Compound for preparation of antibody-payload conjugate and use thereof

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 preparing site-specifically coupled antibody-payload conjugates, the antibody-payload conjugates prepared therewith, and a method for preparing antibody-payload conjugates. More specifically, the present invention relates to a linker that contains a carbonyl group carbon with two or more different partial positive charges and functional groups at both ends, and can link compounds, peptides, and / or proteins to biological (target) molecules through a substitution reaction. [Background technology]

[0002] Bioconjugation is the process of linking at least two or more molecules, where bioconjugation refers to a process where at least one or more of the molecules are biologically active molecules. A biologically active molecule may sometimes be referred to as a "target molecule" or "molecule of interest" and may be, for example, a protein (or peptide), a glycan, a nucleic acid (or oligonucleotide), a lipid, a hormone, or a natural drug (or a fragment thereof, or a combination thereof), etc. Such linkers linking two or more molecules can be widely used for detection, diagnosis, biomarkers, etc., by binding a target-specific protein, such as an antibody, to a fluorescent material, etc.

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

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

[0005] To solve these problems, the present inventors have invented a linker that can link a payload to a target molecule without affecting the biological activity of the target molecule, comprising a carbonyl group with two or more different partial positive charges (δ+) and a leaving group and / or click compound at both ends. The linker can increase reactivity by increasing the water solubility for the target molecule through length tuning as well as site specificity. Such a linker is intended to provide a more stable and economically highly effective linker, since it does not have harsh reaction conditions and has high conjugation yields for molecules including the target molecule as bioconjugation. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] WO / 2015 / 122478 Summary of the Invention [Problem to be solved by the invention]

[0007] The present application aims to provide a novel trans-configured linker comprising two or more electrophilic carbons of a carbonyl group and one or more click chemistry moieties, as well as a method for preparing the same.

[0008] The present application aims to provide a novel cis-configured linker comprising two or more electrophilic carbons of a carbonyl group and one or more click chemistry moieties, as well as a method for preparing the same. [Means for solving the problem]

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

[0010] In one embodiment, the present application provides a compound represented by formula 2:

[0011] [ka]

[0012] In formula 2, R' is an ester group activating moiety, R" is any one of acetylene, transcyclooctene, cyclooctyne, diarylcyclooctyne, methyl ester phosphine, norbornene, methylcyclopropene, azetine, and cyanide, and R"' is a substituted or unsubstituted 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 Aryl alkylene, substituted or unsubstituted C 8~16 Arylalkenylene, substituted or unsubstituted C 3~10 Cycloalkylene, substituted or unsubstituted C 3~10 Heterocycloalkylene or substituted or unsubstituted C 5~12Heteroaryl, where the heteroalkylene, heterocycloalkylene or heteroaryl comprises at least one or more selected from the group consisting of N, O and S, and the substituents 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, where Ra is H, C 1~6 Alkyl, C 5~12 Aryl, C 7~12 R" is arylalkyl or heterocycle, Rb is F, Cl, Br or I, and X is O, N or S. Additionally, the present application provides compounds where R" is selected from the group consisting of norbornene, transcyclooctene, cyclooctyne and methylcyclopropene. Further, the present application provides compounds where R" is norbornene.

[0013] Furthermore, the present application provides that R''' is a substituted or unsubstituted C 1~10 Alkylene, substituted or unsubstituted C 1~10 Heteroalkylene, and C 1~10and polymethylene, the heteroalkylene comprises at least one or more selected from the group consisting of N, O and S, and the substituents are substituted with non-hydrogen substituents, the non-hydrogen substituents being -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 )ORa, -S(=O)NRa, -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, wherein Ra is C 1~6 Alkyl, C 5~12 Aryl, C 7~12 arylalkyl or heterocycle, Rb is F, Cl, Br or I, and X is O, N or S. Additionally, the present application provides compounds in which R''' is an unsubstituted C 1~5 Alkylene or unsubstituted C 1~5 A compound that is polymethylene is provided.

[0014] Further, the present application provides compounds where X is O.

[0015] Furthermore, the present application provides a compound represented by the following formula 2-1-2:

[0016] [ka]

[0017] In another aspect, the present application provides a method for producing an antibody-payload conjugate, the method comprising: reacting a linker having a structure of Formula 2 with an Fc binding peptide to prepare a linker-Fc binding peptide conjugate; reacting the linker-Fc binding peptide conjugate with an antibody to obtain an antibody comprising a first click chemistry functional group; and reacting the antibody comprising the first click chemistry functional group with a payload comprising a second click chemistry functional group capable of undergoing a click chemistry reaction with the first click chemistry functional group to prepare an antibody-payload conjugate having a structure of Formula 8 below.

[0018] [ka]

[0019] where Ab is an antibody and R′″ is an unsubstituted C 1~5 Alkylene or unsubstituted C 1~5 Y4 is polymethylene, Y4 is N, Fp is an Fc-binding peptide, B is any structure formed by a click chemical reaction between a first click chemistry functional group and a second click chemistry functional group, Am is an active moiety or a structure including an active moiety, the active moiety being any one selected from the group consisting of a drug molecule, an imaging moiety, an optical agent, a vitamin and a toxin, and n is an integer greater than or equal to 1 and less than or equal to 4.

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

[0021] [ka]

[0022] wherein G is glycine, E is glutamate, L is leucine, V is valine, T is threonine, K is lysine, m is an integer equal to or greater than 1 and equal to or less than 4, the N-terminal cysteine ​​and the C-terminal cysteine ​​are optionally 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, the present application provides an antibody-payload conjugate of formula 8:

[0024] [ka]

[0025] where Ab is an antibody and R′″ is an unsubstituted C 1~5 Alkylene or unsubstituted C 1~5 wherein A is polymethylene, Y4 is N, Fp is an Fc-binding peptide, B is any one structure formed by a click chemical reaction of the first click chemistry functional group and the second click chemistry functional group, Am is an active moiety or a structure including an active moiety, the active moiety being any one selected from the group consisting of a drug molecule, an imaging moiety, an optical agent, a vitamin and a toxin, and n is an integer of 1 or more and 4 or less.

[0026] Furthermore, the present application relates to a peptide, wherein Fp is selected from the group consisting of the following formulas 13 and 14: [Formula 13] DCAWH-Xa-GELVWCT [Formula 14] DCAWHKGELVWCT (wherein D is aspartic acid, C is cysteine, A is alanine, W is tryptophan, H is histidine, and Xa is

[0027] [ka]

[0028] wherein G is glycine, E is glutamate, L is leucine, V is valine, T is threonine, K is lysine, and m is an integer equal to or greater than 1 and equal to or less than 4, and the N-terminal cysteine ​​and the C-terminal cysteine ​​are optionally linked to each other), and Fp is linked via Y4 at amino acid residue 6.

[0029] Furthermore, the present application provides that B:

[0030] [ka]

[0031] wherein 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, the present application provides that B:

[0033] [ka]

[0034] The present invention provides an antibody-payload conjugate, wherein

[0035] The present application further provides an antibody-payload conjugate, wherein Am comprises an anti-cancer drug. The present application further provides an antibody-payload conjugate, wherein the anti-cancer drug is mertansine (DM1). The present application further provides an antibody-payload conjugate, wherein Am comprises two or more anti-cancer drugs.

[0036] Additionally, the present application provides an antibody-payload conjugate, wherein the nitrogen atom linked to the Ab is contained at lysine 246 or lysine 248 of the Fc of the antibody.

[0037] Additionally, the present application provides an antibody-payload conjugate wherein n is 2 and the nitrogen atom linked to Ab is contained at lysine 246 or lysine 248 of both Fc of the antibody.

[0038] In another aspect, the present application provides a pharmaceutical composition for treating cancer, comprising an antibody-payload that comprises an anti-cancer drug.

[0039] Furthermore, the present application provides a pharmaceutical composition, wherein the cancer is breast cancer. Effect of the Invention

[0040] The scientific technology disclosed in this specification provides the following advantages:

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

[0042] In addition, compound 2 disclosed herein provides a linker that can site-specifically link payload to antibody. The linker can affect the biological activity of target molecule, for example, has the effect of reducing the half-life or promoting excretion of target molecule and / or payload. The linker can be usefully used as bioconjugation for detection, diagnosis and biomarker.

