FL (75 μΜ) was added to the mixture and incubated in HEPES buffer (50 mM, pH 7.2) at 37 °C for 4 hours, and then quenched in the same way as the above reaction. Washing was performed using PD SpinTrap G-5 for removal of unreacted NASA-FL.

[0258] 3) Optimization of NASA probe labeling for Fc fragment and trastuzumab

[0259] Z-M variant containing PyOx (60 μΜ) was mixed with Fc fragment or trastuzumab (5 μΜ). NASA-probe (250 μΜ) was added to the mixture and incubated in HEPES buffer (50 mM, pH 8.0) at 37 °C for 6 hours, and then quenched in the same way as the above reaction. Washing was performed using PD SpinTrap G-5 for removal of unreacted NASA-probe.

[0260] 4) Manufacture of ADC

[0261] Trastuzumab (5 μΜ) was mixed with Z-M M8AzK-PyOx (60 μΜ) for the production of ADCs. NASA-N3 (60 μΜ) was added to the mixture and incubated in HEPES buffer (50 mM, pH 8.0) at 37 °C for 6 h to produce trastuzumab-N3. The mixture was quenched after the reaction and exchanged to phosphate buffered saline (PBS, pH 7.4) for additional SPAAC reaction. Trastuzumab-N3 (10 μΜ) was mixed with BCN-MMAE (50 μΜ) and reacted at 25 °C for 36 h, and the product was exchanged to PBS for additional purification using fast protein liquid chromatography (FPLC).

[0262] 2.3. Analysis of labeled residues

[0263] 1) Enzymatic digestion of WT and FL labeled STZ

[0264] WT and FL labeled STZ (15 μg) were washed using PDSpin Trap G-25 containing ammonium bicarbonate buffer (50 mM). Urea (final concentration 6 M) was added to the reaction mixture and incubated at 50 °C for 45 min. Endoproteinase Glu-C (Worthington) was added to a final concentration of 2 μΜ and incubated at 37 °C overnight. The reaction mixture was quenched with 0.4% TFA and purified using Pierce C18 ZipTips. The purified product was concentrated by solvent evaporation using N2 blowing and mixed with DHB homogeneously. MALDI-TOF (Bruker Daltonics) was used to find the labeled peptide fragments, and MALDI-TOF / TOF was used to determine the accurate labeled residues. TM Additional purification was performed using C18 spin columns. The purified product was concentrated by solvent evaporation using N2 blowing and mixed with DHB homogeneously. MALDI-TOF (Bruker ) was used to find the labeled peptide fragments, and MALDI-TOF / TOF was used to determine the accurate labeled residues.

[0265] 2) Enzymatic digestion of WT and probe labeled Fc fragments

[0266] The WT and probe-labeled Fc fragments (30 g) were washed using PD SpinTrap G-25 with ammonium bicarbonate buffer (50 mM) containing 0.1% SDS. DTT was added to a final concentration of 20 mM and incubated at 95°C for 20 min. Next, iodoacetamide (IAA) was added to a final concentration of 50 mM and incubated at 25°C for 30 min in the dark, and the excess IAA was quenched with 5 mM DTT. One-twentieth of the Fc fragment was added sequencing grade modified trypsin and incubated overnight at 37°C. The reaction mixture was quenched with 0.4% TFA and purified using C18 spin columns. In addition, the sample was concentrated by solvent evaporation using N2blow and mixed homogeneously with CHCA for investigation. MALDI-TOF was used to find the labeled peptide fragments and MALDI-TOF / TOF was used to determine the exact labeled residues.

[0267] 3) Enzymatic digestion of WT and probe-labeled antibodies

[0268] The WT and probe-labeled trastuzumab (30 g) was washed using PD SpinTrap G-25 with ammonium bicarbonate buffer (50 mM) and treated with 30 units of Ide S enzyme. Next, 0.1% SDS was added to the sample and one-twentieth of the antibody was added sequencing grade modified trypsin. The sample preparation for MALDI-TOF and MALDI-TOF / TOF was performed in the same way as for the Fc fragment analysis.

[0269] 2.4. Binding affinity determination using surface plasmon resonance (SPR)

[0270] 1) Binding affinity analysis of Z-M proteins to antibodies

[0271] The binding constant (K D) were determined. HBS-EP+ buffer (150 mM NaCl, 10 mM HEPES, 3 mM EDTA and 0.05% (v / v) surfactant P20, pH 7.4) was used as running buffer. Blank channels of the chip were used as negative controls while FcRn was immobilized on the chip. Two-fold serial dilutions of antibody were flowed over the chip surface at different concentrations (0.78 nM, 1.56 nM, 3.13 nM, 6.25 nM and 12.50 nM). The chip surface was regenerated after each cycle using 10 mM glycine-HCl buffer (pH 1.5) and affinity was calculated using BIAevaluation software and a 1:1 (Langmuir) binding fit model.

[0272] 2) Binding affinity analysis of the neonatal Fc receptor (FcRn) to antibodies or ADCs

[0273] Surface plasmon resonance (SPR) analysis was performed using a BIAcore T200 system for the evaluation of binding kinetics. Binding constants (K D ) were determined by direct immobilization method and using an amino -bound CM5 chip. pH 6.0 or pH 7.4 phosphate buffer (50 mM sodium phosphate, 150 mM NaCl and 0.05% (v / v) surfactant P20) was used as running buffer. Blank channels of the chip were used as negative controls while FcRn was immobilized on the chip. Two-fold serial dilutions of antibody were flowed over the chip surface at different concentrations (1.96 nM, 3.91 nM, 7.81 nM, 15.63 nM and 31.25 nM). The chip surface was regenerated after each cycle using Tris-HCl buffer (0.1 M Tris-HCl, pH 8.0) and affinity was calculated using BIAevaluation software and a 1:1 (Langmuir) binding fit model.

