Compounds for the preparation of antibody-payload conjugates and their use

Abstract

To provide a linker, having a novel trans structure, including two or more electrophilic carbons of carbonyl groups and one or more click chemical moieties, and a method for preparing the same; and to provide a linker, having a novel cis structure, including two or more electrophilic carbons of carbonyl groups and one or more click chemical moieties, and a method for preparing the same.SOLUTION: The present application relates to a novel linker for use in bioconjugation, comprising two or more electrophilic carbon atoms of a carbonyl group, and a click chemistry functional group and, more specifically, to a linker through which a compound, a peptide, and / or a protein can be directly and / or indirectly linked by a substitution reaction to a desired target molecule, that is, a target molecule.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to the field of bioconjugation. The present invention relates to a linker for the preparation of site-specifically bound antibody-payload conjugates, an antibody-payload conjugate prepared using the same, and a method for preparing an antibody-payload conjugate. More specifically, the present invention relates to a linker comprising two or more carbonyl carbons having two or more different partially positive charges and functional groups at both ends, which can link compounds, peptides, and / or proteins to biological (target) molecules by substitution reactions. [Background technology]

[0002] Bioconjugation is a process of linking at least two molecules, in which case bioconjugation refers to a process in which at least one or more molecules are bioactive molecules. Bioactive molecules are sometimes referred to as "target molecules" or "molecules of interest," and may include, for example, proteins (or peptides), glycans, nucleic acids (or oligonucleotides), lipids, hormones, or natural drugs (or fragments thereof, or combinations thereof). Linkers that link two or more molecules can be widely used for detection, diagnosis, biomarkers, etc., by conjugating target-specific proteins such as antibodies to fluorescent materials.

[0003] Currently, polyethylene glycol (PEG), for example, is widely used commercially as a linker for detection, diagnosis, and treatment as a bioconjugation because it is highly water-soluble, non-toxic, non-antigenic, and does not aggregate. In recent years, with the growing interest in therapeutic drugs that treat specific diseases by linking cytotoxic drugs (anticancer drugs) to antibodies, research into linkers that can bind cytotoxic drugs to target molecules such as antibodies has also become active. For these linkers that can link two or more molecules, all factors such as in vivo blood stability, compatibility, solubility, and target specificity must be considered.

[0004] On the other hand, linkers currently being studied in the field of antibody-payload conjugates have the problem that the biological activity of the antibody is reduced by its length and size. For example, there are problems such as a decrease in half-life due to blocking FcRn receptor binding, and difficulties in generating homogeneous antibody-drug conjugates due to difficulties in site-specific binding. Therefore, there is an urgent need to develop linkers that maintain the activity of the target molecule while possessing excellent in vivo blood stability, compatibility, solubility, and site specificity.

[0005] To solve these problems, the inventors of the present invention have invented a linker capable of linking a payload to a target molecule without affecting the biological activity of the target molecule, the linker comprising two or more carbonyl groups having two or more different partial positive charges (δ+), and leaving groups and / or click compounds at both ends. The linker can increase reactivity not only through site specificity but also by increasing water solubility for the target molecule through length adjustment. Since such linkers do not have harsh reaction conditions and have a high conjugation yield for molecules including the target molecule as a bioconjugation, it is intended to provide a more stable and economically efficient linker. [Prior art documents] [Patent Documents]

[0006] [Patent Document 1] WO / 2015 / 122478 [Overview of the Initiative] [Problems that the invention aims to solve]

[0007] This application aims to provide a novel linker having a trans structure comprising two or more electrophilic carbons of a carbonyl group and one or more click chemical moieties, as well as a method for preparing the same.

[0008] This application aims to provide a linker having a novel cis structure containing two or more electrophilic carbons of a carbonyl group and one or more click chemistry moieties, and a method for preparing the same. **Means for Solving the Problems**

[0009] To solve the problems described above in this application, this specification provides a linker that aids in transport and binding reactions to directly and / or indirectly link a payload to a target molecule.

[0010] In one aspect, this application provides a compound represented by the following formula 2:

[0011] **[Chemical Formula]**

[0012] In formula 2, R' is an ester group activating moiety, R" is any one of acetylene, trans-cyclooctene, cyclooctyne, diarylcyclooctyne, methyl ester phosphine, norbornene, methylcyclopropene, azetine and cyanide, and R''' is a substituted or unsubstituted C 1~20 alkylene, a substituted or unsubstituted C 2~20 alkenylene, a substituted or unsubstituted C 1~10 alkynylene, a substituted or unsubstituted C 1~10 polymethylene, a substituted or unsubstituted C 5~12 aryl, a substituted or unsubstituted C 5~14 arylalkylene, a substituted or unsubstituted C 8~16 arylalkenylene, a substituted or unsubstituted C 3~10 cycloalkylene, a substituted or unsubstituted C 3~10 heterocycloalkylene, or a substituted or unsubstituted C 5~12The heteroaryl is a heteroalkylene, heterocycloalkylene, or heteroaryl, which contains at least one or more selected from the group N, O, and S, and the substitution is substituted with a non-hydrogen substituent, the non-hydrogen substituent being -Ra, -O-, =O, -ORa, -SRa, -S-, -N(Ra)2, =NRa, -C(Rb)3, -N=C=O, -NCS, -NO, -NO2, =N-OH, =N2, -N3, -NHC(=O)Ra, -C(=O)Ra, -C(=O)NRaRa-S(=O)2O-, -S(=O)2OH, -S(=O )2Ra, -OS(=O)2ORa, -S(=O)2NRa, -S(=O)Ra, -C(=O)Ra, alkylene-C(=O)Ra, -C(=S)Ra, -C(=O)ORa, alkylene-C(=O)ORa, -C(=O)O-, alkylene-C(=O)O-, -C(=S)ORa, -C(=O)SRa, -C(=S)SRa, -C(=O)NRaRa, alkylene-C(=O)NRaRa, -C(=S)NRaRa, -C(-NRa)NRaRa, and Rb, one or more selected from the group, where Ra is H, C 1~6 Alkyl, C 5~12 Ariel, C 7~12 The compound is an arylalkyl or heterocyclic compound, where Rb is F, Cl, Br, or I, and X is O, N, or S. In addition, this application provides compounds in which R'' is selected from the group consisting of norbornene, transcyclooctene, cyclooctine, and methylcyclopropene. Furthermore, this application provides compounds in which R'' is norbornene.

[0013] Furthermore, this application is valid if R''' is a substituted or unsubstituted C 1~10 Alkylene, substituted or unsubstituted C 1~10 Heteroalkylenes, and C 1~10Selected from the group of polymethylenes, the heteroalkylene contains at least one or more selected from the group consisting of N, O, and S, and the substitution is substituted with a non-hydrogen substituent, where the non-hydrogen substituent is -O-, =O, -ORa, -SRa, -S-, -N(Ra)2, =NRa, -N=C=O, -NCS, -NO, -NO2, =N-OH, =N2, -N3, -NHC(=O)Ra, -C(=O)Ra, -C(=O)NRaRa-S(=O)2O-, -S(=O)2OH, -S(=O)2Ra, -OS(=O )2ORa, -S(=O)2NRa, -S(=O)Ra, -C(=O)Ra, alkylene-C(=O)Ra, -C(=S)Ra, -C(=O)ORa, alkylene-C(=O)ORa, -C(=O)O-, alkylene-C(=O)O-, -C(=S)ORa, -C(=O)SRa, -C(=S)SRa, -C(=O)NRaRa, alkylene-C(=O)NRaRa, -C(=S)NRaRa, and -C(-NRa)NRaRa, one or more selected from the group consisting of C 1~6 Alkyl, C 5~12 Ariel, C 7~12 The present invention provides compounds that are arylalkyl or heterocyclic, where Rb is F, Cl, Br, or I, and X is O, N, or S. Furthermore, the present invention provides compounds where R''' is unsubstituted C 1~5 Alkylene or unsubstituted carbon 1~5 The present invention provides a compound that is polymethylene.

[0014] Furthermore, this application provides a compound in which X is O.

[0015] Furthermore, this application provides a compound in which the compound of formula 2 is represented by the following formula 2-1-2:

[0016] [ka]

[0017] In another embodiment, the present application relates to a method for producing an antibody-payload conjugate, comprising: the steps of: preparing a linker-Fc-binding peptide conjugate by reacting a linker having the structure of formula 2 with an Fc-binding peptide; obtaining an antibody containing a first click chemistry functional group by reacting the linker-Fc-binding peptide conjugate with an antibody; and preparing an antibody-payload conjugate having the structure of formula 8 by reacting the antibody containing the first click chemistry functional group with a payload containing a second click chemistry functional group capable of performing a click chemical reaction with the first click chemistry functional group.

[0018] [ka]

[0019] (In the formula, Ab is an antibody, and R''' is an unsubstituted C) 1~5 Alkylene or unsubstituted carbon 1~5 The material is polymethylene, Y4 is N, Fp is an Fc-binding peptide, B is any one structure formed by a click chemical reaction between a first click chemistry functional group and a second click chemistry functional group, Am is the active site or a structure including the active site, the active site is any one selected from the group consisting of a drug molecule, an imaging site, an optical agent, a vitamin, and a toxin, and n is an integer between 1 and 4).

[0020] Furthermore, this application relates to a method for generating an antibody-payload conjugate, wherein the Fc-binding peptide is a peptide selected from the group consisting of the following formulas 13 and 14. [Formula 13] DCAWH-Xa-GELVWCT [Formula 14] DCAWHKGELVWCT (In the formula, D is aspartic acid, C is cysteine, A is alanine, W is tryptophan, H is histidine, and Xa is

[0021] [ka]

[0022] The present invention provides a method in which G is glycine, E is glutamate, L is leucine, V is valine, T is threonine, K is lysine, m is an integer between 1 and 4, and the N-terminal cysteine ​​and C-terminal cysteine ​​are selectively linked to each other), n=2, and the nitrogen atom linked to Ab is contained in lysine 246 or lysine 248 of both Fc of the antibody.

[0023] In another embodiment, this application relates to the antibody-payload conjugate of the following formula 8:

[0024] [ka]

[0025] (In the formula, Ab is an antibody, and R''' is an unsubstituted C) 1~5 Alkylene or unsubstituted carbon 1~5 The material is polymethylene, Y4 is N, Fp is an Fc-binding peptide, B is any one structure formed by a click chemical reaction between a first click chemistry functional group and a second click chemistry functional group, Am is the active site or a structure including the active site, the active site is any one selected from the group consisting of a drug molecule, an imaging site, an optical agent, a vitamin, and a toxin, and n is an integer between 1 and 4).

[0026] Furthermore, this application is based on the premise that Fp is a peptide selected from the group consisting of the following formulas 13 and 14. [Formula 13] DCAWH-Xa-GELVWCT [Formula 14] DCAWHKGELVWCT (In the formula, D is aspartic acid, C is cysteine, A is alanine, W is tryptophan, H is histidine, and Xa is

[0027] [ka]

[0028] The present invention provides an antibody-payload conjugate in which G is glycine, E is glutamate, L is leucine, V is valine, T is threonine, K is lysine, m is an integer between 1 and 4, and the N-terminal cysteine ​​and C-terminal cysteine ​​are selectively linked to each other), and Fp is linked via Y4 at amino acid residue 6.

[0029] Furthermore, this application is based on the fact that B,

[0030] [ka]

[0031] The present invention provides an antibody-payload conjugate in which (in the formula, A1 is linked to the antibody and A2 is linked to Am, or A1 is linked to Am and A2 is linked to the antibody)

[0032] Furthermore, this application is based on the fact that B,

[0033] [ka]

[0034] This provides an antibody-payload conjugate.

[0035] Furthermore, this application provides an antibody-payload conjugate in which Am contains an anticancer drug. Furthermore, this application provides an antibody-payload conjugate in which the anticancer drug is meltansine (DM1). Furthermore, this application provides an antibody-payload conjugate in which Am contains two or more anticancer drugs.

[0036] Furthermore, this application provides an antibody-payload conjugate in which the nitrogen atom linked to Ab is contained in lysine 246 or lysine 248 of the antibody's Fc.

[0037] Furthermore, this application provides an antibody-payload conjugate in which n is 2 and the nitrogen atom linked to Ab is contained in lysine 246 or lysine 248 of both Fc of the antibody.

[0038] In another embodiment, the present application provides a pharmaceutical composition for treating cancer, comprising an antibody-payload containing an anticancer drug.

[0039] Furthermore, this application provides a pharmaceutical composition in which the cancer is breast cancer. [Effects of the Invention]

[0040] According to the scientific and technological information disclosed herein, the following effects occur:

[0041] Compound 1 disclosed herein provides a linker capable of site-specifically linking a payload to an antibody. The linker has the effect of not affecting the biological activity of the antibody, such as its half-life, and can be usefully used as a bioconjugation for detection, diagnosis, biomarkers, and anticancer therapeutics.

[0042] In addition, Compound 2 disclosed herein provides a linker capable of site-specifically linking a payload to an antibody. The linker can influence the biological activity of a target molecule, for example, by reducing the half-life of the target molecule and / or the payload, or by promoting its excretion. The linker can be usefully used as a bioconjugation for detection, diagnosis, and biomarker applications.

[0043] Furthermore, the antibody-payload conjugates provided by compounds 1 and 2 have the advantage of high uniformity due to uniform binding sites. [Brief explanation of the drawing]

[0044] [Figure 1] This diagram illustrates the entire synthesis process of compound I (trans). [Figure 2] This diagram illustrates the entire synthesis process of compound II (cis). [Figure 3] This figure illustrates the results of HPLC spectral analysis of the isomer structures of compound 6. [Figure 4] This figure illustrates the results of the molecular weight analysis of compound 6 (cis). [Figure 5] This figure illustrates the results of the molecular weight analysis of compound 6 (trans). [Figure 6] This figure illustrates the results of confirming the isomer structure of compound II via HPLC. [Figure 7] This figure illustrates the results obtained by obtaining compound II via HPLC. [Figure 8] This figure illustrates the HPLC results for FcBP(6Lys)-norbornene. [Figure 9] This figure illustrates the LC mass results for FcBP(6Lys)-norbornene. [Figure 10] This figure illustrates the HPLC results for the compound I-FcBP(6Lys)-norbornene. [Figure 11]This figure illustrates the LC mass results for the compound I-FcBP(6Lys)-norbornene. [Figure 12] This figure illustrates the HPLC results for compound II-FcBP(6Lys)-norbornene. [Figure 13] This figure illustrates the mass spectrometry results for compound II-FcBP(6Lys)-norbornene. [Figure 14] This diagram illustrates the reaction between the compound I-FcBP(6Lys)-norbornene and an antibody. [Figure 15] This figure illustrates the structure of Ab (Lys246 / 248)-norbornene, which is produced by the reaction of the compound I-FcBP(6Lys)-norbornene with an antibody. [Figure 16] This figure illustrates the results of reaction monitoring of the reaction between the compound I-FcBP(6Lys)-norbornene and an antibody via HIC-HPLC. [Figure 17] This diagram illustrates the reaction between the compound II-FcBP(6Lys)-norbornene and an antibody. [Figure 18] This figure illustrates the structure of Ab(Lys246 / 248)-norbornene, which is produced by the reaction of the compound I-FcBP(6Lys)-norbornene with an antibody. [Figure 19] This figure illustrates the results of reaction monitoring of the reaction between the compound II-FcBP(6Lys)-norbornene and an antibody via HIC-HPLC. [Figure 20] This figure illustrates the results of mass spectrometry of herceptin-norbornene. [Figure 21] Figure 18 illustrates the structure of an antibody-payload conjugate generated using an antibody containing the first click chemistry functional group. The enlarged structure is the payload structure, while the unenlarged portion is the same as the structure illustrated in Figure 18. [Figure 22] This figure illustrates the results of reaction monitoring of the reaction between Ab(Lys246 / 248)-norbornene and tetrazine-PEG8-DM1 via HIC-HPLC, as shown in Figure 18. [Figure 23] This figure illustrates the results of mass spectrometry of an antibody-payload conjugate. [Figure 24] This figure shows the results of cytotoxicity experiments against NCI-N87. [Figure 25] This figure shows the results of cytotoxicity experiments against BT474. [Figure 26] This figure shows the results of cytotoxicity experiments against MDA-MB-468. [Figure 27] This figure shows a table summarizing the results of cytotoxicity experiments against NCI-N87, BT474, and MDA-MB-468. [Figure 28] This figure illustrates the results of tumor growth inhibition experiments using Herceptin and antibody-payload conjugates according to this application. [Figure 29] This figure illustrates the results of tumor growth inhibition experiments using Herceptin and antibody-payload conjugates according to this application. [Figure 30] This figure illustrates the HPLC results for compound II-FcBP(L6Dap)-norbornene. [Figure 31] This figure illustrates the mass spectrometry results for compound II-FcBP(L6Dap)-norbornene. [Figure 32] This figure illustrates the HPLC results for compound II-FcBP(L6Dab)-norbornene. [Figure 33] This figure illustrates the mass spectrometry results for compound II-FcBP(L6Dab)-norbornene. [Figure 34] This figure illustrates the HPLC results for compound II-FcBP(L6Orn)-norbornene. [Figure 35] This figure illustrates the mass spectrometry results for compound II-FcBP(L6Orn)-norbornene. [Figure 36] This figure illustrates the HPLC results for compound II-FcBP(L6Lys)-norbornene. [Figure 37] This figure illustrates the mass spectrometry results for compound II-FcBP(L6Lys)-norbornene. [Figure 38] This figure illustrates HIC-HPLC results for monitoring the binding reaction with compound II-FcBP(L6Dap)-norbornene-based antibodies. [Figure 39] This figure illustrates HIC-HPLC results for monitoring the binding reaction with compound II-FcBP(L6Dab)-norbornene-based antibodies. [Figure 40] This figure illustrates HIC-HPLC results for monitoring the binding reaction with compound II-FcBP(L6Orn)-norbornene-based antibodies. [Figure 41] This figure illustrates HIC-HPLC results for monitoring the binding reaction with compound II-FcBP(L6Lys)-norbornene-based antibodies. [Modes for carrying out the invention]

[0045] The term "heteroalkyl" refers to an alkyl group in which one or more carbon atoms are substituted with heteroatoms such as O, N, or S. For example, when the carbon atoms of an alkyl group attached to a parent molecule are substituted with heteroatoms (e.g., O, N, or S), the resulting heteroalkyl group is an alkoxy group (e.g., -OCH3), an amine (e.g., -NHCH3, -N(CH3)2), or a thioalkyl group (e.g., -SCH3), respectively. When the non-terminal carbon atoms of an alkyl group not attached to a parent molecule are substituted with heteroatoms (e.g., O, N, or S), the resulting heteroalkyl group is an alkyl ether (e.g., -CH2CH2-O-CH3), an alkylamine (e.g., -CH2NHCH3, -CH2N(CH3)2), or a thioalkyl ether (e.g., -CH2-S-CH3), respectively. When the terminal carbon atoms of an alkyl group are substituted with a heteroatom (e.g., O, N, or S), the resulting heteroalkyl group is a hydroxyalkyl group (e.g., -CH2CH2-OH), an aminoalkyl group (e.g., -CH2NH2), or an alkylthiol group (e.g., -CH2CH2-SH), respectively. Heteroalkyl groups can have, for example, 1 to 20 carbon atoms, 1 to 10 carbon atoms, or 1 to 6 carbon atoms. C1-C6 heteroalkyl groups refer to heteroalkyl groups having 1 to 6 carbon atoms.