[0043] Furthermore, the antibody-payload conjugates provided by compounds 1 and 2 have the advantage of being highly homogeneous due to uniform attachment sites. [Brief description of the drawings]

[0044] [Figure 1] FIG. 1 illustrates the overall synthesis process of compound I (trans). [Diagram 2] FIG. 1 illustrates the overall synthesis process of compound II (cis). [Diagram 3] FIG. 1 illustrates the results of HPLC spectrum analysis of the isomeric structures of compound 6. [Figure 4] FIG. 1 illustrates the results of molecular weight analysis of compound 6 (cis). [Diagram 5] FIG. 1 illustrates the results of molecular weight analysis of compound 6 (trans). [Figure 6] FIG. 2 illustrates the results of confirming the isomeric structure of Compound II via HPLC. [Figure 7] FIG. 1 illustrates the results of obtaining compound II via HPLC. [Figure 8] FIG. 1 shows an example of the HPLC result of FcBP(6Lys)-norbornene. [Figure 9] FIG. 1 illustrates the LC mass results of FcBP(6Lys)-norbornene. [Figure 10] FIG. 1 illustrates the HPLC results of compound I-FcBP(6Lys)-norbornene. [Figure 11]FIG. 1 illustrates the LC mass result of compound I-FcBP(6Lys)-norbornene. [Figure 12] FIG. 1 illustrates the HPLC results of compound II-FcBP(6Lys)-norbornene. [Figure 13] FIG. 1 illustrates the mass spectrometry results of compound II-FcBP(6Lys)-norbornene. [Figure 14] FIG. 1 illustrates the reaction of compound I-FcBP(6Lys)-norbornene with an antibody. [Figure 15] FIG. 1 illustrates the structure of Ab(Lys246 / 248)-norbornene produced by reaction of compound I-FcBP(6Lys)-norbornene with an antibody. [Figure 16] FIG. 1 illustrates the results of reaction monitoring of the reaction of compound I-FcBP(6Lys)-norbornene with an antibody via HIC-HPLC. [Figure 17] FIG. 1 illustrates the reaction of compound II-FcBP(6Lys)-norbornene with an antibody. [Figure 18] FIG. 1 illustrates the structure of Ab(Lys246 / 248)-norbornene produced by reaction of compound I-FcBP(6Lys)-norbornene with an antibody. [Figure 19] FIG. 1 illustrates the results of reaction monitoring of the reaction of compound II-FcBP(6Lys)-norbornene with antibody via HIC-HPLC. [Figure 20] FIG. 1 illustrates the mass spectrometry results of Herceptin-norbornene. [Figure 21] FIG. 19 illustrates the structure of an antibody-payload conjugate generated using an antibody containing the first click chemistry functional group in FIG. 18. The expanded structure is that of the payload, and the non-expanded portion is the same as the structure illustrated in FIG. [Figure 22] FIG. 19 illustrates the results of reaction monitoring of the reaction of Ab(Lys246 / 248)-norbornene with tetrazine-PEG8-DM1 in FIG. 18 via HIC-HPLC. [Figure 23] FIG. 1 illustrates mass spectrometry results of antibody-payload conjugates. [Figure 24] FIG. 1 shows the results of a cytotoxicity experiment on NCI-N87. [Diagram 25] FIG. 1 shows the results of a cytotoxicity experiment on BT474. [Figure 26] FIG. 1 shows the results of a cytotoxicity experiment against MDA-MB-468. [Figure 27] FIG. 1 shows a table summarizing the results of cytotoxicity experiments for NCI-N87, BT474 and MDA-MB-468. [Figure 28] FIG. 1 illustrates the results of tumor growth inhibition experiments of Herceptin and antibody-payload conjugates according to the present application. [Figure 29] FIG. 1 illustrates the results of tumor growth inhibition experiments of Herceptin and antibody-payload conjugates according to the present application. [Diagram 30] FIG. 1 illustrates the HPLC results of compound II-FcBP(L6Dap)-norbornene. [Diagram 31] FIG. 1 illustrates the mass spectrometry results of compound II-FcBP(L6Dap)-norbornene. [Diagram 32] FIG. 1 illustrates the HPLC results of compound II-FcBP(L6Dab)-norbornene. [Diagram 33] FIG. 1 illustrates the mass spectrometry results of compound II-FcBP(L6Dab)-norbornene. [Diagram 34] FIG. 1 illustrates the HPLC results of compound II-FcBP(L6Orn)-norbornene. [Diagram 35] FIG. 1 illustrates the mass spectrometry results of compound II-FcBP(L6Orn)-norbornene. [Diagram 36] FIG. 1 illustrates the HPLC results of compound II-FcBP(L6Lys)-norbornene. [Figure 37] FIG. 1 illustrates the mass spectrometry results of compound II-FcBP(L6Lys)-norbornene. [Figure 38] FIG. 1 illustrates the HIC-HPLC results for monitoring the binding reaction with Compound II-FcBP(L6Dap)-norbornene-based antibody. [Figure 39] FIG. 1 illustrates the HIC-HPLC results for monitoring the binding reaction with Compound II-FcBP(L6Dab)-norbornene-based antibody. [Diagram 40] FIG. 1 illustrates the HIC-HPLC results for monitoring the binding reaction with Compound II-FcBP(L6Orn)-norbornene-based antibody. [Diagram 41] FIG. 1 illustrates the HIC-HPLC results for monitoring the binding reaction with Compound II-FcBP(L6Lys)-norbornene-based antibody. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0045] The term "heteroalkyl" refers to an alkyl group in which one or more carbon atoms are replaced with a heteroatom, such as O, N, or S. For example, when a carbon atom of an alkyl group that is attached to a parent molecule is replaced with a heteroatom (e.g., O, N, or S), the resulting heteroalkyl group is an alkoxy group (e.g., -OCH), an amine (e.g., -NHCH, -N(CH), or a thioalkyl group (e.g., -SCH), respectively. When a non-terminal carbon atom of an alkyl group that is not attached to a parent molecule is replaced with a heteroatom (e.g., O, N, or S), the resulting heteroalkyl group is an alkyl ether (e.g., -CHCH-O-CH), an alkylamine (e.g., -CHNHCH, -CHN(CH), or a thioalkyl ether (e.g., -CH-S-CH), respectively. When the terminal carbon atom of an alkyl group is replaced 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. The heteroalkyl group can have, for example, 1 to 20 carbon atoms, 1 to 10 carbon atoms, or 1 to 6 carbon atoms. C1-C6 heteroalkyl group means a heteroalkyl group having 1 to 6 carbon atoms.

[0046] The term "alkylene" refers to a branched, straight chain, or cyclic saturated hydrocarbon group containing two monovalent radical 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 (-CH-), 1,1-ethyl (-CH(CH)-), 1,2-ethyl (-CHCH-), 1,1-propyl (-CH(CHCH)-), 1,2-propyl (-CHCH(CH)-), 1,3-propyl (-CHCHCH-), 1,4-butyl (-CHCHCHCH-), and the like.

[0047] The term "alkenylene" refers to a branched, straight chain or cyclic unsaturated hydrocarbon group containing two monovalent radical 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, straight chain or cyclic unsaturated hydrocarbon group containing two monovalent radical 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 (-CHC≡C-), and 4-pentynyl (-CHCHCHC≡C-).

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

[0050] Those of skill in the art will recognize that when moieties such as "alkyl", "aryl" and "heterocyclyl" are substituted with one or more substituents, they can alternatively be referred to as moieties such as "alkylene", "arylene" and "heterocyclylene" (i.e., which means that one or more hydrogen atoms of the parent bodies "alkyl", "aryl" and "heterocyclyl" moieties are replaced with the described substituents). When moieties such as "alkyl", "aryl" and "heterocyclyl" are referred to as "substituted" in this application or illustrated in the drawings as substituted (or optionally substituted, e.g., where the number of substituents is zero to a positive number), terms such as "alkyl", "aryl" and "heterocyclyl" should be understood to be interchangeable with "alkylene", "arylene", "heterocyclylene", etc.

[0051] The term "acyl" refers to -C(=O)-alkyl, -C(=O)-carbocycle (substituted or unsubstituted), -C(=O)-heterocycle (substituted or unsubstituted), where the alkyl, carbocycle, or heterocycle moiety is 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), and the like.

[0052] The term "substituted", e.g., "substituted alkyl", "substituted alkylene", "substituted aryl", "substituted arylalkyl", "substituted heterocyclyl" and "substituted carbocyclyl (e.g., substituted cycloalkyl)" means alkyl, alkylene, aryl, arylalkyl, heterocyclyl, and carbocyclyl (e.g., cycloalkyl), in which one or more hydrogen atoms are each independently replaced with a non-hydrogen substituent. 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, -S(=O)2N Examples of alkylene-C(=O)R include, but are not limited to, R, -S(=O)R, -OP(=O)(OR), -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, -C(-NR)NRR (each X is independently a halogen: F, Cl, Br, or I, and R is independently H, alkyl, aryl, arylalkyl, or heterocycle). Alkylene, alkenylene, and alkynylene groups may be similarly substituted.

[0053] "Optionally substituted" refers to certain moieties of a compound of Formula 1 having one, two or more substituents (eg, an optionally substituted aryl group).

[0054] "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" refers to a molecule that is intended to have a payload linked thereto. By way of example, the target molecule may be a biologically active molecule, such as, but not limited to, a protein (or peptide), a glycan, a nucleic acid (or oligonucleotide), a lipid, a hormone, or a natural drug (or a fragment thereof, or a combination thereof).

[0056] The term "payload" refers to a molecule that is intended to be linked to a target molecule. By way of example, the payload may be a compound, a peptide, a polypeptide, a protein and / or a 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] cycloadditions, thiol-ene reactions, Diels-Alder reactions, inverse electron demand Diels-Alder reactions, [4+1] cycloadditions, and the like. More specifically, click chemistry reactions include, but are not limited to, copper(I)-catalyzed azide-alkyne cycloadditions (CuAAC), strain-promoted azide-alkyne cycloadditions (SPAAC), strain-promoted alkyne-nitrone cycloadditions (Span C), [3+2] cycloadditions of alkenes and azides, inverse demand Diels-Alder of alkenes and tetrazines, photoclick reactions of alkenes and tetrazoles, and Huisgen cycloadditions of azides and alkynes. Click chemistry functional groups include, but are not limited to, alkynes, cycloalkynes, cyclooctynes ​​and cyclononynes (e.g., cycloalkynes such as bicyclo[6.1.0]non-4-yn-9-ylmethanol), trans-cyclooctene, nitrones, nitrile oxides, azides, conjugated dienes, dienophiles, and cycloalkynes such as cyclooctynes, cyclononynes, dibenzocyclooctynes ​​(DIBO), biarylazacyclooctynes ​​(BARAC), aryllessoctynes ​​(ALO), difluorinated cyclooctynes ​​(DIFO), monofluorinated (MOFO), dibenzo-aza-cyclooctynes ​​(DIBAC), and dimethoxyazacyclooctynes ​​(DIMAC).