[0274] 3) Binding affinity analysis of human epidermal growth factor receptor type 2 (HER2) to antibodies or ADCs

[0275] Binding constants (K ) were determined by direct immobilization method and using an amino -bound CM5 chip. pH 6.0 or pH 7.4 phosphate buffer (50 mM sodium phosphate, 150 mM NaCl and 0.05% (v / v) surfactant P20) was used as running buffer. Blank channels of the chip were used as negative controls while FcRn was immobilized on the chip. Two-fold serial dilutions of antibody were flowed over the chip surface at different concentrations (1.96 nM, 3.91 nM, 7.81 nM, 15.63 nM and 31.25 nM). The chip surface was regenerated after each cycle using Tris-HCl buffer (0.1 M Tris-HCl, pH 8.0) and affinity was calculated using BIAevaluation software and a 1:1 (Langmuir) binding fit model.D ) was performed. HBS-EP+ buffer (150 mM NaCl, 10 mM HEPES, 3 mM EDTA and 0.05% (v / v) surfactant P20, pH 7.4) was used as the running buffer. Blank channels of the chip were used as negative controls, while trastuzumab or trastuzumab-ADC were immobilized on the chip. Antibody was serially diluted 2-fold at different concentrations (0.78 nM, 1.56 nM, 3.13 nM, 6.25 nM and 12.50 nM) and flowed over the chip surface. After each cycle, the chip surface was regenerated using 3 M magnesium chloride buffer, and affinity was calculated using BIAevaluation software and 1:1 (Langmuir) binding fit model.

[0276] 2.5. Cytotoxicity confirmation of ADC

[0277] 1) Cell culture

[0278] MDA-MB-231, MDA-MB-453 and SK-BR-3 were purchased from KCLB (Korea Cell Line Bank, Seoul, Korea). Cells were cultured in RPMI1640 supplemented with 10% (v / v) fetal bovine serum (FBS) and 1% (v / v) penicillin / streptomycin (Welgene, Korea) and incubated at 37°C in a humidified 5% CO2 incubator.

[0279] 2) Anti-proliferation assay

[0280] Cells were seeded into 96-well plates at a cell density of 3,000 per well and treated with 4-fold serially diluted ADCs in PBS after cell attachment. After 72 hours of incubation at 37°C, cell viability was observed using CellTiter Glo (G7572, Promega, Madison, WI, USA). Dose-response curves were fitted and GI50 values were obtained using Graphpad Prism 6.0 software, and all experiments were performed in duplicate.

[0281] 3) Western blot

[0282] The primary antibodies for HER2 (#2165) and GAPDH (#5174) were purchased from Cell Signaling Technology. The HRP-conjugated goat anti-Rabbit secondary antibody (SA002-500) was purchased from Gendepot. The cells were washed twice with cold PBS and lysed using NP40 buffer (50 mM Tris-HCl pH 7.4, 1% NP40, 2 mM EDTA, 150 mM NaCl) containing protease inhibitor cocktail (#11873580001, Roche) and phosphatase inhibitor cocktail (#04906837001, Roche). Equal amount of protein was loaded in each sample and separated by SDS-PAGE gel. After the samples were transferred to nitrocellulose membrane, blocking was performed with 5% skim milk in TBS / T. The membrane was incubated with primary antibody diluted uniformly in TBS / T at 1:1,000 (v / v) at 4 °C overnight. After incubation with secondary antibody (1:10,000, v / v) at room temperature for 1 hour, the membrane was treated with enhanced chemiluminescence (ECL) solution and visualized using ImageQuant LAS4000 (GE Healthcare, USA). The western blot images were quantified using ImageJ (N=3). TM LAS4000 (GE Healthcare, USA) to detect chemiluminescent signals, followed by quantification of western blot images using ImageJ (N=3).

[0283] 3. Synthesis of each compound

[0284] 3.1. Synthesis of PyOx-alkyne

[0285]

[0286] To a stirred solution of pyridine-4-carboxaldoxime (370 mg, 3.03 mmol, 1 equiv) dissolved in dry CH3CN (1.5 mL) was added 4-bromo-1-butyne (0.60 mL, 6.6 mmol, 2.2 equiv) and the mixture was refluxed for 24 h. The solution was cooled to room temperature and cooled and filtered to obtain PyOx-alkyne as a yellow solid (518 mg, 2.03 mmol, 67%).

[0287] 1H NMR (400 MHz, D20) δ 8.86 (d, J = 6.4 Hz, 2H), 8.24 (s, 1H), 8.15 (d, J = 6.4 Hz, 2H), 4.64 (t, J = 6.4 Hz, 2H), 2.88 (m, 2H), 2.48 (m, 1H).

[0288] HRMS: [C 10 H 11 N2O1 +H] + The accurate mass calculation requires m / z = 175.0866, the found value is m / z = 175.0868 (HESI+).

[0289] 3.2. Synthesis of PyOx_C6-alkyne

[0290]

[0291] To a stirred solution of pyridine-4-carboxaldoxime (370 mg, 3.03 mmol, 1 equiv) dissolved in dry CH3CN (1.5 mL) was added 6-chloro-1-hexyne (0.73 mL, 6.06 mmol, 2.2 equiv). The mixture was heated to reflux (85 °C, charged in a small vial) and stirred for 25 h. The precipitate was collected and stirred in dry CH3CN (1.5 mL) for 24 h. The solution was cooled to room temperature and cooled and filtered to obtain PyOx_C6-alkyne as a yellow solid (210 mg, 1.04 mmol, 34%).

[0292] 1 H NMR (400 MHz, D20) δ 8.86 (d, J = 6.4 Hz, 2H), 8.24 (s, 1H), 8.15 (d, J = 6.4 Hz, 2H), 4.64 (t, J = 6.4 Hz, 2H), 2.88 (m, 2H), 2.48 (m, 1H).

[0293] HRMS: [C 12 H 15 N2O1 +H] + The accurate mass calculation requires m / z = 203.1179, the found value is m / z = 203.1182 (HESI+).

[0294] 3.3. Synthesis of tert-butyl (15-((4-nitrophenyl)sulfonamido)-15-oxo-3,6,9,12- tetraoxapentadecyl)carbamate (S1)

[0295]

[0296] To a solution of Boc-NH-PEG4-COOH (1.00 g, 2.74 mmol, 1.2 eq) dissolved in dry N,N-dimethylformamide (DMF) (10 mL) was added diisopropylethylamine (1.05 mL, 6.02 mmol), 4-nitrobenzenesulfonamide (0.61 g, 3.01 mmol, 1 eq) and 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (HATU) (1.25 g, 3.28 mmol) and stirred for 6 h at room temperature. The resulting mixture was diluted with ethyl acetate and washed twice with 0.5 M aqueous HC1 and brine. After drying the organic phase over MgS04and evaporation under reduced pressure, the residue was purified by flash column chromatography on silica gel (CH2Cl2:MeOH = 20:1) to give S1 (0.83 g, 55% yield) as a colorless oil.