[0046] The term "alkylene" refers to a branched, linear, or cyclic saturated hydrocarbon group containing two monovalent centers, derived by removing two hydrogen atoms from the same or two different carbon atoms of a parent alkane. For example, an alkylene group can have 1 to 20 carbon atoms, 1 to 10 carbon atoms, or 1 to 6 carbon atoms. Typical alkylene groups include, but are not limited to, methylene (-CH2-), 1,1-ethyl (-CH(CH3)-), 1,2-ethyl (-CH2CH2-), 1,1-propyl (-CH(CH2CH3)-), 1,2-propyl (-CH2CH(CH3)-), 1,3-propyl (-CH2CH2CH2-), and 1,4-butyl (-CH2CH2CH2CH2-).

[0047] The term "alkenylene" refers to a branched, linear, or cyclic unsaturated hydrocarbon group containing two monovalent centers, derived by removing two hydrogen atoms from the same or two different carbon atoms of a parent alkene. For example, an alkenylene group can have 1 to 20 carbon atoms, 1 to 10 carbon atoms, or 1 to 6 carbon atoms. Typical alkenylene groups include, but are not limited to, 1,2-ethylene (-CH=CH-).

[0048] The term "alkynylene" refers to a branched, linear, or cyclic unsaturated hydrocarbon group containing two monovalent centers, derived by removing two hydrogen atoms from the same or two different carbon atoms of a parent alkyne. For example, an alkynylene group can have 1 to 20 carbon atoms, 1 to 10 carbon atoms, or 1 to 6 carbon atoms. Typical alkynylene groups include, but are not limited to, acetylene (-C≡C-), propargyl (-CH2C≡C-), and 4-pentinyl (-CH2CH2CH2C≡C-).

[0049] The term "polymethylene" means alkylene having one or more carbon atoms, and includes methylene, ethylene, trimethylene, tetramethylene, pentamethylene, hexamethylene, and heptamethylene.

[0050] Those skilled in the art will recognize that when a “alkyl,” “aryl,” and “heterocyclyl” moiety is substituted with one or more substituents, these can be selectively referred to as “alkylene,” “arylene,” and “heterocyclylene” moiety (i.e., it means that one or more hydrogen atoms of the parent “alkyl,” “aryl,” and “heterocyclyl” moiety are substituted with the substituents described). When a “alkyl,” “aryl,” and “heterocyclyl” moiety is referred to as “substituted” in this application or is exemplified in the drawings as being substituted (or, for example, substituted by choice if the number of substituents is between zero and a positive number), the terms “alkyl,” “aryl,” and “heterocyclyl” should be understood to be interchangeable with “alkylene,” “arylene,” and “heterocyclylene.”

[0051] The term "acyl" refers to -C(=O)-alkyl, -C(=O)-carbon ring (substituted or unsubstituted), and -C(=O)-heterocyclic (substituted or unsubstituted), where the alkyl, carbon ring, or heterocyclic portion is the same as defined in this application. Non-limiting examples of "acyl" include -C(=O)CH3, -C(=O)CH2CH3, -C(=O)CH(CH3)2, -C(=O)C(CH3)3, -C(=O)-phenyl (substituted or unsubstituted), -C(=O)-cyclopropyl (substituted or unsubstituted), -C(=O)-cyclobutyl (substituted or unsubstituted), -C(=O)-cyclopentyl (substituted or unsubstituted), -C(=O)-cyclohexyl (substituted or unsubstituted), -C(=O)-pyridyl (substituted or unsubstituted), etc.

[0052] The terms “substituted,” for example, “substituted alkyl,” “substituted alkylene,” “substituted aryl,” “substituted arylalkyl,” “substituted heterocyclyl,” and “substituted carbocyclyl (e.g., substituted cycloalkyl),” mean alkyl, alkylene, aryl, arylalkyl, heterocyclyl, and carbocyclyl (e.g., cycloalkyl) in which one or more hydrogen atoms are independently substituted by non-hydrogen substituents. Typical substituents include -X, -R, -O-, =O, -OR, -SR, -S-, -NR2, -N+R3, =NR, -C(X)3, -CN, -OCN, -SCN, -N=C=O, -NCS, -NO, -NO2, =N-OH, =N2, -N3, -NHC(=O)R, -C(=O)R, -C(=O)NRR-S(=O)2O-, -S(=O)2OH, -S(=O)2R, -OS(=O)2OR, and -S(=O)2N Examples of substitutions include, but are not limited to, R, -S(=O)R, -OP(=O)(OR)2, -C(=O)R, alkylene-C(=O)R, -C(S)R, -C(=O)OR, alkylene-C(=O)OR, -C(=O)O-, alkylene-C(=O)O-, -C(=S)OR, -C(=O)SR, -C(=S)SR, -C(=O)NRR, alkylene-C(=O)NRR, -C(=S)NRR, and -C(-NR)NRR (where X is independently a halogen: F, Cl, Br, or I, and R is independently H, alkyl, aryl, arylalkyl, or heterocycle). Alkylene groups, alkenylene groups, and alkynylene groups may be substituted in the same manner.

[0053] "Optionally substituted" refers to a special part of a compound of formula 1 having one, two, or more substituents (e.g., an optionally substituted aryl group).

[0054] A “leaving group” refers to a chemical moiety that can be removed or replaced by another chemical group. Throughout this specification of the present invention, the term leaving group includes, but is not limited to, click chemistry functional groups, N-hydroxysuccinimide (NHS), or maleimide.

[0055] The term “target molecule” or “molecule of interest” means a molecule intended to have a payload to which it is attached. For example, a target molecule may be a bioactive molecule, such as a protein (or peptide), glycan, nucleic acid (or oligonucleotide), lipid, hormone, or natural drug (or fragment or combination thereof).

[0056] The term "payload" refers to a molecule intended to be linked to a target molecule. For example, a payload may be a compound, peptide, polypeptide, protein, and / or drug molecule.

[0057] The term "click chemistry functional group" collectively refers to functional groups involved in click chemistry reactions. The types of click chemistry and the functional groups involved are typically well known. Examples of click chemistry reactions include, but are not limited to, [3+2] cycloaddition, thiol-ene reactions, Diels-Alder reactions, reverse electron-demand Diels-Alder reactions, and [4+1] cycloaddition. More specifically, examples of click chemistry reactions include, but are not limited to, copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC), strain-enhanced azide-alkyne cycloaddition (SPAAC), strain-enhanced alkyne-nitrone cycloaddition (Span C), [3+2] cycloaddition of alkenes and azides, reverse-demand Diels-Alder reactions of alkenes and tetrazines, photoclick reactions of alkenes and tetrazoles, and huisgene cycloaddition of azides and alkynes. Examples of click chemistry functional groups include, but are not limited to, alkynes, cycloalkynes, cyclooctin and cyclononines (e.g., cycloalkynes such as bicyclo[6.1.0]nonano-4-in-9-ylmethanol), trans-cyclooctenes, nitrones, nitrile oxides, azides, conjugated dienes, dienophiles, and cycloalkynes, such as cyclooctin, cyclononine, dibenzocyclooctin (DIBO), biarylazacyclooctinone (BARAC), aryllessoctin (ALO), difluorinated cyclooctin (DIFO), monofluorinated (MOFO), dibenzo-aza-cyclooctin (DIBAC), and dimethoxyazacyclooctin (DIMAC).

[0058] The term "ester-activating moiety" collectively refers to the portion of the ester group that is linked to the oxygen atom and therefore capable of converting the ester group into an active ester. For example,

[0059] [ka]

[0060] When an ester group having the structure shown is an active ester, Z is the ester-activating moiety. Examples of ester-activating moieties include, but are not limited to, the N-hydroxysuccinimide group (NHS), the p-nitrophenyl group, and the pentafluorophenyl group. Examples of ester-activating moieties in the present invention include, but are not limited to, those described in WO / 2015 / 122478. The term "active ester" means an ester that is sensitive to nucleophilic substitution reactions.

[0061] In this specification, terms such as "1" and "2" are used to describe various components, and these terms are used solely for the purpose of distinguishing one component from another.

[0062] Furthermore, it should be noted that the terms used herein are solely for illustrative purposes and are not intended to limit the invention. Singular expressions include plural expressions unless otherwise clearly indicated by the context.

[0063] In this specification, terms such as “comprise,” “include,” or “have” are intended to indicate the presence of realized features, numbers, steps, operations, components, or any combination thereof, and should be understood that the possibility of the presence or addition of one or more other features, numbers, steps, operations, components, or any combination thereof is not excluded.

[0064] This specification can be modified in various ways and may include various illustrative embodiments, so specific illustrative embodiments are illustrated and described in detail below. However, this description is not intended to limit the invention to any particular disclosure, and all variations, equivalents, and substitutions that fall within the concept and art of the invention should be understood to be included in the invention.

[0065] The present invention will be described in detail in the following paragraphs.

[0066] This application can provide a linker that assists transport and binding reactions so that a payload can be linked to a desired target molecule.

[0067] The linker may contain two or more functional groups that can react with the payload and / or target molecule.

[0068] The above reactions mean that two or more identical or different molecules, or parts of functional groups of such molecules, are linked to one another, or that elimination reactions such as E1, E2, SN1, SN2, and nucleophilic substitution reactions occur. Such linking includes all direct or indirect covalent and / or noncovalent bonds.

[0069] In the example, the first functional group of two or more functional groups may be a leaving group.

[0070] In the example, the second functional group of two or more functional groups may be a click chemistry functional group.

[0071] The linker may contain two or more electrophilic carbon atoms of the carbonyl group.

[0072] The electrophilic carbon atom of the carbonyl group may be linked to a functional group.

[0073] For example, the electrophilic carbon atom and functional group of the carbonyl group may be covalently bonded.

[0074] As another example, one or more atoms may be included between the electrophilic carbon atom of the carbonyl group and the functional group. For example, a nucleophilic atom may be included between the electrophilic carbon atom of the carbonyl group and the functional group. In this case, the nucleophilic atom may be O (oxygen), N (nitrogen), or S (sulfur).

[0075] The linker may contain two or more electrophilic carbon atoms of the carbonyl group having different partial positive charges (δ+).

[0076] Among the two or more electrophilic carbon atoms of a carbonyl group, the carbonyl group with the largest partial positive charge (δ+) can be the first carbonyl group carbon atom.

[0077] Among the two or more electrophilic carbon atoms of a carbonyl group, the carbonyl group having the second largest partial positive charge (δ+) can be the second carbonyl group carbon atom.

[0078] For example, the electrophilic carbon atom of the first carbonyl group may be linked to the first functional group. In this case, the nucleophilic atom may be located between the electrophilic carbon atom of the first carbonyl group and the first functional group.

[0079] As another example, the electrophilic carbon atom of the second carbonyl group may be linked to the second functional group. In this case, the electrophilic carbon atom of the second carbonyl group can form a covalent bond with the first functional group.

[0080] The linker can adjust the position to which the payload, which is to be covalently bound to the target molecule, is attached.

[0081] For example, the payload may be linked to the electrophilic carbon atom of the first carbonyl group.

[0082] The water solubility of the linker can be adjusted by length adjustment. For example, the water solubility of the linker can be increased by increasing the number of alkyl groups, including substituents that can increase water solubility.

[0083] The linker structure is described in detail in the following section.

[0084] According to one embodiment disclosed herein, a linker compound represented by the following formula 1 and / or formula 2 may be provided.

[0085] [ka]

[0086] In Equation 1, R' is the ester-activating moiety. Examples of ester-activating moieties include, but are not limited to, the N-hydroxysuccinimide group (NHS), the p-nitrophenyl group, and the pentafluorophenyl group. Furthermore, R' is,

[0087] [ka]

[0088] It can be any one of the following. R" is one of the click chemistry functional groups, The click chemistry functional group may be, but is not limited to, one or more of the following: alkynes, cycloalkynes, e.g., cyclooctin and cyclononine (e.g., bicyclo[6.1.0]nona-4-in-9-ylmethanol), trans-cyclooctene, nitrones, nitrile oxides, azides, conjugated dienes, and dienophiles.

[0089] When the click chemistry functional group is a cycloalkyne, the cycloalkyne may be any one of the following: cyclooctin, cyclononine, dibenzocyclooctin (DIBO), biarylazacyclooctinone (BARAC), aryl-less octyne (ALO), difluorinated cyclooctin (DIFO), monofluorinated cyclooctin (MOFO), dibenzo-aza-cyclooctin (DIBAC), and dimethoxyazacyclooctin (DIMAC). The cycloalkyne is not limited to these.

[0090] If the click chemistry functional group is a conjugated diene, the conjugated diene may be an alkene or a cycloalkane. For example, the conjugated diene may be a tetrazine (e.g., 1,2,3,4-tetrazine and / or 1,2,4,5-tetrazine).

[0091] When the click chemistry functional group is a dienophile, the dienophile may be an alkene or a cycloalkane, and the cycloalkane may have a bicyclic or fused ring structure. For example, the dienophile may be norbornene.

[0092] R''' is a substituted or non-substituted C 1~20 Alkylene, substituted or unsubstituted C 2~20 Alkenylene, substituted or unsubstituted C 1~10 Alkynylene, substituted or unsubstituted C 1~10 Polymethylene, substituted or unsubstituted C 5~12 Aryl, substituted, or unsubstituted C 5~14 Arylalkylenes, substituted or unsubstituted C 8~16 Arylalkenylenes, substituted or unsubstituted C 3~10 Cycloalkylene, substituted or unsubstituted C 3~10 Heterocycloalkylenes, or substituted or unsubstituted C 5~12 A heteroaryl is a heteroalkylene, heterocycloalkylene, or heteroaryl that contains at least one or more N, O, or S. The substituted products are substituted with non-hydrogen substituents, and the non-hydrogen substituents are -Ra, -O-, =O, -ORa, -SRa, -S-, -N(Ra)2, =NRa, -C(Rb)3, -N=C=O, -NCS, -NO, -NO2, =N-OH, =N2, -N3, -NHC(=O)Ra, -C(=O)Ra, -C(=O)NRaRa-S(=O)2O-, -S(=O)2OH, -S(=O)2Ra, -OS(=O)2ORa, -S(=O)2NRa, -S(=O )Ra, -C(=O)Ra, alkylene-C(=O)Ra, -C(=S)Ra, -C(=O)ORa, alkylene-C(=O)ORa, -C(=O)O-, alkylene-C(=O)O-, -C(=S)ORa, -C(=O)SRa, -C(=S)SRa, -C(=O)NRaRa, alkylene-C(=O)NRaRa, -C(=S)NRaRa, -C(-NRa)NRaRa, and Rb, one or more selected from this group. Ra is H, C 1~6 Alkyl, C 5~12 Ariel, C7~12 It is an arylalkyl or heterocyclic, Rb is F, Cl, Br, or I. X is O, N, or S.

