Compounds for preparing antibody-loaded moiety conjugates and uses thereof

Abstract

The present application relates to a novel linker for bioconjugation, wherein the linker comprises two or more electrophilic carbon atoms of a carbonyl group and a click chemistry functional group; more specifically, to a linker through which a compound, peptide and / or protein can be directly and / or indirectly linked to a desired target molecule (i.e., target molecule) by a substitution reaction.

Description

Technical Field

[0001] The present invention relates to the field of bioconjugation. The present invention relates to a linker for preparing site-specifically bound antibody-load conjugates, an antibody-load conjugate prepared using the linker, and a method for preparing the antibody-load conjugate. More specifically, the present invention relates to a linker, wherein the linker comprises a carbonyl carbon having two or more different partial positive charges and having functional groups at both ends, and can be used to connect a compound, peptide, and / or protein to a biological (target) molecule through a substitution reaction. Background Art

[0002] Bioconjugation is the process of connecting at least two or more molecules, in this context, where at least one or more of the molecules is a bioactive molecule. Bioactive molecules are sometimes referred to as "target molecules" or "molecules of interest" and can be, for example, proteins (or peptides), glycans, nucleic acids (or oligonucleotides), lipids, hormones, or natural drugs (or fragments thereof, or combinations thereof). Linkers connecting two or more molecules are widely used in detection, diagnosis, biomarkers, and the like, by conjugating target-specific proteins, such as antibodies, to fluorescent materials.

[0003] At present, for the joint that is used for detection, diagnosis, treatment etc. as bioconjugate, for example, polyethylene glycol (PEG) is widely used in commerce, and this is because PEG is highly water-soluble, non-toxic, non-antigenic and does not cohere.Recently, along with the interest that increases by the therapeutic agent that cytotoxic drugs (anticancer drugs) are connected to antibodies to treat specific diseases, the research about the joint that can be bound to cytotoxic drugs to target molecule (such as antibody) is also being carried out actively.For the joint that can connect these two or more molecules, blood stability, compatibility, solubility, target specificity etc. in vivo all need to be considered.

[0004] At the same time, current linkers studied in the field of antibody-payload conjugates face challenges with their bioactivity, which can be reduced by the length and size of the antibody. For example, these can reduce half-life by blocking FcRn receptor binding, and hinder the production of similar antibody-drug conjugates due to difficulties in site-specific binding. Therefore, there is an urgent need to develop linkers that maintain the activity of the target molecule while exhibiting excellent in vivo blood stability, compatibility, solubility, and site-specificity.

[0005] In order to solve these problems, the inventors of the present invention have invented a linker that can connect the payload portion to the target molecule without affecting the biological activity of the target molecule. The linker comprises a carbonyl group having two or more different partial positive charges (δ+) and a leaving group and / or a click compound at both ends. The reactivity of the linker can be increased not only by increasing site specificity but also by increasing the water solubility of the target molecule by length adjustment. Since such a linker does not have harsh reaction conditions and has a high coupling rate for molecules containing target molecules as a bioconjugate, it is intended to provide a more stable and cost-effective linker. Summary of the Invention

[0006] Technical issues

[0007] The present application provides a linker with a novel trans structure and a preparation method thereof, wherein the linker has one or more click chemistry moieties and two or more electrophilic carbons of a carbonyl group.

[0008] The present application provides a linker with a novel cis structure and a preparation method thereof, wherein the linker with the novel cis structure comprises one or more click chemistry moieties and two or more electrophilic carbons of a carbonyl group.

[0009] Technical means

[0010] In order to solve the above problems of the present application, the present specification provides a linker that facilitates transfer and binding reactions to directly and / or indirectly connect a payload moiety to a target molecule.

[0011] In one aspect, the present application provides a compound represented by the following Formula 2:

[0012] [Formula 2]

[0013]

[0014] In Formula 2, R' is an ester activation moiety, R" is any one of an acetylene group, a trans-cyclooctene group, a cyclooctyne group, a diarylcyclooctyne group, a methyl ester phosphine group, a norbornene group, a methylcyclopropene group, an azetidinyl group, and a cyanide group, and R"' is a substituted or unsubstituted C 1-20 Alkylene, substituted or unsubstituted C 2-20 Alkenylene, substituted or unsubstituted C 1-10 Alkynylidene, substituted or unsubstituted C 1-10 Polymethylene, substituted or unsubstituted C 5-12 Aryl, substituted or unsubstituted C 5-14 Arylalkylene, substituted or unsubstituted C 8-16Arylalkenylene, substituted or unsubstituted C 3-10 Cycloalkylene, substituted or unsubstituted C 3-10 Heterocycloalkylene, or substituted or unsubstituted C 5-12 heteroaryl, and the heteroalkylene, heterocycloalkylene or heteroaryl contains at least one or more selected from the group consisting of N, O and S, and the substitution is substituted by a non-hydrogen substituent, and the non-hydrogen substituent is selected from the group consisting of -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, wherein Ra is H, C 1-6 Alkyl, C 5-12 Aryl, C 7-12 An aralkyl group or a heterocyclic group, Rb is F, Cl, Br or I, and X is O, N or S. In addition, the present application provides a compound wherein R" is selected from the group consisting of a norbornene group, a trans-cyclooctene group, a cyclooctyne group and a methylcyclopropene group. In addition, the present application provides a compound wherein R" is a norbornene group.

[0015] In addition, the present application provides a compound, wherein R'' is selected from substituted or unsubstituted C 1-10 Alkylene, substituted or unsubstituted C 1-10 Heteroalkylene and C 1-10The group consisting of polymethylene, the heteroalkylene contains at least one or more selected from the group consisting of N, O and S, and the substitution is substituted by a non-hydrogen substituent, and the non-hydrogen substituent is selected from -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( Any one or more of the group consisting of: -C(=O)2ORa, -S(=O)2NRa, -S(=O)Ra, -C(=O)Ra, alkylene-C(=O)Ra, -C(=S)Ra, -C(=O)ORa, alkylene-C(=O)ORa, -C(=O)O-, alkylene-C(=O)O-, -C(=S)ORa, -C(=O)SRa, -C(=S)SRa, -C(=O)NRaRa, alkylene-C(=O)NRaRa, -C(=S)NRaRa and -C(-NRa)NRaRa, Ra is C 1-6 Alkyl, C 5-12 Aryl, C 7-12 Aralkyl or heterocyclic group, Rb is F, Cl, Br or I, X is O, N or S. In addition, the present application provides wherein R "' is unsubstituted C 1-5 Alkylene or unsubstituted C 1-5 Polymethylene compounds.

[0016] In addition, the present application provides compounds wherein X is O.

[0017] In addition, the present application provides a compound, wherein the compound of Formula 2 is represented by the following Formula 2-1-2:

[0018] [Formula 2-1-2]

[0019]

[0020] In another aspect, the present application provides a method for preparing an antibody-loaded moiety conjugate, the method comprising: preparing a linker-Fc-binding peptide conjugate by reacting a linker having a structure of Formula 2 with an Fc-binding peptide; 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 loaded moiety 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-loaded moiety conjugate having the following structure of Formula 8:

[0021] [Formula 8]

[0022]

[0023] Wherein, Ab is an antibody, R'' is an unsubstituted C 1-5 Alkylene or unsubstituted C 1-5 Polymethylene, Y4 is N, Fp is an Fc-binding peptide, B is any structure formed by a click chemistry reaction of a first click chemistry functional group and a second click chemistry functional group, Am is an active portion or a structure containing an active portion, wherein the active portion is any one selected from the group consisting of a drug molecule, an imaging portion, 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.

[0024] In addition, the present application provides a method for preparing an antibody-loaded portion conjugate, wherein the Fc binding peptide is a peptide selected from the group consisting of the following formula 13 and formula 14,

[0025] in,

[0026] [Equation 13]

[0027] DCAWH-Xa-GELVWCT

[0028] [Equation 14]

[0029] DCAWHKGELVWCT

[0030] Among them, D is aspartic acid, C is cysteine, A is alanine, W is tryptophan, H is histidine, and Xa is G is glycine, E is glutamic acid, L is leucine, V is valine, T is threonine, and K is lysine, wherein m is an integer of 1 to 4, the cysteine ​​at the N-terminus and the cysteine ​​at the C-terminus are selectively linked to each other, n=2, and the nitrogen atom linked to the Ab is contained in lysine at position 246 or lysine at position 248 of both Fc residues of the antibody.

[0031] In another aspect, the present application provides an antibody-loaded moiety conjugate of the following formula 8:

[0032] [Formula 8]

[0033]

[0034] Wherein, Ab is an antibody, R″′ is an unsubstituted C 1-5 Alkylene or unsubstituted C 1-5Polymethylene, Y4 is N, Fp is an Fc-binding peptide, B is any structure formed by a click chemistry reaction of a first click chemistry functional group and a second click chemistry functional group, Am is an active portion or a structure containing an active portion, wherein the active portion is any one selected from the group consisting of a drug molecule, an imaging portion, 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.

[0035] In addition, the present application provides an antibody-loaded portion conjugate, wherein Fp is a peptide selected from the group consisting of the following formula 13 and formula 14,

[0036] in,

[0037] [Equation 13]

[0038] DCAWH-Xa-GELVWCT

[0039] [Equation 14]

[0040] DCAWHKGELVWCT

[0041] Among them, D is aspartic acid, C is cysteine, A is alanine, W is tryptophan, H is histidine, and Xa is G is glycine, E is glutamic acid, L is leucine, V is valine, T is threonine, and K is lysine, wherein m is an integer of 1 to 4, the cysteine ​​at the N-terminus and the cysteine ​​at the C-terminus are selectively linked to each other, and Fp is linked through Y4 at amino acid residue 6.

[0042] In addition, the present application provides an antibody-loaded portion conjugate, wherein B is wherein A1 is connected to the antibody and A2 is connected to Am, or A1 is connected to Am and A2 is connected to the antibody. In addition, the present application provides an antibody-loaded moiety conjugate, wherein B is

[0043] Furthermore, the present application provides an antibody-payload conjugate, wherein Am comprises an anticancer drug. Furthermore, the present application provides an antibody-payload conjugate, wherein the anticancer drug is maytansine (DM1). Furthermore, the present application provides an antibody-payload conjugate, wherein Am comprises two or more anticancer drugs.

[0044] Furthermore, the present application provides an antibody-loaded moiety conjugate, wherein the nitrogen atom attached to the Ab is contained in lysine 246 or lysine 248 of the Fc of the antibody.

[0045] Furthermore, the present application provides an antibody-loaded portion conjugate, wherein n is 2, and the nitrogen atom connected to the Ab is contained in lysine 246 or lysine 248 of the two Fcs of the antibody.

[0046] In another aspect, the present application provides a pharmaceutical composition for treating cancer, wherein the pharmaceutical composition comprises an antibody-load portion containing an anti-cancer drug.

[0047] In addition, the present application provides a pharmaceutical composition, wherein the cancer is breast cancer.

[0048] Beneficial effects

[0049] According to the technology disclosed in this specification, the following effects are achieved.

[0050] Compound 1 disclosed herein provides a linker capable of site-specifically attaching a payload moiety to an antibody. The linker has no effect on biological activity, such as antibody half-life, and can be useful as a bioconjugate for detection, diagnosis, biomarkers, and anti-cancer therapeutics.

[0051] In addition, compound 2 disclosed herein provides a linker capable of site-specifically linking a payload moiety to an antibody. The linker can affect the biological activity of the target molecule and has, for example, the effect of reducing the half-life of the target molecule and / or the payload moiety or promoting secretion. The linker can be used as a bioconjugate for detection, diagnosis, and biomarkers.

[0052] Furthermore, the antibody-loaded moiety conjugates provided by Compound 1 and Compound 2 have the advantage of being highly homogeneous due to their identical binding sites. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] Figure 1 The entire synthetic process of compound I (trans) is shown.

[0054] Figure 2 The entire synthetic process of compound II (cis) is shown.

[0055] Figure 3 The results of HPLC spectrum analysis of the isomer structure of Compound 6 are shown.

[0056] Figure 4 The results of molecular weight analysis of compound 6 (cis form) are shown.

[0057] Figure 5 The results of molecular weight analysis of compound 6 (trans) are shown.

[0058] Figure 6The results of confirming the isomeric structure of Compound II by HPLC are shown.

[0059] Figure 7 The results of obtaining Compound II by HPLC are shown.

[0060] Figure 8 The HPLC results of FcBP(6Lys)-norbornene are shown.

[0061] Figure 9 The LC mass results of FcBP(6Lys)-norbornene are shown.

[0062] Figure 10 The HPLC results of compound I-FcBP(6Lys)-norbornene are shown.

[0063] Figure 11 The LC mass results of compound I-FcBP(6Lys)-norbornene are shown.

[0064] Figure 12 The HPLC results of Compound II-FcBP(6Lys)-norbornene are shown.

[0065] Figure 13 Shown are the mass spectrometry results for Compound II-FcBP(6Lys)-norbornene.

[0066] Figure 14 The reaction of Compound I-FcBP(6Lys)-norbornene with antibodies is shown.

[0067] Figure 15 The structure of Ab(Lys 246 / 248)-norbornene produced by the reaction of compound I-FcBP(6Lys)-norbornene with an antibody is shown.

[0068] Figure 16 The results of reaction monitoring of the reaction of compound I-FcBP(6Lys)-norbornene with antibodies by HIC-HPLC are shown.

[0069] Figure 17 The reaction of Compound II-FcBP(6Lys)-norbornene with antibodies is shown.

[0070] Figure 18 The structure of Ab(Lys 246 / 248)-norbornene produced by the reaction of compound I-FcBP(6Lys)-norbornene with an antibody is shown.

[0071] Figure 19The results of reaction monitoring of the reaction of Compound II-FcBP(6Lys)-norbornene with antibodies by HIC-HPLC are shown.

[0072] Figure 20 The mass analysis results of Herceptin-norbornene are shown.

[0073] Figure 21 Shows the use Figure 18 The structure of the antibody-loaded moiety conjugate generated by an antibody containing a first click chemistry functional group is shown in Figure 1. The magnified structure is the structure of the load moiety, and the non-magnified part is the same as the Figure 18 The structure shown in is the same.

[0074] Figure 22 The HIC-HPLC analysis of Figure 18 Results of reaction monitoring of the reaction of Ab(Lys 246 / 248)-norbornene with tetrazine-PEG8-DM1.

[0075] Figure 23 The results of mass analysis of the antibody-loaded moiety conjugates are shown.

[0076] Figure 24 、 25 , 26 and 27 are the results of cytotoxicity experiments of NCI-N87, BT474 and MDA-MB-468, respectively, and a table summarizing the results.

[0077] Figure 28 and Figure 29 Shown are the results of tumor growth inhibition experiments with Herceptin and antibody-loaded moiety conjugates described herein.