[0297] 1 H NMR (400 MHz, CDC13) δ 8.32 (d, J = 8.8 Hz, 2H), 8.25 (d, J = 8.8 Hz, 2H), 3.71 - 3.51 (m, 18H), 3.32 (m, 2H), 2.56 (m, 2H), 1.42 (s, 9H).

[0298] 13C NMR (100 MHz, CDC13) δ 156.4, 150.3, 129.7 (2C), 123.9 (2C), 70.69, 70.62, 70.58, 70.5, 70.35, 70.32, 70.2, 70.11, 70.06, 66.7, 53.6, 40.4, 37.8, 28.5 (3C).

[0299] 3.4. Synthesis of tert-butyl (l-(4-nitrophenyl)-2-((4-nitrophenyl)sulfonyl)-3-oxo-6,9,12,15- tetraoxa-2-azaheptadecan-17-yl)carbamate (S2)

[0300]

[0301] To a solution of compound S1 (1.0 g, 1.8 mmol, 1 equiv) dissolved in dry DMF (6.3 mL, 0.3 M) was added 4-nitrobenzyl bromide (472 mg, 2.2 mmol, 1.2 equiv) and N,N-diisopropylethylamine (DIPEA) (0.6 mL, 3.6 mmol, 2 equiv) and stirred for 24 h at room temperature. After removal of the solvent, the reaction mixture was diluted with ethyl acetate (EtOAc) and washed with brine. After drying the organic phase over MgS04and evaporation under reduced pressure, the residue was purified by flash column chromatography on silica gel (hexane:EtOAc = 1:2) to give compound S2 (731 mg, 59% yield) as a colorless oil.

[0302] 1 H NMR (400 MHz, CDC13) δ 8.39 (d, J = 9.0 Hz, 2H), 8.24 (d, J = 8.8 Hz, 2H), 8.13 (d, J = 8.9 Hz, 2H), 7.56 (d, J = 8.8 Hz, 2H), 5.20 (s, 2H), 3.70-3.65 (m, 2H), 3.64-3.57 (m, 8H), 3.56-3.52 (m, 2H), 3.52-3.48 (m, 4H), 3.34-3.23 (m, 2H), 2.82 (t, J = 6.0 Hz, 2H), 1.43 (s, 9H).

[0303] 13C NMR (175 MHz, CDC13) δ 171.5, 151, 147.9, 144.7, 143.4, 129.5 (2C), 128.4 (2C), 124.6 (2C), 124.2 (2C), 70.8, 70.7 (2C), 70.5, 70.39, 70.37, 66.5, 49.5, 40.5, 37.0, 31.7, 28.6 (3C), 22.8, 14.2.

[0304] 3.5. Synthesis of NASA-FL

[0305]

[0306] To a solution of compound S2 (8.2 mg, 12.0 pmol, 1 equiv) dissolved in CH2Cl2(0.5 mL) was added TFA (30 pL) and stirred for 1 h at room temperature, then the solvent was removed under vacuum. After adding 5(6)-carboxyfluorescein (5.4 mg, 14.4 pmol, 1.2 equiv), 4-(4,6-dimethoxy-l,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM) (3.9 mg, 14.1 pmol, 1.2 equiv), and N-methylmorpholine (NMM) (3.9 pL, 35.5 pmol, 3 equiv) to a solution of the residue dissolved in dry DMF (0.5 mL) and stirred for 1 h at room temperature, then stirred overnight at 4 °C. After removing the solvent, the resulting mixture was purified on silica gel by flash column chromatography (CH2Cl2:MeOH = 10:1) to obtain compound NASA-FL (8.5 mg, 75% yield).

[0307] 1 H NMR (400 MHz, CD3OD) δ 8.39 (d, J = 8.4 Hz, 2H), 8.20-8.10 (m, 6H), 7.61-7.22 (m, 3H), 6.66-6.50 (m, 6H), 5.25 (s, 2H), 3.63-3.33 (m, 18H), 2.79-2.75 (m, 2H).

[0308] HRMS: [C 45 H 42 N4O 17 S+H] +The accurate mass calculation requires m / z = 943.2338, the found value is m / z = 943.2339 (HESI+).

[0309] 3.6. Synthesis of NASA-Bt

[0310]

[0311] To a solution of compound S2 (8.2 mg, 12.0 pmol, 1 equiv) dissolved in CH2Cl2(0.5 mL) was added TFA (30 pL). The mixture was stirred for 1 h at room temperature and the residue of solvent was removed under vacuum, the residue was dissolved in dry DMF (0.5 mL) and after addition of N-succinimidyl D-biotinate (4.92 mg, 14.4 pmol, 1.2 equiv), DMTMM (3.9 mg, 14.1 pmol, 1.2 equiv) and N-methylmorpholine (NMM) (3.9 pL, 35.5 pmol, 3 equiv) and stirred for 1 h at room temperature, it was stirred overnight at 4 °C. After removal of the solvent, the resulting mixture was purified by flash column chromatography on silica gel (CH2Cl2:MeOH = 10:1) to obtain compound NASA-Bt (5.4 mg, 55% yield).

[0312] 1 H NMR (400 MHz, CD3OD) d 8.34 (d, J = 8.4 Hz, 2H), 8.15 (m, 4H), 8.11 (d, J = 8.9 Hz, 2H), 7.53 (d, J = 8.8 Hz, 2H), 5.20 (s, 2H), 4.38 (m, 1H), 4.19 (m, 1H), 3.56-3.38 (m, 18H), 3.09 (m, 1H), 2.76 (m, 2H), 2.59 (dd, J = 12.8 Hz, 1H), 2.10 (m, 2H), 1.55-1.32 (m, 6H).

[0313] HRMS: [C 34 H 46 N6O 13 S2+Na] + The accurate mass calculation requires m / z = 943.2338, the found value is m / z = 943.2339 (HESI+).