[0093] In Equation 2, R' is the ester-activating moiety. Examples of ester-activating moieties include, but are not limited to, the N-hydroxysuccinimide group (NHS), the p-nitrophenyl group, and the pentafluorophenyl group. Furthermore, R' is,

[0094] [ka]

[0095] It can be any one of the following. R" is one of the click chemistry functional groups, The click chemistry functional group may be, but is not limited to, one or more of the following: alkynes, cyclooctines and cyclononines (e.g., cycloalkynes, e.g., bicyclo[6.1.0]nona-4-in-9-ylmethanol), trans-cyclooctenes, nitrones, nitrile oxides, azides, conjugated dienes, and dienophiles.

[0096] When the click chemistry functional group is a cycloalkyne, the cycloalkyne may be, but is not limited to, any one of the following: cyclooctin, cyclononine, dibenzocyclooctin (DIBO), BARAC (biarylazacyclooctinone), ALO (aryllessoctin), DIFO (difluorinated cyclooctin), MOFO (monofluorinated), DIBAC (dibenzo-aza-cyclooctin), and DIMAC (dimethoxyazacyclooctin).

[0097] If the click chemistry functional group is a conjugated diene, the conjugated diene may be an alkene or a cycloalkane. For example, the conjugated diene may be a tetrazine (e.g., 1,2,3,4-tetrazine and / or 1,2,4,5-tetrazine).

[0098] When the click chemistry functional group is a dienophile, the dienophile may be an alkene or a cycloalkane, and the cycloalkane may have a bicyclic or fused ring structure. For example, the dienophile may be transcyclooctene (TCO) or norbornene.

[0099] R''' is a substituted or non-substituted C 1~20 Alkylene, substituted or unsubstituted C 2~20 Alkenylene, substituted or unsubstituted C 1~10 Alkynylene, substituted or unsubstituted C 1~10 Polymethylene, substituted or unsubstituted C 5~12 Aryl, substituted, or unsubstituted C 5~14 Arylalkylenes, substituted or unsubstituted C 8~16 Arylalkenylenes, substituted or unsubstituted C 3~10 Cycloalkylene, substituted or unsubstituted C 3~10 Heterocycloalkylenes, or substituted or unsubstituted C 5~12 A heteroaryl is a heteroalkylene, heterocycloalkylene, or heteroaryl that contains at least one or more N, O, or S. The substituted products are substituted with non-hydrogen substituents, and the non-hydrogen substituents are -Ra, -O-, =O, -ORa, -SRa, -S-, -N(Ra)2, =NRa, -C(Rb)3, -N=C=O, -NCS, -NO, -NO2, =N-OH, =N2, -N3, -NHC(=O)Ra, -C(=O)Ra, -C(=O)NRaRa-S(=O)2O-, -S(=O)2OH, -S(=O)2Ra, -OS(=O)2ORa, -S(=O)2NRa, -S(=O )Ra, -C(=O)Ra, alkylene-C(=O)Ra, -C(=S)Ra, -C(=O)ORa, alkylene-C(=O)ORa, -C(=O)O-, alkylene-C(=O)O-, -C(=S)ORa, -C(=O)SRa, -C(=S)SRa, -C(=O)NRaRa, alkylene-C(=O)NRaRa, -C(=S)NRaRa, -C(-NRa)NRaRa, and Rb, one or more selected from this group. Ra is H, C 1~6 Alkyl, C 5~12 Ariel, C 7~12 It is an arylalkyl or heterocyclic, Rb is F, Cl, Br, or I. X is O, N, or S.

[0100] In the formula of the present invention,

[0101] [ka]

[0102] This is used to indicate a bond that is an attachment point for a part or substituent to a nucleus or skeletal structure.

[0103] According to the illustrative embodiments disclosed herein, the compound represented by Formula 1 may be a trans compound.

[0104] Equation 1 is expressed by Equation 1-1 below if Equation 1 is equivalent to the following: R' is

[0105] [ka]

[0106] And, R" is one of the click chemistry functional groups, The click chemistry functional group is one or more of the following: alkynes, cycloalkynes, e.g., cyclooctin and cyclononine (e.g., bicyclo[6.1.0]nonano-4-in-9-ylmethanol), trans-cyclooctene, nitrone, nitrile oxide, azide, conjugated diene, and dienophile, e.g., alkene, in which case cycloalkynes, conjugated dienes, and dienes are the same as those described above.

[0107] R''' is a substituted or non-substituted C 1~20 Alkylene, substituted or unsubstituted C 1~20 Heteroalkylenes, substituted or unsubstituted C 1~20 Haloalkyl, or C 1~10 The polymethylene is a heteroalkylene, and the heteroalkylene comprises at least one or more selected from the group consisting of N, O, and S. The substituted products are substituted with non-hydrogen substituents, and the non-hydrogen substituents are -Ra, -O-, =O, -ORa, -SRa, -S-, -N(Ra)2, =NRa, -C(Rb)3, -N=C=O, -NCS, -NO, -NO2, =N-OH, =N2, -N3, -NHC(=O)Ra, -C(=O)Ra, -C(=O)NRaRa-S(=O)2O-, -S(=O)2OH, -S(=O)2Ra, -OS(=O)2ORa, -S(=O)2NRa, -S(=O )Ra, -C(=O)Ra, alkylene-C(=O)Ra, -C(=S)Ra, -C(=O)ORa, alkylene-C(=O)ORa, -C(=O)O-, alkylene-C(=O)O-, -C(=S)ORa, -C(=O)SRa, -C(=S)SRa, -C(=O)NRaRa, alkylene-C(=O)NRaRa, -C(=S)NRaRa, -C(-NRa)NRaRa, and Rb, one or more selected from this group. Ra is H, C 1~6 Alkyl, C 5~12Ariel, C 7~12 It is an arylalkyl or heterocyclic, Rb is F, Cl, Br, or I. X is one of O, N, or S.

[0108] [ka]

[0109] Specifically, R''' is,

[0110] [ka]

[0111] It is one of them, R1 is H, R2 is either H or C (=O), R3 is either H or C (=O), R4 is either H or C (=O), R5 is either H or C (=O), R6 is either H or C (=O), Y1 may be any one of C, N, O, and S. n may be any integer from 1 to 20, but is not limited to these values.

[0112] R5 and R6 cannot both be C (=O) at the same time. If Y1 is one of N, O, or S, then R4, R5, and R6 cannot be C (=O).

[0113] More specifically, R''' is,

[0114] [ka]

[0115] And, R4 is either H or C (=O), R5 is either H or C (=O), R6 is either H or C (=O), Y1 may be any one of C, N, O, and S. n can be any integer from 1 to 10.

[0116] In an illustrative embodiment, if Equation 1-1 is the same as below, then Equation 1-1 is represented by the following Equation 1-1-1: R' is

[0117] [ka]

[0118] And, R" is one of the click chemistry functional groups, R''' is,

[0119] [ka]

[0120] And, n is 1, R4 is either H or C (=O), R5 is either H or C (=O), R6 is either H or C (=O), Y1 is C, X is one of N, O, or S.

[0121] [ka]

[0122] In another illustrative embodiment, Equation 1-1 is represented by Equation 1-1-2 below, where Equation 1-1 is the same as below: R' is

[0123] [ka]

[0124] And, R" stands for Norbornen, R''' is,

[0125] [ka]

[0126] And, R4 is H, R5 is H, R6 is H, n is 1, Y1 is C, X is O.

[0127] [ka]

[0128] As another illustrative embodiment, compounds that can be represented by Formula 1 may be those listed in Table 1 below.

[0129] [Table 1-1]

[0130] [Table 1-2]

[0131] Equation 1 can be expressed by Equation 1-2 below if Equation 1 is the same as below: R' is

[0132] [ka]

[0133] And, R" is one of the click chemistry functional groups, The click chemistry functional group is one or more of the following: alkynes, cycloalkynes, e.g., cyclooctin and cyclononine (e.g., bicyclo[6.1.0]nonano-4-in-9-ylmethanol), trans-cyclooctene, nitrone, nitrile oxide, azide, conjugated diene, and dienophile, e.g., alkene, in which case the cycloalkynes, conjugated dienes and dienes are the same as those described above.

[0134] R''' is a substituted or non-substituted C 1~20 Alkylene, substituted or unsubstituted C 1~20 Heteroalkylenes, substituted or unsubstituted C 1~20 Haloalkyl, or C 1~10 The polymethylene is a heteroalkylene, and the heteroalkylene comprises at least one or more selected from the group consisting of N, O, and S. The substituted products are substituted with non-hydrogen substituents, and the non-hydrogen substituents are -Ra, -O-, =O, -ORa, -SRa, -S-, -N(Ra)2, =NRa, -C(Rb)3, -N=C=O, -NCS, -NO, -NO2, =N-OH, =N2, -N3, -NHC(=O)Ra, -C(=O)Ra, -C(=O)NRaRa-S(=O)2O-, -S(=O)2OH, -S(=O)2Ra, -OS(=O)2ORa, -S(=O)2NRa, -S(=O )Ra, -C(=O)Ra, alkylene-C(=O)Ra, -C(=S)Ra, -C(=O)ORa, alkylene-C(=O)ORa, -C(=O)O-, alkylene-C(=O)O-, -C(=S)ORa, -C(=O)SRa, -C(=S)SRa, -C(=O)NRaRa, alkylene-C(=O)NRaRa, -C(=S)NRaRa, -C(-NRa)NRaRa, and Rb, one or more selected from this group. Ra is H, C 1~6 Alkyl, C 5~12 Ariel, C 7~12 It is an arylalkyl or heterocyclic, Rb is F, Cl, Br, or I. X is any one of O, N, and S.

[0135] [Chemical formula]

[0136] Specifically, R''' is

[0137] [Chemical formula]

[0138] one of R1 is H, R2 is H or C(=O), R3 is H or C(=O), R4 is H or C(=O), R5 is H or C(=O), R6 is H or C(=O), Y1 may be any one of C, N, O, and S, n may be any one of the integers from 1 to 20, but is not limited thereto.

[0139] In this case, both R5 and R6 shall not be C(=O), When Y1 is any one of N, O, and S, R4, R5, and R6 cannot be C(=O).

[0140] More specifically, R''' is

[0141] [Chemical formula]

[0142] and R4 is H or C(=O), R5 is H or C(=O), R6 is H or C(=O), Y1 may be any one of C, N, O, and S. n can be any integer from 1 to 10.

[0143] In an illustrative embodiment, if formula 1-2 is the same as below, then formula 1-2-1 is represented by the following formula: R''' is,

[0144] [ka]

[0145] And, R" is one of the click chemistry functional groups, n is 1, R4 is either H or C (=O), R5 is either H or C (=O), R6 is either H or C (=O), Y1 is C, X is one of N, O, or S.

[0146] [ka]

[0147] In another illustrative embodiment, Equation 1-2 is represented by Equation 1-2-2 below, if Equation 1-2 is the same as below: R' is

[0148] [ka]

[0149] And, R" stands for Norbornen, R''' is,

[0150] [ka]

[0151] and R4 is H, R5 is H, R6 is H, n is 1, Y1 is C, X is O.

[0152]

Chemical formula

[0153] As an illustrative embodiment, the compound represented by Formula 1 can be a compound described in Table 2 below.

[0154]

Table 2-1

[0155]

Table 2-2

[0156] According to the illustrative embodiments disclosed herein, the compound represented by Formula 2 may be a cis-type compound.

[0157] Formula 2 is represented by Formula 2-1 below when Formula 2 is the same as follows: R' is

[0158]

Chemical formula

[0159] and R" is any one of click chemistry functional groups, The click chemistry functional group is one or more of the following: alkynes, cycloalkynes, e.g., cyclooctin and cyclononine (e.g., bicyclo[6.1.0]nonano-4-in-9-ylmethanol), trans-cyclooctene, nitrone, nitrile oxide, azide, conjugated diene, and dienophile, e.g., alkene, in which case the cycloalkynes, conjugated dienes and dienes are the same as those described above. R''' is a substituted or non-substituted C 1~20 Alkylene, substituted or unsubstituted C 1~20 Heteroalkylenes, substituted or unsubstituted C 1~20 Haloalkyl, or C 1~10 The polymethylene is a heteroalkylene, and the heteroalkylene comprises at least one or more selected from the group consisting of N, O, and S. The substituted products are substituted with non-hydrogen substituents, and the non-hydrogen substituents are -Ra, -O-, =O, -ORa, -SRa, -S-, -N(Ra)2, =NRa, -C(Rb)3, -N=C=O, -NCS, -NO, -NO2, =N-OH, =N2, -N3, -NHC(=O)Ra, -C(=O)Ra, -C(=O)NRaRa-S(=O)2O-, -S(=O)2OH, -S(=O)2Ra, -OS(=O)2ORa, -S(=O)2NRa, -S(=O )Ra, -C(=O)Ra, alkylene-C(=O)Ra, -C(=S)Ra, -C(=O)ORa, alkylene-C(=O)ORa, -C(=O)O-, alkylene-C(=O)O-, -C(=S)ORa, -C(=O)SRa, -C(=S)SRa, -C(=O)NRaRa, alkylene-C(=O)NRaRa, -C(=S)NRaRa, -C(-NRa)NRaRa, and Rb, one or more selected from this group. Ra is H, C 1~6 Alkyl, C 5~12 Ariel, C 7~12 It is an arylalkyl or heterocyclic, Rb is F, Cl, Br, or I. X is one of O, N, or S.

[0160] [ka]

[0161] Specifically, R''' is,

[0162] [ka]

[0163] It is one of them, R7 is H, R8 is either H or C (=O), R9 is either H or C (=O), R 10 is either H or C (=O), R 11 is either H or C (=O), R 12 is either H or C (=O), Y2 may be one of C, N, O, and S. n may be any integer from 1 to 20, but is not limited to these values.

[0164] In this case, R 11 and R 12 Both must not be C (=O), If Y2 is one of N, O, or S, then R 10 , R 11 and R 12 C (=O) is not possible.

[0165] More specifically, R''' is,

[0166] [ka]

[0167] And, R 10 is either H or C (=O), R 11 is either H or C (=O), R12 is either H or C (=O), Y2 may be one of C, N, O, and S. n can be any integer from 1 to 10.

[0168] In an illustrative embodiment, if Equation 2-1 is the same as below, then Equation 2-1 is represented by Equation 2-1-1 below: R' is

[0169] [ka]

[0170] And, R" is one of the click chemistry functional groups, R''' is,

[0171] [ka]

[0172] And, n is 1, R 10 is either H or C (=O), R 11 is either H or C (=O), R 12 is either H or C (=O), Y2 is C, X is one of N, O, or S.

[0173] [ka]

[0174] In another illustrative embodiment, formula 2-1 is represented by formula 2-1-2 below, where formula 2-1 is the same as below: R' is

[0175] [ka]

[0176] And, R" stands for Norbornen, R''' is,

[0177] [ka]

[0178] And, n is 1, R 10 H is, R 11 H is, R 12 H is, Y2 is C, n is 1, X is O.

[0179] [ka]

[0180] As an illustrative embodiment, compounds that can be represented by Formula 2 may be those listed in Table 3 below.

[0181] [Table 3-1]

[0182] [Table 3-2]

[0183] [Table 3-3]

[0184] Formula 2 is represented by the following Formula 2-2 when Formula 2 is the same as follows: R' is

[0185] [Chemical formula]

[0186] and R" is any one of click chemistry functional groups, The click chemistry functional groups are any one or more of alkyne, cycloalkyne such as cyclooctyne and cyclononyne (e.g., bicyclo[6.1.0]nona-4-in-9-ylmethanol), trans-cyclooctene, nitrone, nitrile oxide, azide, conjugated diene, and dienophile such as alkene. In this case, the cycloalkyne, conjugated diene, and diene are the same as those described above.

[0187] R''' is substituted or unsubstituted C 1~20 alkylene, substituted or unsubstituted C 1~20 heteroalkylene, substituted or unsubstituted C 1~20 haloalkyl, or C 1~10 polymethylene, and heteroalkylene contains at least one or more selected from the group consisting of N, O, and S. The substituent is substituted with a non-hydrogen substituent, and the non-hydrogen substituent is -Ra, -O-, =O, -ORa, -SRa, -S-, -N(Ra)2, =NRa, -C(Rb)3, -N=C=O, -NCS, -NO, -NO2, =N-OH, =N2, -N3, -NHC(=O)Ra, -C(=O)Ra, -C(=O)NRaRa - S(=O)2O-, -S(=O)2OH, -S(=O)2Ra, -OS(=O)2ORa, -S(=O)2NRa, -S(=O)Ra, -C(=O)Ra, alkylene-C(=O)Ra, -C(=S)Ra, -C(=O)ORa, alkylene-C(=O)ORa, -C(=O)O-, alkylene-C(=O)O-, -C(=S)ORa, -C(=O)SRa, -C(=S)SRa, -C(=O)NRaRa, alkylene-C(=O)NRaRa, -C(=S)NRaRa, -C(-NRa)NRaRa and any one or more selected from the group consisting of Rb, Ra is H, C 1~6 alkyl, C 5~12 aryl, C 7~12 arylalkyl or heterocycle, Rb is F, Cl, Br or I, X is any one of O, N and S.