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

[0059] [ka]

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

[0061] As used herein, terms such as first and second are used to describe various components, and such terms are used only to distinguish one component from another.

[0062] Furthermore, it should be noted that the terminology used herein is for the purpose of describing illustrative embodiments only and is not intended to limit the invention. The singular terms include the plural terms unless the context clearly indicates otherwise.

[0063] In this specification, terms such as "comprise", "include" or "having" are intended to indicate the presence of implemented features, numbers, steps, operations, components, or any combination thereof, and should be understood to mean not excluding the possible presence or addition of one or more other features, numbers, steps, operations, components, or any combination thereof.

[0064] Because the present specification can be modified in various forms and can include various exemplary embodiments, certain exemplary embodiments are illustrated and described in detail below. However, the description is not intended to limit the present invention to the specific disclosure, and all changes, equivalents, and substitutions included within the concept and technical scope of the present invention should be understood to be included in the present invention.

[0065] In the following the invention will be described in detail.

[0066] The present application can provide linkers that aid in the transport and binding reactions so that the payload can be linked to a desired target molecule.

[0067] The linker can include two or more functional groups capable of reacting with a payload and / or a targeting molecule.

[0068] The above reaction means that two or more identical or different molecules or functional groups of parts of the molecules are linked to each other or elimination reactions occur, such as E1, E2, SN1, SN2 and nucleophilic substitution reactions, etc. The linkage includes all direct or indirect covalent and / or non-covalent bonds.

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

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

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

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

[0073] As an example, the electrophilic carbon atom of a carbonyl group and a functional 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 a carbonyl group that have different partial positive charges (δ+).

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

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

[0078] As an example, the electrophilic carbon atom of the first carbonyl group may be linked to a first functional group, in which case a nucleophilic atom may be included 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 can be linked to a second functional group, in which case the electrophilic carbon atom of the second carbonyl group can form a covalent bond with the first functional group.

[0080] The linker can control the position at which a payload is attached that is to be covalently attached to a target molecule.

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

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

[0083] Below, linker structures are described in detail.

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

[0085] [ka]

[0086] In formula 1, R' is an ester group activating moiety. Ester group activating moieties include, but are not limited to, N-hydroxysuccinimide (NHS), p-nitrophenyl, and pentafluorophenyl groups. Further, R' is

[0087] [ka]

[0088] It may be any one of the following. R″ is one of the click chemistry functional groups; The click chemistry functional group may be any one or more of, but is not limited to, an alkyne, a cycloalkyne, such as cyclooctyne and cyclononyne (e.g., bicyclo[6.1.0]non-4-yn-9-ylmethanol), trans-cyclooctene, a nitrone, a nitrile oxide, an azide, a conjugated diene, and a dienophile.

[0089] When the click chemistry functional group is a cycloalkyne, the cycloalkyne may be any one of cyclooctyne, cyclononyne, dibenzocyclooctyne (DIBO), biarylazacyclooctynone (BARAC), aryl-less octyne (ALO), difluorinated cyclooctyne (DIFO), monofluorinated (MOFO), dibenzo-aza-cyclooctyne (DIBAC), and dimethoxyazacyclooctyne (DIMAC). The cycloalkyne is not limited thereto.

[0090] When 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, the cycloalkane including a bicyclic or fused ring structure. As an example, the dienophile may be norbornene.

[0092] R''' is a substituted or unsubstituted 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 Aryl alkylene, substituted or unsubstituted C 8~16 Arylalkenylene, substituted or unsubstituted C 3~10 Cycloalkylene, substituted or unsubstituted C 3~10 Heterocycloalkylene or substituted or unsubstituted C 5~12 heteroaryl, where the heteroalkylene, heterocycloalkylene or heteroaryl contains at least one or more of N, O or S; The substituents 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; Ra is H, C 1~6 Alkyl, C 5~12 Aryl, C7~12 arylalkyl or heterocycle; Rb is F, Cl, Br or I; X is O, N or S.

[0093] In formula 2, R' is an ester group activating moiety. Ester group activating moieties include, but are not limited to, N-hydroxysuccinimide (NHS), p-nitrophenyl, and pentafluorophenyl groups. Further, R' is

[0094] [ka]

[0095] It may be any one of the following. R″ is one of the click chemistry functional groups; The click chemistry functional group may be any one or more of, but is not limited to, alkynes, cyclooctynes ​​and cyclononynes (e.g., cycloalkynes such as bicyclo[6.1.0]non-4-yn-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 cyclooctyne, cyclononyne, dibenzocyclooctyne (DIBO), BARAC (biarylazacyclooctynone), ALO (arylless octyne), DIFO (difluorinated cyclooctyne), MOFO (monofluorinated), DIBAC (dibenzo-aza-cyclooctyne), and DIMAC (dimethoxyazacyclooctyne).

[0097] When 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, the cycloalkane including a bicyclic or fused ring structure. By way of example, the dienophile may be transcyclooctene (TCO) or norbornene.

[0099] R''' is a substituted or unsubstituted 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 Aryl alkylene, substituted or unsubstituted C 8~16 Arylalkenylene, substituted or unsubstituted C 3~10 Cycloalkylene, substituted or unsubstituted C 3~10 Heterocycloalkylene or substituted or unsubstituted C 5~12 heteroaryl, where the heteroalkylene, heterocycloalkylene or heteroaryl contains at least one or more of N, O or S; The substituents 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; 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 O, N or S.

[0100] In the formula of the present invention,

[0101] [ka]

[0102] is used to indicate the bond that is the point of attachment of a moiety or substituent to a core or backbone structure.

[0103] According to exemplary embodiments disclosed herein, the compound represented by formula 1 can be a trans compound.

[0104] Formula 1 is represented by Formula 1-1 below when Formula 1 is equal to: R' is

[0105] [ka]

[0106] and R″ is one of the click chemistry functional groups; The click chemistry functional group can be any one or more of an alkyne, a cycloalkyne, such as cyclooctyne and cyclononyne (e.g., bicyclo[6.1.0]non-4-yn-9-ylmethanol), a trans-cyclooctene, a nitrone, a nitrile oxide, an azide, a conjugated diene, and a dienophile, such as an alkene, wherein the cycloalkyne, the conjugated diene, and the diene are the same as described above.

[0107] R''' is a 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 the heteroalkylene contains at least one or more selected from the group consisting of N, O, and S; The substituents 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; Ra is H, C 1~6 Alkyl, C 5~12Aryl, C 7~12 arylalkyl or heterocycle; Rb is F, Cl, Br or I; X is any one of O, N and S.

[0108] [ka]

[0109] Specifically, R''' is

[0110] [ka]

[0111] It is one of the 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.

[0112] R5 and R6 may not both be C(=O) at the same time; When Y1 is any one of N, O, and S, R4, R5, and R6 cannot be C(=O).

[0113] More specifically, R''' is

[0114] [ka]

[0115] 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 may be any one of the integers from 1 to 10.

[0116] In an illustrative embodiment, formula 1-1 is represented by formula 1-1-1 below, where formula 1-1 is the same as: 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 H or C(=O); R5 is H or C(=O); R6 is H or C(=O); Y1 is C; X is any one of N, O and S.

[0121] [ka]

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

[0123] [ka]

[0124] and R" is norbornene; 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, the compound that can be represented by Formula 1 can be a compound set forth in Table 1 below.

[0129] [Table 1-1]

[0130] [Table 1-2]

[0131] Formula 1 is represented by Formula 1-2 below when Formula 1 is the same as: R' is

[0132] [ka]

[0133] and R″ is one of the click chemistry functional groups; The click chemistry functional group can be any one or more of an alkyne, a cycloalkyne, such as cyclooctyne and cyclononyne (e.g., bicyclo[6.1.0]non-4-yn-9-ylmethanol), trans-cyclooctene, a nitrone, a nitrile oxide, an azide, a conjugated diene, and a dienophile, such as an alkene, where the cycloalkyne, the conjugated diene, and the diene are the same as described above.

[0134] R''' is a 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 the heteroalkylene contains at least one or more selected from the group consisting of N, O, and S; The substituents 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; 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.

[0135] [ka]

[0136] Specifically, R''' is

[0137] [ka]

[0138] It is one of the 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, R5 and R6 cannot both 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] [ka]

[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 may be any one of the integers from 1 to 10.

[0143] In an illustrative embodiment, Formula 1-2 is represented by Formula 1-2-1 below, where Formula 1-2 is equal to: R''' is

[0144] [ka]

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

[0146] [ka]

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

[0148] [ka]

[0149] and R" is norbornene; 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] [ka]

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

[0154] [Table 2-1]

[0155] [Table 2-2]

[0156] According to exemplary embodiments disclosed herein, the compound represented by formula 2 may be a cis compound.

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

[0158] [ka]

[0159] and R″ is one of the click chemistry functional groups; The click chemistry functional group is any one or more of an alkyne, a cycloalkyne, such as cyclooctyne and cyclononyne (e.g., bicyclo[6.1.0]non-4-yn-9-ylmethanol), a trans-cyclooctene, a nitrone, a nitrile oxide, an azide, a conjugated diene, and a dienophile, such as an alkene, wherein the cycloalkyne, the conjugated diene, and the diene are the same as described above; R''' is a 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 the heteroalkylene contains at least one or more selected from the group consisting of N, O, and S; The substituents 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; 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.