[0078] Figure 30 The HPLC results of Compound II-FcBP(L6Dap)-norbornene are shown.

[0079] Figure 31 Shown are the mass spectrometry results for Compound II-FcBP(L6Dap)-norbornene.

[0080] Figure 32 The HPLC results of Compound II-FcBP(L6Dab)-norbornene are shown.

[0081] Figure 33 Shown are the mass spectrometry results for Compound II-FcBP(L6Dab)-norbornene.

[0082] Figure 34 The HPLC results of Compound II-FcBP(L6Orn)-norbornene are shown.

[0083] Figure 35 Shown are the mass spectrometry results for Compound II-FcBP(L6Orn)-norbornene.

[0084] Figure 36 The HPLC results of Compound II-FcBP(L6Lys)-norbornene are shown.

[0085] Figure 37 Shown are the mass spectrometry results for Compound II-FcBP(L6Lys)-norbornene.

[0086] Figure 38 Shown are the results of HIC-HPLC for monitoring the binding reaction with Compound II-FcBP(L6Dap)-norbornene-based antibody.

[0087] Figure 39 Shown are the results of HIC-HPLC for monitoring the binding reaction with Compound II-FcBP(L6Dab)-norbornene-based antibody.

[0088] Figure 40 Shown are the results of HIC-HPLC for monitoring the binding reaction with Compound II-FcBP(L6Orn)-norbornene-based antibody.

[0089] Figure 41 Shown are the results of HIC-HPLC for monitoring the binding reaction with Compound II-FcBP(L6Lys)-norbornene-based antibody. DETAILED DESCRIPTION

[0090] The term "heteroalkyl" refers to an alkyl group in which one or more carbon atoms are replaced by a heteroatom (e.g., O, N, or S). For example, when a carbon atom of an alkyl group attached to the parent molecule is replaced by a heteroatom (e.g., O, N, or S), the resulting heteroalkyl group is an alkoxy group (e.g., -OCH3, etc.), an amine (e.g., -NHCH3, -N(CH3)2, etc.), or a sulfanyl group (e.g., -SCH3), respectively. When a non-terminal carbon atom of an alkyl group not attached to the parent molecule is replaced by a heteroatom (e.g., O, N, or S), the resulting heteroalkyl group is an alkyl ether (e.g., -CH2CH2-O-CH3, etc.), an alkylamine (e.g., -CH2NHCH3, -CH2N(CH3)2, etc.), or an alkyl sulfide (e.g., -CH2-S-CH3), respectively. When the terminal carbon atom of an alkyl group is substituted with a heteroatom (e.g., O, N, or S), the resulting heteroalkyl group is a hydroxyalkyl group (e.g., -CH2CH2-OH), an aminoalkyl group (e.g., -CH2NH2), or an alkylthiol group (e.g., -CH2CH2-SH). The heteroalkyl group may have, for example, 1 to 20 carbon atoms, 1 to 10 carbon atoms, or 1 to 6 carbon atoms. A C1-C6 heteroalkyl group refers to a heteroalkyl group having 1 to 6 carbon atoms.

[0091] The term "alkylene" refers to a branched, straight-chain, or cyclic saturated hydrocarbon radical derived from the same or two different carbon atoms of a parent alkane, comprising two monovalent radical centers. For example, an alkylene group may have 1 to 20 carbon atoms, 1 to 10 carbon atoms, or 1 to 6 carbon atoms. Typical alkylene groups include, but are not limited to, methylene (-CH2-), 1,1-ethyl (-CH(CH3)-), 1,2-ethyl (-CH2CH2-), 1,1-propyl (-CH(CH2CHV)-), 1,2-propyl (-CH2CH(CH3)-), 1,3-propyl (-CH2CH2CH2-), 1,4-butyl (-CH2CH2CH2CH2-), and the like.

[0092] The term "alkenylene" refers to a branched, straight-chain, or cyclic unsaturated hydrocarbon group derived from the same or two different carbon atoms of a parent olefin, comprising two monovalent radical centers. For example, an alkenylene group may 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-).

[0093] The term "alkynylene" refers to a branched, straight-chain, or cyclic unsaturated hydrocarbon radical derived from the same or two different carbon atoms of a parent alkyne, comprising two monovalent radical centers. For example, an alkynylene group may 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, ethynyl (-C≡C-), propargyl (-CH2C≡C-), and 4-pentynyl (-CH2CH2CH2C≡C-).

[0094] The term "polymethylene" refers to an alkylene group having one or more carbon atoms, including methylene, ethylene, trimethylene, tetramethylene, pentamethylene, hexamethylene, and heptamethylene.

[0095] Those skilled in the art will recognize that when moieties such as "alkyl," "aryl," and "heterocyclyl" are substituted with one or more substituents, they may alternatively be referred to as moieties such as "alkylene," "arylene," and "heterocyclyl" (i.e., it means that one or more hydrogen atoms of the parent "alkyl," "aryl," and "heterocyclyl" moieties are replaced with the substituents.) When moieties such as "alkyl," "aryl," and "heterocyclyl" are referred to herein as "substituted" or illustrated in the accompanying drawings as substituted (or optionally substituted, e.g., when the number of substituents is from 0 to a positive number), terms such as "alkyl," "aryl," and "heterocyclyl" should be understood to be interchangeable with "alkylene," "arylene," "heterocyclyl," etc.

[0096] The term "acyl" refers to -C(=O)-alkyl, -C(=O)-carbocycle (substituted or unsubstituted), -C(=O)-heterocycle (substituted or unsubstituted), wherein the alkyl, carbocycle or heterocycle moiety is the same as defined herein. 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.

[0097] The term "substituted", for example, "substituted alkyl", "substituted alkylene", "substituted aryl", "substituted aralkyl", "substituted heterocyclyl" and "substituted carbocyclyl (e.g., substituted cycloalkyl)" refers to alkyl, alkylene, aryl, aralkyl, heterocyclyl and carbocyclyl (e.g., cycloalkyl) groups in which one or more hydrogen atoms are each independently replaced by 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)N RR, -S(=O)2O-, -S(=O)2OH, -S(=O)2R, -OS(=O)2OR, -S(=O)2NR, -S(=O)R, -OP(=O)(OR)2, -C(=O)R, alkylene-C(= -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 (wherein each X is independently a halogen: F, Cl, Br or I, and R is independently H, an alkyl group, an aryl group, an aralkyl group or a heterocyclic group), but is not limited thereto. Alkylene, alkenylene and alkynylene groups may also be similarly substituted.

[0098] "Optionally substituted" refers to a particular portion of the compound of Formula 1 having one, two, or more substituents (eg, optionally substituted aryl).

[0099] "Leaving group" represents a chemical moiety that can be removed or replaced by another chemical group. Throughout the specification of the present invention, the term leaving group includes click chemistry functional groups, N-hydroxysuccinimide (NHS) or maleimide, but is not limited thereto.

[0100] The term "target molecule" or "molecule of interest" refers to a molecule to which a payload moiety is intended to be attached. For example, the target molecule can be a biologically active molecule and can 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), but is not limited thereto.

[0101] The term "load moiety" refers to a molecule that is intended to be attached to a target molecule. For example, the load moiety can be a chemical compound, a peptide, a polypeptide, a protein and / or a drug molecule.

[0102] The term "click chemistry functional group" refers to a functional group that participates in a click chemistry reaction. The types of click chemistry and the functional groups involved therein are generally known. Examples of click chemistry reactions include [3+2] cycloadditions, thiol-ene reactions, Diels-Alder reactions, reverse electron demand Diels-Alder reactions, [4+1] cycloadditions, but are not limited thereto. More specifically, click chemistry reactions include copper (I)-catalyzed azide-alkyne cycloadditions (CuAAC), force-induced azide-alkyne cycloadditions (SPAAC), force-induced alkyne-nitro cycloadditions (SPANC), [3+2] cycloadditions of alkenes and azides, reverse demand Diels-Alder reactions of alkenes and tetrazines, photoclick reactions of alkenes and tetrazoles, and Huisgen cycloadditions of azides and alkynes, but are not limited thereto. 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-cyclooctenes, nitrones, nitrile oxides, azides, conjugated dienes, dienophiles, and cycloalkynes such as cyclooctyne, cyclononyne, dibenzocyclooctyne (DIBO), biarylazacyclooctynone (BARAC), aryl-free octyne (ALO), difluorinated cyclooctyne (DIFO), monofluorinated cyclooctyne (MOFO), dibenzoazacyclooctyne (DIBAC), and dimethoxyazacyclooctynone (DIMAC).

[0103] The term "ester activating moiety" refers to a moiety that is attached to the oxygen of an ester group and thus can convert the ester group into an activated ester. For example, when When the ester group of the structure is an activated ester, Z is an ester-activating moiety. Examples of ester-activating moieties include, but are not limited to, N-hydroxysuccinimide (NHS), p-nitrophenyl, and pentafluorophenyl. Ester-activating moieties of the present invention include, but are not limited to, those described in WO / 2015 / 122478. The term "activated ester" refers to an ester that is susceptible to nucleophilic substitution reactions.

[0104] In this specification, terms such as first and second are used to describe various components, and the above terms are used only for the purpose of distinguishing one component from another.

[0105] Furthermore, it should be noted that the terms used in this specification are only used to describe exemplary embodiments and are not intended to limit the present invention. Unless the context clearly indicates otherwise, a singular expression includes a plural expression.

[0106] In this specification, terms such as "comprises", "includes" or "has" are intended to indicate the presence of applicable features, numbers, steps, operations, constituent elements or any combination thereof, and should be understood to mean that the possibility of the presence or addition of one or more other features, numbers, steps, operations, constituent elements or any combination thereof is not excluded.

[0107] Since this specification can be modified into various forms and include various exemplary embodiments, specific exemplary embodiments will be described in detail below. However, the specification is not intended to limit the present invention to the specific disclosure, and it should be understood that all changes, equivalents and replacements included in the spirit and technical scope of the present invention are included in the present invention.

[0108] Hereinafter, the present invention will be described in detail.

[0109] The present application can provide linkers that facilitate transfer and conjugation reactions so that the cargo moiety can be attached to the desired target molecule.

[0110] A linker may include two or more functional groups capable of reacting with a cargo moiety and / or a target molecule.

[0111] The above-mentioned reactions refer to the mutual connection of two or more identical or different molecules or certain functional groups of molecules, or the occurrence of leaving reactions, i.e., E1, E2, SN1, SN2 and nucleophilic substitution reactions, etc. The connection includes all direct or indirect covalent and / or non-covalent bonds.

[0112] In one example, the first functional group of the two or more functional groups can be a leaving group.

[0113] In one example, the second functional group of the two or more functional groups can be a click chemistry functional group.

[0114] The linker may include two or more electrophilic carbon atoms of a carbonyl group.

[0115] The electrophilic carbon atom of the carbonyl group can be attached to a functional group.

[0116] As an example, the electrophilic carbon atom of a carbonyl group and a functional group can be covalently bonded.

[0117] As another example, one or more atoms can be included between the electrophilic carbon atom of the carbonyl group and the functional group. For example, a nucleophilic atom can be included between the electrophilic carbon atom of the carbonyl group and the functional group. In this case, the nucleophilic atom can be O (oxygen), N (nitrogen) or S (sulfur).

[0118] A linker may include two or more electrophilic carbon atoms of a carbonyl group having different partial positive charges (δ+).

[0119] Among the two or more electrophilic carbon atoms in the carbonyl group, the carbonyl group having the largest partial positive charge (δ+) may be the first carbonyl carbon atom.

[0120] Of the two or more electrophilic carbon atoms in a carbonyl group, the carbonyl group having the second most positive charge (δ+) can be the second carbonyl carbon atom.

[0121] As an example, the electrophilic carbon atom of the first carbonyl group may be connected to the first functional group. In this case, the nucleophilic atom may be included between the electrophilic carbon atom of the first carbonyl group and the first functional group.

[0122] As another example, the electrophilic carbon atom of the second carbonyl group can be attached to the second functional group. In this case, the electrophilic carbon atom of the second carbonyl group can form a covalent bond with the first functional group.

[0123] The linker can adjust the binding position of the cargo moiety to be covalently bound to the target molecule.

[0124] For example, the payload moiety can be attached to the electrophilic carbon atom of the first carbonyl group.

[0125] The water solubility of the linker can be adjusted by adjusting its length. For example, the water solubility of the linker can be increased by increasing the number of alkyl groups containing substituents that increase water solubility.

[0126] Hereinafter, the linker structure will be described in detail.

[0127] According to one aspect disclosed herein, a linker compound represented by the following Formula 1 and / or Formula 2 may be provided.

[0128] [Formula 1]

[0129]

[0130] [Formula 2]

[0131]

[0132] In formula 1,

[0133] R' is an ester-activated moiety. Ester-activated moieties include, but are not limited to, N-hydroxysuccinimide (NHS), p-nitrophenyl, and pentafluorophenyl. In addition, R' can be Any of.

[0134] R" is any one of the click chemistry functional groups, and

[0135] The click chemistry functional group can be any one or more of alkynes, cycloalkynes 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, but is not limited thereto.

[0136] When the click chemistry functional group is a cycloalkyne group, the cycloalkyne group can be any one of cyclooctyne, cyclononyne, dibenzocyclooctyne (DIBO), diarylazacyclooctyne ketone (BARAC), aryl-free octyne (ALO), difluorinated cyclooctyne (DIFO), monofluorinated cyclooctyne (MOFO), dibenzoazacyclooctyne (DIBAC), and dimethoxyazacyclooctyne (DIMAC). The cycloalkyne group is not limited thereto.

[0137] When the chemical functional group is a conjugated diene group, the conjugated diene group can be an olefin group and a cycloolefin group. As an example, the conjugated diene group can be a tetrazine (e.g., 1,2,3,4-tetrazine and / or 1,2,4,5-tetrazine) group.

[0138] When the click chemistry functional group is a dienophile group, the dienophile group can be an olefin group and a cycloolefin group, and the cycloolefin group includes a bicyclic or fused ring structure. As an example, the dienophile group can be a norbornene group.

[0139] R'' is substituted or unsubstituted C 1-20 Alkylene, substituted or unsubstituted C 2-20 Alkenylene, substituted or unsubstituted C 1-10 Alkynylidene, substituted or unsubstituted C 1-10 Polymethylene, substituted or unsubstituted C 5-12 Aryl, substituted or unsubstituted C 5-14 Arylalkylene, 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, and the heteroalkylene, heterocycloalkylene or heteroaryl group contains at least one or more of N, O or S, and

[0140] The substitution is a non-hydrogen substituent selected from -Ra, -O-, =O, -ORa, -SRa, -S-, -N(Ra)2, =NRa, -C(Rb)3, -N=C=O, -NCS, -NO, -NO2, =N-OH, =N2, -NV, -NHC(=O)Ra, -C(=O)Ra, -C(=O)NRaRa, -S(=O)2O-, -S(=O)2OH, -S(=O)2Ra, -OS(=O)2ORa, -S(=O) any one or more of the group consisting of: -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,

[0141] Ra is H, C 1-6 Alkyl, C 5-12 Aryl, C 7-12 an aralkyl group or a heterocyclic group,

[0142] Rb is F, Cl, Br or I,

[0143] X is O, N or S.