[0188]

Chemical formula

[0189] Specifically, R''' is

[0190]

Chemical formula

[0191] one of R7 is H, R8 is H or C(=O), R9 is H or C(=O), R 10 is H or C(=O), R 11 is H or C(=O), R 12 is either H or C (=O), Y2 may be one of C, N, O, and S. n may be any integer from 1 to 20, but is not limited to these values.

[0192] In this case, R 11 and R 12 Both must not be C (=O), If Y2 is one of N, O, or S, then R 10 , R 11 and R 12 C (=O) is not possible.

[0193] More specifically, R''' is,

[0194] [ka]

[0195] And, R 10 is either H or C (=O), R 11 is either H or C (=O), R 12 is either H or C (=O), Y2 may be one of C, N, O, and S. n can be any integer from 1 to 10.

[0196] In an illustrative embodiment, if Equation 2-2 is the same as below, then Equation 2-2 is represented by Equation 2-2-1 below: R' is

[0197] [ka]

[0198] And, R" is one of the click chemistry functional groups, R''' is,

[0199] [ka]

[0200] And, n is 1, R 10 is either H or C (=O), R 11 is either H or C (=O), R 12 is either H or C (=O), Y2 is C, X is one of N, O, or S.

[0201] [ka]

[0202] In another illustrative embodiment, Equation 2-2 is represented by the following Equation 2-2-2 if Equation 2-2 is the same as below: R' is

[0203] [ka]

[0204] And, R" stands for Norbornen, R''' is,

[0205] [ka]

[0206] And, n is 1, R 10 H is, R 11 H is, R 12 H is, Y2 is C, X is O.

[0207] [ka]

[0208] As an illustrative embodiment, compounds that can be represented by Formula 2 may be those listed in Table 4 below.

[0209] [Table 4-1]

[0210] [Table 4-2]

[0211] [Table 4-3]

[0212] In the following section, when the target molecule is an antibody, a method for preparing an antibody-payload conjugate using the linker described above is described. A method for preparing an antibody-payload conjugate comprising: (1) preparing a linker-Fc-binding peptide conjugate by reacting the linker with an Fc-binding peptide; (2) obtaining an antibody containing a first click chemistry functional group by reacting the linker-Fc-binding peptide conjugate with an antibody; and (3) preparing an antibody-payload conjugate by reacting the antibody containing the first click chemistry functional group with a payload containing a second click chemistry functional group complementary to the first click chemistry functional group. In the following section, each process is described separately.

[0213] Methods for preparing antibody-payload conjugates may include: (1) preparing a linker-Fc-binding peptide conjugate by reacting a linker with an Fc-binding peptide.

[0214] Fc-binding peptides are a collective term for peptides that have the property of binding to the Fc domain of an antibody. A well-known example of an Fc-binding peptide is the 13mer peptide discovered by DeLano et al., which is known to have the property of binding to the FcRn domain. The inventors of this invention have invented Fc-binding peptides SEQ ID NOs: 1 to 5, in which specific residues of the 13mer peptide are substituted and the Fc-binding peptides are able to react with the linker. As can be seen in the following structure, the Fc-binding peptides SEQ ID NOs: 1 to 5 can react with the linker via the free amine group contained in residue 6.

[0215] The Fc-binding peptide according to Sequence ID No. 1 is the peptide represented by formula 13: [Formula 13] DCAWH-Xa-GELVWCT (Sequence ID: 1) D is aspartic acid, C is cysteine, A is alanine, W is tryptophan, H is histidine, and Xa is

[0216] [ka]

[0217] Here, G is glycine, E is glutamate, L is leucine, V is valine, and T is threonine. In this case, m is an integer between 1 and 4, and the N-terminal cysteine ​​and C-terminal cysteine ​​may be selectively linked to each other. When Xa is m=1 (SEQ ID NO: 3), it is 2,3-diaminopropionic acid (Dap); when Xa is m=2 (SEQ ID NO: 4), it is 2,4-diaminobutyric acid (Dab); when Xa is m=3 (SEQ ID NO: 5), it is ornithine; and when Xa is m=4 (SEQ ID NO: 2), it is lysine.

[0218] The Fc-binding peptide according to Sequence ID No. 2 is the peptide represented by formula 14: [Formula 14] DCAWHKGELVWCT (Sequence ID: 2) D is aspartic acid, C is cysteine, A is alanine, W is tryptophan, H is histidine, K is lysine, G is glycine, E is glutamate, L is leucine, V is valine, and T is threonine. The N-terminal and C-terminal cysteines may be selectively linked to each other.

[0219] Examples of the linker include compounds according to chemical formula 1 or chemical formula 2 and specific examples thereof.

[0220] The linker and the Fc-binding peptide can be bonded by a nucleophilic substitution reaction. This nucleophilic substitution reaction may occur when a nucleophilic atom present in the Fc-binding peptide attacks a positively charged or partially positively charged atom of the linker. The nucleophilic atom may be the nitrogen atom of lysine. In addition, the positively charged or partially positively charged atom may be the electrophilic carbon of the carbonyl group.

[0221] In the following, it is shown, for example, that the compounds represented by formulas 1 and / or 2 disclosed herein, i.e., linkers, can form linker-Fc-binding peptide conjugates by binding to Fc-binding peptides.

[0222] For example, a linker-Fc-binding peptide conjugate may have the structure of formula 3.

[0223] [ka]

[0224] In Equation 3, R" is one of the click chemistry functional groups, The click chemistry functional group is one or more of the following: alkynes, cycloalkynes, e.g., cyclooctin and cyclononine (e.g., bicyclo[6.1.0]nona-4-in-9-ylmethanol), trans-cyclooctene, nitrone, nitrile oxide, azide, conjugated diene, and dienophile, in which case cycloalkynes, conjugated dienes and dienes are the same as those described above.

[0225] R''' is a substituted or non-substituted C 1~20 Alkylene, substituted or unsubstituted C 2~20 Alkenylene, substituted or unsubstituted C 1~10 Alkynylene, substituted or unsubstituted C 1~10 Polymethylene, substituted or unsubstituted C 5~12 Aryl, substituted, or unsubstituted C 5~14 Arylalkylenes, substituted or unsubstituted C 8~16 Arylalkenylenes, substituted or unsubstituted C 3~10 Cycloalkylene, substituted or unsubstituted C 3~10 Heterocycloalkylenes, or substituted or unsubstituted C 5~12A heteroaryl is a heteroalkylene, heterocycloalkylene, or heteroaryl that contains at least one or more N, O, or S. The substituted products are substituted with non-hydrogen substituents, and the non-hydrogen substituents are -Ra, -O-, =O, -ORa, -SRa, -S-, -N(Ra)2, =NRa, -C(Rb)3, -N=C=O, -NCS, -NO, -NO2, =N-OH, =N2, -N3, -NHC(=O)Ra, -C(=O)Ra, -C(=O)NRaRa-S(=O)2O-, -S(=O)2OH, -S(=O)2Ra, -OS(=O)2ORa, -S(=O)2NRa, -S(=O )Ra, -C(=O)Ra, alkylene-C(=O)Ra, -C(=S)Ra, -C(=O)ORa, alkylene-C(=O)ORa, -C(=O)O-, alkylene-C(=O)O-, -C(=S)ORa, -C(=O)SRa, -C(=S)SRa, -C(=O)NRaRa, alkylene-C(=O)NRaRa, -C(=S)NRaRa, -C(-NRa)NRaRa, and Rb, one or more selected from this group. Ra is H, C 1~6 Alkyl, C 5~12 Ariel, C 7~12 It is an arylalkyl or heterocyclic, Rb is F, Cl, Br, or I. X may be O, N, or S.

[0226] Y3 may be any one of O, N, or S.

[0227] Fp is an Fc-binding peptide. Furthermore, Fp may be one peptide selected from formula 13 or 14. Details of formulas 13 and 14 are the same as already described.

[0228] Fp may be linked via Y3 of amino acid residue 6. In this case, amino acid residue 6 may be lysine, ornithine, 2,3-diaminopropionic acid, or 2,4-diaminobutyric acid.

[0229] The compound represented by Equation 3 can be produced, for example, by reaction scheme 1.

[0230] [ka]

[0231] Reaction scheme 1 is a reaction to produce a compound represented by formula 3 by reacting the compound represented by formula 1 with an Fc-binding peptide containing 1 to 50 amino acids.

[0232] The binding reaction between the Fc-binding peptide and formula 1 can be carried out by a substitution reaction.

[0233] The substitution reaction may be a nucleophilic acyl substitution reaction.

[0234] The substitution reaction can be carried out by a reaction between the compound of formula 1 and one of the amine group (-NH2), thiol group (-SH), or hydroxyl group (-OH) of the Fc-binding peptide.

[0235] For example, when the compound of formula 1 reacts with the amine group (-NH2) of an Fc-binding peptide, Y3 can be N(H). If the peptides of sequence numbers 1 to 5 contain 2,3-diaminopropionic acid, 2,4-diaminobutyric acid, ornithine, or lysine at residue 6, then all corresponding residues contain a free amine group, and a substitution reaction can occur.

[0236] As another example, when the compound of formula 1 reacts with the thiol group (-SH) of an Fc-binding peptide, Y3 can be S.

[0237] For example, when the compound of formula 1 reacts with any one of the hydroxyl groups (-OH) of an Fc-binding peptide, Y3 can be O.

[0238] For example, a linker-Fc-binding peptide conjugate may have the structure of formula 4.

[0239] [ka]

[0240] In Equation 4, R" is one of the click chemistry functional groups, The click chemistry functional groups are one or more of the following: alkynes, cycloalkynes, e.g., cyclooctin and cyclononine (e.g., bicyclo[6.1.0]nona-4-in-9-ylmethanol), trans-cyclooctene, nitrone, nitrile oxide, azide, conjugated diene, and dienophile, where cycloalkynes, conjugated dienes, and dienes are the same as those described above.

[0241] R''' is a substituted or non-substituted C 1~20 Alkylene, substituted or unsubstituted C 2~20 Alkenylene, substituted or unsubstituted C 1~10 Alkynylene, substituted or unsubstituted C 1~10 Polymethylene, substituted or unsubstituted C 5~12 Aryl, substituted, or unsubstituted C 5~14 Arylalkylenes, substituted or unsubstituted C 8~16 Arylalkenylenes, substituted or unsubstituted C 3~10 Cycloalkylene, substituted or unsubstituted C 3~10 Heterocycloalkylenes, or substituted or unsubstituted C 5~12 A heteroaryl is a heteroalkylene, heterocycloalkylene, or heteroaryl that contains at least one or more N, O, or S. The substituted products are substituted with non-hydrogen substituents, and the non-hydrogen substituents are -Ra, -O-, =O, -ORa, -SRa, -S-, -N(Ra)2, =NRa, -C(Rb)3, -N=C=O, -NCS, -NO, -NO2, =N-OH, =N2, -N3, -NHC(=O)Ra, -C(=O)Ra, -C(=O)NRaRa-S(=O)2O-, -S(=O)2OH, -S(=O)2Ra, -OS(=O)2ORa, -S(=O)2NRa, -S(=O )Ra, -C(=O)Ra, alkylene-C(=O)Ra, -C(=S)Ra, -C(=O)ORa, alkylene-C(=O)ORa, -C(=O)O-, alkylene-C(=O)O-, -C(=S)ORa, -C(=O)SRa, -C(=S)SRa, -C(=O)NRaRa, alkylene-C(=O)NRaRa, -C(=S)NRaRa, -C(-NRa)NRaRa, and Rb, one or more selected from this group. Ra is H, C 1~6 Alkyl, C 5~12 Ariel, C 7~12 It is an arylalkyl or heterocyclic, Rb is F, Cl, Br, or I. X may be O, N, or S.

[0242] Y4 may be any one of O, N, or S.

[0243] Fp is an Fc-binding peptide. Furthermore, Fp may be one peptide selected from formula 13 or 14. Details of formulas 13 and 14 are the same as already described.

[0244] Fp may be linked via Y4 of amino acid residue 6. In this case, amino acid residue 6 may be lysine, ornithine, 2,3-diaminopropionic acid, or 2,4-diaminobutyric acid.

[0245] The compound represented by Equation 4 can be produced, for example, by reaction scheme 2.

[0246] [ka]

[0247] Reaction scheme 2 is a reaction to produce the compound represented by formula 4 by reacting the compound represented by formula 2 with an Fc-binding peptide containing 1 to 50 amino acids.

[0248] The reaction between the Fc-binding peptide and formula 2 can be carried out by substitution.

[0249] Substitution reactions can include nucleophilic acyl substitution reactions.

[0250] The substitution reaction can be carried out by a reaction between the compound of formula 2 and one of the amine group (-NH2), thiol group (-SH), or hydroxyl group (-OH) of the Fc-binding peptide.

[0251] For example, when the compound of formula 2 reacts with the amine group (-NH2) of an Fc-binding peptide, Y4 can be N. If the peptides of sequence numbers 1 to 5 contain 2,3-diaminopropionic acid, 2,4-diaminobutyric acid, ornithine, or lysine at residue 6, all corresponding residues contain a free amine group, and therefore a substitution reaction can occur.

[0252] As another example, when the compound of formula 2 reacts with the thiol group (-SH) of an Fc-binding peptide, Y4 can be S.

[0253] For example, if the compound of formula 2 reacts with any one of the hydroxyl groups (-OH) of an Fc-binding peptide, Y4 can be O.

[0254] In reaction schemes 1 and 2, the compounds represented by formula 3 and / or formula 4 can be produced by reacting the amine group (-NH2), thiol group (-SH), and hydroxyl group (-OH) residues of the Fc-binding peptide with the carbon atom of the first carbonyl group.

[0255] In this case, the carbon atom of the carbonyl group most closely bonded to R', i.e., the electrophilic carbon atom of the first carbonyl group, can have the largest partial positive charge (δ+).

[0256] A method for preparing an antibody-payload conjugate may include the step of (2) reacting a linker-Fc binding peptide with an antibody to obtain an antibody containing a first click chemistry functional group.

[0257] In addition, in this case, if the Fc-binding peptide has affinity for a specific site on the Fc domain, the linker-Fc-binding peptide conjugate can be induced to react with a specific site on the antibody. The inventors of the present invention have developed a technology that allows a first click chemistry functional group to be moved to a specific site on the antibody, in particular at the lysine 246 (Fc-Lys246) or lysine 248 (Fc-248) position of Fc, using the Fc-binding peptides SEQ ID NOs: 1 to 5, which have affinity for the FcRn domain of the antibody.

[0258] The reaction between a linker-Fc-binding peptide conjugate and an antibody may be a nucleophilic substitution reaction. In this case, the nucleophilic substitution reaction may occur when a nucleophilic atom present in the antibody attacks a positively charged or partially positively charged atom of the linker-Fc-binding peptide conjugate. In this case, the nucleophilic atom may be the nitrogen atom of lysine. In addition, the positively charged or partially positively charged atom may be the electrophilic carbon of the carbonyl group contained in the linker structure.

[0259] In the example, an antibody containing the first click chemistry functional group can be represented by formula 5.

[0260] [ka]

[0261] In Equation 5, Ab is an antibody. In certain embodiments, Ab may be trastuzumab.

[0262] R" is one of the click chemistry functional groups, The click chemistry functional group is one or more of the following: alkynes, cycloalkynes, e.g., cyclooctin and cyclononine (e.g., bicyclo[6.1.0]nona-4-in-9-ylmethanol), trans-cyclooctene, nitrone, nitrile oxide, azide, conjugated diene, and dienophile, in which case cycloalkynes, conjugated dienes and dienes are the same as those described above.

[0263] In addition, the nitrogen atom linked to Ab may be contained in lysine 246 or lysine 248 of the antibody's Fc. In certain embodiments, the nitrogen linked to Ab may be contained in lysine 246 of the antibody's Fc. In certain embodiments, the nitrogen atom linked to Ab may be contained in lysine 248 of the antibody's Fc.

[0264] n is an integer between 1 and 4. For example, n could be 2. In this case, R'' can be generated by being linked to either lysine 246 or lysine 248, respectively, in two Fc cells in one antibody. In another example, n could be 4. In this case, R'' can be generated by being linked to lysine 246 and lysine 248, respectively, in two Fc cells in one antibody.

[0265] n is 2, and the nitrogen atom linked to Ab may be contained in lysine 246 of both Fc of the antibody. Alternatively, n is 2, and the nitrogen atom linked to Ab may be contained in lysine 248 of both Fc of the antibody.

[0266] The antibody of formula 5 can be produced by the reaction represented by reaction scheme 3 below.

[0267] [ka]

[0268] In this case, the details of equations 3 and 5 are the same as those already described.

[0269] In this case, if the peptides of SEQ ID NOs: 1 to 5 contain 2,3-diaminopropionic acid, 2,4-diaminobutyric acid, ornithine, or lysine at residue 6, then all corresponding residues have similar structures, differing only in the length of the carbon skeleton containing the free amine group. Therefore, all peptides of SEQ ID NOs: 1 to 5 exhibit the characteristic of binding to the Fc domain of the antibody.

[0270] In another example, an antibody containing the first click chemistry functional group can be represented by formula 6.

[0271] [ka]

[0272] In Equation 6, Ab is an antibody. In certain embodiments, Ab may be trastuzumab.