[0160] [ka]

[0161] Specifically, R''' is

[0162] [ka]

[0163] It is one of the 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 H or C(=O), Y2 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.

[0164] In this case, R 11 and R 12 cannot both be C(=O), When Y2 is any one of N, O, and S, R 10 , R 11 and R 12 cannot be C(=O).

[0165] More specifically, R''' is

[0166] [ka]

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

[0168] In an illustrative embodiment, formula 2-1 is represented by formula 2-1-1 below, where formula 2-1 is the same as: 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 H or C(=O), R 11 is H or C(=O), R 12 is H or C(=O), Y2 is C; X is any one of N, O and S.

[0173] [ka]

[0174] In another illustrative embodiment, Formula 2-1 is represented by Formula 2-1-2 below, where Formula 2-1 is equal to: R' is

[0175] [ka]

[0176] and R" is norbornene; R''' is

[0177] [ka]

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

[0179] [ka]

[0180] As an illustrative embodiment, the compound that can be represented by Formula 2 can be a compound set forth in Table 3 below.

[0181] [Table 3-1]

[0182] [Table 3-2]

[0183] [Table 3-3]

[0184] Equation 2 is represented by Equation 2-2 below, where Equation 2 is the same as: R' is

[0185] [ka]

[0186] and R″ is one of the click chemistry functional groups; The click chemistry functional group can be any one or more of an alkyne, a cycloalkyne, such as cyclooctyne and cyclononyne (e.g., bicyclo[6.1.0]non-4-yn-9-ylmethanol), trans-cyclooctene, a nitrone, a nitrile oxide, an azide, a conjugated diene, and a dienophile, such as an alkene, where the cycloalkyne, the conjugated diene, and the diene are the same as described above.

[0187] R''' is a 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 the heteroalkylene contains at least one or more selected from the group consisting of N, O, and S; The substituents 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; 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] [ka]

[0189] Specifically, R''' is

[0190] [ka]

[0191] It is one of the 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 H or C(=O), Y2 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.

[0192] In this case, R 11 and R 12 cannot both be C(=O), When Y2 is any one of N, O, and S, R 10 , R 11 and R 12 cannot be C(=O).

[0193] More specifically, R''' is

[0194] [ka]

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

[0196] In an illustrative embodiment, Formula 2-2 is represented by Formula 2-2-1 below, where Formula 2-2 is the same as: 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 H or C(=O), R 11 is H or C(=O), R 12 is H or C(=O), Y2 is C; X is any one of N, O and S.

[0201] [ka]

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

[0203] [ka]

[0204] and R" is norbornene; R''' is

[0205] [ka]

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

[0207] [ka]

[0208] As an illustrative embodiment, the compound that can be represented by Formula 2 can be a compound set forth in Table 4 below.

[0209] [Table 4-1]

[0210] [Table 4-2]

[0211] [Table 4-3]

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

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

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

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

[0216] [ka]

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

[0218] The Fc binding peptide according to SEQ ID NO:2 is a peptide represented by formula 14: [Formula 14] DCAWHKGELVWCT (SEQ ID NO: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, T is threonine, and the N-terminal cysteine ​​and the C-terminal cysteine ​​may optionally be linked to each other.

[0219] The linker includes compounds according to Formula 1 or Formula 2 and specific examples thereof.

[0220] The linker and the Fc binding peptide can be bonded by nucleophilic substitution reaction. Nucleophilic substitution reaction can 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 can be the nitrogen atom of lysine. In addition, the positively charged or partially positively charged atom can be the electrophilic carbon of a carbonyl group.

[0221] In the following, as an example, the compounds represented by formula 1 and / or 2 disclosed herein, i.e., the linkers, are shown to be bound to an Fc binding peptide to form a linker-Fc binding peptide conjugate.

[0222] As an example, the linker-Fc binding peptide conjugate can 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 any one or more of an alkyne, a cycloalkyne, such as cyclooctyne and cyclononyne (e.g., bicyclo[6.1.0]non-4-yn-9-ylmethanol), a trans-cyclooctene, a nitrone, a nitrile oxide, an azide, a conjugated diene, and a dienophile, where the cycloalkyne, the conjugated diene, and the diene are the same as described above.

[0225] R''' is a substituted or unsubstituted 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 Aryl alkylene, substituted or unsubstituted C 8~16 Arylalkenylene, substituted or unsubstituted C 3~10 Cycloalkylene, substituted or unsubstituted C 3~10 Heterocycloalkylene or substituted or unsubstituted C 5~12heteroaryl, where the heteroalkylene, heterocycloalkylene or heteroaryl contains at least one or more of N, O or S; The substituents 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; 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 can be O, N or S.

[0226] Y3 can be any one of O, N and S.

[0227] Fp is an Fc binding peptide. Furthermore, Fp may be any one peptide selected from formula 13 or 14. The details of formula 13 and 14 are the same as those described above.

[0228] Fp may be linked via Y3 at 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 formula 3 can be produced, for example, by reaction scheme 1.

[0230] [ka]

[0231] Reaction scheme 1 is a reaction for producing a compound represented by formula 3 by reacting a 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 reaction between the compound of formula 1 and any one of the amine group (-NH2), thiol group (-SH) and hydroxy group (-OH) of the Fc binding peptide.

[0235] As an example, if a compound of Formula 1 is to be reacted with an amine group (-NH2) of an Fc binding peptide, Y3 can be N(H). 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, all corresponding residues contain a free amine group and thus a displacement reaction can be performed.

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

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

[0238] As an example, the linker-Fc binding peptide conjugate can 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 group can be any one or more of an alkyne, a cycloalkyne, such as cyclooctyne and cyclononyne (e.g., bicyclo[6.1.0]non-4-yn-9-ylmethanol), a trans-cyclooctene, a nitrone, a nitrile oxide, an azide, a conjugated diene, and a dienophile, where the cycloalkyne, the conjugated diene, and the diene are the same as those described above.

[0241] R''' is a substituted or unsubstituted 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 Aryl alkylene, substituted or unsubstituted C 8~16 Arylalkenylene, substituted or unsubstituted C 3~10 Cycloalkylene, substituted or unsubstituted C 3~10 Heterocycloalkylene or substituted or unsubstituted C 5~12 heteroaryl, where the heteroalkylene, heterocycloalkylene or heteroaryl contains at least one or more of N, O or S; The substituents 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; 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 can be O, N or S.

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

[0243] Fp is an Fc binding peptide. Furthermore, Fp may be any one peptide selected from formula 13 or 14. The details of formula 13 and 14 are the same as those described above.

[0244] Fp may be linked via Y4 at 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 formula 4 can be produced, for example, by reaction scheme 2.

[0246] [ka]

[0247] Reaction Scheme 2 is a reaction for producing a compound represented by formula 4 by reacting a 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 a substitution reaction.

[0249] The substitution reaction may be a nucleophilic acyl substitution reaction.

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

[0251] As an example, when a compound of formula 2 is to be reacted with an amine group (-NH2) of an Fc binding peptide, Y4 can be N. When the peptides of SEQ ID NOs: 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 thus a displacement reaction can be performed.

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

[0253] As an example, Y4 can be O when the compound of formula 2 is reacted with any one of the hydroxy groups (-OH) of an Fc binding peptide.

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

[0255] In this case, the carbon of the carbonyl group that is most closely linked to R', ie, the electrophilic carbon atom of the first carbonyl group, will bear the largest partial positive charge (δ+).

[0256] The method for preparing an antibody-payload conjugate can include (2) reacting a linker-Fc binding peptide with an antibody to obtain an antibody that includes a first click chemistry functional group.

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

[0258] The reaction between the linker-Fc binding peptide conjugate and the 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 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 or partially positively charged atom may be the electrophilic carbon of a carbonyl group contained in the linker structure.

[0259] In an example, an antibody comprising a first click chemistry functional group can be represented by Formula 5:

[0260] [ka]

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

[0262] R″ is one of the click chemistry functional groups; The click chemistry functional group is any one or more of an alkyne, a cycloalkyne, such as cyclooctyne and cyclononyne (e.g., bicyclo[6.1.0]non-4-yn-9-ylmethanol), a trans-cyclooctene, a nitrone, a nitrile oxide, an azide, a conjugated diene, and a dienophile, where the cycloalkyne, the conjugated diene, and the diene are the same as described above.

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

[0264] n is an integer equal to or greater than 1 and equal to or less than 4. As an example, n can be 2. In this case, R" can be linked to lysine 246 or lysine 248 contained in each of the two Fc residues in one antibody to generate such a structure. In another example, n can be 4. In this case, R" can be linked to lysine 246 and lysine 248 contained in each of the two Fc residues in one antibody to generate such a structure.

[0265] n may be 2 and the nitrogen atom linked to Ab may be contained in lysine 246 of both Fc of the antibody. Alternatively, n may be 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 for formulas 3 and 5 are the same as previously described.

[0269] In this case, if the peptides of SEQ ID NO:1 to 5 contain 2,3-diaminopropionic acid, 2,4-diaminobutyric acid, ornithine or lysine, respectively, at residue 6, all the corresponding residues have similar structures, differing only in the length of the carbon backbone containing the free amine group, and therefore all the peptides of SEQ ID NO:1 to 5 exhibit the property of binding to the Fc domain of an antibody.