[0144] In formula 2,

[0145] R' is an ester activation moiety. The ester activation moiety includes, but is not limited to, N-hydroxysuccinimide (NHS), p-nitrophenyl and pentafluorophenyl. In addition, R' can be Any of.

[0146] R" is any one of the click chemistry functional groups, and

[0147] The click chemistry functional group can be any one or more of alkynes, cyclooctynes ​​and cyclononynes (e.g., cycloalkynes such as bicyclo[6.1.0]non-4-yn-9-ylmethanol), trans-cyclooctene, nitrone, nitrile oxide, azide, conjugated diene, and dienophile, but is not limited thereto.

[0148] When the click chemistry functional group is a cycloalkyne group, the cycloalkyne group can be any one of cyclooctyne, cyclononyne, dibenzocyclooctyne (DIBO), BARAC (diarylazacyclooctyne ketone), ALO (aryl-free octyne), DIFO (difluorinated cyclooctyne), MOFO (monofluorinated cyclooctyne), DIBAC (dibenzoazacyclooctyne) and DIMAC (dimethoxyazacyclooctyne), but is not limited thereto.

[0149] When the click chemistry functional group is a conjugated diene group, the conjugated diene group can be an olefin group and a cycloolefin group. As an example, the conjugated diene group can be a tetrazine (e.g., 1,2,3,4-tetrazine and / or 1,2,4,5-tetrazine) group.

[0150] When the click chemistry functional group is a dienophile group, the dienophile can be an olefin group and a cycloolefin group, and the cycloolefin group includes a bicyclic or fused ring structure. As an example, the dienophile group can be a trans-cyclooctene (TCO) group or a norbornene group.

[0151] R'' is substituted or unsubstituted C 1-20 Alkylene, substituted or unsubstituted C 2-20 Alkenylene, substituted or unsubstituted C 1-10 Alkynylidene, substituted or unsubstituted C 1-10 Polymethylene, substituted or unsubstituted C 5-12 Aryl, substituted or unsubstituted C 5-14 Arylalkylene, 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, and the heteroalkylene, heterocycloalkylene or heteroaryl group contains at least one or more of N, O or S, and

[0152] The substitution is a non-hydrogen substituent selected from -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) any one or more of the group consisting of: -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,

[0153] Ra is H, C 1-6 Alkyl, C 5-12 Aryl, C 7-12 an aralkyl group or a heterocyclic group,

[0154] Rb is F, Cl, Br or I,

[0155] X is O, N or S.

[0156] In the formula of the present invention, Used to indicate the bond that is the point of attachment of a moiety or substituent to a core or backbone structure.

[0157] According to exemplary embodiments disclosed in this specification, the compound represented by Formula 1 may be a trans compound.

[0158] When Formula 1 is the same as the following, Formula 1 is expressed as the following Formula 1-1:

[0159] R' is

[0160] R" is any one of the click chemistry functional groups, and

[0161] 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 (e.g., an alkene), in which case the cycloalkyne, conjugated diene, and diene are the same as those described above.

[0162] R'' is substituted or unsubstituted C 1-20Alkylene, substituted or unsubstituted C 1-20 Heteroalkylene, substituted or unsubstituted C 1-20 Haloalkyl or C 1-10 polymethylene, and the heteroalkylene group contains at least one or more selected from the group consisting of N, O and S,

[0163] The substitution is a non-hydrogen substituent selected from -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) any one or more of the group consisting of: -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,

[0164] Ra is H, C 1-6 Alkyl, C 5-12 Aryl, C 7-12 an aralkyl group or a heterocyclic group,

[0165] Rb is F, Cl, Br or I,

[0166] and X is any one of O, N and S.

[0167] [Formula 1-1]

[0168]

[0169] Specifically, R'' is One of

[0170] R1 is H,

[0171] R2 is H or C(=O),

[0172] R3 is H or C(=O),

[0173] R4 is H or C(=O),

[0174] R5 is H or C(=O),

[0175] R6 is H or C(=O),

[0176] Y1 can be any one of C, N, O and S, and

[0177] n may be any integer from 1 to 20, but is not limited thereto.

[0178] R5 and R6 cannot be C(=O) at the same time, and

[0179] When Y1 is any one of N, O and S, R4, R5 and R6 may not be C(=O).

[0180] More specifically, R'' is

[0181] R4 is H or C(=O),

[0182] R5 is H or C(=O),

[0183] R6 is H or C(=O),

[0184] Y1 can be any one of C, N, O and S, and

[0185] n may be any integer from 1 to 10.

[0186] In an exemplary embodiment, when Formula 1-1 is the same as the following, Formula 1-1 is represented by the following Formula 1-1-1:

[0187] R' is

[0188] R" is any click chemistry functional group,

[0189] R"' is

[0190] n is 1,

[0191] R4 is H or C(=O),

[0192] R5 is H or C(=O),

[0193] R6 is H or C(=O),

[0194] Y1 is C,

[0195] and X is any one of N, O and S.

[0196] [Formula 1-1-1]

[0197]

[0198] In another exemplary embodiment, when Formula 1-1 is the same as the following, Formula 1-1 is represented by the following Formula 1-1-2:

[0199] R' is

[0200] R" is a norbornene group,

[0201] R"' is

[0202] R4 is H,

[0203] R5 is H,

[0204] R6 is H,

[0205] n is 1,

[0206] Y1 is C,

[0207] And X is O.

[0208] [Formula 1-1-2]

[0209]

[0210] As another exemplary embodiment, the compound that may be represented by Formula 1 may be a compound described in Table 1 below.

[0211] [Table 1]

[0212]

[0213]

[0214] When Formula 1 is the same as the following, Formula 1 is represented by the following Formula 1-2:

[0215] R' is

[0216] R" is any one of the click chemistry functional groups,

[0217] 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 (e.g., an alkene), in which case the cycloalkyne group, the conjugated diene group, and the diene group are the same as those described above.

[0218] R'' is substituted or unsubstituted C 1-20 Alkylene, substituted or unsubstituted C 1-20 Heteroalkylene, substituted or unsubstituted C 1-20 Haloalkyl or C 1-10polymethylene, and the heteroalkylene group contains at least one or more selected from the group consisting of N, O and S,

[0219] The substitution is a non-hydrogen substituent selected from -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) any one or more of the group consisting of: -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,

[0220] Ra is H, C 1-6 Alkyl, C 5-12 Aryl, C 7-12 an aralkyl group or a heterocyclic group,

[0221] Rb is F, Cl, Br or I,

[0222] and X is any one of O, N and S.

[0223] [Formula 1-2]

[0224]

[0225] Specifically, R'' is One of

[0226] R1 is H,

[0227] R2 is H or C(=O),

[0228] R3 is H or C(=O),

[0229] R4 is H or C(=O),

[0230] R5 is H or C(=O),

[0231] R6 is H or C(=O),

[0232] Y1 can be any one of C, N, O and S, and

[0233] n may be any integer from 1 to 20, but is not limited thereto.

[0234] In this case, R5 and R6 cannot be C(=O) at the same time, and

[0235] When Y1 is any one of N, O and S, R4, R5 and R6 may not be C(=O).

[0236] More specifically, R'' is

[0237] R4 is H or C(=O),

[0238] R5 is H or C(=O),

[0239] R6 is H or C(=O),

[0240] Y1 can be any one of C, N, O and S, and

[0241] n may be any integer from 1 to 10.

[0242] In an exemplary embodiment, when Formula 1-2 is the same as the following, Formula 1-2 is represented by the following Formula 1-2-1:

[0243] R"' is

[0244] R" is any click chemistry functional group,

[0245] n is 1,

[0246] R4 is H or C(=O),

[0247] R5 is H or C(=O),

[0248] R6 is H or C(=O),

[0249] Y1 is C,

[0250] and X is any one of N, O and S.

[0251] [Formula 1-2-1]

[0252]

[0253] In another exemplary embodiment, when Formula 1-2 is the same as the following, Formula 1-2 is represented by the following Formula 1-2-2:

[0254] R' is

[0255] R" is a norbornene group,

[0256] R"' is

[0257] R4 is H,

[0258] R5 is H,

[0259] R6 is H,

[0260] n is 1,

[0261] Y1 is C,

[0262] And X is O.

[0263] [Formula 1-2-2]

[0264]

[0265] As an exemplary embodiment, the compound that may be represented by Formula 1 may be the compounds described in Table 2 below.

[0266] [Table 2]

[0267]

[0268]

[0269] According to exemplary embodiments disclosed in this specification, the compound represented by Formula 2 may be a cis-form compound.

[0270] When Formula 2 is the same as the following, Formula 2 is represented by the following Formula 2-1:

[0271] R' is

[0272] R" is any one of the click chemistry functional groups,

[0273] 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 (e.g., an alkene), in which case the cycloalkyne group, the conjugated diene group, and the diene group are the same as those described above.

[0274] R'' is substituted or unsubstituted C 1-20 Alkylene, substituted or unsubstituted C 1-20 Heteroalkylene, substituted or unsubstituted C 1-20 Haloalkyl or C 1-10 polymethylene, and the heteroalkylene group contains at least one or more selected from the group consisting of N, O and S,

[0275] The substitution is a non-hydrogen substituent selected from -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) any one or more of the group consisting of: -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,

[0276] Ra is H, C 1-6 Alkyl, C 5-12 Aryl, C 7-12 an aralkyl group or a heterocyclic group,

[0277] Rb is F, Cl, Br or I,

[0278] and X is any one of O, N and S.

[0279] [Formula 2-1]

[0280]

[0281] Specifically, R'' is one,

[0282] R7 is H,

[0283] R8 is H or C(=O),

[0284] R9 is H or C(=O),

[0285] R 10 is H or C(=O),

[0286] R 11 is H or C(=O),

[0287] R 12 is H or C(=O),

[0288] Y2 can be any one of C, N, O and S, and

[0289] n may be any integer from 1 to 20, but is not limited thereto.

[0290] In this case, R 11 and R 12 cannot be C(=O) at the same time, and

[0291] When Y2 is any one of N, O and S, R 10 、R 11 and R 12 It may not be C(=O).

[0292] More specifically, R'' is

[0293] R 10 is H or C(=O),

[0294] R 11 is H or C(=O),

[0295] R 12 is H or C(=O),

[0296] Y2 can be any one of C, N, O and S, and

[0297] n may be any integer from 1 to 10.

[0298] In an exemplary embodiment, when Formula 2-1 is the same as the following, Formula 2-1 is represented by the following Formula 2-1-1:

[0299] R' is

[0300] R" is any click chemistry functional group,

[0301] R"' is

[0302] n is 1,

[0303] R 10 is H or C(=O),

[0304] R 11 is H or C(=O),

[0305] R 12 is H or C(=O),

[0306] Y2 is C,

[0307] and X is any one of N, O and S.

[0308] [Formula 2-1-1]

[0309]

[0310] In another exemplary embodiment, when Formula 2-1 is the same as the following, Formula 2-1 is represented by the following Formula 2-1-2:

[0311] R' is

[0312] R" is a norbornene group,

[0313] R"' is

[0314] n is 1,

[0315] R 10 For H,

[0316] R 11 For H,

[0317] R 12 For H,

[0318] Y2 is C,

[0319] n is 1, and

[0320] X is O.

[0321] [Formula 2-1-2]

[0322]

[0323] As an exemplary embodiment, the compound that may be represented by Formula 2 may be the compounds described in Table 3 below.

[0324] [Table 3]

[0325]

[0326]

[0327]

[0328]

[0329] When Formula 2 is the same as the following, Formula 2 is represented by the following Formula 2-2:

[0330] R' is

[0331] R" is any one of the click chemistry functional groups,

[0332] 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 (e.g., an alkene), in which case the cycloalkyne group, the conjugated diene group, and the diene group are the same as those described above.

[0333] R'' is substituted or unsubstituted C 1-20 Alkylene, substituted or unsubstituted C 1-20 Heteroalkylene, substituted or unsubstituted C 1-20 Haloalkyl or C 1-10 polymethylene, and the heteroalkylene group contains at least one or more selected from the group consisting of N, O and S,

[0334] The substitution is a non-hydrogen substituent selected from -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) any one or more of the group consisting of: -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,

[0335] Ra is H, C 1-6 Alkyl, C 5-12 Aryl, C 7-12 an aralkyl group or a heterocyclic group,

[0336] Rb is F, Cl, Br or I,

[0337] and X is any one of O, N and S.

[0338] [Formula 2-2]

[0339]

[0340] Specifically, R'' is One of

[0341] R7 is H,

[0342] R8 is H or C(=O),

[0343] R9 is H or C(=O),

[0344] R 10 is H or C(=O),

[0345] R 11 is H or C(=O),

[0346] R 12 is H or C(=O),

[0347] Y2 can be any one of C, N, O and S, and

[0348] n may be any integer from 1 to 20, but is not limited thereto.

[0349] In this case, R 11 and R 12 cannot be C(=O) at the same time, and

[0350] When Y2 is any one of N, O and S, R 10 、R 11 and R 12 It may not be C(=O).

[0351] More specifically, R'' is

[0352] R 10 is H or C(=O),

[0353] R 11 is H or C(=O),

[0354] R 12 is H or C(=O),

[0355] Y2 can be any one of C, N, O and S, and

[0356] n may be any integer from 1 to 10.

[0357] In an exemplary embodiment, when Formula 2-2 is the same as the following, Formula 2-2 is represented by the following Formula 2-2-1:

[0358] R' is

[0359] R" is any click chemistry functional group,

[0360] R"' is

[0361] n is 1,

[0362] R 10 is H or C(=O),

[0363] R 11 is H or C(=O),

[0364] R 12 is H or C(=O),

[0365] Y2 is C,

[0366] and X is any one of N, O and S.

[0367] [Formula 2-2-1]

[0368]

[0369] In another exemplary embodiment, when Formula 2-2 is the same as the following, Formula 2-2 is represented by the following Formula 2-2-2:

[0370] R' is

[0371] R" is a norbornene group,

[0372] R"' is

[0373] n is 1,

[0374] R 10 For H,

[0375] R 11 For H,

[0376] R 12 For H,

[0377] Y2 is C, and

[0378] X is O.

[0379] [Formula 2-2-2]

[0380]

[0381] As an exemplary embodiment, the compound that may be represented by Formula 2 may be the compounds described in Table 4 below.