[0273] R''' is a substituted or non-substituted C 1~20 Alkylene, substituted or unsubstituted C 2~20 Alkenylene, substituted or unsubstituted C 1~10 Alkynylene, substituted or unsubstituted C 1~10 Polymethylene, substituted or unsubstituted C 5~12 Aryl, substituted, or unsubstituted C 5~14 Arylalkylenes, substituted or unsubstituted C 8~16 Arylalkenylenes, substituted or unsubstituted C 3~10 Cycloalkylene, substituted or unsubstituted C 3~10 Heterocycloalkylenes, or substituted or unsubstituted C 5~12A heteroaryl is a heteroalkylene, heterocycloalkylene, or heteroaryl that contains at least one or more N, O, or S. The substituted products are substituted with non-hydrogen substituents, and the non-hydrogen substituents are -Ra, -O-, =O, -ORa, -SRa, -S-, -N(Ra)2, =NRa, -C(Rb)3, -N=C=O, -NCS, -NO, -NO2, =N-OH, =N2, -N3, -NHC(=O)Ra, -C(=O)Ra, -C(=O)NRaRa-S(=O)2O-, -S(=O)2OH, -S(=O)2Ra, -OS(=O)2ORa, -S(=O)2NRa, -S(=O One or more selected from the group consisting of )Ra, -C(=O)Ra, alkylene-C(=O)Ra, -C(=S)Ra, -C(=O)ORa, alkylene-C(=O)ORa, -C(=O)O-, alkylene-C(=O)O-, -C(=S)ORa, -C(=O)SRa, -C(=S)SRa, -C(=O)NRaRa, alkylene-C(=O)NRaRa, -C(=S)NRaRa, -C(-NRa)NRaRa, and Rb.

[0274] In addition, the nitrogen atom linked to Ab may be contained in lysine 246 or lysine 248 of the antibody's Fc. In certain embodiments, the nitrogen linked to Ab may be contained in lysine 246 of the antibody's Fc. In certain embodiments, the nitrogen atom linked to Ab may be contained in lysine 248 of the antibody's Fc.

[0275] Furthermore, in this case, the Fc-binding peptide may contain a first click chemistry functional group. The click chemistry functional group is one or more of the following: alkynes, cycloalkynes, e.g., cyclooctin and cyclononine (e.g., bicyclo[6.1.0]nona-4-in-9-ylmethanol), trans-cyclooctene, nitrone, nitrile oxide, azide, conjugated diene, and dienophile, in this case, cycloalkynes, conjugated dienes, and dienes are the same as those described above. In addition, the Fc-binding peptide may contain a first click chemistry functional group at an amino acid residue adjacent to the N-terminus of its sequence. Furthermore, the Fc-binding peptide may contain a first click chemistry functional group at the N-terminus of its sequence. Alternatively, the Fc-binding peptide may contain a first click chemistry functional group at an amino acid residue adjacent to the C-terminus of its sequence. Furthermore, the Fc-binding peptide may contain a first click chemistry functional group at the C-terminus of its sequence.

[0276] Fp is an Fc-binding peptide. Furthermore, Fp may be one peptide selected from formula 13 or 14. Details of formulas 13 and 14 are the same as already described.

[0277] Fp may be linked via Y3 of amino acid residue 6. In this case, amino acid residue 6 may be lysine, ornithine, 2,3-diaminopropionic acid, or 2,4-diaminobutyric acid.

[0278] n is an integer between 1 and 4, inclusive. For example, n could be 2. In this case, Fp can generate this structure by being linked to lysine 246 or lysine 248, which are present in two Fc molecules in one antibody, respectively. In another example, n could be 4. In this case, R'' can generate this structure by being linked to lysine 246 and lysine 248, which are present in two Fc molecules in one antibody, respectively.

[0279] n is 2, and the nitrogen atom linked to Ab may be contained in lysine 246 of both Fc of the antibody. In another example, n is 2, and the nitrogen atom linked to Ab may be contained in lysine 248 of both Fc of the antibody.

[0280] The antibody of formula 6 can be produced by the reaction represented by the following reaction scheme 4.

[0281] [ka]

[0282] In this case, the details of equations 4 and 6 are the same as those already described.

[0283] In this case, if the peptides of SEQ ID NOs: 1 to 5 contain 2,3-diaminopropionic acid, 2,4-diaminobutyric acid, ornithine, or lysine at residue 6, then all corresponding residues have similar structures, differing only in the length of the carbon skeleton including the free amine group. Therefore, all peptides of SEQ ID NOs: 1 to 5 exhibit the characteristic of binding to the Fc domain of the antibody.

[0284] A method for preparing an antibody-payload conjugate may include the step of (3) preparing the antibody-payload conjugate by reacting an antibody containing a first click chemistry functional group with a payload containing a second click chemistry functional group complementary to the first click chemistry functional group.

[0285] The payload may include an active moiety. The active moiety may be one or more selected from nucleic acids, peptides, and compounds. The active moiety may be any one selected from the group consisting of drug molecules, imaging moieties, optical agents, vitamins, and toxins. In certain embodiments, the active moiety may be a drug molecule. The drug molecule may be a prodrug, a precursor, or a drug. The drug molecule may be an anticancer drug, an anti-inflammatory agent, another anti-disease agent, and an antimicrobial agent (antibacterial agent, antifungal agent, antiviral agent). In certain embodiments, the drug molecule may be meltansine (DM1). In certain embodiments, the payload may include two or more drug molecules. In certain embodiments, the active moiety may be an imaging moiety. The imaging moiety may be a contrast agent, a radioisotope, or a fluorescent material. In certain embodiments, the active moiety may be, but is not limited to, an optical agent, a vitamin, or a toxin.

[0286] In addition, the payload includes a second click chemistry functional group. The click chemistry functional group is one or more of the following: alkynes, cycloalkynes, e.g., cyclooctin and cyclononine (e.g., bicyclo[6.1.0]nona-4-in-9-ylmethanol), trans-cyclooctene, nitrone, nitrile oxide, azide, conjugated diene, and dienophile, in which case cycloalkynes, conjugated dienes and dienes are the same as those described above.

[0287] In the example, the antibody-payload conjugate can be represented by Equation 7.

[0288] [ka]

[0289] In Equation 7, Ab is an antibody. In certain embodiments, Ab may be trastuzumab.

[0290] The nitrogen atom linked to Ab may be contained in lysine 246 or lysine 248 of the antibody's Fc. In certain embodiments, the nitrogen linked to Ab may be contained in lysine 246 of the antibody's Fc. In certain embodiments, the nitrogen atom linked to Ab may be contained in lysine 248 of the antibody's Fc.

[0291] B may be any one of the structures formed by the click chemistry of a first click chemistry functional group and a second click chemistry functional group. For example, B may be any one of the structures that can be produced by two reactions selected from alkynes, cycloalkynes, e.g., cyclooctin and cyclononine (e.g., bicyclo[6.1.0]nona-4-in-9-ylmethanol), trans-cyclooctene, nitrones, nitrile oxides, azides, conjugated dienes, and dienophiles. In certain embodiments, B is

[0292] [ka]

[0293] This is possible. In the formula, A1 may be linked to the antibody, and A2 may be linked to Am. Alternatively, A1 may be linked to Am, and A2 may be linked to the antibody.

[0294] Am is an active site or a structure containing it. In one embodiment, Am may contain two or more active sites. The details of the active sites are the same as those described in the payload description.

[0295] n is an integer between 1 and 4. For example, n could be 2. In this case, the payload can generate this structure by being linked to lysine 246 or lysine 248 contained in two Fc cells in one antibody, respectively. In another example, n could be 4. In this case, R" can generate this structure by being linked to lysine 246 and lysine 248 contained in two Fc cells in one antibody, respectively.

[0296] n is 2, and the nitrogen atom linked to Ab may be contained in lysine 246 of both Fc of the antibody. In another embodiment, n is 2, and the nitrogen atom linked to Ab may be contained in lysine 248 of both Fc of the antibody.

[0297] The compound represented by Equation 7 can be produced, for example, by reaction scheme 5.

[0298] [ka]

[0299] In this case, R'' is the first click chemistry functional group, and is the same as the one described above when formula 5 is described above.

[0300] Furthermore, R'''' is a second click chemistry functional group, and if a payload is specified, it is the same as the one specified.

[0301] In the example, the antibody-payload conjugate can be represented by Equation 8.

[0302] [ka]

[0303] In Equation 8, Ab is an antibody. In certain embodiments, Ab may be trastuzumab.

[0304] The nitrogen atom linked to Ab may be contained in lysine 246 or lysine 248 of the antibody. In certain embodiments, the nitrogen atom linked to Ab may be contained in lysine 246 of the antibody. In certain embodiments, the nitrogen atom linked to Ab may be contained in lysine 248 of the antibody.

[0305] B may be any one of the structures formed by the click chemistry of a first click chemistry functional group and a second click chemistry functional group. For example, B may be any one of the structures that can be produced by two reactive moieties selected from alkynes, cycloalkynes, e.g., cyclooctin and cyclononine (e.g., bicyclo[6.1.0]nona-4-in-9-ylmethanol), trans-cyclooctene, nitrones, nitrile oxides, azides, conjugated dienes, and dienophiles. In certain embodiments, B is

[0306] [ka]

[0307] This is possible. In the formula, A1 may be linked to the antibody, and A2 may be linked to Am. Alternatively, A1 may be linked to Am, and A2 may be linked to the antibody.

[0308] Fp is an Fc-binding peptide. Furthermore, Fp may be one peptide selected from formula 13 or 14. Details of formulas 13 and 14 are the same as already described.

[0309] Fp may be linked via Y4 of amino acid residue 6. In this case, amino acid residue 6 may be lysine, ornithine, 2,3-diaminopropionic acid, or 2,4-diaminobutyric acid.

[0310] Am is an active site or a structure containing it. In certain embodiments, Am may contain two or more active sites. The details of the active sites are the same as those described in the payload description.

[0311] B may be bound to an amino acid residue adjacent to the N-terminus of the Fc-binding peptide sequence. In another embodiment, B may be bound to the N-terminus of the Fc-binding peptide. In another embodiment, B may be bound to an amino acid residue adjacent to the C-terminus of the Fc-binding peptide sequence. In another embodiment, B may be bound to the C-terminus of the Fc-binding peptide.

[0312] n is an integer between 1 and 4. For example, n could be 2. In this case, the payload can generate this structure by being linked to lysine 246 or lysine 248 contained in two Fc cells in one antibody, respectively. In another example, n could be 4. In this case, the payload can generate this structure by being linked to lysine 246 and lysine 248 contained in two Fc cells in one antibody, respectively.

[0313] n is 2, and the nitrogen atom linked to Ab may be contained in lysine 246 of both Fc of the antibody. Alternatively, n is 2, and the nitrogen atom linked to Ab may be contained in lysine 248 of both Fc of the antibody.

[0314] The compound represented by Equation 8 can be produced, for example, by reaction scheme 6.

[0315] [ka]

[0316] In this case, R'''''' is the first click chemistry functional group, which is the same as the one described above when formula 6 is described above. The first click chemistry functional group may be involved in an amino acid residue in the process of synthesizing the Fc-binding peptide. Any one residue can be used as the amino acid residue in which the first click chemistry functional group is involved, but preferably the amino acid residue is not residue 6. This is because residue 6 is a residue designed for substitution reactions with the linker. Through the process of artificial synthesis of the peptide, the Fc-binding peptide may contain the first click chemistry functional group at an amino acid residue adjacent to the N-terminus of its sequence. In certain embodiments, the Fc-binding peptide may contain the first click chemistry functional group at the N-terminus of its sequence. In certain embodiments, the Fc-binding peptide may contain the first click chemistry functional group at an amino acid residue adjacent to the C-terminus of its sequence. In certain embodiments, the Fc-binding peptide may contain the first click chemistry functional group at the C-terminus of its sequence.

[0317] Furthermore, R'''' is a second click chemistry functional group, and if a payload is specified, it is the same as the one specified.

[0318] This application can provide a linker that assists transport and binding reactions so that a payload can be linked to a desired target molecule.

[0319] The linker or linker portion can be directly linked to a target molecule. The linker portion directly linked to the target molecule may be linked to a payload.

[0320] The linker portion may be part of a linker residue or part of a functional group.

[0321] For example, the linker portion may be part of a residue containing R'' or R''.

[0322] For example, the linker portion may be part of the linker residues that exclude R''.

[0323] Target molecules to which linker portions are linked can be linked to a payload by a click reaction, i.e., a click chemistry reaction between functional groups, but are not limited to these reactions.

[0324] In an illustrative embodiment, if the linker has the structure of formula 1, The linker portion attached to the target molecule may contain click chemistry functional groups.

[0325] In this case, the linker portion attached to the target molecule can participate in the click reaction along with the payload.

[0326] These linkers cannot affect the biological activity of the target molecule. In other words, linkers have little to no effect on the half-life, excretion, or blood stability of the target molecule.

[0327] The half-life of a target molecule can be determined by its FcRn binding affinity. For example, a target molecule can be recycled in vivo by binding to FcRn, and its half-life can be increased in vivo.

[0328] The excretion of a target molecule can be determined by the extent to which the target molecule is excreted by the kidney as a result of the aggregation of the protein itself.

[0329] For example, linkers can increase the half-life of drugs in the blood by linking proteins and compounds with short intracellular half-lives. For instance, the drug may be linked to a target molecule with a long intracellular half-life.

[0330] In another illustrative embodiment, if the linker has the structure of formula 2, The linker portion attached to the target molecule cannot contain click chemistry functional groups.

[0331] The linker portion linked to the target molecule may be linked to a peptide, polypeptide, protein, and / or compound containing a click chemistry functional group.

[0332] The peptide, polypeptide, protein, and / or compound can be linked to the payload by a click reaction.

[0333] Such linkers can affect the biological activity of the target molecule. When a linker affects the biological activity of the target molecule, it can affect the half-life, excretion, or blood stability of the target molecule. For example, if a linker is indirectly linked to the target molecule, the linker can rapidly release the payload in vivo by reducing the half-life of the target molecule.

[0334] The linker can be linked to a peptide or polypeptide having a binding affinity to a specific target molecule. Such a linker can site-specifically link the payload to the target molecule.

[0335] For example, the target molecule could be an antibody.

[0336] For example, the antibody may be an IgG antibody or a partial fragment of an IgG antibody. The IgG antibody may be human IgG (IgG1, IgG2, IgG3, or IgG4) and / or rabbit IgG. The IgG antibody may be a human-derived CH2-CH3 domain. The target molecule may be an antibody or a partial fragment of an antibody, but is not limited to these.

[0337] For example, Fc-binding peptides can have binding affinity to antibodies.

[0338] For example, an Fc-binding peptide may have binding affinity to an IgG antibody. An Fc-binding peptide may also have specific binding affinity to a particular domain of an IgG antibody.

[0339] For example, an Fc-binding peptide can have specificity for the heavy chain or light chain variable region of an IgG antibody.

[0340] For example, an Fc-binding peptide can have specificity with the constant domain of the heavy chain of an IgG antibody. In this case, the heavy chain constant domain may be the CH2 domain and / or the CH3 domain.

[0341] The Fc-binding peptide may be a peptide or polypeptide to which a compound is linked.

[0342] For example, Fc-binding peptides can contain click chemistry functional groups.

[0343] The present invention provides a method for treating cancer, comprising administering an antibody-payload conjugate. In this case, the cancer can be selected from bladder cancer, bone cancer, brain cancer, breast cancer, heart cancer, cervical cancer, colorectal cancer, rectal cancer, esophageal cancer, fibrosarcoma, stomach cancer, gastrointestinal cancer, head and neck cancer, Kaposi's sarcoma, kidney cancer, leukemia, liver cancer, lung cancer, lymphoma, melanoma, myeloma, ovarian cancer, pancreatic cancer, penile cancer, prostate cancer, testicular germ cell carcinoma, thymoma, and thymic carcinoma. Furthermore, the cancer may be breast cancer.

[0344] The present invention provides a pharmaceutical composition for treating cancer, comprising an antibody-payload conjugate. In this case, the cancer can be selected from bladder cancer, bone cancer, brain cancer, breast cancer, heart cancer, cervical cancer, colorectal cancer, rectal cancer, esophageal cancer, fibrosarcoma, stomach cancer, gastrointestinal cancer, head and neck cancer, Kaposi's sarcoma, kidney cancer, leukemia, liver cancer, lung cancer, lymphoma, melanoma, myeloma, ovarian cancer, pancreatic cancer, penile cancer, prostate cancer, testicular germ cell carcinoma, thymoma, and thymic carcinoma. Furthermore, the cancer may be breast cancer.

[0345] The present invention will be described in more detail later in this document through examples.

