Antibody-drug conjugates containing anti-claudin-18.2 antibody and their therapeutic applications for cancer

Abstract

This application relates to antibody-drug conjugates and their use.

Description

Technical Field

[0001] The present application relates to antibody-drug conjugates. The antibody-drug conjugates provided according to some embodiments of the present application have therapeutic uses for cancer or tumors.

[0002] Furthermore, the present application provides a pharmaceutical composition comprising an antibody-drug conjugate, a treatment method using an antibody-drug conjugate, a therapeutic use of an antibody-drug conjugate, and the use of an antibody-drug conjugate for the manufacture of a medicament for treating cancer.

Background Art

[0003] An antibody is a biomolecule having a function of recognizing a specific molecule and is used in various industrial applications. As an example, an antibody can be used to detect or search for (select) a specific component, identify the pathway by which a specific component moves in the body or within cells, and induce an immune response to a specific component, whereby the antibody can be used for therapeutic purposes.

[0004] Attempts have been made to improve such antibodies so as to expand their functionality. Typically, attempts have been made to label or conjugate various components (such as drugs or radioactive moieties, etc.) to supplement or expand the functionality of the antibody. Typically, an antibody is labeled with a fluorescent component and can be used in a fluorescence assay, or an antibody is labeled with or conjugated to an agent for treating a specific disease to maximize the therapeutic efficacy of the antibody. These attempts and techniques can be referred to as antibody labeling or antibody-target moiety conjugates, and the present application relates to antibody labeling or antibody-target moiety conjugates.

[0005] In previous studies, antibody-target substructure conjugates were prepared using highly reactive amino acid residues (e.g., amine or thiol groups) among the amino acid residues that make up the antibody. Specifically, a reactive group capable of reacting with the residue was introduced into the target substructure, and then a target substructure capable of reacting with the reactive residue of the antibody (more specifically, a modified target substructure with the reactive group introduced) was prepared. The antibody-target substructure conjugate was then prepared by reacting the modified target substructure with the antibody.

[0006] Previous studies like these involved randomly attaching target substructure components to antibodies, and these past methods had many problems.

[0007] Essentially, previous methods could not precisely control the "site" where the target substructure binds to the antibody, nor could they precisely control the "number" of target substructures that bind to the antibody. In other words, antibody-target substructure conjugates prepared by conventional methods suffer from the problem of heterogeneity in drug structure.

[0008] Such drug structural heterogeneity inevitably leads to heterogeneity in drug effects caused by "differences" in drug structure. These problems pose a significant obstacle to the development of antibody-drug conjugate (ADC) technologies, which require high safety and reproducibility.

[0009] Furthermore, the problem of drug structural heterogeneity leads to the problem of impaired antibody function. Antibodies contain a Fab region, which includes an antigen-binding domain that recognizes the antigen, and an Fc region, which is involved in antibody crystallization. Non-site-specific conjugates / labels make it impossible to precisely control the binding site of the target substructure to the antibody, making it impossible to prevent the target substructure from binding to the antibody's antigen-binding domain or a site adjacent to the antigen-binding domain, thereby interfering with the antibody's recognition function.

[0010] As a result, in this field, techniques for site-specific antibody labeling are required to ensure the structural uniformity of antibody-target substructure conjugates. Although several techniques have been developed, most of them lack technical and economic effectiveness, such as those involving genetic manipulation or modification of antibodies.

[0011] Accordingly, the inventors have developed a novel antibody-drug conjugate using a technique that allows for the transfer of a click chemical functional group, which is a target substance, to an antibody using a compound containing an Fc binding unit. [Overview of the project] [Problems that the invention aims to solve]

[0012] In the generation of antibody-target substructure conjugates, if the "position" where the target substructure binds to the antibody and the "number" of target substructures bound to the antibody cannot be precisely controlled, a problem of heterogeneity in drug structure arises. In particular, in drug manufacturing, this problem of heterogeneity in drug structure inevitably leads to a problem of heterogeneity in drug effects. In addition, the problem of heterogeneity in drug structure causes a problem of inhibiting antibody function. Accordingly, the present invention provides an antibody-drug conjugate generated by a method for site-specific delivery of a target substance (e.g., a drug) to an antibody, and its use. [Means for solving the problem]