[0270] In another example, an antibody comprising a first click chemistry functional group can be represented by Formula 6:

[0271] [ka]

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

[0273] R''' is a substituted or unsubstituted 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 Aryl alkylene, substituted or unsubstituted C 8~16 Arylalkenylene, substituted or unsubstituted C 3~10 Cycloalkylene, substituted or unsubstituted C 3~10 Heterocycloalkylene or substituted or unsubstituted C 5~12heteroaryl, where the heteroalkylene, heterocycloalkylene or heteroaryl contains at least one or more of N, O or S; The substituents 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.

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

[0275] Further, in this case, the Fc binding peptide can comprise a first click chemistry functional group. The click chemistry functional group is any one or more of alkyne, cycloalkyne, such as cyclooctyne and cyclononyne (e.g., bicyclo[6.1.0]non-4-yn-9-ylmethanol), trans-cyclooctene, nitrone, nitrile oxide, azide, conjugated diene, and dienophile, where the cycloalkyne, conjugated diene, and diene are the same as those described above. In addition, the Fc binding peptide can comprise a first click chemistry group at an amino acid residue adjacent to the N-terminus of the sequence. Furthermore, the Fc binding peptide can comprise a first click chemistry group at the N-terminus of the sequence. Alternatively, the Fc binding peptide can comprise a first click chemistry functional group at an amino acid residue adjacent to the C-terminus of the sequence. Furthermore, the Fc binding peptide can comprise a first click chemistry functional group at the C-terminus of the sequence.

[0276] Fp is an Fc binding peptide. Furthermore, Fp can be any one peptide selected from formula 13 or 14. The details of formula 13 and 14 are the same as those described above.

[0277] Fp may be linked via Y3 at 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 equal to or greater than 1 and equal to or less than 4. As an example, n can be 2. In this case, Fp can be linked to lysine 246 or lysine 248 contained in each of the two Fcs in one antibody to generate such a structure. In another example, n can be 4. In this case, R" can be linked to lysine 246 and lysine 248 contained in each of the two Fcs in one antibody to generate such a structure.

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

[0280] The antibody of Formula 6 can be produced by the reaction represented by Reaction Scheme 4 below.

[0281] [ka]

[0282] In this case, the details for Equations 4 and 6 are the same as previously described.

[0283] In this case, if the peptides of SEQ ID NO:1 to 5 contain 2,3-diaminopropionic acid, 2,4-diaminobutyric acid, ornithine or lysine, respectively, at residue 6, all the corresponding residues have similar structures, differing only in the length of the carbon backbone including the free amine group, and therefore all the peptides of SEQ ID NO:1 to 5 exhibit the property of binding to the Fc domain of an antibody.

[0284] The method for preparing an antibody-payload conjugate can include (3) reacting an antibody that includes a first click chemistry functional group with a payload that includes a second click chemistry functional group that is complementary to the first click chemistry functional group, thereby preparing the antibody-payload conjugate.

[0285] The payload can include an active moiety. The active moiety can be one or more selected from a nucleic acid, a peptide, and a chemical compound. The active moiety can be any one selected from the group consisting of a drug molecule, an imaging moiety, an optical agent, a vitamin, and a toxin. In certain embodiments, the active moiety can be a drug molecule. The drug molecule can be a prodrug, a precursor drug, or a drug. The drug molecule can be an anti-cancer drug, an anti-inflammatory agent, another anti-disease agent, and an anti-microbial agent (anti-bacterial agent, anti-fungal agent, anti-viral agent). In certain embodiments, the drug molecule can be mertansine (DM1). In certain embodiments, the payload can include two or more drug molecules. In certain embodiments, the active moiety can be an imaging moiety. The imaging moiety can be a contrast agent, a radioisotope, a fluorescent material. In certain embodiments, the active moiety can be, but is not limited to, an optical agent, a vitamin, a toxin, and the like.

[0286] In addition, the payload comprises a second click chemistry functional group, which is any one or more of alkyne, cycloalkyne, such as cyclooctyne and cyclononyne (e.g., bicyclo[6.1.0]non-4-yn-9-ylmethanol), trans-cyclooctene, nitrone, nitrile oxide, azide, conjugated diene, and dienophile, wherein the cycloalkyne, conjugated diene, and diene are the same as those described above.

[0287] In an example, the antibody-payload conjugate can be represented by Formula 7:

[0288] [ka]

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

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

[0291] B may be any one of the structures formed by click chemistry of the first click chemistry functional group and the second click chemistry functional group. For example, B may be any one of the structures that can be produced by the reaction of two selected from alkynes, cycloalkynes, such as cyclooctynes ​​and cyclononynes (e.g., bicyclo[6.1.0]non-4-yn-9-ylmethanol), trans-cyclooctenes, nitrones, nitrile oxides, azides, conjugated dienes, and dienophiles. In certain embodiments, B is

[0292] [ka]

[0293] where A1 may be linked to the antibody and A2 may be linked to Am, or A1 may be linked to Am and A2 may be linked to the antibody.

[0294] Am is an active moiety or a structure containing it. In one embodiment, Am can contain two or more active moieties. The content regarding the active moiety is the same as that described in the description of the payload.

[0295] n is an integer equal to or greater than 1 and equal to or less than 4. As an example, n can be 2. In this case, the payload can be linked to lysine 246 or lysine 248 contained in each of the two Fcs in an antibody to generate such a structure. In another example, n can be 4. In this case, R" can be linked to lysine 246 and lysine 248 contained in each of the two Fcs in an antibody to generate such a structure.

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

[0297] The compound represented by formula 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 that described when formula 5 is described above.

[0300] Additionally, R'''' is a second click chemistry functional group, which is the same as the payload depicted, if any.

[0301] In an example, the antibody-payload conjugate can be represented by Formula 8:

[0302] [ka]

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

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

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

[0306] [ka]

[0307] where A1 may be linked to the antibody and A2 may be linked to Am, or A1 may be linked to Am and A2 may be linked to the antibody.

[0308] Fp is an Fc binding peptide. Furthermore, Fp can be any one peptide selected from formula 13 or 14. The details of formula 13 and 14 are the same as those described above.

[0309] Fp may be linked via Y4 at 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 moiety or a structure containing it. In certain embodiments, Am can contain two or more active moieties. The content regarding the active moiety is the same as that described in the description of the payload.

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

[0312] n is an integer equal to or greater than 1 and equal to or less than 4. As an example, n can be 2. In this case, such a structure can be generated by linking the payload to lysine 246 or lysine 248 contained in each of the two Fc's in one antibody. In another example, n can be 4. In this case, such a structure can be generated by linking the payload to lysine 246 and lysine 248 contained in each of the two Fc's in one antibody.

[0313] n may be 2 and the nitrogen atom linked to Ab may be contained in lysine 246 of both Fc of the antibody. Alternatively, n may be 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 formula 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 described 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 involved in the first click chemistry functional group, but it is preferred that the amino acid residue is not residue 6. This is because residue 6 is the residue designed for the substitution reaction with the linker. Through the process of artificial synthesis of the peptide, the Fc binding peptide can include a first click chemistry functional group at the amino acid residue adjacent to the N-terminus of the sequence. In certain embodiments, the Fc binding peptide can include a first click chemistry functional group at the N-terminus of the sequence. In certain embodiments, the Fc binding peptide can include a first click chemistry functional group at the amino acid residue adjacent to the C-terminus of the sequence. In certain embodiments, the Fc binding peptide can include a first click chemistry functional group at the C-terminus of the sequence.

[0317] Additionally, R'''' is a second click chemistry functional group, which is the same as the payload depicted, if any.

[0318] The present application can provide linkers that aid in the transport and binding reactions so that the payload can be linked to a desired target molecule.

[0319] The linker or linker moiety can be directly linked to the targeting molecule. A linker moiety that is directly linked to the targeting molecule can be linked to a payload.

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

[0321] For example, the linker moiety can be part of an R" or an R"-containing residue.

[0322] For example, the linker moiety can be that portion of the residue of the linker excluding R″.

[0323] The targeting molecule to which the linker moiety is attached can be linked to the payload via a click reaction, ie, a reaction between click chemistry functional groups, including but not limited to a click reaction.

[0324] In an exemplary embodiment, when the linker has the structure of Formula 1: The linker moiety that is linked to the target molecule can include a click chemistry functional group.

[0325] In this case, the linker moiety linked to the target molecule can participate in the click reaction together with the payload.

[0326] Such linkers may not affect the biological activity of the target molecule, i.e., the linker has little or no effect on the half-life, excretion, blood stability, etc. of the target molecule.

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

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

[0329] For example, a linker can increase the half-life of a drug in the blood by linking a protein and a compound that has a short intracellular half-life, e.g., a drug may be linked to a targeting molecule that has a long intracellular half-life.

[0330] In another illustrative embodiment, when the linker has the structure of Formula 2: The linker moiety that is linked to the target molecule cannot contain a click chemistry functional group.

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

[0332] The peptides, polypeptides, proteins and / or compounds can be linked to a payload by a click reaction.

[0333] Such linkers can affect the biological activity of target molecules. When linkers affect the biological activity of target molecules, they affect the half-life, excretion or blood stability of target molecules. For example, when linkers are indirectly linked to target molecules, they can reduce the half-life of target molecules, thereby rapidly releasing payload in vivo.