[0382] [Table 4]

[0383]

[0384]

[0385]

[0386] Hereinafter, when the target molecule is an antibody, a method for preparing an antibody-payload conjugate using the above-mentioned linker will be described. The method for preparing an antibody-payload conjugate comprises: (1) preparing a linker-Fc-binding peptide conjugate by reacting a linker with an Fc-binding peptide; (2) reacting the linker-Fc-binding peptide conjugate with an antibody to obtain an antibody comprising a first click chemistry functional group, and (3) reacting the antibody comprising the first click chemistry functional group with a payload comprising a second click chemistry functional group complementary to the first click chemistry functional group to prepare the antibody-payload conjugate. Hereinafter, each process will be described separately.

[0387] The method of preparing an antibody-loaded moiety conjugate may comprise: (1) preparing a linker-Fc-binding peptide conjugate by reacting a linker with an Fc-binding peptide.

[0388] Fc-binding peptides are generally referred to as peptides that have the property of binding to the Fc domain of an antibody. As a representative example of an Fc-binding peptide, 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 developed Fc-binding peptides of SEQ ID NOS: 1 to 5, wherein specific residues of the 13-mer peptides are substituted, thereby enabling the Fc-binding peptides to react with a linker. As can be seen from the following structures, 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.

[0389] The Fc-binding peptide of SEQ ID NO: 1 is a peptide represented by Formula 13:

[0390] [Equation 13]

[0391] DCAWH-Xa-GELVWCT (SEQ ID NO: 1)

[0392] Among them, D is aspartic acid, C is cysteine, A is alanine, W is tryptophan, H is histidine, and Xa is G is glycine, E is glutamic acid, L is leucine, V is valine, and T is threonine. In this case, m is an integer from 1 to 4, and the cysteine ​​at the N-terminus and the cysteine ​​at the C-terminus can be selectively linked to each other. When m = 1 (SEQ ID NO: 3), Xa is 2,3-diaminopropionic acid (Dap); when m = 2 (SEQ ID NO: 4), Xa is 2,4-diaminobutyric acid (Dab); when m = 3 (SEQ ID NO: 5), Xa is ornithine; and when m = 4 (SEQ ID NO: 2), Xa is lysine.

[0393] The Fc-binding peptide of SEQ ID NO: 2 is a peptide represented by Formula 14:

[0394] [Equation 14]

[0395] DCAWHKGELVWCT (SEQ ID NO: 2)

[0396] Among them, D is aspartic acid, C is cysteine, A is alanine, W is tryptophan, H is histidine, K is lysine, G is glycine, E is glutamic acid, L is leucine, V is valine, T is threonine, and the cysteine ​​at the N-terminus and the cysteine ​​at the C-terminus can be selectively linked to each other.

[0397] The linker includes the compound described in Chemical Formula 1 or Chemical Formula 2 and specific examples thereof.

[0398] The linker and Fc-binding peptide can bind via a nucleophilic substitution reaction. This reaction occurs when a nucleophilic atom present in the Fc-binding peptide attacks a positively charged or partially positively charged atom in the linker. The nucleophilic atom can be a nitrogen atom of lysine. Furthermore, the positively charged or partially positively charged atom can be an electrophilic carbon atom of a carbonyl group.

[0399] Hereinafter, compounds represented by Formula 1 and / or Formula 2 exemplarily disclosed in this specification are shown, ie, linkers are bound to Fc-binding peptides to form linker-Fc-binding peptide conjugates.

[0400] For example, the linker-Fc binding peptide conjugate can have the structure of Formula 3.

[0401] [Formula 3]

[0402]

[0403] In formula 3,

[0404] R" is any one of the click chemistry functional groups,

[0405] 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, in which case the cycloalkyne group, the conjugated diene group, and the diene group are the same as those described above.

[0406] R'' is substituted or unsubstituted C 1-20 Alkylene, substituted or unsubstituted C 2-20 Alkenylene, substituted or unsubstituted C 1-10 Alkynylidene, substituted or unsubstituted C 1-10Polymethylene, substituted or unsubstituted C 5-12 Aryl, substituted or unsubstituted C 5-14 Arylalkylene, 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, and the heteroalkylene, heterocycloalkylene or heteroaryl group contains at least one or more of N, O or S, and

[0407] The substitution is a non-hydrogen substituent selected from -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) any one or more of the group consisting of: -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,

[0408] Ra is H, C 1-6 Alkyl, C 5-12 Aryl, C 7-12 an aralkyl group or a heterocyclic group,

[0409] Rb is F, Cl, Br or I,

[0410] X can be O, N or S.

[0411] Y3 can be any one of O, N and S.

[0412] Fp is an Fc binding peptide. In addition, Fp may be any peptide selected from Formula 13 or Formula 14. The details of Formula 13 and Formula 14 are the same as those already described.

[0413] Fp may be attached 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.

[0414] As an example, the compound shown in Formula 3 can be prepared by Reaction Scheme 1.

[0415] [Reaction Scheme 1]

[0416]

[0417] Reaction Scheme 1 is a reaction for producing a compound represented by Formula 3 by reacting the compound represented by Formula 1 with an Fc-binding peptide comprising 1 to 50 amino acids.

[0418] The binding reaction between the Fc binding peptide and Formula 1 can be performed by a substitution reaction.

[0419] The substitution reaction may be a nucleophilic acyl substitution reaction.

[0420] The substitution reaction can be performed by reacting the compound of Formula 1 with any of the amine group (—NH 2 ), sulfhydryl group (—SH) and hydroxyl group (—OH) of the Fc-binding peptide.

[0421] As an example, when the compound of Formula 1 reacts with an amine group (-NH2) of an Fc-binding peptide, Y3 can be N(H). Since 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 of the corresponding residues contain free amine groups to allow for substitution reactions.

[0422] As another example, when the compound of Formula 1 reacts with a sulfhydryl group (—SH) of an Fc-binding peptide, Y 3 can be S.

[0423] As an example, when the compound of Formula 1 reacts with any hydroxyl group (—OH) of an Fc-binding peptide, Y 3 may be O.

[0424] For example, the linker-Fc binding peptide conjugate can have the structure of Formula 4.

[0425] [Formula 4]

[0426]

[0427] In formula 4,

[0428] R" is any one of the click chemistry functional groups,

[0429] 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, wherein the cycloalkyne group, the conjugated diene group, and the diene group are the same as those described above.

[0430] R'' is substituted or unsubstituted C1-20 Alkylene, substituted or unsubstituted C 2-20 Alkenylene, substituted or unsubstituted C 1-10 Alkynylidene, substituted or unsubstituted C 1-10 Polymethylene, substituted or unsubstituted C 5-12 Aryl, substituted or unsubstituted C 5-14 Arylalkylene, 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, and the heteroalkylene, heterocycloalkylene or heteroaryl group contains at least one or more of N, O or S, and

[0431] The substitution is a non-hydrogen substituent selected from -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) any one or more of the group consisting of: -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,

[0432] Ra is H, C 1-6 Alkyl, C 5-12 Aryl, C 7-12 an aralkyl group or a heterocyclic group,

[0433] Rb is F, Cl, Br or I,

[0434] X can be O, N or S.

[0435] Y4 can be any one of O, N and S.

[0436] Fp is an Fc binding peptide. In addition, Fp may be any peptide selected from Formula 13 or Formula 14. The details of Formula 13 and Formula 14 are the same as those already described.

[0437] Fp may be attached 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.

[0438] As an example, the compound shown in Formula 4 can be prepared by Reaction Scheme 2.

[0439] [Reaction Scheme 2]

[0440]

[0441] Reaction Scheme 2 is a reaction for producing a compound represented by Formula 4 by reacting the compound represented by Formula 2 with an Fc-binding peptide comprising 1 to 50 amino acids.

[0442] The binding reaction between the Fc binding peptide and Formula 2 can be performed by a substitution reaction.

[0443] The substitution reaction may be a nucleophilic acyl substitution reaction.

[0444] The substitution reaction can be performed by reacting the compound of Formula 2 with any of the amine group (—NH 2 ), sulfhydryl group (—SH) and hydroxyl group (—OH) of the Fc-binding peptide.

[0445] As an example, when the compound of Formula 2 reacts with an amine group (-NH2) of an Fc-binding peptide, Y4 can be N. Since 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 of the corresponding residues contain free amine groups so that the substitution reaction can be performed.

[0446] As another example, when the compound of Formula 2 reacts with a sulfhydryl group (—SH) of an Fc-binding peptide, Y 4 can be S.

[0447] As an example, when the compound of Formula 2 reacts with any hydroxyl group (—OH) of an Fc-binding peptide, Y 4 can be O.

[0448] In Reaction Scheme 1 and Reaction Scheme 2, compounds represented by Formula 3 and / or Formula 4 can be prepared by reacting the amine (-NH2), sulfhydryl (-SH) and hydroxyl (-OH) residues of the Fc-binding peptide with the carbon atom of the first carbonyl group.

[0449] In this case, the carbonyl carbon most closely attached to R' (ie, the electrophilic carbon atom of the first carbonyl group) can have the largest partial positive charge (δ+).

[0450] The method of preparing an antibody-loaded moiety conjugate may comprise (2) reacting a linker-Fc binding peptide with an antibody to obtain an antibody comprising a first click chemistry functional group.

[0451] Furthermore, in this case, when the Fc-binding peptide has an 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 an antibody, in particular, to the position of lysine 246 (Fc-Lys246) or lysine 248 (Fc-Lys248) of Fc, using Fc-binding peptides of SEQ ID NOS: 1 to 5 that have an affinity for the FcRn domain of an antibody.

[0452] The reaction between the linker-Fc binding peptide conjugate and the antibody can be a nucleophilic substitution reaction. In this case, a nucleophilic substitution reaction can occur when a nucleophilic atom present in the antibody attacks a positively charged or partially positively charged atom of the linker-Fc binding peptide conjugate. In this case, the nucleophilic atom can be a nitrogen atom of lysine. In addition, the positively charged or partially positively charged atom can be an electrophilic carbon of a carbonyl group contained in the linker structure.

[0453] In one example, an antibody comprising a first click chemistry functional group can be represented by Formula 5.

[0454] [Formula 5]

[0455]

[0456] In formula 5,

[0457] Ab is an antibody. In a specific embodiment, Ab can be trastuzumab.

[0458] R" is any one of the click chemistry functional groups,

[0459] The click chemistry functional group can be any one or more groups of alkynes, cycloalkynes 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, and in this case, the cycloalkyne group, conjugated diene group, and diene group are the same as those described above.

[0460] In addition, the nitrogen atom attached to Ab may be contained in lysine 246 or lysine 248 of the antibody's Fc. In a specific embodiment, the nitrogen attached to Ab may be contained in lysine 246 of the antibody's Fc. In a specific embodiment, the nitrogen attached to Ab may be contained in lysine 248 of the antibody's Fc.

[0461] n is an integer of 1 to 4. For example, n may be 2. In this case, R″ may be linked to lysine 246 or lysine 248, which are respectively contained in two Fc residues in one antibody, to produce such a structure. In another example, n may be 4. In this case, R″ may be linked to lysine 246 or lysine 248, which are respectively contained in two Fc residues in one antibody, to produce such a structure.

[0462] n is 2, and the nitrogen atom linked to Ab may be contained in lysine 246 of two Fc residues of the antibody. Alternatively, n is 2, and the nitrogen atom linked to Ab may be contained in lysine 248 of two Fc residues of the antibody.

[0463] The antibody of Formula 5 can be prepared by the reaction represented by the following Reaction Scheme 3.

[0464] [Reaction Scheme 3]

[0465]

[0466] In this case, details about Formula 3 and Formula 5 are the same as those already described.

[0467] In this case, since the peptides of SEQ ID NOS: 1 to 5 contain 2,3-diaminopropionic acid, 2,4-diaminobutyric acid, ornithine, or lysine, respectively, at residue 6, all corresponding residues differ only in the length of the carbon skeleton containing a free amine group and have similar structures. Therefore, all peptides of SEQ ID NOS: 1 to 5 exhibit the property of binding to the Fc domain of an antibody.

[0468] In another example, an antibody comprising a first click chemistry functional group can be represented by Formula 6.

[0469] [Formula 6]

[0470]

[0471] In Equation 6,

[0472] Ab is an antibody. In a specific embodiment, Ab can be trastuzumab.

[0473] R'' is substituted or unsubstituted C 1-20 Alkylene, substituted or unsubstituted C 2-20 Alkenylene, substituted or unsubstituted C 1-10 Alkynylidene, substituted or unsubstituted C 1-10 Polymethylene, substituted or unsubstituted C 5-12 Aryl, substituted or unsubstituted C 5-14 Arylalkylene, substituted or unsubstituted C 8-16Arylalkenylene, substituted or unsubstituted C 3-10 Cycloalkylene, substituted or unsubstituted C 3-10 Heterocycloalkylene or substituted or unsubstituted C 5-12 heteroaryl, and the heteroalkylene, heterocycloalkylene or heteroaryl group contains at least one or more of N, O or S, and

[0474] The substitution is a non-hydrogen substituent selected from -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) any one or more of the group consisting of 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.

[0475] In addition, the nitrogen atom attached to Ab may be contained in lysine 246 or lysine 248 of the antibody's Fc. In a specific embodiment, the nitrogen attached to Ab may be contained in lysine 246 of the antibody's Fc. In a specific embodiment, the nitrogen attached to Ab may be contained in lysine 248 of the antibody's Fc.

[0476] Further, in this case, the Fc-binding peptide may include a first click chemistry functional group. The click chemistry functional group can be any one or more groups of alkynes, 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, and in this case, the cycloalkyne group, conjugated diene group and diene group are the same as those described above. In addition, the Fc-binding peptide may include a first click chemistry group at an amino acid residue near the N- end of its sequence. In addition, the Fc-binding peptide may include a first click chemistry group at the N- end of its sequence. Alternatively, the Fc-binding peptide may include a first click chemistry functional group at an amino acid residue near the C- end of its sequence. In addition, the Fc-binding peptide may include a first click chemistry functional group at the C- end of its sequence.

[0477] Fp is an Fc binding peptide. In addition, Fp may be any peptide selected from Formula 13 or Formula 14. The details of Formula 13 and Formula 14 are the same as those already described.

[0478] Fp may be attached 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.

[0479] n is an integer of 1 to 4. For example, n may be 2. In this case, Fp may be linked to lysine 246 or lysine 248, each of which is contained in two Fc residues in one antibody, to produce such a structure. In another example, n may be 4. In this case, R" may be linked to lysine 246 and lysine 248, each of which is contained in two Fc residues in one antibody, to produce such a structure.

[0480] n is 2, and the nitrogen atom attached to Ab can be contained in lysine 246 of two Fc of antibody. In another example, n is 2, and the nitrogen atom attached to Ab can be contained in lysine 248 of two Fc of antibody.

[0481] The antibody of Formula 6 can be prepared by the reaction represented by the following Reaction Scheme 4.