[0346] These examples are provided solely to illustrate the present invention in more detail, and it will be apparent to those skilled in the art that the scope of the invention is not limited by these examples. The numbers indicated in the names of the compounds in the following examples are written with reference to the drawings. [Examples]

[0347] Synthesis method and structural confirmation of compound I (Trans linker: NHS & norbornene, formula 1-1-2) Example 1-1. Synthesis and structural confirmation of compound 1 10 g (68.4 mmol, 1.0 equivalent) of monomethyl glutarate was dissolved in 250 mL of dichloromethane (DCM), and the resulting solution was stirred. 17.7 g (92.3 mmol, 1.34 equivalents) of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCi) and 24 mL (138 mmol, 2.0 equivalents) of N,N-diisopropylethylamine (DIPEA) were slowly added dropwise, and the resulting solution was stirred for 20 minutes. Then, 8 mL (68.7 mmol, 1.0 equivalent) of O-benzylhydroxyamine was slowly added dropwise. After 18 hours, the reaction solution was removed by concentration under reduced pressure, and the residue was redissolved in ethyl acetate (EA). The organic layer was washed three times with 10% citric acid solution and dried using saturated salt solution and sodium sulfate. The target compound was used in the following reaction without purification (crude yield: 12.9 g, 88%). TLC (EA:Hex = 2:1); R f = 0.5 1H NMR (300 MHz, DMSO) δ 7.35 (d, J = 3.5 Hz, 5H), 4.75 (d, J = 3.5 Hz, 2H), 3.56 (d, J = 4.0 Hz, 3H), 2.26 (td, J = 7.3, 3.8 Hz, 2H), 1.96 (t, J = 5.6 Hz, 2H), 1.78 - 1.61 (m, 2H).

[0348] Examples 1-2 Synthesis and structural confirmation of compound 2 12.9 g of compound 1 was dissolved in 200 mL of N,N-dimethylformamide (DMF), and the resulting solution was stirred at 0°C. 24 mL (160 mmol, 3.08 equivalents) of 1,8-diazabicyclo(5.4.0)undec-7-ene (DBU) and 10 mL (160 mmol, 3.08 equivalents) of methyl iodide (MeI) were slowly added dropwise. 0.2 mL of diethylamine was slowly added dropwise. After stirring the resulting solution for 20 hours, the reaction solvent was mixed with Celite and removed by concentration under reduced pressure. The target compound was purified by column chromatography (EA:Hex = 1:1) to obtain 7.6 g (yield: 56%). TLC (EA:Hex = 1:1); R f = 0.5 1H NMR (300 MHz, cdcl3) δ 7.44 - 7.33 (m, 5H), 4.82 (s, 2H), 3.65 (s, 3H), 3.20 (s, 3H), 2.43 (d, J = 7.2 Hz, 2H), 2.34 (t, J = 7.3 Hz, 2H), 1.98 - 1.85 (m, 2H).

[0349] Examples 1-3 Synthesis and structural confirmation of compound 3 4.18 g (15.8 mmol, 1.0 equivalent) of compound 2 was dissolved in 100 mL of tetrahydrofuran (THF), and the resulting solution was stirred. 5 mL of 2N lithium hydroxide (LiOH) was slowly added dropwise at 0°C, and the resulting solution was stirred for 3 hours. The reaction was stopped by adding 60 mL of H2O, and the aqueous layer was washed twice with 150 mL of EA. The aqueous layer was then titrated to pH 3.0 with 2N HCl solution, and the target compound was extracted three times with 50 mL of EA. The organic layer was dried using saturated salt solution and sodium sulfate to obtain 2.46 g (yield: 62%). TLC (EA:Hex = 1:1); R f= 0.1 1H NMR (300 MHz, DMSO) δ12.06 (s, 1H), 7.51 - 7.29 (m, 5H), 4.86 (s, 2H), 3.13 (s, 3H), 2.38 (t, J = 7.3 Hz, 2H), 2.22 (dt, J = 11.7, 7.4Hz, 4H).

[0350] Examples 1-4 Synthesis and structural confirmation of compound 4 2.46 g of compound 3 was dissolved in 40 mL of methanol (MeOH), and the hydrogenation reaction was carried out in the presence of palladium on carbon (Pd / C) for 18 hours. After stopping the reaction, Pd / C was removed via a short-path column, and then the target compound was purified and concentrated to obtain 1.2 g (yield: 76%). 1H NMR (300 MHz, DMSO) δ 12.06 (s, 1H), 7.51 - 7.29 (m, 5H), 4.86 (s, 2H), 3.13 (s, 3H), 2.38 (t, J = 7.3 Hz, 2H), 2.22 (dt, J = 11.7, 7.4Hz, 4H).

[0351] Examples 1-5 Synthesis and structural confirmation of compound 5 0.17 g (1.06 mmol, 1.0 equivalent) of compound 4 was dissolved in 10 mL of DCM, and the resulting solution was stirred. 0.22 g (1.73 mmol, 1.63 equivalent) of exo-5-norbornenate chloride and 0.2 mL (1.1 mmol, 1.0 equivalent) of DIPEA were slowly added dropwise at 0°C. The resulting solution was stirred for 2 hours, after which the organic layer was washed three times with 10% citrate solution and dried using saturated salt solution and sodium sulfate. The target compound was used in the next reaction without purification (crude yield: 0.24 g, 68%). TLC (DCM:MeOH = 10:1); R f = 0.3 Predicted MW (M+H)+: 282.13 g / mol Measured MW (M+H)+: 282.1g / mol

[0352] Examples 1-6 Synthesis and structural confirmation of compound I 0.24 g (0.72 mmol, 1.0 equivalent) of compound 5 was dissolved in 3 mL of DCM, and the resulting solution was stirred. 0.26 g (0.87 mmol, 1.2 equivalents) of N,N,N',N'-tetramethyl-O-(N-succinimidyl)uronium tetrafluororate (TSTU) and 0.15 mL (0.87 mmol, 1.2 equivalents) of DIPEA were slowly added dropwise. The resulting solution was stirred for 1 hour, after which the organic layer was washed three times with 10% citrate solution and dried using saturated salt solution and sodium sulfate. The target compound was purified by column chromatography (EA:Hex = 1:1) to obtain 0.04 g (yield: 15%). TLC (EA:Hex = 1:1); R f = 0.3 (See Figure 1) 1H NMR (300 MHz, DMSO) δ6.18 (ddd, J = 16.9, 5.5, 3.0 Hz, 2H), 2.94 (s, 1H), 2.80 (s, 4H), 2.71 (s, 1H), 2.67 (q, J = 1.0 Hz, 3H), 2.25 (dd, J = 25.9, 18.6 Hz, 4H), 1.86 (ddd, J = 18.9, 11.5, 5.8 Hz, 4H), 1.45 - 1.26 (m, 4H). [Examples]

[0353] Synthetic method and structural confirmation of compound II (Cis linker: NHS & norbornene, formula 2-1-2) Example 2-1 Synthesis and structural confirmation of compound 6 0.1 g (0.88 mmol, 1.0 equivalent) of N-methylhydroxyamine hydrochloride was dissolved in 2 mL of tetrahydrofuran (THF) at -25°C, and the resulting solution was stirred. 0.07 g (0.88 mmol, 1.0 equivalent) of glutaric anhydride was added thereto, and the resulting solution was stirred for 10 minutes. Then, 0.25 mL (1.76 mmol, 2.0 equivalent) of triethylamine was slowly added dropwise. After 1 hour, 0.14 g (0.88 mmol, 1.0 equivalent) of (1S,2R,4S)-bicyclo[2.2.1]hepta-5-ene-2-carbonyl chloride and 0.12 mL (0.88 mmol, 1.0 equivalent) of triethylamine were slowly added dropwise, and the reaction was carried out at room temperature for 1 hour. After removing the reaction solution by concentration under reduced pressure, the residue was redissolved in ethyl acetate (EA), and the resulting solution was washed three times with a 10% citric acid solution and dried using a saturated salt solution and sodium sulfate. The target compound was used in the next reaction without purification. TLC (DCM:MeOH = 3:1, 1 drop of acetic acid); R f = 0.4 Calculated mass (M+H)+: 282.13g / mol Measured mass (M+H)+: 282.1g / mol

[0354] Example 2-2 Synthesis of Compound II Compound 6 was dissolved in 2 mL of dichloromethane (DCM), and 0.22 g (0.73 mmol, 0.8 equivalents) of TSTU and 0.15 mL (0.88 mmol, 1.0 equivalent) of N,N-diisopropylethylamine (DIPEA) were slowly added dropwise. After a reaction of 1 hour, the reaction solution was washed three times with 10% citric acid solution and dried using saturated sodium bicarbonate solution, saturated salt solution, and sodium sulfate. The target compound was purified by column chromatography (EA:Hex = 1:1). TLC (EA:Hex = 2:1); R f = 0.4 (See Figure 3)

[0355] Examples 2-3 Confirmation of the structure of compound II The final compound II was synthesized by the methods specified in Examples 2-1 and 2-2, and the presence of isomers was confirmed by HPLC analysis as illustrated in Figure 6. Based on the analytical conditions illustrated in Figure 6, HPLC purification was performed to obtain compound II (cis) with the purity shown in Figure 7 via HPLC analysis. The analysis of the final compound II was completed by confirming the structure of the purified compound II by mass spectrometry. Calculated mass (M+H)+: 379.14g / mol Measured mass (M+H)+: 379.0g / mol [Examples]

[0356] Confirmation of the structure of isomer compound 6 (Cis linker or Trans linker: NHS & norbornene) The cis and trans structures of compound 6, an isomer, were confirmed using a high-performance liquid chromatography (HPLC) apparatus. For HPLC analysis, a Waters 2695 HPLC model from Waters was used, with an Xbridge C18 (4.6 × 250 mm, 5 μm; Waters) column. For the mobile phase solvent, water containing 0.1% trifluoroacetic acid was used as solvent A, and acetonitrile containing 0.075% trifluoroacetic acid was used as solvent B. As described above, the characteristics of each structure were analyzed by absorbance at a wavelength of 220 nm.

[0357] As illustrated in Figure 3, peaks were observed at 14.89 mins for the cis-form structure of compound 6 and at 15.32 mins for the trans-form structure of compound 6. Analysis of each structure was confirmed by mass spectrometry (Shimadzu, LCMS-8050), and Figures 4 and 5 illustrate the results of mass spectrometry for the cis and trans structures of compound 6. As a result of confirming the molecular weight, it was confirmed that the material corresponding to each peak had a molecular weight of 282, to which one hydrogen molecule was bonded. Calculated mass (M+H)+ : 282.13 g / mol Measured mass (M+H) + : 282.1 g / mol [Examples]

[0358] Synthesis and structural confirmation of Fc-binding peptides Example 4-1. Synthesis and structural confirmation of FcBP(6Lys)-norbornene.

[0359] [ka]

[0360] Example 4-1-1: FcBP(6Lys) - Synthesis of norbornene List of Fmoc amino acids used, and the order in which the Fmoc amino acids were introduced. Fmoc-L-Thr(tBu)-OH, Fmoc-Cys(Trt)-OH, Fmoc-L-Trp(Boc)-OH, Fmoc-L-Val-OH, Fmoc-L-Leu-OH, Fmoc-L-Glu(OtBu)-OH, Fmoc- Gly-OH, Fmoc-Lys(Boc)-OH, Fmoc-L-His(Trt)-OH, Fmoc-L-Trp(Boc)-OH, Fmoc-Ala-OH, Fmoc-Cys(Trt)-OH, Fmoc-Asp(tBu)-OH.

[0361] Generation method (a) Introduction of amino acids The amounts of reagents used in the following process were based on 0.25 mM. 0.5 g of clear amide resin (0.48 mM / g, Peptides International, USA) was placed in a synthesis reactor, and 1 mM of each Fmoc-amino acid block was weighed and prepared in the order of the peptide amino acid sequence from C-terminus to N-terminus.

[0362] The reaction to attach the activated residue to a transparent amide resin by activating Fmoc-amino acids was carried out sequentially, starting from the C-terminal amino acid.

[0363] Fmoc was removed in 20% piperidine-containing DMF. To activate and introduce the residues, amino acids prepared according to the sequence were mixed with 2 mL of 0.5 M HOBt-containing DMF solution, 2 mL of 0.5 M HBTU-containing DMF solution, and 174 μL of DIPEA for 5 minutes. The resulting mixture was then poured into a reactor containing resin and mixed for 2 hours.

[0364] The introduction reaction was confirmed using the Kaiser test method. If no reaction was observed, the introduction reaction was repeated, or capping was performed with a 20% Ac2O-containing DMF solution. After thoroughly washing the resin with DMF and DCM, the next step in each introduction reaction and Fmoc removal process was carried out. This process was repeated until the target peptide sequence was complete.

[0365] (b) Introduction of H-PEG8-OH After all amino acid introduction was complete, in order to introduce H-PEG8-OH to the N-terminus, 1 mL of 0.5 M Fmoc-N-amide-dPEG8-acid, 1 mL of 0.5 M HBTU-containing DMF solution, 1 mL of 0.5 M HOBt-containing DMF solution, and 87 μL of DIPEA were mixed for 5 minutes, and then the resulting mixture was poured into a reactor containing resin and mixed for 2 hours.

[0366] The progress of the reaction was confirmed by the Kaiser test method, and if it was determined that unreacted amine remained, the reaction time was extended for a further 1 to 3 hours, or the reaction solution was emptied and the reaction process described above was repeated. The N-terminal Fmoc protecting group was removed using 20% ​​piperidine-containing DMF, and then the resin to which the peptide was attached was dried and weighed.

[0367] (c) Introduction of Norbornene To remove the N-terminal Fmoc protecting group, 4 equivalents of norbornene carboxylic acid, 2 mL of 0.5 M HOBt-containing DMF solution, 2 mL of 0.5 M HBTU-containing DMF solution, and 174 μL of DIPEA were mixed with the resin for 5 minutes. The resulting mixture was then poured into a reactor containing the resin and mixed for 2 hours. The introduction reaction was confirmed by the Kaiser test, and if no reaction was confirmed, the introduction reaction was repeated.

[0368] (d) The peptide was cleaved from the 250 mg peptide-attached resin prepared in step (c) by stirring with 2 ml of a mixture of TFA, TIS, water, and EDT (94:1.0:2.5:2.5) at room temperature for 120 minutes. The cleaved mixture was filtered, the filtrate was concentrated by about half with nitrogen gas, and then the peptide was precipitated by pouring in ether. The precipitated peptide was further washed three times with ether and dried with nitrogen gas. The dried precipitate was dissolved in water containing 0.1% TFA and 30% ACN, the resulting solution was stirred for 6 hours, and then concentrated.

[0369] The concentrate was dissolved in a 0.01 M ammonium acetate buffer (pH 6.5) solution containing 0.1 mg / mL of 5% DMSO-20% ACN, and the resulting solution was stirred for 3 days in an air-exposed state. The progress of the disulfide bond formation reaction was observed by HPLC, and when it was determined that the reaction would not proceed any further, the reaction solution was freeze-dried to obtain a peptide precipitate.

[0370] (e) Purification The peptide precipitate obtained by freeze-drying in step (d) was purified under the prep-LC conditions shown in Table 5 and then freeze-dried. Each of the obtained peptides was confirmed to have a purity of 90% or higher by analytical HPLC, and the results are illustrated in Figure 8. Norbornene-PEG8-Asp-Cys * -Ala-Trp-His-Lys-Gly-Glu-Leu-Val-Trp-Cys *-Thr-NH2(Cys * (Disulfide bond site)

[0371] [Table 5]

[0372] Example 4-1-2: Confirmation of the structure of FcBP(6Lys)-norbornene (oxidized form) The synthesis of FcBP (6Lys) was confirmed by LC mass-based molecular weight measurement. Measuring instrument: Waters Quattro Premier XE Calculated molecular weight: 2088.40g / mol Measured molecular weight (M / 2+H) 2+ : 1044.84 g / mol

[0373] The results are illustrated in Figure 9.

[0374] Example 4-2. Synthesis and structural confirmation of compound I-FcBP (6Lys)-norbornene

[0375] [ka]

[0376] Example 4-2-1: Synthesis of compound I-FcBP (6Lys)-norbornene Compound I (trans-norbornene Weinreb amide)-FcBP was synthesized in DMF. To introduce compound I into FcBP (6Lys)-norbornene, 3 equivalents of DIPEA and 3 μmol of compound I were dissolved in 2.5 μmol of FcBP (6Lys)-norbornene dissolved in DMF, and the resulting solution was stirred.

[0377] To confirm the introduction reaction, analysis was performed by HPLC. If the reaction did not stop, DIPEA was added one equivalent at a time, and the cessation of the reaction was observed.

[0378] After confirming that the reaction had stopped, the reaction solution was concentrated, and I-FcBP (6Lys)-norbornene was purified by preparative HPLC. 2.07 μmol was obtained by lyophilization after purification, and its purity was also confirmed using HPLC (purity: >95% (HPLC), yield: 83%).

[0379] The results are illustrated in Figure 10.

[0380] Example 4-2-2: Confirmation of the structure of compound I-FcBP(6Lys)-norbornene The synthesis of the compound I-FcBP(6Lys)-norbornene was confirmed by LC mass-based molecular weight measurement. Measuring instrument: Waters Quattro Premier XE Calculated molecular weight: 2351.69g / mol Measured molecular weight (M / 2+H) 2+ : 1176.42 g / mol

[0381] The results are illustrated in Figure 11.