[0314] 3.7. Synthesis of NASA-N3

[0315]

[0316] To a solution of compound S2 (100 mg, 0.15 mmol, 1 eq) in CH2Cl2(7 mL, 0.02 M) was added TFA (0.02 mL, 0.30 mmol, 2 eq). The mixture was stirred at room temperature for 1 h, and the residue of solvent was removed under vacuum, the residue was dissolved into dry DMF (7 mL, 0.02 M), then 2-azidoacetic acid (18 mg, 0.18 mmol, 1.2 eq), DMTMM (49 mg, 0.18 mmol, 1.2 eq) and N-methylmorpholine (NMM) (0.05 mL, 0.35 mmol, 2.3 eq) were added and stirred at room temperature for 8 h. After removing the solvent, the resulting mixture was purified by flash column chromatography on silica gel (CH2Cl2:MeOH = 20:1) to give compound NASA-N3 (83 mg, yield 86%) as a colorless oil.

[0317] 1 H NMR (400 MHz, CDC13) δ 8.37 (d, J = 9.0 Hz, 2H), 8.20 (d, J = 8.8 Hz, 2H), 8.11 (d, J = 8.9 Hz, 2H), 7.54 (d, J = 8.7 Hz, 2H), 6.94 (s, 1H), 5.18 (s, 2H), 3.91 (s, 2H), 3.66 (t, J = 6.1 Hz, 2H), 3.63-3.57 (m, 8H), 3.56-3.46 (m, 6H), 3.43 (m, 2H), 2.81 (t, J = 6.1 Hz, 2H).

[0318] 13 CNMR (175 MHz, CDC13) δ 171.5, 166.9, 150.9, 147.9, 144.7, 143.4, 129.5 (2C), 128.4 (2C), 124.6 (2C), 124.2 (2C), 70.70, 70.66 (3C), 70.5, 70.4, 69.7, 66.5, 52.8, 49.5, 39.3, 37.0.

[0319] IR (FT-ATR, cm -1 , CH2Cl2) v max3064, 2873, 2359, 2338, 2106, 1837, 1818, 1779, 1756, 1707, 1689, 1669, 1647, 1641, 1629, 1623, 1606, 1576, 1570, 1559, 1526, 1496, 1481, 1465, 1442, 1424, 1404, 1346, 1267, 1168, 1086, 1012, 854, 731, 700, 682.

[0320] HRMS: [C 26 H 33 N7O 12 S+H] + The accurate mass calculation requires m / z = 668.1981, the found value is m / z = 668.1980 (FAB+).

[0321] 3.8. Synthesis of BCN-VC-PABC-MMAE

[0322] 1) Synthesis of Fmoc-VC-PABC-MMAE

[0323]

[0324] Fmoc-VC-PABC-PNP (200 mg, 0.26 mmol, 1 equiv) and MMAE (280 mg, 0.39 mmol, 1.5 equiv) were dissolved in DMF (40 mL, 6.5 mM). Next, hydroxybenzotriazole (HOBt) (8 mg, 0.52 mmol, 2 equiv) and pyridine (0.6 mL, 7.5 mmol, 28 equiv) were added to the solution and stirred at room temperature for 30 hours of stirring, monitored by reverse phase high performance liquid chromatography (RP-HPLC). The product was purified by preparative chromatography column to obtain a white powder (232 mg, 66% yield).

[0325] 2) Synthesis of NH2-VC-PABC-MMAE

[0326]

[0327] Fmoc-VC-PABC-MMAE (125 mg, 0.09 mmol) was dissolved in DMF containing 20.0% piperidine (0.9 mL, 0.1 M) and stirred at room temperature for 1 hour. RP-HPLC analysis was performed after completion of the de-Fmoc reaction, the solution was concentrated under reduced pressure to obtain a white powder (88.0 mg, 88% yield).

[0328] 3) Synthesis of BCN-VC-PABC-MMAE

[0329]

[0330] BCN-PEG4-COOH (13 mg, 0.03 mmol, 1 eq), NH2-VC-PABC-MMAE (40 mg, 0.036 mmol, 1.2 eq), DMTMM (10 mg, 0.036 mmol, 1.2 eq) and N-methylmorpholine (NMM) (0.01 mL, 0.09 mmol, 3 eq) were dissolved in DMF (0.14 mL, 0.2 M) and stirred at room temperature for 8 hours. After removing the solvent, the resulting mixture was purified by a preparative chromatography column to obtain a white powder (22 mg, 48% yield).

[0331] HRMS: [C 80 H 127 N 11 O 19 + Na] + The accurate mass calculation requires m / z = 1568.9202, and the actual value is m / z = 1568.9223 (HESI+).

[0332] 4. Site-selective labeling of proteins (see Figure 3 and Figure 4 )

[0333] To perform site-selective labeling of undamaged proteins, a proximity-based labeling system was used that employs a protein that can be incorporated into a binding protein. The binding protein acts as a catalyst, and the labeling reagent forms a highly reactive intermediate by selectively reacting with the binding protein near the binding protein, thereby facilitating the formation of a covalent bond with a proximal lysine residue of the binding protein.

[0334] A B-domain variant of staphylococcal protein A, Z-domain protein (Z-DM), was used as the target protein, and a Z-domain-specific affibody (Z-AFB) was used as the binding protein (see Figure 3). The two proteins are small in size (8 kDa or less) and can be suitable for complex structures with high resolution, and thus are suitable for use as a model system. To secure the catalytic function of the binding protein as a catalyst that can form an intermediate with a high reactivity to a labeling reagent, pyridinium aldoxime (PyOx) was used (see Figure 4 ). It has been known that PyOx can efficiently form an amide bond with an amine after rapidly reacting with N-alkylsulfonamide (NASA) to form a reactive ester.

[0335] To introduce PyOx into a specific position of Z-AFB, a gene code expansion method, which is an extension technique that can introduce ncAA containing an azido group into Z-AFB by position, was used, and then Cu-catalyzed azido-alkyne cycloaddition (CuAAC) reaction was used to connect PyOx to a specific position of Z-AFB. By simply mixing Z-DM containing PyOx, Z-AFB, and a probe derivatized with NASA, and the like, three components, site-specific connection of the probe to the binding protein can be effectively achieved (see Figure 5 ).

[0336] 4.1. Labeling test on intact Z-DM

[0337] 1) Introduction of ncAA into Z-AFB

[0338] Non-canonical amino acids (ncAA) were introduced into Z-AFB in order to introduce PyOx into a specific position of Z-AFB. The ncAA introduction positions (F32, K28, and K4) were selected based on the X-ray crystal structure of the Z-AFB and Z-DM complex, and did not affect the binding of Z-AFB and Z-DM and were located adjacent to the Lys of Z-DM (see Figure 6 ) were used as the introduction material. 4-azido-L-phenylalanine (AzF) is widely used in protein labeling by bioorthogonal reactions (see Figure 7 ).