[0482] [Reaction Scheme 4]

[0483]

[0484] In this case, details about Formula 4 and Formula 6 are the same as those already described.

[0485] In this case, since the peptides of SEQ ID NOS: 1 to 5 contain 2,3-diaminopropionic acid, 2,4-diaminobutyric acid, ornithine, or lysine, respectively, at residue 6, all corresponding residues differ only in the length of the carbon skeleton with a free amine group and have similar structures. Therefore, all peptides of SEQ ID NOS: 1 to 5 exhibit the property of binding to the Fc domain of an antibody.

[0486] The method of preparing an antibody-cargo moiety conjugate may comprise (3) preparing the antibody-cargo moiety conjugate by reacting an antibody comprising a first click chemistry functional group with a cargo moiety comprising a second click chemistry functional group complementary to the first click chemistry functional group.

[0487] The load portion may include an active portion. The active portion may be selected from one or more of nucleic acids, peptides, and compounds. The active portion may be any one selected from the group consisting of a drug molecule, an imaging portion, an optical agent, a vitamin, and a toxin. In a specific embodiment, the active portion may be a drug molecule. The drug molecule may be a prodrug, a precursor drug, or a drug. The drug molecule may be an anticancer drug, an anti-inflammatory agent, another anti-disease agent, and an antimicrobial agent (antibacterial agent, antifungal agent, antiviral agent). In a specific embodiment, the drug molecule may be maytansine (DM1). In a specific embodiment, the load portion may include two or more drug molecules. In a specific embodiment, the active portion may be an imaging portion. The imaging portion may be a contrast agent, a radioisotope, or a fluorescent substance. In a specific embodiment, the active portion may be an optical agent, a vitamin, a toxin, etc., but is not limited thereto.

[0488] In addition, the payload portion comprises a second click chemistry functional group, wherein 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), trans-cyclooctene, a nitrone, a nitrile oxide, an azide, a conjugated diene, and a dienophile, and in this case, the cycloalkyne group, the conjugated diene group, and the diene group are the same as those described above.

[0489] In one example, the antibody-loaded moiety conjugate can be represented by Formula 7.

[0490] [Formula 7]

[0491]

[0492] In Equation 7,

[0493] Ab is an antibody. In a specific embodiment, Ab can be trastuzumab.

[0494] The nitrogen atom attached to Ab may be contained within lysine 246 or lysine 248 of the antibody's Fc. In a specific embodiment, the nitrogen attached to Ab may be contained within lysine 246 of the antibody's Fc. In a specific embodiment, the nitrogen attached to Ab may be contained within lysine 248 of the antibody's Fc.

[0495] B can be any 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 can be any of the structures generated by two reactants selected from alkynes, cycloalkynes 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. In a specific embodiment, B can be Here, A1 can be linked to the antibody, and A2 can be linked to Am. Alternatively, A1 can be linked to Am, and A2 can be linked to the antibody.

[0496] Am is an active moiety or a structure comprising the active moiety. In one embodiment, Am may include two or more active moieties. The content of the active moiety is the same as that described in the description of the payload moiety.

[0497] n is an integer of 1 to 4. For example, n may be 2. In this case, the payload can be linked to lysine 246 or lysine 248, which are contained in both Fc regions of an antibody, to produce such a structure. In another example, n may be 4. In this case, R" can be linked to lysine 246 and lysine 248, which are contained in both Fc regions of an antibody, to produce such a structure.

[0498] n is 2, and the nitrogen atom linked to Ab may be contained in lysine 246 of two Fcs of the antibody. In another embodiment, n is 2, and the nitrogen atom linked to Ab may be contained in lysine 248 of two Fcs of the antibody.

[0499] As an example, the compound shown in Formula 7 can be prepared by Reaction Scheme 5.

[0500] [Reaction Scheme 5]

[0501]

[0502] In this case, R" is the first click chemistry functional group and is the same as described above for Formula 5.

[0503] In addition, R"" is the second click chemistry functional group and is the same as described when describing the payload part.

[0504] In one example, the antibody-load moiety conjugate can be represented by Formula 8.

[0505] [Formula 8]

[0506]

[0507] In formula 8,

[0508] Ab is an antibody. In a specific embodiment, Ab can be trastuzumab.

[0509] The nitrogen atom attached to Ab may be contained within the antibody's lysine 246 or lysine 248. In a specific embodiment, the nitrogen atom attached to Ab may be contained within the antibody's lysine 246. In a specific embodiment, the nitrogen atom attached to Ab may be contained within the antibody's lysine 248.

[0510] B can be any 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 can be any of the structures generated by two reactive moieties selected from alkynes, cycloalkynes 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. In a specific embodiment, B can be Here, A1 can be linked to the antibody, and A2 can be linked to Am. Alternatively, A1 can be linked to Am, and A2 can be linked to the antibody.

[0511] Fp is an Fc-binding peptide. In addition, Fp may be any peptide selected from Formula 13 or Formula 14. The details of Formula 13 and Formula 14 are the same as those already described.

[0512] Fp may be attached 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.

[0513] Am is an active moiety or a structure containing the active moiety. In a specific embodiment, Am may include two or more active moieties. The content of the active moiety is the same as that described in the description of the payload moiety.

[0514] B may bind to an amino acid residue near the N-terminus of the Fc-binding peptide sequence. In another embodiment, B may bind to the N-terminus of the Fc-binding peptide. In another embodiment, B may bind to an amino acid residue near the C-terminus of the Fc-binding peptide sequence. In another embodiment, B may bind to the C-terminus of the Fc-binding peptide.

[0515] n is an integer of 1 to 4. For example, n may be 2. In this case, the payload moiety may be linked to lysine 246 or lysine 248, which are respectively contained in both Fc regions of an antibody, to produce such a structure. In another example, n may be 4. In this case, the payload moiety may be linked to lysine 246 and lysine 248, which are respectively contained in both Fc regions of an antibody, to produce such a structure.

[0516] n is 2, and the nitrogen atoms linked to Ab may be contained in lysine 246 of two Fcs of the antibody. Alternatively, n is 2, and the nitrogen atoms linked to Ab may be contained in lysine 248 of two Fcs of the antibody.

[0517] As an example, the compound shown in Formula 8 can be prepared by Reaction Scheme 6.

[0518] [Reaction Scheme 6]

[0519]

[0520] In this case, R""' is the first click chemistry functional group and is the same as described above when describing Formula 6. In the process of synthesizing the Fc-binding peptide, the first click chemistry functional group may be contained in an amino acid residue. Any residue can be used as the amino acid residue containing the first click chemistry functional group, but preferably the amino acid residue is not residue 6, because residue 6 is a residue designed for substitution reaction with the linker. Through the process of artificially synthesizing peptides, the Fc-binding peptide may contain a first click chemistry functional group at an amino acid residue near the N-terminus of its sequence. In a specific embodiment, the Fc-binding peptide may contain a first click chemistry functional group at the N-terminus of its sequence. In a specific embodiment, the Fc-binding peptide may contain a first click chemistry functional group at an amino acid residue near the C-terminus of its sequence. In a specific embodiment, the Fc-binding peptide may contain a first click chemistry functional group at the C-terminus of its sequence.

[0521] In addition, R"" is a second click chemistry functional group and is the same as the functional group described when describing the payload portion.

[0522] The present application can provide linkers that facilitate transfer and conjugation reactions so that the cargo moiety can be attached to the desired target molecule.

[0523] The linker or linker moiety can be directly attached to the target molecule.The linker moiety that is directly attached to the target molecule can be attached to the cargo moiety.

[0524] The linker moiety can be part of a residue or part of a functional group in the linker.

[0525] For example, the linker moiety may be R" or part of a residue comprising R".

[0526] For example, the linker moiety can be a portion of the residue of the linker that does not include R".

[0527] The target molecule connected to the linker portion can be linked to the payload portion via a click reaction (ie, a reaction between click chemistry functional groups), but is not limited thereto.

[0528] In an exemplary embodiment, when the linker has the structure of Formula 1, the linker portion that is attached to the target molecule may include a click chemistry functional group.

[0529] In this case, the linker moiety attached to the target molecule can participate in the click reaction together with the payload moiety.

[0530] Such a linker will not affect the biological activity of the target molecule. That is, the linker will have little effect on the half-life, secretion, blood stability, etc. of the target molecule.

[0531] The half-life of the target molecule can be determined by the FcRn binding affinity of the target molecule. For example, the target molecule can be recycled in the body by binding to FcRn, and the half-life can be increased in the body.

[0532] The secretion of the target molecule as a result of proteosome aggregation can be determined by the extent to which the target molecule is secreted by the kidney.

[0533] As an example, a linker can increase the half-life of a drug in the blood by connecting a compound with a short intracellular half-life to a protein. For example, a drug can be linked to a target molecule with a long intracellular half-life.

[0534] In another exemplary embodiment, when the linker has the structure of Formula 2, the linker portion that is connected to the target molecule may not include a click chemistry functional group.

[0535] The linker moiety that is attached to the target molecule can be attached to a peptide, polypeptide, protein, and / or a compound comprising a click chemistry functional group.

[0536] Peptides, polypeptides, proteins and / or compounds can be linked to cargo moieties via click reactions.

[0537] Such linkers may affect the biological activity of the target molecule. When a linker affects the biological activity of the target molecule, the linker affects the half-life, secretion, or blood stability of the target molecule. For example, when the linker is indirectly attached to the target molecule, the linker can rapidly release the payload in vivo by shortening the half-life of the target molecule.

[0538] The linker can be attached to a peptide or polypeptide that has binding affinity for a specific target molecule. Such a linker can attach the payload moiety to the target molecule in a site-specific manner.

[0539] As an example, the target molecule may be an antibody.

[0540] 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 have a human CH2-CH3 domain. The target molecule may be an antibody or a partial fragment of an antibody, but is not limited thereto.

[0541] As an example, an Fc binding peptide can have binding affinity to an antibody.

[0542] For example, an Fc-binding peptide can have binding affinity to an IgG antibody. An Fc-binding peptide can have specific binding affinity to a specific domain of an IgG antibody.

[0543] For example, an Fc-binding peptide can be specific for the heavy or light chain variable region of an IgG antibody.

[0544] For example, the Fc binding peptide can be specific for the constant domain of the heavy chain of an IgG antibody. In this case, the heavy chain constant domain can be a CH2 domain and / or a CH3 domain.

[0545] The Fc binding peptide can be a peptide or polypeptide linked to a compound.

[0546] For example, the Fc-binding peptide may comprise a click chemistry functional group.

[0547] The present invention provides a method for treating cancer, comprising administering an antibody-loaded moiety 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, kidney cancer, leukemia, liver cancer, lung cancer, lymphoma, melanoma, myeloma, ovarian cancer, pancreatic cancer, penile cancer, prostate cancer, testicular germ cell cancer, thymoma, and thymic carcinoma. In addition, the cancer can be breast cancer.

[0548] The present invention provides a pharmaceutical composition for treating cancer, comprising an antibody-loaded moiety conjugate. In this case, the cancer can be selected from bladder cancer, bone cancer, brain cancer, breast cancer, heart cancer, cervical cancer, colorectal cancer, rectal cancer, esophageal cancer, fibrosarcoma, stomach cancer, gastrointestinal cancer, head and neck cancer, Kaposi's sarcoma, kidney cancer, leukemia, liver cancer, lung cancer, lymphoma, melanoma, myeloma, ovarian cancer, pancreatic cancer, penile cancer, prostate cancer, testicular germ cell cancer, thymoma, and thymic carcinoma. In addition, the cancer can be breast cancer.

[0549] Example

[0550] Hereinafter, the present invention will be described in more detail through examples.

[0551] These examples are provided only to more specifically describe the present invention, and it is obvious to those skilled in the art that the scope of the present invention is not limited by these examples. The numbers given for the compound names in the following examples are written with reference to the accompanying drawings.

[0552] [Example 1] Synthesis of Compound I (trans-linker: NHS & norbornene, Formula 1-1-2) and its structural confirmation

[0553] Example 1-1. Synthesis and structure confirmation of compound 1

[0554] 10 g (68.4 mmol, 1.0 eq) 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 eq) of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCi) and 24 mL (138 mmol, 2.0 eq) of N,N-diisopropylethylamine (DIPEA) were slowly added dropwise thereto, and the resulting solution was stirred for 20 minutes, and then 8 mL (68.7 mmol, 1.0 eq) 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 with 10% citric acid solution and dried over saturated saline 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.

[0555] 1H NMR (300MHz, dmso) δ7.35 (d, J=3.5Hz, 5H), 4.75 (d, J=3.5Hz, 2H), 3.56 (d, J=4. 0Hz, 3H), 2.26 (td, J=7.3, 3.8Hz, 2H), 1.96 (t, J=5.6Hz, 2H), 1.78-1.61 (m, 2H).

[0556] Example 1-2 Synthesis and structure confirmation of compound 2

[0557] After dissolving 12.9 g of compound 1 in 200 mL of N, N-dimethylformamide (DMF), the resulting solution was stirred at 0°C. 24 mL (160 mmol, 3.08 eq) of 1,8-diazabicyclo (5.4.0) undec-7-ene (DBU) and 10 mL (160 mmol, 3.08 eq) of iodomethane (MeI) were slowly added dropwise. 0.2 mL of diethylamine was slowly added dropwise. After stirring the resulting solution for 20 hours, the reaction solvent was mixed with celite and concentrated under reduced pressure to remove it, and the target compound was purified by column chromatography (EA: Hex = 1: 1) to obtain 7.6 g (yield: 56%). TLC (EA: Hex = 1: 1); R f =0.5.

[0558] 1H NMR (300MHz, cdcl3) δ7.44-7.33 (m, 5H), 4.82 (s, 2H), 3.65 (s, 3H), 3.20 (s, 3H), 2.43 (d, J=7.2Hz x 2H), 2.34(t, J=7.3Hz, 2H), 1.98-1.85(m, 2H).

[0559] Synthesis and structure confirmation of compound 3 in Example 1-3

[0560] After 4.18g (15.8mmol, 1.0eq) of compound 2 was dissolved in 100mL of tetrahydrofuran (THF), the resulting solution was stirred. 5mL of 2N lithium hydroxide (LiOH) was slowly added dropwise thereto at 0°C, and the resulting solution was then stirred for 3 hours. The reaction was terminated by adding 60mL of H2O, and the water layer was washed twice with 150mL of EA. The water layer was then titrated to pH 3.0 with 2N HCl solution, and the target compound was extracted three times with 50mL of EA. The organic layer was dried over saturated saline solution and sodium sulfate to obtain 2.46g (yield: 62%). TLC (EA: Hex = 1: 1); R f =0.1.

[0561] 1H NMR (300MHz, dmso) δ12.06 (s, 1H), 7.51-7.29 (m, 5H), 4.86 (s, 2H), 3.13 (s, 3H), 2.38 (t, J=7.3Hz, 2H), 2.22 (dt, J=11.7, 7.4Hz, 4H).