[0382] Example 4-3. Synthesis and structural confirmation of compound II-FcBP(6Lys)-norbornene

[0383] [ka]

[0384] Example 4-3-1: Synthesis of compound II-FcBP (6Lys)-norbornene Compound II (cis-norborneneWeinrebamide)-FcBP was synthesized in DMF. To introduce compound II into FcBP (6Lys)-norbornene, 3 equivalents of DIPEA and 3 μmol of compound II were dissolved in 2.5 μmol of FcBP (6Lys)-norbornene dissolved in DMF, and the resulting solution was stirred.

[0385] To confirm the introduction reaction, analysis was performed by HPLC. If the reaction did not stop, DIPEA was added one equivalent at a time, and the cessation of the reaction was observed.

[0386] After confirming that the reaction had stopped, the reaction solution was concentrated, and II-FcBP (6Lys)-norbornene was purified by preparative HPLC. 2.02 μmol was obtained by lyophilization after purification, and its purity was also confirmed using HPLC (purity: >95% (HPLC), yield: 81%).

[0387] The results are illustrated in Figure 12.

[0388] Example 4-3-2: Confirmation of compound II-FcBP (6Lys)-norbornene Measuring instrument: Waters Quattro Premier XE Calculated molecular weight: 2351.69g / mol Measured molecular weight (M / 2+H) 2+ : 1176.42 g / mol The results are illustrated in Figure 13. [Examples]

[0389] Antibody-click chemistry: Synthesis methods and structural confirmation of chemical substances. Example 5-1: Antibody-norbornene Example 5-1-1: Method for synthesizing trastuzumab-norbornene (1) Introduction reaction using compound I-FcBP (6Lys)-norbornene Ab(Lys246 / 248)-norbornene was synthesized using compound I-FcBP(6Lys)-norbornene in phosphate-buffered saline (PBS) buffer at pH 7.4. To introduce norbornene into two specific sites of the antibody, 6 equivalents of compound I-FcBP(6Lys)-norbornene per antibody were added to the reaction solution, and the reaction was then carried out. The reaction took more than one week at room temperature, and the reaction was monitored and stopped by HIC-HPLC. For purification, the compound was purified by three dialysis cycles (pH 5.5, 20 mM histidine acetate buffer) and size exclusion chromatography (molecular weight cutoff 40 kDa).

[0390] The reaction between the compound I-FcBP(6Lys)-norbornene and the antibody is illustrated in Figure 14, the structure of the final product Ab(Lys246 / 248)-norbornene is illustrated in Figure 15, and reaction monitoring by HIC-HPLC is illustrated in Figure 16.

[0391] Example 5-1-2: Method for synthesizing trastuzumab-norbornene (2) Introduction reaction using compound II-FcBP(6Lys)-norbornene Ab(Lys246 / 248)-norbornene was synthesized using compound II-FcBP (6Lys)-norbornene in phosphate-buffered saline (PBS) buffer at pH 7.4. To introduce norbornene into two specific sites of the antibody, 6 equivalents of compound II-FcBP (6Lys)-norbornene per antibody were added to the reaction solution, and the reaction was then carried out. The reaction was carried out at room temperature for 12 hours, and reaction monitoring and termination were confirmed by HIC-HPLC. Purification was performed via three dialysis cycles (pH 5.5, 20 mM histidine acetate buffer) and size exclusion chromatography (molecular weight cutoff 40 kDa) to obtain 135 mg of trastuzumab from 150 mg of trastuzumab. (Yield = 90%)

[0392] The reaction between compound II-FcBP(6Lys)-norbornene and the antibody is illustrated in Figure 17, and the structure of the product Ab(Lys246 / 248)-norbornene is illustrated in Figure 18. Reaction monitoring by HIC-HPLC is illustrated in Figure 19.

[0393] Example 5-2-1: Confirmation of the Herceptin-norbornene bond The antibody intermediate containing the FcBP norbornene linker (Figure 18) was verified by mass spectrometry. Measuring instrument: Ultraflex III (TOF / TOF) Analysis mode: Linear mode Polarity: Positive Detection: m / z 2,000 to 300,000 Laser repetition rate: 100Hz Number of shots: 1,000 shots Deflection: On, 5,000 Da Voltage: Ion source I 25.00kV, Ion source II 23.00kV, Lens 9.00kV Calculated molecular weight: 152,385g / mol Measured molecular weight: 152,407g / mol (M+Na)

[0394] The analysis results are illustrated in Figure 20. [Examples]

[0395] Synthesis and confirmation of antibody-drug conjugates Example 6-1: Antibody-drug conjugation Example 6-1-1: Method for synthesizing trastuzumab-DM1 Antibody-payload conjugates were synthesized using trastuzumab into which two molecules of norbornene were introduced by compound II-FcBP(6Lys)-norbornene (Figure 18). The reaction was carried out using 25 mL of tetrazine-PEG8-DM1 drug at a concentration of 4.5 mg / mL, and biorthogonal chemistry was attempted for the conjugation of the tetrazine-PEG8-DM1 drug with norbornene conjugated to the antibody. Four equivalents of the drug were used compared to the antibody, and the conjugation reaction was carried out in 20 mM histidine acetate solution at pH 5.5 at room temperature for 24 hours. The conjugation reaction was confirmed by HIC-HPLC, and the formation of the antibody-payload conjugate was observed by observing that a peak in the 9.4 min range, which appears only when the FcBP linker is bound to trastuzumab, changed to the 11.2 min range when the antibody-FcBP linker reacted with the drug.

[0396] The structure of the product antibody-payload conjugate is illustrated in Figure 21, and reaction monitoring by HIC-HPLC is illustrated in Figure 22. In Figure 21, the enlarged structure is the payload structure, while the unenlarged portion is the same as the structure illustrated in Figure 18.

[0397] Example 6-1-2: Trastuzumab-DM1 purification method To obtain high-purity antibody-payload conjugates, dialysis using a 20 mM histidine acetate solution at pH 5.5 and HIC purification using high-performance protein liquid chromatography (FPLC) were performed.

[0398] The HIC purification conditions are as follows: FPLC Model: AKTA Pure Flow rate: 1mL / min Column: HiPrep butyl FF16 / 10 column Elution solvent: (A) 1.5M ammonium sulfate + 50mM phosphate pH 7.0 (B) 50 mM phosphate pH 7.0 Elution conditions 0:00~10:00 A: 40%, B: 60% 10:00~20:00 A: 65%, B: 35% 20:00~102.5:00 A: 75%, B: 25% 102:5~115:00 A:100%, B:0% 115:00~135:00 A: 100%, B: 0%

[0399] The HIC chromatogram of the purified antibody-payload conjugate is illustrated in Figure 19.

[0400] Using the method described above, 67 mg of trastuzumab-DM1 was obtained in which the drug was conjugated at two sites (drug-antibody ratio = 2). (Yield = 60%)

[0401] Example 6-1-3: Confirmation of trastuzumab-DM1 binding The FcBP linker and the antibody-payload conjugate containing the drug were verified using mass spectrometry. Measuring instrument: Ultraflex III (TOF / TOF) Analysis mode: Linear mode Polarity: Positive Detection: m / z 2,000 to 300,000 Laser repetition rate: 100Hz Number of shots: 1,000 shots Deflection: On, 5,000 Da Voltage: Ion source I 25.00kV, Ion source II 23.00kV, Lens 9.00kV Calculated molecular weight: 155,493g / mol Molecular weight measured: 155,523 g / mol (approximately M+Na)

[0402] The analysis results are illustrated in Figure 23. [Examples]

[0403] Evaluation of the pharmacokinetic efficacy of novel antibody-drug conjugates (ADCs, trastuzumab-DM1 conjugates) at the cellular level (in vitro cytotoxicity studies). The efficacy of antibody-payload conjugates (ADCs, trastuzumab-DM1 conjugates) was evaluated based on the target marker levels of cancer cells. Cytotoxicity experiments were performed using NCI-N87 and BT474 cell lines for Her2-positive expression and MDA-MB-468 cell line for Her2-negative expression. In Her2-overexpressing cells NCI-N87 and BT474, observations after treating cells with the generated antibody-drug conjugates at various concentrations revealed excellent anticancer effects with IC50 values ​​of 82.1 ng / mL and 29.6 ng / mL, respectively. Compared to commercially available Herceptin ADCs and Kadcyla, comparable efficacy was observed, confirming the usefulness of this new ADC.

[0404] The results of the experiment are illustrated in Figures 24 to 27. [Examples]

[0405] Evaluation of the pharmacokinetic efficacy of novel antibody-payload conjugates (ADCs, trastuzumab-DM1 conjugates) at the animal level (in vivo cytotoxicity studies). Xenograft models were prepared by subcutaneously transplanting NCI-N87 gastric cancer cell lines that overexpress the target antigen, and anticancer efficacy tests were performed on the administered material by classifying the xenograft models into four groups. The BALB / c nude mice used in this experiment were chosen as a suitable model for anticancer efficacy tests using rodents because they lack T cells and cancer cells are easily transplanted into them.

[0406] (a) Preparation of cell lines RPMI1640 medium (Gibco, 22400-089) containing heat-inactivated 10% fetal bovine serum (FBS, Gibco, 10082-742) was placed in a cell culture flask, and one vial of human tumor cell line (NCI-N87 cell line) was added thereto. The cells were cultured at 37°C in a 5% CO2 incubator. The culture flask was washed with PBS, and the cells were isolated by diluting 2.5% trypsin-EDTA (Gibco, 15090) tenfold and then adding the diluted trypsin-EDTA thereto. The cells were then centrifuged (1,000 rpm, 5 min), the supernatant was discarded, and the cell suspension was obtained in fresh medium. After confirming viability using a microscope, 1.25 × 10⁶ cells were collected. 7 Cell lines were prepared by diluting the cell suspension in a solution containing a 1:1 mixture of culture medium and Matrigel at a concentration of cells / mL.

[0407] (b) Transplantation of cell lines The cell line was prepared according to the method described in "4.3) (4) Preparation of Cell Line". Upon preparation of the cell line, it was resuspended and homogenized, and the prepared cell line was immediately administered to the animals. When transplanting the cell line, the back of the animal was disinfected with 70% alcohol, a space was created between the skin and muscle by pulling the skin on the back of the neck with the thumb and index finger, and then the cell line was transplanted by inserting a needle equipped with a 26 gauge needle from the front of the animal into the subdermal space between the thumb and index finger. 6 The cells were administered subcutaneously at a dose of 0.2 mL per animal. During the acclimatization period, healthy animals were selected and inoculated with the cell line. Therefore, the size of the cell line at the transplantation site was approximately 100 mm. 3 From 150mm 3 If this is reached, the tumors are distributed according to their ranked size so that the tumor sizes in each group are distributed as evenly as possible.

[0408] (c) Determination of test group composition, dosage, and administration method Cell line = NCI-N87 Mouse type = BALB / cNude(CAnN.Cg-Foxn1nu / CrljOri) Number of groups = 5 Method of administration = Intravenous injection (using a 26-gauge needle syringe) Dosage = 5 mg / kg Dosage frequency = 1 dose / 2 days, 3 doses per day Observation period = 5 weeks Group 1: PBS, Group 2: Herceptin (trastuzumab), Group 3: Newly manufactured ADC (Herceptin-DM1 conjugate, 1.5st ADC)

[0409] (d) Observation and examination items General symptoms During the administration and observation period, the type of general symptoms, including death, the date of onset, and the severity of symptoms were observed and recorded once daily for each individual. Individuals experiencing worsening general symptoms were isolated.

[0410] body weight Body weight is measured on the day of group separation or at the start of administration of the test material, and then twice a week thereafter.

[0411] Tumor size measurement Tumor size was measured twice a week for 5 weeks from the start of administration of the test material. The long and short diameters of the tumor were measured using calipers, and the tumor size was calculated using the following formula. Tumor size = ab 2 / 2 (a: major axis length, b: minor axis length)

[0412] (e) Results The group injected with phosphate-buffered saline (PBS) and Herceptin (trastuzumab) did not show a tendency to suppress tumor growth during the 5-week observation period. The antibody-payload conjugate (Herceptin-DM1) generated by this application was confirmed to have a superior ability to suppress tumor growth compared to Herceptin, thus confirming that the antibody-payload conjugate functioned successfully as an ADC.

[0413] The results related to the above are illustrated in Figures 28 and 29. [Examples]

[0414] Synthesis and confirmation of site-specific interactomes according to carbon length. Example 9-1. Synthesis and structural confirmation of compound II-FcBP(L6Dap, L6Dab, L6Orn, L6Lys)-norbornene

[0415] [ka]

[0416] Example 9-1-1: FcBP(L6Dap, L6Dab, L6Orn, L6Lys) - Synthesis of norbornene FcBP(L6Dap): List of Fmoc amino acids used and the order of introduction of the Fmoc amino acids used (n=1) Fmoc-L-Thr(tBu)-OH, Fmoc-Cys(Trt)-OH, Fmoc-L-Trp(Boc)-OH, Fmoc-L-Val-OH, Fmoc-L-Leu-OH, Fmoc-L-Glu(OtBu)-OH, Fmoc- Gly-OH, Fmoc-Dap(Boc)-OH, Fmoc-L-His(Trt)-OH, Fmoc-L-Trp(Boc)-OH, Fmoc-Ala-OH, Fmoc-Cys(Trt)-OH, Fmoc-Asp(tBu)-OH.

[0417] FcBP(L6Dab): List of Fmoc amino acids used and the order of introduction of the Fmoc amino acids used (n=2) Fmoc-L-Thr(tBu)-OH, Fmoc-Cys(Trt)-OH, Fmoc-L-Trp(Boc)-OH, Fmoc-L-Val-OH, Fmoc-L-Leu-OH, Fmoc-L-Glu(OtBu)-OH, Fmoc- Gly-OH, Fmoc-Dab(Boc)-OH, Fmoc-L-His(Trt)-OH, Fmoc-L-Trp(Boc)-OH, Fmoc-Ala-OH, Fmoc-Cys(Trt)-OH, Fmoc-Asp(tBu)-OH.

[0418] FcBP(L6Orn): List of Fmoc amino acids used and the order of introduction of the Fmoc amino acids used (n=3) Fmoc-L-Thr(tBu)-OH, Fmoc-Cys(Trt)-OH, Fmoc-L-Trp(Boc)-OH, Fmoc-L-Val-OH, Fmoc-L-Leu-OH, Fmoc-L-Glu(OtBu)-OH, Fmoc- Gly-OH, Fmoc-Orn(Boc)-OH, Fmoc-L-His(Trt)-OH, Fmoc-L-Trp(Boc)-OH, Fmoc-Ala-OH, Fmoc-Cys(Trt)-OH, Fmoc-Asp(tBu)-OH.

[0419] FcBP(L6Lys): List of Fmoc amino acids used and the order of introduction of the Fmoc amino acids used (n=4) Fmoc-L-Thr(tBu)-OH, Fmoc-Cys(Trt)-OH, Fmoc-L-Trp(Boc)-OH, Fmoc-L-Val-OH, Fmoc-L-Leu-OH, Fmoc-L-Glu(OtBu)-OH, Fmoc- Gly-OH, Fmoc-Lys(Boc)-OH, Fmoc-L-His(Trt)-OH, Fmoc-L-Trp(Boc)-OH, Fmoc-Ala-OH, Fmoc-Cys(Trt)-OH, Fmoc-Asp(tBu)-OH.

[0420] Generation method (a) Introduction of amino acids The amounts of reagents used in the following process were based on 0.25 mM. 0.5 g of clear amide resin (0.48 mM / g, Peptides International, USA) was placed in a synthesis reactor, and 1 mM of each Fmoc-amino acid block was weighed and prepared in the order of the peptide amino acid sequence from C-terminus to N-terminus.

[0421] The reaction involves activating Fmoc amino acids to attach the activated residues to a transparent amide resin, with the reaction proceeding sequentially from the C-terminal amino acid.

[0422] Fmoc was removed in DMF containing 20% ​​piperidine. Then, for residue activation and introduction, amino acids prepared according to the sequence were mixed with 2 mL of 0.5 M HOBt-containing DMF solution, 2 mL of 0.5 M HBTU-containing DMF solution, and 174 μL of DIPEA for 5 minutes. The resulting mixture was then poured into a reactor containing resin and mixed for 2 hours.

[0423] The introduction reaction was confirmed using the Kaiser test method. If no reaction was observed, the introduction reaction was repeated, or capping was performed with a 20% Ac2O-containing DMF solution. After thoroughly washing the resin with DMF and DCM, the next step in each introduction reaction and Fmoc removal process was carried out. This process was repeated until the target peptide sequence was complete.

[0424] (b) Introduction of H-PEG8-OH After all amino acid introduction was complete, in order to introduce H-PEG8-OH to the N-terminus, 1 mL of 0.5 M Fmoc-N-amide-dPEG8-acid, 1 mL of 0.5 M HBTU-containing DMF solution, 1 mL of 0.5 M HOBt-containing DMF solution, and 87 μL of DIPEA were mixed for 5 minutes in DMF solution, and then the resulting mixture was poured into a reactor containing resin and mixed for 2 hours.

[0425] The progress of the reaction was confirmed by the Kaiser test method, and if it was determined that unreacted amine remained, the reaction time was extended for a further 1 to 3 hours, or the reaction solution was emptied and the reaction process described above was repeated. The N-terminal Fmoc protecting group was removed using 20% ​​piperidine-containing DMF, and then the peptide-coated resin was dried and weighed.