[0339] To introduce AzF into Z-AFB, the codons corresponding to F32, K28, and K4 of Z-AFB gene were modified to amber codons (TAG). The modified genes were expressed with aminoacyl-tRNA and aminoacyl-tRNA synthetase (AzFRS) in the presence of AzF, thereby synthesizing Z-AFB variants containing AzF (Z-AFB_K4AzF, Z-AFB_K28AzF, and Z-AFB_F32AzF). By analysis using SDS-PAGE and MALDI-TOF MS, it was confirmed that AzF was quantitatively incorporated.

[0340] 2) Introduction of PyOx into Z-AFB containing AzF

[0341] PyOx was introduced into Z-AFB containing AzF by CuAAC reaction. The alkyne-substituted PyOx compound (see Example 3.1 above) was reacted with Z-AFB under the previously reported conditions, and it was confirmed by SDS-PAGE and MALDI-TOF analysis that it was introduced into all Z-AFB variants (Z-AFB_K4AzF-PyOx, Z-AFB_K28AzF-PyOx, and Z-AFB_F32AzF-PyOx).

[0342] 3) Production of NASA-FL as a labeling reagent

[0343] Using the previously reported method, fluorescein (FL) was linked to NASA, thereby synthesizing NASA-FL as a labeling reagent (see Example 3.5 above). The possibility of site-specific labeling in the basic state of Z-DM using Z-AFB-PyOx and NASA-FL was tested. The labeling test was performed by simply mixing Z-DM with Z-AFB-PyOx and 0 NASA-FL, and confirming the labeling efficiency by MALDI-TOF analysis after the labeling reaction. As a result of performing the labeling reaction using three Z-AFB-PyOx variants (Z-AFB_K4AzF-PyOx, Z-AFB_K28AzF-PyOx, and Z-AFB_F32AzF-PyOx), Z-AFB_K4AzF-PyOx, Z-AFB_K28AzF-PyOx, and Z-AFB_F32AzF-PyOx achieved labeling efficiencies of 35%, 70%, and 96%, respectively (see Figure 7 and Figure 8 ).

[0344] With regard to the difference in labeling efficiency observed between the three variants, the closer the distance from the Z-AFB position where PyOx is introduced to the nearest Lys residue of Z-DM, the higher the labeling efficiency. To confirm this, Z-AFB_K28AzK-PyOx, which contains N6-[(2-azidoe thoxy)c arbonyl]-L-ly sine (Az K) having a longer alkyl chain instead of AzF, was synthesized and labeling experiments were performed under the same conditions, and as a result, it was confirmed that the labeling efficiency was improved by 93% (see Figure 7 ).

[0345] As described above, the labeling method exhibits high efficiency in labeling of native Z-DM protein (96% in the labeling experiment using Z-AFB_F32AzF-PyOx), and the labeling efficiency can be optimized by screening the position of PyOx using genetic code expansion (GCE) technology and adjusting the length of the linker in Z-AFB-PyOx.

[0346] 4.2. Confirmation of labeling residue in Z-DM

[0347] The labeling position of Z-DM was confirmed to be consistent with the Lys residue predicted based on the crystal structure. In the labeling experiment using Z-AFB_F32AzF-PyOx, the FL-labeled Z-DM was treated with a protease (Glu-C). As a result of analyzing the generated peptides by using MALDI-TOF / TOF tandem MS to analyze the labeling position, it was confirmed that labeling occurred at K18 of Z-DM, which was consistent with the position predicted based on the crystal structure (see Figure 9 to Figure 11 ). This indicates that the designed labeling system can effectively label the entire protein, and the labeling position can be predicted by structural analysis.

[0348] In addition, as a result of measuring the Z-DM labeling efficiency according to different times, it was confirmed that the labeling efficiency increased as time increased up to a maximum of 4 hours after the reaction began. More than half of Z-DM completed labeling within 1 hour, and 96% of the protein was labeled within 4 hours (see Figure 12a and Figure 12b ). When the reaction time was further extended, a small amount of labeling was observed at the N-terminal amine (unmarked data), but the labeling was mainly concentrated at K18 within 4 hours. When the non-binding protein was subjected to the labeling reaction, no or almost no labeling occurred, which indicates that the labeling reaction occurs only in the presence of the binding protein containing PyOx (see Figure 13a andFigure 13b ).

[0349] 4.3. System design for Fc fragment labeling

[0350] To adapt the labeling system to antibody labeling, the Fc fragment of human immunoglobulin G1 (IgG1) was labeled (see Figure 14 ). Since the Fc fragment is preserved in various therapeutic antibodies, the labeling method developed for the Fc fragment can be directly applied to antibodies having the same Fc fragment. To achieve site-specific labeling of the Fc fragment, a modified 2-helix Fc binding protein was selected after searching and evaluating Fc binding proteins with high affinity and selectivity, and further optimized for superior protein stability and expression by introducing an additional helix. The design of the additional helix was based on the sequence of the existing 3-helix Fc binding protein. Through computational analysis using the AlphaFold2 program, it was confirmed that the additional helix maintained a good 3-helix structure (see Figure 15 ), and the generated 3-helix protein (Z-M) was designed to stably and specifically bind to the Fc fragment.

[0351] To determine the appropriate position for introducing PyOx using the Fc binding platform, Z-M protein, the crystal structure of the complex formed by the 2-helix Fc binding protein and the Fc fragment was referred to. M8, H19, K33, and D37 of the Z-M protein were selected as the optimal positions for introducing PyOx. It was confirmed that these positions are very close to the Fc fragment while minimizing interference with Fc binding, thereby enabling efficient labeling (see Figure 16 ). It was confirmed that the distance between the selected residues in Z-M and the nearest Lys of the Fc fragment was approximately to This is significantly farther than the distance measured in Z-DM / Z-AFB. In considering the correlation between labeling efficiency and the distance from the position of PyOx introduction to the nearest Lys, since AzK, which replaces AzF, has a longer chain length, it is expected to be more suitable for Fc fragment labeling.