[0013] The present invention provides an antibody-drug conjugate comprising an anti-claudin 18.2 antibody. The antibody-drug conjugate of the present invention is characterized in that the drug is site-specifically linked to the antibody. [Effects of the Invention]

[0014] The present invention provides a pharmaceutical composition for treating cancer, comprising an antibody-drug conjugate containing an anti-claudin 18.2 antibody.

[0015] Furthermore, the present invention provides a method for treating cancer using an antibody-drug conjugate comprising an anti-claudin 18.2 antibody.

[0016] Furthermore, the present invention provides the use of an antibody-drug conjugate comprising an anti-claudin 18.2 antibody for treating cancer.

[0017] Furthermore, the present invention provides the use of an antibody-drug conjugate comprising an anti-claudin 18.2 antibody for the manufacture of a drug for treating cancer. [Brief explanation of the drawing]

[0018] [Figure 1] The graph illustrates the absorbance measured after treating CHO-K1 cell lines (MOCK CHO-K1), which were transiently transfected with only a MOCK vector (empty vector), with antibody-A and ADC-A, respectively.

[0019] [Figure 2] The graph illustrates the absorbance measured after treating CHO-K1 cell lines (Claudin18.1CHO-K1), which were transiently transfected with the gene encoding the claudin 18.1 protein (CLDN18.1) (SEQ ID NO: 18), with antibody-A and ADC-A, respectively.

[0020] [Figure 3] The graph illustrates the absorbance measured after treating CHO-K1 cell lines (Claudin18.2CHO-K1), which were transiently transfected with the gene encoding the claudin 18.2 protein (CLDN18.2) (SEQ ID NO: 19), with antibody-A and ADC-A, respectively.

[0021] [Figure 4] The graph shows examples of absorbance measured after treating CLDN18.2-virus-like particles (VLPs) expressing claudin 18.2 protein (CLDN18.2) with antibody-A and ADC-A, respectively.

[0022] [Figure 5] The graph shows examples of absorbance measured after treating the MIA PaCa-2~CLDN18.2 cell line with antibody-A, ADC-A, antibody-B, and ADC-B, respectively.

[0023] [Figure 6] The graph shows examples of absorbance measured after treating the SNU601 cell line with antibody-A, ADC-A, antibody-B, and ADC-B, respectively.

[0024] [Figure 7] The graph shows examples of absorbance measured after treating the PATU8988S cell line with antibody-A, ADC-A, antibody-B, and ADC-B, respectively.

[0025] [Figure 8] The graph shows examples of absorbance measured after treating the MIA PaCa-2 (CLDN18.2-) cell line with antibody-A, ADC-A, antibody-B, and ADC-B, respectively.

[0026] [Figure 9] The graph illustrates the level of internalization measured hourly after treatment of the MIA PaCa-2~CLDN18.2 cell line with antibody-A and ADC-A, respectively. The red area on the vertical axis represents the percentage of the total area occupied by the cell that was occupied by antibody-A or ADC-A (measured as red dots).

[0027] [Figure 10] The graph illustrates the level of internalization measured hourly after treatment of the MIA PaCa-2 (CLDN18.2-) cell line with antibody-A and ADC-A, respectively. The red area on the vertical axis represents the percentage of the total area occupied by the cell that was occupied by antibody-A or ADC-A (measured as red dots).

[0028] [Figure 11] The graph illustrates the level of internalization measured hourly after treatment of the SNU601 cell line with antibody-A and ADC-A, respectively. The red area on the vertical axis represents the percentage of the total area occupied by the cells that was occupied by antibody-A or ADC-A (measured as red dots).