[0426] (c) Introduction of Norbornene To remove the N-terminal Fmoc protecting group, 4 equivalents of norbornene carboxylic acid, 2 mL of 0.5 M HOBt-containing DMF solution, 2 mL of 0.5 M HBTU-containing DMF solution, and 174 μL of DIPEA were mixed with the resin for 5 minutes. The resulting mixture was then poured into a reactor containing the resin and mixed for 2 hours. The introduction reaction was confirmed by the Kaiser test, and if no reaction was confirmed, the introduction reaction was repeated.

[0427] (d) The peptide was cleaved from the 250 mg peptide-attached resin prepared in step (c) by stirring with 2 mL of a mixed solution of TFA, TIS, water, and EDT (94:1.0:2.5:2.5) at room temperature for 120 minutes. The cleaved mixture was filtered, the filtrate was concentrated by about half with nitrogen gas, and then the peptide was precipitated by pouring ether. The precipitated peptide was further washed three times with ether and dried with nitrogen gas. The dried precipitate was dissolved in water containing 0.1% TFA-30% ACN, and the resulting solution was stirred for 6 hours and then concentrated.

[0428] The concentrate was dissolved in a 0.01 M ammonium acetate buffer (pH 6.5) solution containing 5%-DMSO-20%-ACN at a concentration of 0.1 mg / mL. The resulting solution was then stirred in an air-exposed environment for 3 days. The progress of the disulfide bond formation reaction was observed by HPLC, and when it was determined that the reaction would not proceed any further, the reaction solution was freeze-dried to obtain a peptide precipitate.

[0429] (e) Purification The peptide precipitate obtained by freeze-drying in step (d) was purified under the prep-LC conditions shown in Table 6 below, and then freeze-dried. Each of the resulting peptides was confirmed to have a purity of 90% or higher.

[0430] (f) array FcBP(L6Dap)-norbornene:norbornene-PEG8-Asp-Cys * -Ala-Trp-His-Dap-Gly-Glu-Leu-Val-Trp-Cys * -Thr-NH2(Cys * (Disulfide bond site) FcBP(L6Dab)-norbornene:norbornene-PEG8-Asp-Cys * -Ala-Trp-His-Dab-Gly-Glu-Leu-Val-Trp-Cys * -Thr-NH2(Cys * (Disulfide bond site) FcBP(L6Orn)-norbornene:norbornene-PEG8-Asp-Cys * -Ala-Trp-His-Orn-Gly-Glu-Leu-Val-Trp-Cys * -Thr-NH2(Cys * (Disulfide bond site) FcBP(L6Lys)-norbornene:norbornene-PEG8-Asp-Cys * -Ala-Trp-His-Lys-Gly-Glu-Leu-Val-Trp-Cys * -Thr-NH2(Cys * (Disulfide bond site)

[0431] [Table 6]

[0432] Example 9-1-2: Synthesis of compound II-FcBP (L6Dap)-norbornene Compound II (cis-norborneneWeinrebamide)-FcBP was synthesized in DMF. To introduce compound II into FcBP (L6Dap)-norbornene, 3 equivalents of DIPEA and 8.4 μmol of compound II were dissolved in 7.3 μmol of FcBP (6Dap)-norbornene dissolved in DMF, and the resulting solution was stirred.

[0433] After confirming that the reaction had stopped, the reaction solution was concentrated, and compound II-FcBP (6Dap)-norbornene was purified by preparative HPLC. 14.1 mg was obtained by lyophilization after purification, and its purity was confirmed using HPLC (purity: >99% (HPLC), yield: 83%).

[0434] The results are illustrated in Figure 30.

[0435] Example 9-1-3: Confirmation of the structure of compound II-FcBP (L6Dap)-norbornene Measuring instrument: Waters Quattro Premier XE Calculated molecular weight: 2309.61g / mol Measured molecular weight (M / 2+H) 2+ : 1155.08 g / mol The results are illustrated in Figure 31.

[0436] Example 9-1-4: Synthesis of compound II-FcBP (L6Dab)-norbornene Compound II (cis-norborneneWeinrebamide)-FcBP was synthesized in DMF. To introduce compound II into FcBP (L6Dab)-norbornene, 3 equivalents of DIPEA and 8.4 μmol of compound II were dissolved in 7.3 μmol of FcBP (6Dab)-norbornene dissolved in DMF, and the resulting solution was stirred.

[0437] After confirming the cessation of the reaction, the reaction solution was concentrated, and compound II-FcBP (6Dab)-norbornene was purified by preparative HPLC. After purification, 13.8 mg was obtained by lyophilization, and its purity was confirmed using HPLC (purity: >99% (HPLC), yield: 82%).

[0438] The results are illustrated in Figure 32.

[0439] Example 9-1-5: Confirmation of the structure of compound II-FcBP (L6Dab)-norbornene Measuring instrument: Waters Quattro Premier XE Calculated molecular weight: 2323.64g / mol Measured molecular weight (M / 2+H) 2+ : 1162.02 g / mol The results are illustrated in Figure 33.

[0440] Example 9-1-6: Synthesis of compound II-FcBP (L6Orn)-norbornene Compound II (cis-norborneneWeinrebamide)-FcBP was synthesized in DMF. To introduce compound II into FcBP (L6Orn)-norbornene, 3 equivalents of DIPEA and 8.3 μmol of compound II were dissolved in 7.2 μmol of FcBP (L6Orn)-norbornene dissolved in DMF, and the resulting solution was stirred.

[0441] After confirming that the reaction had stopped, the reaction solution was concentrated, and compound II-FcBP (L6Orn)-norbornene was purified by preparative HPLC. 14.8 mg was obtained by lyophilization after purification, and its purity was confirmed using HPLC (purity: >99% (HPLC), yield: 88%).

[0442] The results are illustrated in Figure 34.

[0443] Example 9-1-7: Confirmation of the structure of compound II-FcBP (L6Orn)-norbornene Measuring instrument: Waters Quattro Premier XE Calculated molecular weight: 2337.66g / mol Measured molecular weight (M / 2+H) 2+ : 1169.03 g / mol The results are illustrated in Figure 35.

[0444] Example 9-1-8: Synthesis of compound II-FcBP (L6Lys)-norbornene Compound II (cis-norborneneWeinrebamide)-FcBP was synthesized in DMF. To introduce compound II into FcBP (L6Lys)-norbornene, 3 equivalents of DIPEA and 8.3 μmol of compound II were dissolved in 7.2 μmol of FcBP (L6Lys)-norbornene dissolved in DMF, and the resulting solution was stirred.

[0445] After confirming that the reaction had stopped, the reaction solution was concentrated, and compound II-FcBP (6Lys)-norbornene was purified by preparative HPLC. 15.4 mg was obtained by lyophilization after purification, and its purity was confirmed using HPLC (purity: >99% (HPLC), yield: 91%).

[0446] The results are illustrated in Figure 36.

[0447] Example 9-1-9: Confirmation of the structure of compound II-FcBP (L6Lys)-norbornene Measuring instrument: Waters Quattro Premier XE Calculated molecular weight: 2351.69g / mol Measured molecular weight (M / 2+H) 2+ : 1176.04 g / mol The results are illustrated in Figure 37. [Examples]

[0448] Verification of antibody binding efficiency of site-specific interactomes according to carbon length. Example 10-1. Generation of site-specific antibody-norbornene conjugates using compound II-FcBP (L6Dap, L6Dab, L6Orn, L6Lys)-norbornene Example 10-1-1: Synthesis of trastuzumab-norbornene based on compound II-FcBP (L6Dap)-norbornene Ab (Lys246 / 248)-norbornene was synthesized using compound II-FcBP (L6Dap)-norbornene in phosphate-buffered saline (PBS) buffer at pH 7.4. To introduce norbornene into two specific sites of the antibody (trastuzumab 4 mg / mL, 1 mL), 6 equivalents of compound II-FcBP (L6Dap)-norbornene per antibody were added to the reaction solution, and the reaction was then carried out. Regarding the reaction temperature and time, the reaction took 12 hours at room temperature, and the reaction monitoring and termination were confirmed by HIC-HPLC. Trastuzumab showed a peak on HIC-HPLC at 6.3 to 6.4 minutes. When the FcBP (L6Dap)-norbornene molecule bound to only one of the two binding sites of trastuzumab, the peak was observed on HIC-HPLC at 8 minutes. When the FcBP (L6Dap)-norbornene molecule binds to both sites of trastuzumab, a peak is observed at 9 minutes on HIC-HPLC. Monitoring of the antibody binding reaction based on compound II-FcBP (L6Dap)-norbornene is illustrated in Figure 38. By observing a peak at 9.074 minutes, it was confirmed that an antibody-payload conjugate with DAR=2 had been synthesized.

[0449] Example 10-1-2: Synthesis of trastuzumab-norbornene based on compound II-FcBP(L6Dab)-norbornene Ab (Lys246 / 248)-norbornene was synthesized using compound II-FcBP (L6Dab)-norbornene in phosphate-buffered saline (PBS) buffer at pH 7.4. To introduce norbornene into two specific sites of the antibody (trastuzumab 4 mg / mL, 1 mL), 6 equivalents of compound II-FcBP (L6Dab)-norbornene per antibody were added to the reaction solution, and the reaction was then carried out. Regarding the reaction temperature and time, the reaction took 12 hours at room temperature, and the reaction monitoring and termination were confirmed by HIC-HPLC. Trastuzumab showed a peak on HIC-HPLC at 6.3 to 6.4 minutes. When the FcBP (L6Dab)-norbornene molecule bound to only one of the two binding sites of trastuzumab, the peak was observed on HIC-HPLC at 8 minutes. When the FcBP (L6Dab)-norbornene molecule binds to both sites of trastuzumab, a peak is observed at 9 minutes on HIC-HPLC. Monitoring of the antibody binding reaction based on compound II-FcBP (L6Dab)-norbornene is illustrated in Figure 39. Observing a peak at 9.231 minutes confirmed the synthesis of an antibody-payload conjugate with DAR=2.

[0450] Example 10-1-3: Synthesis of trastuzumab-norbornene based on compound II-FcBP(L6Orn)-norbornene Ab (Lys246 / 248)-norbornene was synthesized using compound II-FcBP (L6Orn)-norbornene in phosphate-buffered saline (PBS) buffer at pH 7.4. To introduce norbornene into two specific sites of the antibody (trastuzumab 4 mg / mL, 1 mL), 6 equivalents of compound II-FcBP (L6Orn)-norbornene per antibody were added to the reaction solution, and the reaction was then carried out. Regarding the reaction temperature and time, the reaction took 12 hours at room temperature, and the reaction monitoring and termination were confirmed by HIC-HPLC. Trastuzumab showed a peak on HIC-HPLC at 6.3 to 6.4 minutes. When the FcBP (L6Orn)-norbornene molecule bound to only one of the two binding sites of trastuzumab, the peak was observed on HIC-HPLC at 8 minutes. When the FcBP (L6Orn)-norbornene molecule binds to both binding sites of trastuzumab, a peak is observed at 9 minutes on HIC-HPLC. Monitoring of the antibody binding reaction based on compound II-FcBP (L6Orn)-norbornene is illustrated in Figure 40. By observing a peak at 8.975 minutes, it was confirmed that an antibody-payload conjugate with DAR=2 had been synthesized.

[0451] Example 10-1-4: Synthesis of trastuzumab-norbornene based on compound II-FcBP (L6Lys)-norbornene Ab (Lys246 / 248)-norbornene was synthesized using compound II-FcBP (L6Lys)-norbornene in phosphate-buffered saline (PBS) buffer at pH 7.4. To introduce norbornene into two specific sites of the antibody (trastuzumab 4 mg / mL, 1 mL), 6 equivalents of compound II-FcBP (L6Lys)-norbornene per antibody were added to the reaction solution, and the reaction was then carried out. Regarding the reaction temperature and time, the reaction took 12 hours at room temperature, and the reaction monitoring and termination were confirmed by HIC-HPLC. Trastuzumab showed a peak on HIC-HPLC at 6.3 to 6.4 minutes. When the FcBP (L6Lys)-norbornene molecule bound to only one of the two binding sites of trastuzumab, the peak was observed on HIC-HPLC at 8 minutes. When the FcBP (L6Lys)-norbornene molecule binds to both sites, a peak is observed at 9 minutes on HIC-HPLC. Monitoring of the antibody binding reaction based on compound II-FcBP (L6Lys)-norbornene is illustrated in Figure 41. Observing a peak at 9.120 minutes confirmed the synthesis of an antibody-payload conjugate with DAR=2.

Claims

1. A method for generating an antibody-payload conjugate, The process involves reacting an antibody containing a first click chemistry functional group with a payload to generate an antibody-payload conjugate. The antibody containing the first click chemistry functional group has the structure of formula 6 below, 【Chemistry 1】 During the ceremony, Ab is an antibody or a partial fragment of an antibody, and the antibody includes the Fc region of an IgG antibody. n is an integer between 1 and 4, R''' is a non-substituted C 1～6 Alkylene, unsubstituted C 2～6 Alkenylene, unsubstituted C 2～6 Alkynylene or unsubstituted C 1～6 A heteroalkylene is a heteroalkylene that contains one or more heteroatoms selected from the group consisting of N, O, and S. Fp comprises an Fc-binding peptide and a first click chemistry functional group. The Fc-binding peptide has the amino acid sequence DCAWHXGELVWCT (SEQ ID NO: 1), X is a 2,3-diaminopropionic acid (Dap) residue, a 2,4-diaminobutyric acid (Dab) residue, an ornithine (Orn) residue, or a lysine (Lys) residue. Y 4 NH and Y 4 It originates from amino acid residue X of the Fc-binding peptide, The nitrogen atom linked to Ab originates from at least one of the lysine 246 and lysine 248 residues of at least one heavy chain of the antibody. The payload contains a second click chemistry functional group that can undergo a click chemistry reaction with a first click chemistry functional group. The antibody-payload conjugate is generated by a click chemistry reaction between a first click chemistry functional group of the antibody containing a first click chemistry functional group and a second click chemistry functional group of the payload. method.

2. The method according to claim 1, wherein n is 2.

3. n is 2, The method according to claim 1, wherein the nitrogen atom linked to Ab is derived from lysine residue 248 of both heavy chains of the antibody.

4. R''' is a non-substituted C 1～5 Alkylene or unsubstituted C 1～5 The method according to any one of claims 1 to 3, wherein the heteroalkylene is used.

5. R''' is unsubstituted C 3 The method according to any one of claims 1 to 4, wherein the alkylene is 3 .

6. The method according to any one of claims 1 to 5, wherein the first click chemistry functional group is selected from the group consisting of acetylene, transcyclooctene, cyclooctin, diarylcyclooctin, oxime, ketone, aldehyde, thiol, free cysteine, amine, maleimide, NHS (N-hydroxysuccinimide), NHS ester (N-hydroxysuccinimide ester), isocyanate, isothiocyanate, methyl ester phosphine, norbornene, tetrazine, methylcyclopropene, azetine, cyanide, azide, and dibenzocyclooctin.

7. The method according to any one of claims 1 to 6, wherein the click chemistry reaction is selected from the group consisting of [3+2] cycloaddition, thiol-ene reaction, Diels-Alder reaction, reverse electron-demand Diels-Alder reaction, and [4+1] cycloaddition.

8. The method according to any one of claims 1 to 7, wherein the second click chemistry functional group is selected from the group consisting of acetylene, transcyclooctene, cyclooctin, diarylcyclooctin, oxime, ketone, aldehyde, thiol, free cysteine, amine, maleimide, NHS (N-hydroxysuccinimide), NHS ester (N-hydroxysuccinimide ester), isocyanate, isothiocyanate, methyl ester phosphine, norbornene, tetrazine, methylcyclopropene, azetine, cyanide, azide, and dibenzocyclooctin.

9. The method according to any one of claims 1 to 8, wherein the first click chemistry functional group is an azide or norbornene.

10. The first click chemistry functional group is an azide, and the second click chemistry functional group is dibenzocyclooctin, or The method according to any one of claims 1 to 9, wherein the first click chemistry functional group is norbornene and the second click chemistry functional group is tetrazine.

11. The method according to any one of claims 1 to 10, wherein the payload further comprises an active portion.

12. The method according to claim 11, wherein the active portion is selected from the group consisting of drug molecules, contrast agents, radioisotopes, fluorescent materials, optical agents, vitamins, and toxins.

13. The method according to any one of claims 1 to 12, wherein the first click chemistry functional group is linked to the N-terminal or C-terminal amino acid residue of the Fc-binding peptide DCAWHXGELVWCT (SEQ ID NO: 1).

14. The method according to any one of claims 1 to 13, wherein a first click chemistry functional group is linked to an Fc-binding peptide of DCAWHXGELVWCT (SEQ ID NO: 1) via polyethylene glycol units.

15. The method according to any one of claims 1 to 14, wherein the first click chemistry functional group is linked to the N-terminal or C-terminal amino acid residue of the Fc-binding peptide DCAWHXGELVWCT (SEQ ID NO: 1) via polyethylene glycol units.

16. The method according to claim 14 or 15, wherein the polyethylene glycol unit comprises one or more ethylene glycol units.

17. The method according to any one of claims 1 to 16, wherein X is a lysine residue.

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