[0352] As a result, AzK can be introduced into Z-M at positions M8, H19, K33, and D37 using the GCE technique, which can be confirmed by SDS-PAGE and MALDI-TOF MS, indicating that it is quantitatively introduced into the Z-M protein. Next, PyOx was introduced into each Z-M variant through the CuAAC reaction, and quantitative conjugation was verified using SDS-PAGE and MALDI-TOF MS.

[0353] 4.4. Evaluation of binding affinity of Z-M variants

[0354] The binding affinity of antibodies with Fc domain (Trastuzumab) to Z-M variants comprising AzK or AzK-PyOx was evaluated using surface plasmon resonance (SPR) analysis.

[0355] [Table 1]

[0356]

[0357] Referring to Table 1, all variants exhibited almost the same binding affinity compared to the Z-M wild-type protein. This indicates that the introduction of PyOx does not cause too much influence on the binding affinity between Z-M protein variants and Fc fragments.

[0358] 4.5. Labeling of intact Fc fragments and characterization of labeled residues

[0359] The labeling efficiency on Fc fragments was tested using Z-M variants comprising PyOx. First, the best Z-M variant exhibiting the highest labeling efficiency was selected. The labeling reaction was performed by mixing each Z-M variant with NASA-FL to Fc fragments, and the labeling efficiency was evaluated using HPLC.

[0360] 1) Analysis results

[0361] Z-M_M8AzK-PyOx exhibited the highest labeling efficiency, and could effectively and quantitatively label the Fc fragment in the absence of un-labeled or partially labeled Fc almost or completely (see Table 2). In contrast, in the case of using other Z-M variants, a large amount of Fc fragments remained in an un-labeled state. Little labeling was observed in the control experiment without Z-M-PyOx, which indicates that background labeling is minimal. Figure 17

[0362] Unlike Z-DM labeling using Z-AFB, the labeling efficiency of Fc fragment labeling using Z-M did not exhibit a strong correlation with the distance between PyOx and the target Ly s. The order of the measured distance between the PyOx position and Ly s was M8 > H19 > D37 > K33, and the order of the labeling efficiency was M8 > D37 > K33 > H19 (see Table 2 and Table 3). Figure 16 Figure 17 ​​). The highest labeling efficiency was observed when PyOx was introduced at the closest position (M8), but the lowest labeling efficiency was observed at the next closest position (H 19). The results can be explained by careful observation of the complex structure of the Fc fragment with the Z-M protein variant. K338 within the Fc fragment is the residue involved in the labeling reaction using Z-M_H 19AzK-PyOx. In the complex structure, K338 is partially buried by the neighboring residues, which can potentially cause structural hindrance to the labeling reaction (see Figure 18 ). Therefore, although the distance is shorter, the efficiency of the labeling reaction using Z-M_H 19AzK-PyOx is relatively low compared to Z-M_D37AzK-PyOx or Z-M_K33AzK-PyOx. This indicates that not only the distance to the labeled lysine but also the surrounding environment of the lysine to be labeled is an important factor when selecting the PyOx introduction position.

[0363] 2) Additional labeling experiment

[0364] An additional labeling experiment was performed using Z-M_M8AzK-PyOx, which showed the best labeling efficiency in the Fc fragment labeling experiment. NASA-Bt was used in addition to NASA-FL in the Fc labeling experiment (see Example 3.6.), and high labeling efficiency similar to that of NASA-FL was confirmed by HPLC analysis (see Fig. 13).

[0365] In addition, the results of preparing Z-M_M8AzK-PyOx_C6, which has a slightly longer alkyl chain on the PyOx linker, and analyzing the labeling efficiency, Z-M_M8AzK-PyOx_C6 showed almost similar quantitative labeling efficiency to Z-M_M8AzK-PyOx. Both variants, Z-M_M8AzK-PyOx and Z-M_M8AzK-PyOx_C6, showed efficient Fc fragment labeling, so no additional optimization of the PyOx linker length was performed.

[0366] Next, the labeled residues of the Fc fragment labeled using Z-M_M8AzK-PyOx were characterized. The complex structure of the Z-M protein variant and the Fc fragment indicates that the lysine closest to M7 of the Z-M in the Fc fragment is K248, and another lysine (K246) is found nearby (see Figure 19) To confirm the labeled residues, the labeled Fc fragment was treated with trypsin and the resulting peptide fragments were analyzed using MALDI-TOF / TOF tandem MS. In the MALDI-TOF analysis, the peptide fragment containing K248 was clearly shifted to the corresponding labeled fragment in both the FL labeled fragment and the Bt labeled fragment (see Figure 20 ) As observed in the HPLC results ( Figure 17 ), no or almost no unlabeled fragment was found. In the MALDI-TOF / TOF tandem MS analysis of the peptide fragments, K248 was unambiguously labeled in the fragment without detecting other labeled residues (e.g. K246) (see Figure 21 to Figure 23 ).

[0367] 4.6. Labeling experiment on intact trastuzumab

[0368] To apply the efficient and selective labeling method of Fc fragment using Z-M_M8AzK-PyOx to antibody labeling, trastuzumab, which can specifically bind to human epidermal growth factor receptor 2 (HER2) and is used for cancer treatment, was used. Trastuzumab was labeled with Z-M_M8AzK-PyOx and NASA-Bt and analyzed by HPLC. Since the resolution power was not sufficient when the whole antibody was analyzed by HPLC, the antibody was decomposed into F(ab)2 and Fc / 2 fragments using Ide S. As a result, only the Fc / 2 peak was shifted, which indicates specific labeling of the Fc fragment (see Figure 24 ). Next, MALDI-TOF / TOF tandem MS analysis showed bio tin labeling in K248, which is consistent with the results of the Fc fragment described above (see Figure 25 to Figure 27 ).

[0369] Next, trastuzumab was labeled with the synthetic ADC drug. An attempt was made to synthesize an ADC using the most widely used drug in FDA-approved ADCs, monomethyl auristatin E (MMAE), and the NASA moiety introduced to the valine-citrulline-p-aminobenzoyloxycarbonyl (VC-PABC) linker, but it was not possible to directly introduce VC-PABC-MMAE to trastuzumab due to solubility problems of NASA-VC-PABC-MMAE. Instead, an azido group was introduced to trastuzumab using NASA-N3 (see Example 3.7 above), and in order to introduce VC-PABC-MMAE by performing strain-promoted azide-alkyne cycloaddition (SPAAC) (see Figure 28 ), a trastuzumab labeling reaction was performed using Z-M_M8Az K-PyOx and NASA-N3. Although trastuzumab could be efficiently labeled with N3, the efficiency was slightly lower than that of NASA-Bt (see Figure 29 to Figure 32 ), and it was confirmed through MS analysis that the labeling occurred at the same position (K248) as the Fc fragment labeling (see Figure 33 to Figure 35 ).