[0562] Synthesis and structure confirmation of compound 4 in Example 1-4

[0563] After dissolving 2.46 g of compound 3 in 40 mL of methanol (MeOH), a hydrogenation reaction was carried out in the presence of palladium on carbon (Pd / C) for 18 hours. After terminating the reaction, Pd / C was removed by a short-path column, and then the target compound was purified and concentrated to obtain 1.2 g (yield: 76%).

[0564] 1H NMR (300MHz, dmso) δ12.06 (s, 1H), 7.51-7.29 (m, 5H), 4.86 (s, 2H), 3.13 (s, 3H), 2.38 (t, J=7.3Hz, 2H), 2.22 (dt, J=11.7, 7.4Hz, 4H)

[0565] Synthesis and structure confirmation of compound 5 in Example 1-5

[0566] 0.17 g (1.06 mmol, 1.0 eq) of compound 4 was dissolved in 10 mL of DCM and the resulting solution was stirred. 0.22 g (1.73 mmol, 1.63 eq) of exo-5-norbornene chloride and 0.2 mL (1.1 mmol, 1.0 eq) of DIPEA were slowly added dropwise at 0°C. After stirring the resulting solution for 2 hours, the organic layer was washed three times with 10% citric acid solution and dried with saturated saline solution and sodium sulfate. The target compound was used in the next reaction without purification (crude yield: 0.24 g, 68%). TLC (DCM: MeOH = 10: 1); R f =0.3.

[0567] Predicted MW (M+H)+: 282.13 g / mol

[0568] Measured MW (M+H) +: 282.1 g / mol

[0569] Synthesis and structure confirmation of compound I in Example 1-6

[0570] 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. After stirring the resulting solution for 1 hour, the organic layer was washed three times with 10% citric acid solution and dried over saturated saline solution and sodium sulfate. The target compound was purified by column chromatography (EA: Hex = 1: 1) to obtain 0.04g (yield: 15%). TLC (EA: Hex = 1: 1); R f =0.3 (see Figure 1 ).

[0571] 1H NMR (300MHz, DMSO) δ6.18 (ddd, J=16.9, 5.53.0Hz, 2H), 2.94 (s1H), 2.80 (s, 4H), 2.71 (s, 1H), 2.67 (q, J=1.0Hz, 3H), 2.25 (dd, J=25.9, 18.6Hz, 4H), 1.86 (ddd, J=18.9, 11.5, 5.8Hz, 4H), 1.45-1.26 (m, 4H).

[0572] [Example 2] Synthesis and structural confirmation of compound II (cis linker: NHS & norbornene, formula 2-1-2)

[0573] Example 2-1 Synthesis and structure confirmation of compound 6

[0574] 0.1 g (0.88 mmol, 1.0 eq) of N-methylhydroxylamine hydrochloride was dissolved in 2 mL of tetrahydrofuran (THF) at -25°C, and the resulting solution was stirred. 0.07 g (0.88 mmol, 1.0 eq) of glutaric anhydride was added thereto, the resulting solution was stirred for 10 minutes, and then 0.25 mL (1.76 mmol, 2.0 eq) of triethylamine was slowly added thereto dropwise. After 1 hour, 0.14 g (0.88 mmol, 1.0 eq) of (1S, 2R, 4S)-bicyclo[2.2.1]hept-5-ene-2-carbonyl chloride and 0.12 mL (0.88 mmol, 1.0 eq) of triethylamine were slowly added thereto dropwise, and the reaction was then carried out at room temperature for 1 hour. After the reaction solution was removed by concentration under reduced pressure, the residue was redissolved in ethyl acetate (EA), and the resulting solution was washed three times with a 10% citric acid solution and dried over a saturated saline 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.

[0575] Calculated mass (M+H)+: 282.13 g / mol

[0576] Measured mass (M+H)+: 282.1 g / mol

[0577] Example 2-2 Synthesis of Compound II

[0578] 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. After reacting for 1 hour, the reaction solution was washed three times with 10% citric acid solution and dried with saturated sodium bicarbonate solution, saturated saline solution, and sodium sulfate. The target compound was purified by column chromatography (EA: Hex = 1: 1); TLC (EA: Hex = 2: 1) R f =0.4 (see Figure 3 ).

[0579] Example 2-3 Structure confirmation of compound II

[0580] The final compound II was synthesized by the method specified in Example 2-1 and Example 2-2, and Figure 6 The HPLC analysis shown confirmed the presence of isomers. Figure 6 The HPLC purification was performed under the analytical conditions shown in the figure, and the purity was obtained by HPLC analysis. Figure 7 The structure of the purified compound II was confirmed by mass spectrometry to complete the analysis of the final compound II.

[0581] Calculated mass (M+H)+: 379.14 g / mol

[0582] Measured mass (M+H)+: 379.0 g / mol

[0583] [Example 3] Confirmation of the structure of isomer compound 6 (cis linker or trans linker: NHS & norbornene)

[0584] The cis- and trans-forms of compound 6 as isomers were confirmed using a high-speed liquid chromatography (HPLC) apparatus. 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. As 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. The characteristics of each structure were analyzed by absorbance at a wavelength of 220 nm, as described above.

[0585] like Figure 3 As shown, it was confirmed that a peak was observed at 14.89 minutes for the cis structure of compound 6 and a peak was observed at 15.32 minutes for the trans structure of compound 6. The analysis of each structure was confirmed by mass spectrometry (Shimadzu, LCMS-8050). Figure 4 and Figure 5 The results of mass spectrometry analysis of the cis and trans structures of Compound 6 are shown. As a result of molecular weight confirmation, a substance corresponding to each peak was observed to have a molecular weight of 282 with one hydrogen molecule bonded thereto.

[0586] Calculated mass (M+H) + :282.13g / mol

[0587] Measurement mass (M+H) + :282.1g / mol

[0588] [Example 4] Synthesis and structure confirmation of Fc-binding peptide

[0589] Example 4-1. Synthesis and structure confirmation of FcBP(6Lys)-norbornene

[0590] [Equation 10]

[0591] FcBP(6Lys)-norbornene

[0592]

[0593] Example 4-1-1: Synthesis of FcBP(6Lys)-norbornene

[0594] List of Fmoc amino acids used and order of introduction of Fmoc amino acids used

[0595] 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.

[0596] Preparation method

[0597] (a) Introduction of amino acids

[0598] The amounts of reagents used in the following procedures are 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.

[0599] By activating the Fmoc-amino acid, the reaction of linking the activated residue to the transparent amide resin is carried out sequentially from the C-terminal amino acid.

[0600] Fmoc was removed in DMF containing 20% ​​piperidine, and for activation and introduction of residues, the amino acid prepared according to the sequence was mixed with 2 mL of a DMF solution containing 0.5 M HOBt, 2 mL of a DMF solution containing 0.5 M HBTU, and 174 μL of DIPEA for 5 minutes, and the resulting mixture was poured into a reactor containing the resin and mixed for 2 hours.

[0601] The incorporation reaction was confirmed by the Kaiser test. If no reaction was confirmed, the incorporation reaction was repeated or capping was performed with a DMF solution containing 20% ​​AcO. The resin was thoroughly washed with DMF and DCM before proceeding to the next step of each incorporation reaction and Fmoc removal process. This process was repeated until the target peptide sequence was obtained.

[0602] (b) Introduction of H-PEG8-OH

[0603] After all amino acids were introduced, H-PEG8-OH was introduced at the N-terminus. 1 mL of a 0.5 M DMF solution of Fmoc-N-amino-dPEG8-acid, 1 mL of a DMF solution containing 0.5 M HBTU, 1 mL of a DMF solution containing 0.5 M HOBt, and 87 μL of DIPEA were mixed for 5 minutes, and the resulting mixture was poured into a reactor containing the resin and mixed for 2 hours.

[0604] The reaction progress was confirmed by the Kaiser test. If unreacted amine remained, the reaction time was further extended by 1 to 3 hours, or the reaction solution was emptied and the above reaction process was repeated. The N-terminal Fmoc protecting group was removed using DMF containing 20% ​​piperidine, and the peptide-attached resin was dried and weighed.

[0605] (c) Introduction of norbornene

[0606] To remove the N-terminal Fmoc protecting group, 4 eq. of norbornene carboxylic acid, 2 mL of a 0.5 M HOBt solution in DMF, 2 mL of a 0.5 M HBTU solution in DMF, and 174 μL of DIPEA were mixed with the resin for 5 minutes. The resulting mixture was then poured into the reactor containing the resin and mixed for 2 hours. The incorporation reaction was confirmed by the Kaiser test. If no reaction was confirmed, the incorporation reaction was repeated.

[0607] (d) The peptide was cleaved from the resin by stirring 250 mg of the 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) for 120 minutes at room temperature. The cleavage mixture was filtered, and the filtrate was concentrated to about half with nitrogen, then poured into diethyl ether to precipitate the peptide. The precipitated peptide was further washed three times with diethyl ether and dried with nitrogen. The dried precipitate was dissolved in water containing 0.1% TFA-30% ACN, and the resulting solution was stirred for 6 hours and then concentrated.

[0608] The concentrate was dissolved in 0.01 M ammonium acetate buffer (pH 6.5) containing 5% DMSO and 20% ACN (concentration: 0.1 mg / mL), and the resulting solution was stirred for 3 days while exposed to air. The progress of the disulfide bond formation reaction was observed by HPLC. When it was determined that the reaction no longer proceeded, the reaction solution was freeze-dried to obtain a peptide precipitate.

[0609] (e) Purification

[0610] The peptide precipitates obtained by lyophilization in step (d) were purified and lyophilized under the prep-LC conditions shown in Table 5 below. Each of the obtained peptides was confirmed to have a purity of more than 90% by analytical HPLC, and the results were as follows: Figure 8 shown.

[0611] Norbornene-PEG8-Asp-Cys*-Ala-Trp-His-Lys-Gly-Glu-Leu-Val-Trp-Cys*-Thr-NH2 (Cys*: disulfide bond binding site)

[0612] [Table 5]

[0613]

[0614] Example 4-1-2: Confirmation of the structure of FcBP(6Lys)-norbornene (oxidized state)

[0615] The synthesis of FcBP(6Lys) was confirmed by LC mass-based molecular weight measurement.

[0616] Measuring instrument: Waters Quattro Premier XE

[0617] Calculated molecular weight: 2088.40 g / mol

[0618] Measure molecular weight (M / 2+H) 2+ :1044.84g / mol

[0619] The results are as follows Figure 9 shown.

[0620] Example 4-2. Synthesis and structure confirmation of compound I-FcBP (6Lys)-norbornene

[0621] [Equation 11]

[0622] Compound I-FcBP(6Lys)-norbornene

[0623]

[0624] Example 4-2-1: Synthesis of Compound I-FcBP(6Lys)-Norbornene

[0625] Compound I (trans-norbornene Weinreb amide)-FcBP was synthesized in DMF. To introduce compound I into FcBP(6Lys)-norbornene, 3 eq 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.

[0626] In order to confirm the introduction reaction, analysis was performed by HPLC, and when the reaction was not terminated, DIPEA was added thereto in 1 eq increments to observe the termination of the reaction.

[0627] After confirming the reaction was complete, the reaction solution was concentrated and I-FcBP(6Lys)-norbornene was purified by preparative HPLC. After purification, the product was freeze-dried to yield 2.07 μmol. Purity was also confirmed by HPLC (purity: >95% (HPLC), yield: 83%).

[0628] The results are as follows Figure 10 shown.

[0629] Example 4-2-2: Structure confirmation of compound I-FcBP(6Lys)-norbornene

[0630] The synthesis of Compound I-FcBP(6Lys)-norbornene was confirmed by LC mass-based molecular weight measurement.

[0631] Measuring instrument: Waters Quattro Premier XE

[0632] Calculated molecular weight: 2351.69 g / mol

[0633] Measure molecular weight (M / 2+H) 2+ :1176.42g / mol

[0634] The results are as follows Figure 11 shown.

[0635] Example 4-3. Synthesis and structure confirmation of compound II-FcBP (6Lys)-norbornene

[0636] [Equation 12]

[0637] Compound II-FcBP(6Lys)-norbornene

[0638]

[0639] Example 4-3-1: Synthesis of Compound II-FcBP(6Lys)-Norbornene

[0640] Compound II (cis-norbornene Weinreb amide)-FcBP was synthesized in DMF, and in order to introduce Compound II into FcBP(6Lys)-norbornene, 3 eq 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.

[0641] In order to confirm the introduction reaction, analysis was performed by HPLC, and when the reaction was not terminated, termination of the reaction was observed by adding DIPEA thereto in 1 eq increments.

[0642] After confirming the reaction was complete, the reaction solution was concentrated and purified by preparative HPLC. After purification, the product II-FcBP(6Lys)-norbornene was freeze-dried to yield 2.02 μmol. The purity was also confirmed by HPLC (purity: >95% (HPLC), yield: 81%).

[0643] The results are as follows Figure 12 shown.

[0644] Example 4-3-2: Confirmation of Compound II-FcBP(6Lys)-Norbornene

[0645] Measuring instrument: Waters Quattro Premier XE

[0646] Calculated molecular weight: 2351.69 g / mol

[0647] Measure molecular weight (M / 2+H) 2+ :1176.42g / mol

[0648] The results are as follows Figure 13 shown.

[0649] [Example 5] Synthesis and structure confirmation of antibody-click chemistry reagents

[0650] Example 5-1: Antibody-Norbornene

[0651] Example 5-1-1: Synthesis of Trastuzumab-Norbornene (1)

[0652] Introduction reaction using compound I-FcBP(6Lys)-norbornene

[0653] Ab (Lys 246 / 248)-norbornene was synthesized using compound I-FcBP (6Lys)-norbornene in phosphate buffered saline (PBS) buffer at pH 7.4. In order to introduce norbornene into the two specific sites of the antibody, 6 eq of compound I-FcBP (6Lys)-norbornene per antibody was added to the reaction solution and then reacted. The reaction was carried out at room temperature for more than 1 week, and reaction monitoring and termination were confirmed by HIC-HPLC. For purification, purification was carried out by three dialysis (pH 5.5, 20 mM histidine acetate buffer) and size exclusion chromatography (molecular weight cutoff 40 kDa).

[0654] The reaction of compound I-FcBP(6Lys)-norbornene with antibody is as follows Figure 14 As shown, the structure of the final product Ab(Lys 246 / 248)-norbornene is as shown Figure 15 As shown, the reaction monitoring by HIC-HPLC was performed as Figure 16 shown.