[0029] [Figure 12] The graph illustrates the changes in cell viability measured after treatment of the MIA PaCa-2~CLDN18.2 cell line with various concentrations of antibody-A, ADC-A, antibody-B, ADC-B, and combinations of antibody-A and MMAE.

[0030] [Figure 13] The graph illustrates the changes in cell viability measured after treatment of the PATU8988S cell line with various concentrations of antibody-A, ADC-A, antibody-B, ADC-B, and combinations of antibody-A and MMAE.

[0031] [Figure 14] The graph illustrates the changes in cell viability measured after treatment of the SNU601 cell line with various concentrations of antibody-A, ADC-A, antibody-B, ADC-B, and combinations of antibody-A and MMAE.

[0032] [Figure 15] The graph illustrates the changes in cell viability measured after treatment of the NUGC4 cell line with various concentrations of antibody-A, ADC-A, antibody-B, ADC-B, and combinations of antibody-A and MMAE.

[0033] [Figure 16]The graph illustrates the changes in cell viability measured after treatment of the MIA PaCa-2 (CLDN18.2-) cell line with various concentrations of antibody-A, ADC-A, antibody-B, ADC-B, and combinations of antibody-A and MMAE.

[0034] [Figure 17] The graph illustrates the changes in cell viability measured after AGS cell lines were treated with various concentrations of antibody-A, ADC-A, antibody-B, ADC-B, and combinations of antibody-A and MMAE.

[0035] [Figure 18] The graph illustrates the relative percentage of total antibody or total ADC measured after extracting the supernatant from human plasma samples treated with antibody-A, ADC-A, and ADC-B, respectively, and incubated for various time periods.

[0036] [Figure 19] The graph illustrates the relative percentages of total antibodies or total ADCs measured after extracting the supernatant from samples treated with antibody-A, ADC-A, and ADC-B respectively, and incubated for various time periods.

[0037] [Figure 20] The graph illustrates the relative percentage of total antibody or total ADC measured after extracting the supernatant from rat plasma treated with antibody-A, ADC-A, and ADC-B, respectively, and incubated for various time periods.

[0038] [Figure 21] The graph illustrates the relative percentage of total antibody or total ADC measured after extracting the supernatant from samples treated with antibody-A, ADC-A, and ADC-B respectively, and incubated for various time periods.

[0039] [Figure 22]The graphs illustrate tumor volume measured after intravenous injection of antibody-A, ADC-A, and ADC-C into tumor model mice (groups G1-G8) under various conditions.

[0040] [Figure 23] The graph illustrates the body weight measured after intravenous injection of antibody-A, ADC-A, and ADC-C into tumor model mice (groups G1-G8) under various conditions.

[0041] [Figure 24] The images show comparative photographs of tumors dissected 28 days after intravenous injection of antibody-A, ADC-A, and ADC-C into tumor model mice (groups G1-G8) under various conditions.

[0042] [Figure 25] The graphs illustrate the weight of tumors dissected 28 days after intravenous injection of antibody-A, ADC-A, and ADC-C into tumor model mice (groups G1-G8) under various conditions.

[0043] [Figure 26] The graph illustrates the total antibody and total ADC concentrations measured after a certain period of time following intravenous administration of ADC-A to rats at various concentrations.

[0044] [Figure 27] The structure of ADC-A is shown.

[0045] [Figure 28] The detailed structures of the linker and drug substructures contained in ADC-A are shown.

[0046] [Figure 29] This shows some of the reaction processes involved in the manufacturing process of ADC-A.

[0047] [Figure 30] The HIC-HPLC analysis results for antibody-A are shown.

[0048] [Figure 31] The HIC-HPLC analysis results for compound 3 are shown.

[0049] [Figure 32] The HIC-HPLC analysis results of crude ADC-A are shown. [Modes for carrying out the invention]

[0050] Some embodiments of the present application provide antibody-drug conjugates.