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

[0335] By way of example, the target molecule may 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, but is not limited to, an antibody or a partial fragment of an antibody.

[0337] By way of example, an Fc binding peptide can have binding affinity with an antibody.

[0338] For example, an Fc binding peptide can have binding affinity with an IgG antibody. An Fc binding peptide can have specific binding affinity with a particular domain of an IgG antibody.

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

[0340] For example, an Fc binding peptide may have specificity for the constant domain of the heavy chain of an IgG antibody, in which 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, the Fc binding peptide can include a click chemistry functional group.

[0343] The present invention provides a method for treating cancer, comprising administering an antibody-payload conjugate, wherein 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, gastric cancer, gastrointestinal cancer, head and neck cancer, Kaposi's sarcoma, renal cancer, leukemia, liver cancer, lung cancer, lymphoma, melanoma, myeloma, ovarian cancer, pancreatic cancer, penile cancer, prostate cancer, testicular germ cell cancer, thymoma, and thymic carcinoma. Additionally, the cancer can 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, gastric cancer, gastrointestinal cancer, head and neck cancer, Kaposi's sarcoma, renal cancer, leukemia, liver cancer, lung cancer, lymphoma, melanoma, myeloma, ovarian cancer, pancreatic cancer, penile cancer, prostate cancer, testicular germ cell cancer, thymoma, and thymic carcinoma. Further, the cancer can be breast cancer.

[0345] Hereinafter, the invention will be described in more detail through examples.

[0346] These examples are provided only to more specifically describe the present invention, and it is obvious to those skilled in the art to which the present invention pertains that the scope of the present invention is not limited by these examples. The numbers shown in the names of compounds in the following examples are written with reference to the drawings. EXAMPLES

[0347] Synthesis method and structure 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 equiv.) 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 equiv.) of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDCi) and 24 mL (138 mmol, 2.0 equiv.) of N,N-diisopropylethylamine (DIPEA) were slowly added dropwise thereto, the resulting solution was stirred for 20 minutes, and then 8 mL (68.7 mmol, 1.0 equiv.) of O-benzylhydroxylamine was slowly added dropwise thereto. 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 using 10% citric acid solution and dried using saturated salt solution and sodium sulfate. The target compound was used in the next 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] Example 1-2 Synthesis and structural confirmation of compound 2 12.9g of compound 1 was dissolved in 200mL of N,N-dimethylformamide (DMF), and the resulting solution was stirred at 0°C. 24mL (160mmol, 3.08eq) of 1,8-diazabicyclo(5.4.0)undec-7-ene (DBU) and 10mL (160mmol, 3.08eq) of methyl iodide (MeI) were slowly added dropwise thereto. 0.2mL of diethylamine was slowly added dropwise thereto. After stirring the resulting solution for 20 hours, the reaction solvent was mixed with Celite and removed by concentration under reduced pressure, and the target compound was purified by column chromatography (EA:Hex=1:1) to obtain 7.6g (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 4.18g (15.8mmol, 1.0eq) of compound 2 was dissolved in 100mL of tetrahydrofuran (THF), and the resulting solution was stirred. 5mL of 2N lithium hydroxide (LiOH) was slowly added thereto dropwise at 0°C, and then the resulting solution was stirred for 3 hours. The reaction was stopped by adding 60mL of H2O thereto, and washing was performed twice on the aqueous layer using 150ml of EA. Then, the aqueous layer was titrated to pH 3.0 using 2N HCl solution, and the target compound was extracted three times using 50ml of EA. The organic layer was dried using saturated salt solution and sodium sulfate to obtain 2.46g (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 2.46g of compound 3 was dissolved in 40mL of methanol (MeOH), and then hydrogenation reaction was carried out in the presence of palladium on carbon (Pd / C) for 18h. After the reaction was stopped, Pd / C was removed via a short-path column, and then the target compound was purified and concentrated to obtain 1.2g (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 0.17g (1.06mmol, 1.0eq) of compound 4 was dissolved in 10mL of DCM, and the resulting solution was stirred. 0.22g (1.73mmol, 1.63eq) of exo-5-norbornene acid chloride and 0.2mL (1.1mmol, 1.0eq) of DIPEA were slowly added thereto dropwise at 0°C. After the resulting solution was stirred for 2 hours, the organic layer was washed three times using 10% citric acid solution, and dried using saturated salt solution and sodium sulfate. The target compound was used in the next reaction without purification (crude yield: 0.24g, 68%). TLC (DCM:MeOH = 10:1); R f = 0.3 Expected MW (M+H)+: 282.13g / mol Measured MW (M+H)+: 282.1g / mol

[0352] Examples 1-6 Synthesis and structural confirmation of compound I 0.24g (0.72mmol, 1.0eq) of compound 5 was dissolved in 3mL of DCM, and the resulting solution was stirred. 0.26g (0.87mmol, 1.2eq) of N,N,N',N'-tetramethyl-O-(N-succinimidyl)uronium tetrafluoroborate (TSTU) and 0.15mL (0.87mmol, 1.2eq) of DIPEA were slowly added dropwise thereto. After the resulting solution was stirred for 1 hour, the organic layer was washed three times using 10% citric acid solution, and dried using saturated salt solution and sodium sulfate. The target compound was purified via column chromatography (EA:Hex = 1:1) to obtain 0.04g (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] Synthesis method and structure confirmation of compound II (Cis linker: NHS & norbornene, formula 2-1-2) Example 2-1 Synthesis and structural confirmation of compound 6 0.1g (0.88mmol, 1.0eq) of N-methylhydroxylamine hydrochloride was dissolved in 2mL of tetrahydrofuran (THF) at -25°C, and the resulting solution was stirred. 0.07g (0.88mmol, 1.0eq) of glutaric anhydride was added thereto, and the resulting solution was stirred for 10 minutes, and then 0.25mL (1.76mmol, 2.0eq) of triethylamine was slowly added thereto by dropwise. After 1 hour, 0.14g (0.88mmol, 1.0eq) of (1S,2R,4S)-bicyclo[2.2.1]hept-5-ene-2-carbonyl chloride and 0.12mL (0.88mmol, 1.0eq) of triethylamine were slowly added thereto by dropwise, and then 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 10% citric acid solution, and dried with 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 eq.) of TSTU and 0.15 mL (0.88 mmol, 1.0 eq.) of N,N-diisopropylethylamine (DIPEA) were slowly added dropwise thereto. After reacting for 1 hour, the reaction solution was washed three times using 10% citric acid solution, and dried using saturated sodium bicarbonate solution, saturated salt solution, and sodium sulfate. The target compound was purified via column chromatography (EA:Hex = 1:1). TLC (EA:Hex = 2:1); R f = 0.4 (see Figure 3)

[0355] Example 2-3 Confirmation of the structure of compound II Final compound II was synthesized by the method specified in Examples 2-1 and 2-2, and the presence of isomers was confirmed through HPLC analysis as illustrated in Figure 6. HPLC purification was performed based on the analysis conditions illustrated in Figure 6, and compound II (cis) was obtained through HPLC analysis with the purity as shown in Figure 7. Analysis of final compound II was completed by confirming the structural analysis of 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 isomeric compound 6 (Cis linker or Trans linker: NHS & norbornene) A high performance liquid chromatograph (HPLC) instrument was used to confirm the cis and trans structures of the isomers of compound 6. For HPLC analysis, a Waters 2695 HPLC model from Waters was used, and an Xbridge C18 (4.6×250 mm, 5 μm; Waters) was used as the analytical column, and in the case of 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. According to the above description, the characteristics of each structure were analyzed by absorbance at a wavelength of 220 nm.

[0357] As illustrated in Figure 3, it was confirmed that a peak was observed at 14.89 minutes for the cis-form structure of compound 6 and at 15.32 minutes 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 a molecular weight of 282 with one hydrogen molecule bound was observed as the material corresponding to each peak. 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: Synthesis of FcBP(6Lys)-norbornene List of Fmoc amino acids used, order of introduction of Fmoc amino acids used 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 mmol. 0.5 g of transparent amide resin (0.48 mmol / g, Peptides International, USA) was placed in a synthesis reactor, and 1 mmol 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 Fmoc-amino acids were activated to attach the activated residues to a clear amide resin, successively starting from the C-terminal amino acid.

[0363] Fmoc removal was performed in 20% piperidine in DMF, and for activation and introduction of the residue, the amino acid prepared according to the sequence was mixed with 2 mL of 0.5 M HOBt in DMF, 2 mL of 0.5 M HBTU in DMF, and 174 μL of DIPEA for 5 min, and then the resulting mixture was poured into the reactor containing the resin and mixed for 2 h.

[0364] The introduction reaction was confirmed by the Kaiser test method, and if no reaction was confirmed, the introduction reaction was repeated once more or capped with 20% Ac2O in DMF solution. The resin was thoroughly washed with DMF and DCM before moving to the next step in each introduction reaction and Fmoc removal process. These processes were repeated until the target peptide sequence was completed.

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

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

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

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

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

[0370] (e) Purification The peptide precipitates obtained by lyophilization in step (d) were purified and lyophilized under the prep-LC conditions shown in Table 5. Each of the obtained peptides was confirmed to have a purity of 90% or more by analytical HPLC, and the results are illustrated in FIG. 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. Measurement equipment: 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 FIG.

[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, and 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, and if the reaction did not stop, DIPEA was added thereto in 1 equivalent amount to observe the termination of the reaction.

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

[0379] The results are illustrated in FIG.