[0655] Example 5-1-2: Synthesis of Trastuzumab-Norbornene (2)

[0656] Introduction reaction of compound II-FcBP(6Lys)-norbornene

[0657] Ab (Lys 246 / 248)-norbornene was synthesized using compound II-FcBP (6Lys)-norbornene in phosphate buffered saline (PBS) buffer at pH 7.4. In order to introduce norbornene into two specific sites of the antibody, 6 eq of compound II-FcBP (6Lys)-norbornene per antibody was added to the reaction solution and then reacted. The reaction was carried out at room temperature for 12 hours, and reaction monitoring and termination were confirmed by HIC-HPLC. Purification was performed by three dialysis (pH 5.5, 20 mM histidine acetate buffer) and size exclusion chromatography (molecular weight cutoff 40 kDa) to obtain 135 mg of product from 150 mg of trastuzumab (yield = 90%).

[0658] The reaction of compound II-FcBP(6Lys)-norbornene with antibody is as follows Figure 17 As shown, and the structure of the product Ab (Lys246 / 248)-norbornene is as shown Figure 18 The reaction was monitored by HIC-HPLC. Figure 19 shown.

[0659] Example 5-2-1: Confirmation of Herceptin-Norbornene Binding

[0660] Verification of the confirmation of antibody intermediates containing FcBP norbornene linkers in antibodies based on mass spectrometry ( Figure 18 ).

[0661] Measuring instrument: Ultraflex III (TOF / TOF)

[0662] Analysis mode: Linear mode

[0663] Polarity: Positive

[0664] Detection: m / z 2,000 to 300,000

[0665] Laser repetition frequency: 100Hz

[0666] Number of launches: 1,000

[0667] Deflection: On, 5,000Da

[0668] Voltage: Ion source I 25.00kV, Ion source II 23.00kV, Lens 9.00kV

[0669] Calculated molecular weight: 152,385 g / mol

[0670] Measured molecular weight: 152,407 g / mol (M+Na)

[0671] The analysis results are as follows Figure 20 shown.

[0672] [Example 6] Synthesis and confirmation of antibody drug conjugates

[0673] Example 6-1: Antibody-drug conjugate

[0674] Example 6-1-1: Synthesis of Trastuzumab-DM1

[0675] Trastuzumab was used to synthesize an antibody-loaded moiety conjugate in which two norbornene molecules were introduced via Compound II-FcBP(6Lys)-norbornene ( Figure 18 The reaction was carried out at a concentration of 4.5 mg / mL in a volume of 25 mL, and an attempt was made to couple the tetrazine-PEG8-DM1 drug to norbornene conjugated to the antibody by biorthogonal chemical coupling. 4 eq of drug was used relative to the antibody, and the coupling reaction was carried out in a 20 mM histidine acetate solution at pH 5.5 for 24 hours at room temperature. Observation of the coupling reaction was confirmed by HIC-HPLC, and the formation of the antibody-load moiety conjugate was observed by observing the peak shift from the 9.4 minute range to the 11.2 minute range (appearing only when the FcBP linker was bound to trastuzumab) as the antibody-FcBP linker reacted with the drug.

[0676] The structure of the product antibody-loaded moiety conjugate is shown in Figure 21 The reaction monitoring by HIC-HPLC is shown in Figure 22 In. Figure 21 In the figure, the amplified structure is the structure of the load part, and the non-amplified part is the same as Figure 18 The structure is the same as shown in .

[0677] Example 6-1-2: Trastuzumab-DM1 purification method

[0678] To obtain highly pure antibody-loaded moiety conjugates, they were dialyzed against 20 mM histidine acetate solution at pH 5.5 and HIC purified using fast protein liquid chromatography (FPLC).

[0679] HIC purification conditions were as follows.

[0680] FPLC mode: AKTA pure

[0681] Flow rate: 1 mL / min

[0682] Column: HiPrep butyl FF16 / 10 column

[0683] Elution solvent: (A) 1.5 M ammonium sulfate + 50 mM phosphate pH 7.0

[0684] (B) 50 mM phosphate pH 7.0

[0685] Elution conditions

[0686] 0:00-10:00A:40%, B:60%

[0687] 10:00-20:00A:65%, B:35%

[0688] 20:00-102.5:00A:75%, B:25%

[0689] 102:5-115:00A:100%, B:0%

[0690] 115:00-135:00A:100%, B:0%

[0691] The HIC chromatogram of the purified antibody-load moiety conjugate is shown in Figure 2. Figure 19 shown.

[0692] By the above method, 67 mg of trastuzumab-DM1 was obtained, in which the drug was conjugated to two sites (drug-antibody ratio = 2) (yield = 60%).

[0693] Example 6-1-3: Confirmation of Trastuzumab-DM1 Binding

[0694] Mass spectrometry-based validation of antibody-loaded moiety conjugates containing an FcBP linker and a drug was performed.

[0695] Measuring instrument: Ultraflex III (TOF / TOF)

[0696] Analysis mode: Linear mode

[0697] Polarity: Positive

[0698] Detection: m / z 2,000 to 300,000

[0699] Laser repetition frequency: 100Hz

[0700] Number of launches: 1,000

[0701] Deflection: On, 5,000Da

[0702] Voltage: Ion source I 25.00kV, Ion source II 23.00kV, Lens 9.00kV

[0703] Calculated molecular weight: 155,493 g / mol

[0704] Measured molecular weight: 155,523 g / mol (approximate value, M+Na)

[0705] The analysis results are as follows Figure 23 shown.

[0706] [Example 7] Evaluation of the efficacy of a novel antibody-drug conjugate (ADC, trastuzumab-DM1 conjugate) at the cellular level (in vitro cytotoxicity test)

[0707] Based on the target marker level of cancer cells, the efficacy of antibody-load part conjugate (ADC, trastuzumab-DM1 conjugate) is evaluated, and cytotoxicity test is carried out using NCI-N87 and BT474 for the positive expression cell line of target antigen Her2 and MDA-MB-468 for negative cell line. In Her2 overexpressing cells NCI-N87 and BT474, as the observation result by treating cells with the antibody drug conjugate prepared at each concentration, excellent anticancer effect is confirmed, and IC50 values ​​are 82.1ng / mL and 29.6ng / mL. When compared with commercially available Herceptin ADC, Kadcyla, the effect of the same level is confirmed, so the practicality as novel ADC can be confirmed.

[0708] The experimental results are as follows Figures 24 to 27 shown.

[0709] [Example 8] Evaluation of the efficacy of a novel antibody-loaded moiety conjugate (ADC, trastuzumab-DM1 conjugate) at the animal level (in vivo cytotoxicity test)

[0710] Xenograft tumor models were created by subcutaneously transplanting the NCI-N87 gastric cancer cell line overexpressing the target antigen. The xenograft tumor models were divided into four groups, and the administered substances were tested for their anticancer efficacy. Since the BALB / c nude mice used in this experiment lack T cells, cancer cells readily engraft into them, making mice a suitable rodent model for anticancer efficacy testing.

[0711] (a) Preparation of cell lines

[0712] RPMI1640 medium (Gibco, 22400-089) containing heat-inactivated 10% fetal bovine serum (FBS, Gibco, 10082-742) was placed in a cell culture flask, and one vial of human tumor cell line (NCI-N87 cell line) was added thereto and cultured in a 5% CO2 incubator at 37°C. The culture flask was washed with PBS, and the cells were separated by diluting 2.5% trypsin-EDTA (Gibco, 15090) 10-fold. After that, the diluted trypsin-EDTA was added thereto, and the cells were centrifuged (1,000 rpm, 5 minutes) and the supernatant was discarded. Then, a cell suspension was obtained with new culture medium. After confirming the cell activity using a microscope, the cells were suspended at a concentration of 1.25×10 7 Cell lines were prepared by diluting the cell suspension into a 1:1 mixture of culture medium and Matrigel at 10 cells / mL.

[0713] (b) Transplantation of cell lines

[0714] The cell line was prepared according to the method described in '4.3)(4) Preparation of cell line'. When the cell line was prepared, the cell line was resuspended and homogenized, and the prepared cell line was immediately administered to the animal. When the cell line was transplanted, the back of the animal was disinfected with 70% alcohol, and a space was created between the skin and muscle by pulling the skin on the back of the neck with the thumb and index finger. Then, an injection needle equipped with a 26-gauge needle was inserted from the front of the animal into the subcutaneous space between the thumb and index finger, and 2.5×10 6 The cell line was administered subcutaneously at a dose of 0.2 mL / head. During the acclimatization period, healthy animals were selected and inoculated with the cell line. Thus, when the size of the cell line transplant site reached approximately 100 mm 3 Up to 150mm 3 When the tumors were sorted, they were distributed according to the tumor size so that the sizes of the tumors in each group were distributed as evenly as possible.

[0715] (c) Determination of experimental group configuration, dosage and administration method

[0716] Cell line = NCI-N87

[0717] Mouse type = BALB / c nude mice (CAnN.Cg-Foxn1nu / CrljOri)

[0718] Number of groups = 5

[0719] Administration method: intravenous injection (26-gauge needle and syringe)

[0720] Dose = 5 mg / kg

[0721] Dosage number = 1 time / 2 days, three times a day

[0722] Observation period = 5 weeks

[0723] Group 1: PBS, Group 2: Herceptin (trastuzumab), Group 3: newly prepared ADC (Herceptin-DM1 conjugate, 1.5st ADC)

[0724] (d) Observation and inspection items

[0725] General symptoms

[0726] During the dosing and observation period, the general symptom type, including death, onset date and symptom severity, will be observed and recorded daily for each individual. Individuals with severe general symptoms will be isolated.

[0727] weight

[0728] Body weight was measured on the day of grouping or the start of test substance administration and twice a week thereafter.

[0729] Measurement of tumor size

[0730] Tumor size was measured twice a week for 5 weeks starting from the start of test substance administration. The long and short axes of the tumor were measured using calipers, and the tumor size was calculated using the following formula.

[0731] Tumor size = ab 2 / 2(a: major axis length, b: minor axis length)

[0732] (e) Results

[0733] The group injected with phosphate-buffered saline (PBS) and Herceptin (trastuzumab) showed no trend in inhibiting tumor growth during the 5-week observation period. Compared with Herceptin, the antibody-loaded moiety conjugate (Herceptin-DM1) prepared by the present application was confirmed to have excellent ability to inhibit tumor growth, thus confirming that the antibody-loaded moiety conjugate successfully functions as an ADC.

[0734] The above related results are as follows Figure 28 and Figure 29 shown.

[0735] [Example 9] Synthesis and confirmation of site-specific interaction groups based on carbon length

[0736] Example 9-1. Synthesis and Structural Confirmation of Compound II-FcBP (L6Dap, L6Dab, L6Orn, L6Lys)-Norbornene

[0737] Compound II - FcBP (L6Dap, L6Dab, L6Orn, L6Lys)-norbornene

[0738]

[0739] Example 9-1-1: Synthesis of FcBP (L6Dap, L6Dab, L6Orn, L6Lys)-norbornene

[0740] FcBP (L6Dap): List of Fmoc amino acids used and order of introduction of the Fmoc amino acids used (n=1)

[0741] Fmoc-L-Thr(tBu)-OH, Fmoc-Cys(Trt)-OH, Fmoc-L-Trp(Boc)-OH, Fmoc-L-Val-OH, Fmc-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.

[0742] FcBP (L6Dab): List of Fmoc amino acids used and order of introduction of the Fmoc amino acids used (n=2)

[0743] 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, Fmc-Asp(tBu)-OH.

[0744] FcBP (L6Orn): List of used Fmoc amino acids and order of introduction of used Fmoc amino acids (n=3)

[0745] Fmoc-L-Thr(tBu)-OH, Fmoc-Cys(Tit)-OH, Fmoc-L-Trp(Boc)-OH, Fmoc-L-Val-OH, Fmoc-L-Leu-OH, FmocL-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.

[0746] FcBP (L6Lys): List of used Fmoc amino acids and order of introduction of used Fmoc amino acids (n=4)

[0747] 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.

[0748] Preparation method

[0749] (a) Introduction of amino acids

[0750] The amounts of reagents used in the following procedures are 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.

[0751] The reaction of linking the activated residue to the transparent amide resin is performed by activating the Fmoc-amino acid and is carried out sequentially starting from the C-terminal amino acid.

[0752] Fmoc was removed in DMF containing 20% ​​piperidine, and for activation and introduction of residues, the amino acid prepared according to the sequence was mixed with 2 mL of a DMF solution containing 0.5 M HOBt, 2 mL of a DMF solution containing 0.5 M HBTU, and 174 μL of DIPEA for 5 minutes, and the resulting mixture was poured into a reactor containing the resin and mixed for 2 hours.

[0753] The incorporation reaction was confirmed using the Kaiser test. If no reaction was confirmed, the incorporation reaction was repeated or capping was performed with a 20% AcO solution in DMF. The resin was thoroughly washed with DMF and DCM before proceeding to the next step of each incorporation reaction and Fmoc removal process. This process was repeated until the target peptide sequence was obtained.

[0754] (b) Introduction of H-PEG8-OH

[0755] After all amino acids were introduced, H-PEG8-OH was introduced at the N-terminus. 1 mL of a 0.5 M DMF solution of Fmoc-N-amino-dPEG8-acid, 1 mL of a DMF solution containing 0.5 M HBTU, 1 mL of a DMF solution containing 0.5 M HOBt, and 87 μL of DIPEA were mixed for 5 minutes, and the resulting mixture was poured into a reactor containing the resin and mixed for 2 hours.

[0756] The reaction progress was confirmed by the Kaiser test. If unreacted amine remained, the reaction time was further extended by 1 to 3 hours, or the reaction solution was emptied and the above reaction process was repeated. The N-terminal Fmoc protecting group was removed using DMF containing 20% ​​piperidine, and the peptide-attached resin was dried and weighed.

[0757] (c) Introduction of norbornene

[0758] To remove the N-terminal Fmoc protecting group, 4 eq. of norbornene carboxylic acid, 2 mL of a 0.5 M HOBt solution in DMF, 2 mL of a 0.5 M HBTU solution in DMF, and 174 μL of DIPEA were mixed with the resin for 5 minutes. The resulting mixture was then poured into the reactor containing the resin and mixed for 2 hours. The incorporation reaction was confirmed using the Kaiser test. If no reaction was confirmed, the incorporation reaction was repeated.

[0759] (d) The peptide was cleaved from the resin by stirring 250 mg of the 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) for 120 minutes at room temperature. The cleavage mixture was filtered, and the filtrate was concentrated to about half with nitrogen, then poured into diethyl ether to precipitate the peptide. The precipitated peptide was further washed three times with diethyl ether and dried with nitrogen. The dried precipitate was dissolved in water containing 0.1% TFA-30% ACN, and the resulting solution was stirred for 6 hours and then concentrated.

[0760] The concentrate was dissolved in 0.01 M ammonium acetate buffer (pH 6.5) containing 5% DMSO-20% ACN (concentration 0.1 mg / mL), and the resulting solution was stirred for 3 days while exposed to air. The progress of the disulfide bond formation reaction was observed by HPLC, and when it was determined that the reaction no longer proceeded, the reaction solution was freeze-dried to obtain a peptide precipitate.