[0370] In order to introduce VC-PABC-MMAE to trastuzumab-N3, bicyclo[6.1.0]non-4-yne (BCN) was used as a modified alkyne group widely used in SPAAC reactions, and BCN-VC-PABC-MMAE was manufactured (see Example 3.8 above) (see Figure 28 ). Trastuzumab-(VC-PABC-MMAE)2 was obtained by performing a SPAAC reaction on trastuzumab-N3 and BCN-VC-PABC-MMAE, and uniform trastuzumab-(VC-PABC-MMAE)2 was purified by FPLC (see Figure 29 ).

[0371] 4.7. Cell viability assay

[0372] To evaluate the cytotoxicity of the synthesized ADCs, cell viability assays were performed using breast cancer cell lines. A total of 3 cell lines were used, 2 of which were HER2-positive cells (SK-BR-3: high; MDA-MB-453: low) and 1 was a HER2-negative cell (MDA-MB-231). The cells were treated with trastuzumab-(VC-PABC-MMAE)2, and for comparison, trastuzumab WT and Kadcyla were also tested.

[0373] Table 2

[0374]

[0375] Referring to Table 2 and Figure 32 The results of the analysis showed that trastuzumab-(VC-PABC-MMAE)2 exhibited high efficacy in HER2-positive SK-BR-3 cells. In addition, the ADC exhibited slightly higher efficacy compared to Kadcyla. In contrast, the HER2-negative MDA-MB-231 cells did not exhibit cytotoxicity, and the HER2-low-expressing MDA-MB-453 cells exhibited moderate cytotoxicity.

[0376] 4.8. Measurement of the binding affinity of trastuzumab-(VC-PABC-MMAE)2 to HER2 and FcRn

[0377] In addition, the binding affinity of trastuzumab-(VC-PABC-MMAE)2 to HER2 and FcRn was measured.

[0378] Table 3

[0379]

[0380] Table 4

[0381]

[0382] Referring to Table 3 and Table 4, it can be confirmed that labeling of the antibody with the drug did not affect the binding affinity, which indicates that the binding affinity is similar to that of trastuzumab WT. The above results can prove that the ADC generated using the labeling system of the present example has high cancer cell-specific cytotoxicity and the binding affinity to Fc Rn is not damaged, thereby indicating that it can be subsequently applied to therapeutic purposes.

[0383] The present application described above is only for illustration, and those skilled in the art should understand that the present application can be easily modified into other specific forms without changing its technical idea or essential characteristics. Therefore, the embodiments described above should be understood in all aspects as exemplary rather than limiting. For example, each component element described in a single form can be implemented in a distributed manner, and similarly, a component element described in a distributed form can be implemented in a combined form.

Claims

1. A conjugation-mediating protein, which is combined by a binding protein into which a non-canonical amino acid is introduced and a pyridinium oxime derivative. 2.The conjugation-mediating protein according to claim 1, the binding protein is a protein derived from Protein A, Protein G, Protein L, or a similar protein that functions identically thereto, which is known to bind to an antibody. 3.The conjugation-mediating protein according to claim 2, the binding protein is a B domain variant derived from Protein A. 4.The conjugation-mediating protein according to claim 3, the B domain variant is represented by SEQ ID NO: 1 or SEQ ID NO:

2. 5.The conjugation-mediating protein according to claim 1, the non-canonical amino acid is selected from the group consisting of amino acids represented by Chemical Formula 1 to Chemical Formula 9 below: 【Chemical Formula 1】 【Chemical Formula 2】 【Chemical Formula 3】 【Chemical Formula 4】 【Chemical Formula 5】 【Chemical Formula 6】 【Chemical Formula 7】 【Chemical Formula 8】 【Chemical Formula 9】 6.The conjugation-mediating protein according to claim 1, the non-canonical amino acid is 4-azido-L-phenylalanine or N6-[(2-azidoethoxy)carbonyl]-L-lysine. 7.The conjugation-mediating protein according to claim 1, the non-canonical amino acid is introduced by substitution with at least one or more amino acids included in the binding protein. 8.The conjugation-mediating protein according to claim 1, the pyridinium oxime derivative is represented by Formula 1 below: 【Formula 1】 in the Formula 1, A is absent or substituted or unsubstituted C 1-10 alkylene, substituted or unsubstituted C 5-10 cycloalkylene, substituted or unsubstituted C 5-10 phenylene, substituted or unsubstituted C 2-10 polyethylene glycol group, C 1-10 alkylene, C 5-10 cycloalkylene, C 5-20 phenylene, selected from the group consisting of R a and R b each independently is absent, substituted or unsubstituted C 1-10 alkylene of 1 to 10 carbon atoms or substituted or unsubstituted C 2-10 polyethylene glycol group of 1 to 10 carbon atoms, B is selected from and W is -F, -Cl, -Br, -NO2, or -CF3, n is 0 to 4. 9.The conjugation-mediating protein according to claim 8, B of the pyridinium oxime derivative of Formula 1 is combined with a terminal functional group of the non-canonical amino acid. 10.A labeling method of an antibody, comprising: a first step of combining the conjugation-mediating protein according to any one of claims 1 to 9 to a native antibody; and a second step of reacting the conjugation-mediating protein with an N-alkyl sulfonamide derivative to which a probe is bound, thereby forming an active ester intermediate; and a third step of reacting a lysine included in an Fc domain of the native antibody with the intermediate, thereby forming an amide bond. 11.The labeling method of an antibody according to claim 10, The probe-bound N-alkylsulfonamide derivative is represented by the following Formula 2: [Formula 2] In the Formula 2, R' is R is absent or an alkylene group selected from substituted or unsubstituted C 1-10 a cycloalkylene group selected from substituted or unsubstituted C 5-10 a cycloalkylene group selected from substituted or unsubstituted C 5-10 a phenylene group selected from substituted or unsubstituted C 2-10 a polyethylene glycol group, a C 1-10 a cycloalkylene group selected from substituted or unsubstituted C 5-10 a cycloalkylene group selected from substituted or unsubstituted C 5-20 a phenylene group selected from substituted or unsubstituted C and combinations of the same, R c and R d each independently is absent, substituted or unsubstituted C 1-10 alkylene of 1 to 6 carbon atoms or substituted or unsubstituted C 2-10 polyethylene glycol group of 1 to 6 carbon atoms, R1is selected from CF3or CH2OH, R2is selected from CF3, R2is selected from CF3, R2is selected from CF3, R2is R3is H, linear or branched C 1-5 alkyl, P is from is selected from the group consisting of X1, X2, and X3substitute at least one hydrogen of the phenyl, and each is independently F, Cl, Br, CF3, or NO2, Y is O or NH, Z is -NR e R f substituted or unsubstituted linear or branched C 1-20 alkyl, R e and R f each independently is C 1-5 alkyl or cyclic C 4-10 alkyl, with or without heteroatoms, N, S or O.