[0051] Some embodiments of this application are given by Formula 1: [Formula 1] [ka] We provide an antibody-drug conjugate having the structure, During the ceremony, Here, Ab is an antibody unit, L is the linker unit, D is a drug unit, n is an integer between 1 and 4. The antibody unit is a conjugated anti-claudin 18.2 antibody. The drug unit is linked to one or more of the lysine residues 246 (K246) and 248 (K248) in the Fc region of the antibody unit. The linker unit is, Equation 2: [Formula 2] [ka] It has a structure, In the formula, b is an integer between 0 and 6. X' is NH-, -C(O)-, or -NHC(O)-, B' is a group formed by a click chemical reaction between click chemical functional groups. PM 1 and PM 2Each of these is independently a polyethylene (PEG) substructure, and each PEG substructure contains 1 to 10 ethylene glycol units, the ethylene glycol units being CH2OCH2-, -OCH2CH2-, or -CH2CH2O-. 1* represents the attachment site with Ab, 2* represents the attachment site with D, The drug unit is monomethyl auristatin E (MMAE).

[0052] In a particular embodiment, B' is [ka] , [ka] , [ka] , [ka] , [ka] , [ka] , [ka] , [ka] , [ka] , [ka] , [ka] , [ka] , [ka] and [ka] It may include one structure selected from, In the formula, R x H, halogen, and C 1~3 Alkyl can be selected, A1 and A2 may represent the attachment points of the linker unit to the rest of the structure, respectively.

[0053] In certain embodiments, b may be 2.

[0054] In certain embodiments, X' may be -C(O)-.

[0055] In a particular embodiment, PM 1 and PM 2 Each of these independently represents a PEG substructure. The PEG substructure may contain 1 to 10 ethylene glycol units. The ethylene glycol unit may be -[CH2OCH2]-, -[OCH2CH2]-, or -[CH2CH2O]-.

[0056] In a particular embodiment, PM 1 and PM 2 Each has the following structure: [ka] It may have.

[0057] In a particular embodiment, the drug unit is given by the following formula 5: [Formula 5] [ka] It may have a structure, In the formula, 3* may represent the attachment site with L.

[0058] In certain embodiments, the structure of the conjugated anti-claudin 18.2 antibody may be the same as that of the anti-claudin 18.2 antibody, except for the linker unit and the conjugated portion. In this case, the conjugated portion is K246 or K248 of the heavy chain of the anti-claudin 18.2 antibody.

[0059] In certain embodiments, the anti-claudin 18.2 antibody may be an IgG antibody, which may be selected from the subclasses IgG1, IgG2, IgG3, and IgG4.

[0060] In certain embodiments, the heavy chain of the anti-claudin 18.2 antibody may include CDRH1 represented by the amino acid sequence of SEQ ID NO: 10, CDRH2 represented by the amino acid sequence of SEQ ID NO: 11, and CDRH3 represented by the amino acid sequence of SEQ ID NO: 12, and the light chain of the anti-claudin 18.2 antibody may include CDRL1 represented by the amino acid sequence of SEQ ID NO: 13, CDRL2 represented by the amino acid sequence of SEQ ID NO: 14, and CDRL3 represented by the amino acid sequence of SEQ ID NO: 15.

[0061] In certain embodiments, the anti-claudin 18.2 antibody may include a heavy chain represented by the amino acid sequence of SEQ ID NO: 16 and a light chain represented by the amino acid sequence of SEQ ID NO: 17.

[0062] In certain embodiments, n may be 2.

[0063] The antibody unit may include two heavy chains (a first heavy chain and a second heavy chain).

[0064] The antibody-drug conjugate may contain two drug units (a first drug unit and a second drug unit).

[0065] The first drug unit may be linked to one of K246 and K248 of the first heavy chain.

[0066] The second drug unit may be linked to one of K246 and K248 of the second heavy chain.

[0067] In a particular embodiment, the antibody-drug conjugate is given by the following formula 7: [Formula 7] [ka] It may have a structure.

[0068] Some embodiments of the present application provide pharmaceutical compositions for treating cancer, comprising a therapeutically effective amount of antibody-drug conjugate.