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

[0381] The results are illustrated in FIG.

[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-norbornene Weinreb amide)-FcBP was synthesized in DMF, and 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, and if the reaction did not stop, DIPEA was added thereto in 1 equivalent amount to observe the termination of the reaction.

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

[0387] The results are illustrated in FIG.

[0388] Example 4-3-2: Identification of compound II-FcBP (6Lys)-norbornene Measurement equipment: Waters Quattro Premier XE Calculated molecular weight: 2351.69g / mol Measured molecular weight (M / 2+H) 2+ : 1176.42g / mol The results are illustrated in FIG. EXAMPLES

[0389] Confirmation of the synthesis and structure of antibody-click chemistry chemicals Example 5-1: Antibody-Norbornene Example 5-1-1: Method for the synthesis of 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 then the reaction was carried out. The reaction took more than one week at room temperature, and reaction monitoring and termination were confirmed by HIC-HPLC. For purification, purification was carried out via three dialysis (pH 5.5, 20 mM histidine acetate buffer) and size exclusion chromatography (molecular weight cutoff 40 kDa).

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

[0391] Example 5-1-2: Synthesis of Trastuzumab-Norbornene (2) Compound II - Introduction reaction using 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 then the reaction was 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 carried out via three dialysis (pH 5.5, 20 mM histidine acetate buffer) and size exclusion chromatography (molecular weight cutoff 40 kDa), yielding 135 mg attributable to 150 mg of trastuzumab. (Yield=90%)

[0392] The reaction of compound II-FcBP(6Lys)-norbornene with 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 Herceptin-Norbornene binding The identity of the antibody intermediate (FIG. 18) in which the FcBP norbornene linker was included in the antibody was verified based on mass spectrometry. Measurement equipment: 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, 5,000Da 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 results of the analysis are illustrated in FIG. EXAMPLES

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

[0397] Example 6-1-2: Trastuzumab-DM1 Purification Method To obtain highly pure antibody-payload conjugates, dialysis using 20 mM histidine acetate solution at pH 5.5 and HIC purification using fast protein liquid chromatography (FPLC) were performed.

[0398] The HIC purification conditions were as follows. FPLC Model: AKTA Pure Flow rate: 1mL / min Column: HiPrep Butyl FF16 / 10 column Dissolution medium: (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 FIG.

[0400] The above method yielded 67 mg of trastuzumab-DM1 with the drug conjugated to two sites (drug-antibody ratio = 2). (Yield = 60%)

[0401] Example 6-1-3: Confirmation of trastuzumab-DM1 binding The identity of the antibody-payload conjugates containing the FcBP linker and drug was verified based on mass spectrometry. Measurement equipment: 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, 5,000Da Voltage: Ion source I 25.00kV, Ion source II 23.00kV, Lens 9.00kV Calculated molecular weight: 155,493g / mol Measured molecular weight: 155,523g / mol (approximately M+Na)

[0402] The results of the analysis are illustrated in FIG. EXAMPLES

[0403] Evaluation of the pharmaceutical efficacy of novel antibody-drug conjugates (ADCs, trastuzumab-DM1 conjugates) at the cellular level (in vitro cytotoxicity tests) The efficacy of the antibody-payload conjugate (ADC, trastuzumab-DM1 conjugate) was evaluated based on the target marker level of cancer cells, and cytotoxicity experiments were performed using NCI-N87 and BT474 for positive expressing cell lines of the target antigen Her2, and MDA-MB-468 for negative cell lines. As a result of observations by treating cells at each concentration with the produced antibody-drug conjugate in Her2-overexpressing cells NCI-N87 and BT474, excellent anti-cancer effects with IC50 values ​​of 82.1ng / mL and 29.6ng / mL were confirmed. When compared with the commercially available Herceptin ADC, Kadcyla, the effect was confirmed at a similar level, so the usefulness as a new ADC could be confirmed.

[0404] The results for the experiments are illustrated in Figures 24 to 27. EXAMPLES

[0405] Evaluation of the pharmaceutical efficacy of novel antibody-payload conjugates (ADCs, trastuzumab-DM1 conjugates) at the animal level (in vivo cytotoxicity tests) The NCI-N87 gastric cancer cell line that overexpresses the target antigen is subcutaneously implanted to prepare xenograft models, and the xenograft models are divided into four groups to carry out anticancer efficacy tests on the administered materials.The BALB / c nude mice used in this experiment are deficient in T cells, and cancer cells are easily implanted in mice, so the mice are used as a suitable model for anticancer efficacy tests using rodents.

[0406] (a) Preparation of cell lines RPMI1640 medium (Gibco, 22400-089) containing 10% heat-inactivated 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 and cultured at 37°C in a 5% CO2 incubator. The culture flask was washed using PBS, and the cells were isolated by diluting 2.5% trypsin-EDTA (Gibco, 15090) 10-fold and then adding the diluted trypsin-EDTA thereto, after which the cells were centrifuged (1,000 rpm, 5 min) and the supernatant was discarded, and then the cell suspension was obtained in fresh medium. After confirming viability using a microscope, 1.25×10 7 Cell lines were prepared by diluting the cell suspension in a 1:1 mixture of medium and Matrigel at a concentration of cells / mL.

[0407] (b) Transplantation of cell lines The cell line is prepared according to the method described in "4. 3) (4) Preparation of cell line". When the cell line is prepared, the cell line is resuspended and homogenized, and the prepared cell line is immediately administered to the animal. When the cell line is transplanted, the back of the animal is disinfected with 70% alcohol, the skin on the back of the neck is pulled with the thumb and index finger to create a gap between the skin and muscle, and then a syringe needle equipped with a 26-gauge needle is inserted from the front of the animal into the subdermal space between the thumb and index finger to inject 2.5 x 10 cell lines. 6 The cells were administered subcutaneously at a dose of 0.2 mL per animal. During the acclimation period, healthy animals were selected and the selected animals were inoculated with the cell line. Thus, the size of the cell line at the site of implantation was approximately 100 mm. 3 From 150mm 3 If 1 is reached, the tumors are distributed according to ranked tumor size such that the tumor sizes in each group are as evenly distributed as possible.

[0408] (c) Determination of study group composition, dosage, and administration method Cell line = NCI-N87 Mouse type = BALB / c nude (CAnN.Cg-Foxn1nu / CrljOri) Number of groups = 5 Method of administration: Intravenous injection (using a 26-gauge needle syringe) Dose = 5mg / kg Number of doses = 1 / 2 days, 3 doses 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 inspection items General symptoms During the dosing and observation period, the type, date of onset, and severity of symptoms, including death, were observed daily and recorded for each animal. Any animal experiencing worsening symptoms was placed in isolation.

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

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

[0412] (e) Results The groups injected with phosphate buffered saline (PBS) and Herceptin (Trastuzumab) showed no tendency to inhibit tumor growth during the 5-week observation period. The antibody-payload conjugate (Herceptin-DM1) produced by the present application was confirmed to have superior ability to inhibit tumor growth compared to Herceptin, thus confirming that the antibody-payload conjugate successfully functioned as an ADC.

[0413] The above related results are illustrated in FIGS. EXAMPLES

[0414] Synthesis and validation 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: Synthesis of FcBP (L6Dap, L6Dab, L6Orn, L6Lys)-norbornene FcBP(L6Dap): List of Fmoc amino acids used and the order of incorporation 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 mmol. 0.5 g of transparent amide resin (0.48 mmol / g, Peptides International, USA) was placed in a synthesis reactor, and 1 mmol 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 Fmoc-amino acids are activated to attach the activated residues to a transparent amide resin, and the reactions are carried out sequentially starting from the C-terminal amino acid.

[0422] Fmoc removal was carried out in 20% piperidine in DMF, and for activation and introduction of the residue, the amino acid prepared according to the sequence was mixed with 2 mL of 0.5 M HOBt in DMF, 2 mL of 0.5 M HBTU in DMF, and 174 μL of DIPEA for 5 min, and then the resulting mixture was poured into the reactor containing the resin and mixed for 2 h.

[0423] The introduction reaction was confirmed by the Kaiser test method, and if no reaction was confirmed, the introduction reaction was repeated once more or capped with 20% Ac2O in DMF solution. The resin was thoroughly washed with DMF and DCM before moving to the next step in each introduction reaction and Fmoc removal process. These processes were repeated until the target peptide sequence was completed.

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

[0425] The progress of the reaction was checked by the Kaiser test method, and if it was determined that unreacted amines remained, the reaction time was further extended from 1 to 3 hours, or the reaction solution was emptied and the reaction process described above was repeated again. Removal of the N-terminal Fmoc protecting group was carried out using 20% ​​piperidine in DMF, and then the peptide-attached resin was dried and weighed.

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

[0427] (d) The peptide was cleaved from the resin by stirring 250 mg of peptide-attached resin prepared in step (c) 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 min. The cleavage mixture was filtered, the filtrate was concentrated to about half with nitrogen gas, and then ether was poured in to precipitate the peptide. The precipitated peptide was further washed three times with ether and dried with nitrogen gas. After dissolving the dried precipitate in water containing 0.1% TFA-30% ACN, the resulting solution was stirred for 6 h and then concentrated.

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

[0429] (e) Purification The peptide precipitates obtained by lyophilization in step (d) were purified and lyophilized under the prep-LC conditions shown in Table 6. Each of the obtained peptides was confirmed to have a purity of 90% or more.

[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-norbornene Weinreb amide)-FcBP was synthesized in DMF, and 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 in DMF, and the resulting solution was stirred.