12. The method of claim 10, The probe-bound N-alkylsulfonamide derivative is selected from the group consisting of N-alkylsulfonamide-fluorescein (NASA-FL), N-alkylsulfonamide-biotin (NASA-Bt), and N-alkylsulfonamide-N3.

13. A method of site-specific synthesis of an antibody-drug conjugate, comprising: a first step of binding a labeling mediator protein to a native antibody according to any one of claims 1 to 9; a second step of reacting the labeling mediator protein with a probe-bound N-alkylsulfonamide derivative, thereby forming an active ester intermediate; a third step of reacting a lysine contained in an Fc domain of the native antibody with the intermediate, thereby forming an amide bond; and a fourth step of labeling by reacting a drug with the probe.

14. The method of site-specific synthesis of an antibody-drug conjugate of claim 13, The probe-bound N-alkylsulfonamide derivative is represented by the following Formula 2: [Formula 2] In the Formula 2, R' is R is absent or an alkylene group from 1 to 10 carbon atoms, substituted or unsubstituted C 1-10 cycloalkylene group from 1 to 10 carbon atoms, substituted or unsubstituted C 5-10 cycloalkylene group from 1 to 10 carbon atoms, substituted or unsubstituted C 5-10 phenylene group, substituted or unsubstituted C 2-10 polyethylene glycol group, C 1-10 alkylene group from 1 to 10 carbon atoms, C 5-10 cycloalkylene group from 1 to 10 carbon atoms, C 5-20 phenylene group, and combinations of the same, R c and R d each independently is absent, substituted or unsubstituted C 1-10 alkylene of 1 to 6 carbon atoms or substituted or unsubstituted C 2-10 polyethylene glycol group of 1 to 6 carbon atoms, R1is selected from CF3or CH2OH, R2is selected from CF3, R2is selected from CF3, R2is selected from CF R3is H, linear or branched C 1-5 alkyl, P is from is selected from the group consisting of X1, X2, and X3substitute at least one hydrogen of the phenyl, and each is independently F, Cl, Br, CF3, or NO2, Y is O or NH, Z is -NR e R f substituted or unsubstituted linear or branched C 1-20 alkyl group, R e and R f Each independently is C 1-5 Alkyl groups or interconnected C 4-10 and may contain N, S or O heteroatoms.

15. The method of site-specific synthesis of an antibody-drug conjugate of claim 13, The probe-bound N-alkylsulfonamide derivative is selected from the group consisting of N-alkylsulfonamide-fluorescein (NASA-FL), N-alkylsulfonamide-biotin (NASA-Bt), and N-alkylsulfonamide-N3.

16. The method of site-specific synthesis of an antibody-drug conjugate of claim 13, The drug is a drug or a drug comprising a cleavable linker.

17. The method of site-specific synthesis of an antibody-drug conjugate of claim 16, The drug is selected from the group consisting of monomethyl auristatin E (MMAE), Maytansinoids, Calicheamicin, exatecan, SN-38, monomethyl auristatin F (MMAF), and derivatives thereof.

18. A method for position-selective synthesis of an antibody-drug conjugate, comprising: a first step of binding a tag-mediated protein to a native antibody according to any one of claims 1 to 9; and a second step of reacting the tag-mediated protein with a drug-conjugated N-alkyl sulfonamide derivative to form an active ester intermediate; and a third step of reacting a lysine (Lysine) contained in an Fc domain of the native antibody with the intermediate to form an amide bond.

19. The method for position-selective synthesis of an antibody-drug conjugate according to claim 18, the drug-conjugated N-alkyl sulfonamide derivative is represented by the following Formula 3: 【Formula 3】 in the Formula 3, R' is R is absent or an alkylene group from 0 to 2 carbon atoms, substituted or unsubstituted, 1-10 a cycloalkylene group from 0 to 2 carbon atoms, substituted or unsubstituted, 5-10 a cycloalkylene group from 0 to 2 carbon atoms, substituted or unsubstituted, 5-10 a phenylene group, substituted or unsubstituted, 2-10 a polyethylene glycol group, a C 1-10 an alkylene group from 0 to 2 carbon atoms, a C 5-10 a cycloalkylene group from 0 to 2 carbon atoms, a C 5-20 a phenylene group, and combinations of the same, R c and R d each independently is absent, substituted or unsubstituted C 1-10 alkylene of 1 to 6 carbon atoms or substituted or unsubstituted C 2-10 polyethylene glycol group of 1 to 6 carbon atoms, R1is selected from CF3or CH2OH, R2is selected from CF3, R2is selected from CF3, R2is selected from CF3, R2is R3is H, linear or branched C 1-5 alkyl, D is a drug or a drug containing a cleavable linker, X1, X2, and X3substitute at least one hydrogen in a phenyl group, and each is independently F, Cl, Br, CF3, or NO2, Y is O or NH, Z is -NR e R f substituted or unsubstituted linear or branched C 1-20 alkyl, R e and R f Each independently is C 1-5 Alkyl groups or interconnected C 4-10 and may contain N, S or O heteroatoms.

20. The method for position-selective synthesis of an antibody-drug conjugate according to claim 19, the drug is selected from monomethyl auristatin E (MMAE), Maytansinoids, Calicheamicin, exatecan, SN-38, monomethyl auristatin F (MMAF), and derivatives thereof.

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