[0761] (e) Purification

[0762] The peptide precipitate obtained by lyophilization in step (d) was purified and lyophilized under the prep-LC conditions shown in Table 6. It was confirmed that each of the obtained peptides had a purity of 90% or more.

[0763] (f) sequence

[0764] FcBP(L6Dap)-norbornene:

[0765] Norbornene-PEG8-Asp-Cys*-Ala-Trp-His-Dap-Gly-Glu-Leu-Val-Trp-Cys*-Thr-NH2

[0766] (Cys*: disulfide bond binding site)

[0767] FcBP(L6Dab)-norbornene:

[0768] Norbornene-PEG8-Asp-Cys*-Ala-Trp-His-Dab-Gly-Glu-Leu-Val-Trp-Cys*-Thr-NH2

[0769] (Cys*: disulfide bond binding site)

[0770] FcBP(L6DOrn) norbornene:

[0771] Norbornene-PEG8-Asp-Cys*-A1a-Trp-His-Orn-Gly-Glu-Leu-Val-Trp-Cys*-Thr-NH2

[0772] (Cys*: disulfide bond binding site)

[0773] FcBP(L6Lys)-norbornene:

[0774] Norbornene-PEG8-Asp-Cys*-Ala-Trp-His-Lys-Gly-Glu-Leu-Val-Trp-Cys*-Thr-NH2

[0775] (Cys*: disulfide bond binding site)

[0776] [Table 6]

[0777]

[0778] Example 9-1-2: Synthesis of Compound II-FcBP(L6Dap)-Norbornene

[0779] Compound II (cis-norbornene Weinreb amide)-FcBP was synthesized in DMF. To introduce compound II into FcBP(L6Dap)-norbornene, 3 eq of DIPEA and 8.4 μmol of compound II were dissolved in 7.3 μmol of FcBP(L6Dap)-norbornene dissolved in DMF, and the resulting solution was stirred.

[0780] After confirming the reaction was complete, the reaction solution was concentrated and purified by preparative HPLC. After purification, the compound II-FcBP(L6Dap)-norbornene was freeze-dried to yield 14.1 mg. The purity was also confirmed by HPLC (purity: >99% (HPLC), yield: 83%).

[0781] The results are as follows Figure 30 shown.

[0782] Example 9-1-3: Structural confirmation of compound II-FcBP(L6Dap)-norbornene

[0783] Measuring instrument: Waters Quattro Premier XE

[0784] Calculated molecular weight: 2309.61 g / mol

[0785] Measure molecular weight (M / 2+H) 2+ :1155.08g / mol

[0786] The results are as follows Figure 31 shown.

[0787] Example 9-1-4: Synthesis of Compound II-FcBP(L6Dab)-Norbornene

[0788] Compound II (cis-norbornene Weinreb amide)-FcBP was synthesized in DMF. To introduce Compound II into FcBP(L6Dab)-norbornene, 3 eq of DIPEA and 8.4 μmol of Compound II were dissolved in 7.3 μmol of FcBP(L6Dab)-norbornene dissolved in DMF, and the resulting solution was stirred.

[0789] After confirming the reaction was complete, the reaction solution was concentrated and purified by preparative HPLC. After purification, the compound II-FcBP(L6Dab)-norbornene was freeze-dried to yield 13.8 mg. The purity was also confirmed by HPLC (purity: >99% (HPLC), yield: 82%).

[0790] The results are as follows Figure 32 shown.

[0791] Example 9-1-5: Structure confirmation of compound II-FcBP(L6Dab)-norbornene

[0792] Measuring instrument: Waters Quattro Premier XE

[0793] Calculated molecular weight: 2323.64 g / mol

[0794] Measure molecular weight (M / 2+H) 2+ :1162.02g / mol

[0795] The results are as follows Figure 33 shown.

[0796] Example 9-1-6: Synthesis of Compound II-FcBP(L6Orn)-Norbornene

[0797] Compound II (cis-norbornene Weinreb amide)-FcBP was synthesized in DMF, and in order to introduce Compound II into FcBP(L6Orn)-norbornene, 3 eq 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.

[0798] After confirming the reaction was complete, the reaction solution was concentrated and purified by preparative HPLC. After purification, the compound II-FcBP(L6Orn)-norbornene was freeze-dried to yield 14.8 mg. The purity was also confirmed by HPLC (purity: >99% (HPLC), yield: 88%).

[0799] The results are as follows Figure 34 shown.

[0800] Example 9-1-7: Structure confirmation of compound II-FcBP(L6Orn)-norbornene

[0801] Measuring instrument: Waters Quattro Premier XE

[0802] Calculated molecular weight: 2337.66 g / mol

[0803] Measure molecular weight (M / 2+H) 2+ :1169.03g / mol

[0804] The results are as follows Figure 35 shown.

[0805] Example 9-1-8: Synthesis of Compound II-FcBP(L6Lys)-Norbornene

[0806] Compound II (cis-norbornene Weinreb amide)-FcBP was synthesized in DMF. To introduce Compound II into FcBP(L6Lys)-norbornene, 3 eq 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.

[0807] After confirming the reaction was complete, the reaction solution was concentrated and purified by preparative HPLC. After purification, the compound II-FcBP(L6Lys)-norbornene was freeze-dried to yield 15.4 mg. The purity was also confirmed by HPLC (purity: >99% (HPLC), yield: 91%).

[0808] The results are as follows Figure 36 shown.

[0809] Example 9-1-9: Structure confirmation of compound II-FcBP(L6Lys)-norbornene

[0810] Measuring instrument: Waters Quattro Premier XE

[0811] Calculated molecular weight: 2351.69 g / mol

[0812] Measure molecular weight (M / 2+H) 2+ :1176.04g / mol

[0813] The results are as follows Figure 37 shown.

[0814] [Example 10] Verification of antibody binding efficiency of site-specific interactomes based on carbon length

[0815] Example 10-1. Preparation of site-specific antibody-norbornene conjugates using compound II-FcBP (L6Dap, L6Dab, L6Orn, L6Lys)-norbornene

[0816] Example 10-1-1: Synthesis of Trastuzumab-Norbornene Based on Compound II-FcBP(L6Dap)-Norbornene

[0817] Ab (Lys 246 / 248) -norbornene was synthesized using compound II-FcBP (L6Dap) -norbornene in phosphate buffered saline (PBS) buffer at pH 7.4. In order to introduce norbornene into two specific sites of the antibody (trastuzumab 4 mg / mL, 1 mL), 6 eq of compound II-FcBP (L6Dap) -norbornene per antibody was added to the reaction solution and then reacted. For the reaction temperature and time, the reaction was carried out at room temperature for 12 hours, and reaction monitoring and termination were confirmed by HIC-HPLC. Trastuzumab showed a peak at 6.3-6.4 minutes on HIC-HPLC. When the FcBP (L6Dap) -norbornene molecule binds to only one of the two binding sites of trastuzumab, a peak was observed at 8 minutes on HIC-HPLC. When the FcBP (L6Dap) -norbornene molecule binds to both sites of trastuzumab, a peak was observed at 9 minutes on HIC-HPLC. Figure 38 The monitoring of the binding reaction with the antibody based on Compound II-FcBP(L6Dap)-norbornene is shown in . The synthesis of an antibody-loaded moiety conjugate with a DAR of 2 was confirmed by observing the peak at 9.074 minutes.

[0818] Example 10-1-2: Synthesis of Trastuzumab-Norbornene Based on Compound II-FcBP(L6Dab)-Norbornene

[0819] Ab (Lys 246 / 248) -norbornene was synthesized using compound II-FcBP (L6Dab) -norbornene in phosphate buffered saline (PBS) buffer at pH 7.4. In order to introduce norbornene into two specific sites of the antibody (trastuzumab 4 mg / mL, 1 mL), 6 eq of compound II-FcBP (L6Dab) -norbornene per antibody was added to the reaction solution and then reacted. For the reaction temperature and time, the reaction was carried out at room temperature for 12 hours, and reaction monitoring and termination were confirmed by HIC-HPLC. Trastuzumab showed a peak at 6.3-6.4 minutes on HIC-HPLC. When the FcBP (L6Dab) -norbornene molecule binds to only one of the two binding sites of trastuzumab, a peak was observed at 8 minutes on HIC-HPLC. When the FcBP (L6Dab) -norbornene molecule binds to both sites of trastuzumab, a peak was observed at 9 minutes on HIC-HPLC. Figure 39 The monitoring of the binding reaction with the antibody based on Compound II-FcBP(L6Dab)-norbornene is shown in . The synthesis of an antibody-loaded moiety conjugate with a DAR of 2 was confirmed by observing the peak at 9.231 minutes.

[0820] Example 10-1-3: Synthesis of Trastuzumab-Norbornene Based on Compound II-FcBP(L6Orn)-Norbornene

[0821] Ab (Lys 246 / 248) -norbornene was synthesized using compound II-FcBP (L6Orn) -norbornene in phosphate buffered saline (PBS) buffer at pH 7.4. In order to introduce norbornene into two specific sites of the antibody (trastuzumab 4 mg / mL, 1 mL), 6 eq of compound II-FcBP (L6Orn) -norbornene per antibody was added to the reaction solution and then reacted. For the reaction temperature and time, the reaction was carried out at room temperature for 12 hours, and reaction monitoring and termination were confirmed by HIC-HPLC. Trastuzumab showed a peak at 6.3-6.4 minutes on HIC-HPLC. When the FcBP (L6Orn) -norbornene molecule binds to only one of the two binding sites of trastuzumab, a peak was observed at 8 minutes on HIC-HPLC. When the FcBP (L6Orn) -norbornene molecule binds to both sites of trastuzumab, a peak was observed at 9 minutes on HIC-HPLC. Figure 40 The monitoring of the binding reaction with the antibody based on Compound II-FcBP(L6Orn)-norbornene is shown in Figure 2. The synthesis of an antibody-loaded moiety conjugate with a DAR of 2 was confirmed by observing the peak at 8.975 minutes.

[0822] Example 10-1-4: Synthesis of Trastuzumab-Norbornene Based on Compound II-FcBP(L6Lys)-Norbornene

[0823] Ab (Lys 246 / 248) -norbornene was synthesized using compound II-FcBP (L6Lys) -norbornene in phosphate buffered saline (PBS) buffer at pH 7.4. In order to introduce norbornene into two specific sites of the antibody (trastuzumab 4 mg / mL, 1 mL), 6 eq of compound II-FcBP (L6Lys) -norbornene per antibody was added to the reaction solution and then reacted. For the reaction temperature and time, the reaction was carried out at room temperature for 12 hours, and reaction monitoring and termination were confirmed by HIC-HPLC. Trastuzumab showed a peak at 6.3-6.4 minutes on HIC-HPLC. When the FcBP (L6Lys) -norbornene molecule binds to only one of the two binding sites of trastuzumab, a peak was observed at 8 minutes on HIC-HPLC. When the FcBP (L6Lys) -norbornene molecule binds to both sites, a peak was observed at 9 minutes on HIC-HPLC. Figure 41The monitoring of the binding reaction with the antibody based on Compound II-FcBP(L6Lys)-norbornene is shown in Figure 2. The synthesis of an antibody-loaded moiety conjugate with a DAR of 2 was confirmed by observing the peak at 9.120 minutes.

Claims

1. An antibody-loaded moiety conjugate comprising: in, Ab is an IgG antibody or a partial fragment of the IgG antibody, Wherein, n is an integer from 1 to 4, Where R″′ is C 1-6 Alkylene or C 1-10 Heteroalkylene, wherein the heteroalkylene comprises at least one selected from the group consisting of N, O and S, Wherein, B is any structure formed by the click chemistry reaction of the first click chemistry functional group and the second click chemistry functional group, wherein the first click chemistry functional group and the second click chemistry functional group are independently selected from the group consisting of a trans-cyclooctene group, a cyclooctyne group, a diarylcyclooctyne group, a norbornene group, a tetrazine group, an azide group, and a dibenzocyclooctyne group, wherein Am is an active moiety or a structure comprising the active moiety, wherein the active moiety comprises any one selected from the group consisting of drug molecules, radioisotopes, optical agents, vitamins and toxins, Wherein, Fp is an Fc-binding peptide comprising an amino acid sequence, and the Fc-binding peptide is represented by the following formula 13: (Equation 13) DCAWH-Xa-GELVWCT, Among them, D is aspartic acid, C is cysteine, A is alanine, W is tryptophan, and H is histidine. wherein Xa is 2,3-diaminopropionic acid, 2,4-diaminobutyric acid, ornithine or lysine, wherein G is glycine, E is glutamic acid, L is leucine, V is valine, and T is threonine, and wherein the cysteine at the N-terminus and the cysteine at the C-terminus are selectively linked to each other, wherein Fp is linked at amino acid residue 6 via Y4, said amino acid residue 6 being Xa, wherein Y4 is N, and The nitrogen atom connected to Ab is at least one of lysine 246 and lysine 248 derived from the Fc region of the IgG antibody or a partial fragment thereof.

2. The antibody-loaded moiety conjugate of claim 1, wherein Xa is lysine.

3. The antibody-loaded moiety conjugate according to claim 1 or 2, wherein R″′ is C 1-6 Alkylene.

4. The antibody-loaded moiety conjugate according to claim 1 or 2, in, B is as well as Wherein, A1 is connected to the IgG antibody or a partial fragment thereof and A2 is connected to Am, or A1 is connected to Am and A2 is connected to the IgG antibody or a partial fragment thereof.

5. The antibody-loaded moiety conjugate according to claim 1 or 2, in, n is 2, and The nitrogen atom connected to Ab is derived from lysine 246 or lysine 248 in the Fc region of the IgG antibody or a partial fragment thereof.

6. The antibody-loaded moiety conjugate according to claim 1 or 2, in, n is 2, Where R″′ is C 1-6 Alkylene, Wherein, Xa is lysine, Among them, B is wherein the nitrogen atom connected to Ab is derived from lysine 246 or lysine 248 in the Fc region of the IgG antibody or a partial fragment thereof, and Wherein, A1 is connected to the IgG antibody or a partial fragment thereof and A2 is connected to Am, or A1 is connected to Am and A2 is connected to the IgG antibody or a partial fragment thereof.

7. The antibody-loaded moiety conjugate according to claim 1 or 2, wherein The active portion of Am is a drug molecule.

8. The antibody-loaded moiety conjugate of claim 7, wherein The drug molecules are more than one anticancer drug molecules.

9. The antibody-loaded moiety conjugate of claim 8, wherein The anticancer drug molecule is maytansine (DM1).

10. The antibody-loaded moiety conjugate of claim 7, wherein The drug molecules are two anticancer drug molecules.

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