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

[0434] The results are illustrated in FIG.

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

[0436] Example 9-1-4: Synthesis of compound II-FcBP (L6Dab)-norbornene Compound II (cis-norbornene Weinreb amide)-FcBP was synthesized in DMF, and 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 termination 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 the purity was also confirmed using HPLC (purity; >99% (HPLC), yield: 82%).

[0438] The results are illustrated in FIG.

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

[0440] Example 9-1-6: Synthesis of compound II-FcBP (L6Orn)-norbornene Compound II (cis-norbornene Weinreb amide)-FcBP was synthesized in DMF, and 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 the reaction was confirmed to have stopped, the reaction solution was concentrated and compound II-FcBP (L6Orn)-norbornene was purified by preparative HPLC. After purification, 14.8 mg was obtained by lyophilization, and the purity was also confirmed using HPLC (purity; >99% (HPLC), yield: 88%).

[0442] The results are illustrated in FIG.

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

[0444] Example 9-1-8: Synthesis of compound II-FcBP (L6Lys)-norbornene Compound II (cis-norbornene Weinreb amide)-FcBP was synthesized in DMF, and 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 the reaction was confirmed to have stopped, the reaction solution was concentrated and compound II-FcBP (6Lys)-norbornene was purified by preparative HPLC. After purification, 15.4 mg was obtained by lyophilization, and the purity was also confirmed using HPLC (purity; >99% (HPLC), yield: 91%).

[0446] The results are illustrated in FIG.

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

[0448] Validation of antibody binding efficiency of site-specific interactomes according to carbon length Example 10-1. Compound II-FcBP (L6Dap, L6Dab, L6Orn, L6Lys)-norbornene for the generation of site-specific antibody-norbornene conjugates Example 10-1-1: Compound II - Synthesis of Trastuzumab-Norbornene Based on FcBP (L6Dap)-Norbornene Compound II-FcBP (L6Dap)-norbornene was used to synthesize Ab (Lys246 / 248)-norbornene in phosphate buffered saline (PBS) buffer at pH 7.4. To introduce norbornene into two specific sites of antibody (Trastuzumab 4mg / mL, 1mL), 6 equivalents of compound II-FcBP (L6Dap)-norbornene per antibody were put into the reaction solution, and then the reaction was carried out. Regarding reaction temperature and time, it took 12 hours to carry out the reaction at room temperature, and reaction monitoring and termination were confirmed by HIC-HPLC. Trastuzumab exhibits a peak at 6.3 to 6.4 minutes on HIC-HPLC. When FcBP (L6Dap)-norbornene molecule is bound to only one of both binding sites of Trastuzumab, a peak is observed at 8 minutes on HIC-HPLC. When FcBP (L6Dap)-norbornene molecules are conjugated to both sites of trastuzumab, a peak is observed at 9 minutes on HIC-HPLC. Monitoring of the conjugation reaction with an antibody based on compound II-FcBP (L6Dap)-norbornene is illustrated in Figure 38. Observing a peak at 9.074 minutes confirmed the synthesis of an antibody-payload conjugate with DAR=2.

[0449] Example 10-1-2: Compound II - Synthesis of Trastuzumab-Norbornene Based on FcBP(L6Dab)-Norbornene Compound II-FcBP (L6Dab)-norbornene was used to synthesize Ab (Lys246 / 248)-norbornene in phosphate buffered saline (PBS) buffer at pH 7.4. To introduce norbornene into two specific sites of antibody (Trastuzumab 4mg / mL, 1mL), 6 equivalents of compound II-FcBP (L6Dab)-norbornene per antibody were put into the reaction solution, and then the reaction was carried out. Regarding reaction temperature and time, it took 12 hours to carry out the reaction at room temperature, and reaction monitoring and termination were confirmed by HIC-HPLC. Trastuzumab exhibits a peak at 6.3 to 6.4 minutes on HIC-HPLC. When FcBP (L6Dab)-norbornene molecule is bound to only one of both binding sites of Trastuzumab, a peak is observed at 8 minutes on HIC-HPLC. When FcBP (L6Dab)-norbornene molecule is conjugated to both sites of trastuzumab, a peak is observed at 9 minutes on HIC-HPLC. Monitoring of the conjugation reaction with antibody 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: Compound II - Synthesis of Trastuzumab-Norbornene Based on FcBP(L6Orn)-Norbornene Compound II-FcBP (L6Orn)-norbornene was used to synthesize Ab (Lys246 / 248)-norbornene in phosphate buffered saline (PBS) buffer at pH 7.4. To introduce norbornene into two specific sites of antibody (Trastuzumab 4mg / mL, 1mL), 6 equivalents of compound II-FcBP (L6Orn)-norbornene per antibody were put into the reaction solution, and then the reaction was carried out. Regarding reaction temperature and time, it took 12 hours to carry out the reaction at room temperature, and reaction monitoring and termination were confirmed by HIC-HPLC. Trastuzumab exhibits a peak at 6.3 to 6.4 minutes on HIC-HPLC. When FcBP (L6Orn)-norbornene molecule is bound to only one of both binding sites of Trastuzumab, a peak is observed at 8 minutes on HIC-HPLC. When FcBP (L6Orn)-norbornene molecules are bound to both binding sites of trastuzumab, a peak is observed at 9 minutes on HIC-HPLC. Monitoring of the binding reaction with an antibody based on compound II-FcBP (L6Orn)-norbornene is illustrated in Figure 40. Observing a peak at 8.975 minutes confirmed the synthesis of an antibody-payload conjugate with DAR=2.

[0451] Example 10-1-4: Compound II - Synthesis of trastuzumab-norbornene based on FcBP (L6Lys)-norbornene Compound II-FcBP (L6Lys)-norbornene was used to synthesize Ab (Lys246 / 248)-norbornene in phosphate buffered saline (PBS) buffer at pH 7.4. To introduce norbornene into two specific sites of antibody (Trastuzumab 4mg / mL, 1mL), 6 equivalents of compound II-FcBP (L6Lys)-norbornene per antibody were put into the reaction solution, and then the reaction was carried out. Regarding reaction temperature and time, it took 12 hours to carry out the reaction at room temperature, and reaction monitoring and termination were confirmed by HIC-HPLC. Trastuzumab exhibits a peak at 6.3 to 6.4 minutes on HIC-HPLC. When FcBP (L6Lys)-norbornene molecule is bound to only one of both binding sites of Trastuzumab, a peak is observed at 8 minutes on HIC-HPLC. When FcBP (L6Lys)-norbornene molecules are attached to both sites, a peak is observed at 9 minutes on HIC-HPLC. Monitoring of the binding reaction with an antibody 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. 1. A method for producing an antibody-payload conjugate, comprising: reacting an antibody comprising a first click chemistry functional group with a payload to produce an antibody-payload conjugate; The antibody comprising the first click chemistry functional group has the structure of Formula 6: 【Chemistry 1】 During the ceremony, Ab is an antibody or a partial fragment of an antibody; n is an integer greater than or equal to 1 and less than or equal to 4, R''' is unsubstituted C 1～6 Alkylene, unsubstituted C 2～6 Alkenylene, unsubstituted C 2～6 Alkynylene or unsubstituted C 1～6 heteroalkylene, the heteroalkylene containing 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 is NH and Y 4 is derived from amino acid residue X of the Fc-binding peptide, the nitrogen atom linked to Ab is derived from at least one of the lysine 246 and lysine 248 residues of at least one heavy chain of the antibody; the payload comprises a second click chemistry functional group capable of undergoing a click chemistry reaction with the first click chemistry functional group; The antibody-payload conjugate is produced by a click chemistry reaction between a first click chemistry functional group of an antibody comprising a first click chemistry functional group and a second click chemistry functional group of a payload. method.

2. The method of claim 1, wherein the antibody comprises the Fc region of an IgG antibody.

3. 3. The method of claim 1 or 2, wherein n is 2.

4. n is 2, The nitrogen atoms linked to Ab are derived from lysine 248 residues in both heavy chains of the antibody. The method according to claim 1 or 2.

5. R''' is unsubstituted C 1～5 Alkylene or unsubstituted C 1～5 5. The method of claim 1, wherein the alkyl group is a heteroalkylene.

6. R''' is unsubstituted C 3 6. The method of claim 1, wherein the alkylene is an alkylene.

7. 7. The method of any one of claims 1 to 6, wherein the first click chemistry functional group is selected from the group consisting of acetylene, transcyclooctene, cyclooctyne, diarylcyclooctyne, 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 dibenzocyclooctyne.

8. 8. The method of claim 1, wherein the click chemistry reaction is selected from the group consisting of a [3+2] cycloaddition, a thiol-ene reaction, a Diels-Alder reaction, an inverse electron demand Diels-Alder reaction, and a [4+1] cycloaddition.

9. 9. The method of claim 1, wherein the second click chemistry functional group is selected from the group consisting of acetylene, transcyclooctene, cyclooctyne, diarylcyclooctyne, 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 dibenzocyclooctyne.

10. 10. The method of claim 1, wherein the first click chemistry functional group is azide or norbornene.

11. the first click chemistry functional group is an azide and the second click chemistry functional group is a tetrazine; or The first click chemistry functional group is norbornene and the second click chemistry functional group is dibenzocyclooctyne; 11. The method according to any one of claims 1 to 10.

12. 12. The method of claim 1, wherein the payload further comprises an active moiety.

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

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

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

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

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

Images