[0069] In this case, the pharmaceutical composition for treating cancer may further contain a pharmaceutically acceptable carrier and / or a pharmaceutically acceptable adjuvant.

[0070] Some embodiments of the present invention are A step of administering a pharmaceutical composition containing a therapeutically effective amount of antibody-drug conjugate. The present invention provides a method for treating cancer, comprising the following:

[0071] Some embodiments of the present invention provide the use of antibody-drug conjugates for treating cancer.

[0072] Some embodiments of the present invention provide the use of antibody-drug conjugates for the manufacture of drugs for treating cancer.

[0073] [Mode of the invention]

[0074] The contents of the present invention will be described in more detail thereafter by means of embodiments and examples. The invention disclosed herein can be carried out in various forms and is not limited to the specific embodiments described herein.

[0075] A person skilled in the art in the field to which the invention disclosed herein pertains will be able to conceive of various modifications and other embodiments of the invention disclosed herein. Therefore, it should be understood that the invention disclosed herein is not limited to the specific embodiments or examples described herein, and that such modifications and other embodiments are also included within the scope of the invention disclosed herein.

[0076] Explanation of terms

[0077] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art to which this application pertains. All publications, patents, and other references referenced herein are cited by reference in their entirety.

[0078] "Halogen" or "halo" refers to groups containing fluorine, chlorine, bromine, and iodine, which are elements in the halogen group of the periodic table.

[0079] As used herein, the term "hetero" refers to a compound or group containing one or more heteroatoms. That is, the term "hetero" can be used with a term used to refer to the molecule itself or a term used to refer to a part of the molecule. For example, heteroalkylene refers to an alkylene group containing one or more heteroatoms in the main chain. As another example, heteroaryl refers to an aryl group containing one or more heteroatoms on the ring (e.g., a C6 aryl group in which one or more carbons on the ring are each substituted with a heteroatom independently selected). The term "heteroatom" refers to an atom other than carbon or hydrogen and includes, for example, B, Si, N, P, O, S, F, Cl, Br, I, and Se. Preferably, the term includes polyvalent elements such as N, O, and S. For example, if the structure contains one or more heteroatoms, each heteroatom may be independently selected from N, O, and S.

[0080] The terms "alkyl" or "alkane", used to refer to the molecule itself or a part of the molecule, are used to mean a fully saturated straight-chain or branched hydrocarbon group. Straight-chain and branched alkyl groups are, for example, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, isobutyl, pentyl, hexyl, heptyl, octyl, nonyl, and decyl. An alkyl group may include a cyclic structure. "C x~y ", when used, for example, with the term "alkyl", is intended to include a residue containing x to y carbon atoms in a chain or a ring. For example, the term "C x~y alkyl" may mean an alkyl group, substituted or unsubstituted, straight-chain, branched, or including a cyclic structure, containing x to y carbon atoms. C0 alkyl means hydrogen. Examples of C 1~4 alkyl include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, and isobutyl. For example, a straight-chain or branched alkyl group may have 1 to about 60, 1 to 20, or 1 to 10 carbon atoms.

[0081] As used herein, the term "heteroalkyl" refers to an alkyl group containing one or more heteroatoms, where each heteroatom is selected independently.

[0082] The term “alkylene,” used to refer to a molecule itself or a part of a molecule, means a divalent radical derived from an alkyl group. The term “alkylene” may be used with the terms “substituted” or “unsubstituted,” as needed. If the term “alkylene” is not used with the terms “substituted” or “unsubstituted,” the term “alkylene” is intended to encompass both substituted and unsubstituted forms of alkylene. For example, alkylene may refer to a group having 1 to 100 carbon atoms in its main chain. Examples of alkylene may include, but are not limited to, -CH2-, -CH2CH2-, -CH2CH2CH2-, and -CH2CH2CH2CH2-. For example, alkylene may be used as C2 alkylene, referring to an alkylene group having 2 carbon atoms in its main chain. Exemplary, “C x~y The term "alkylene" is used herein to mean an alkylene having x to y carbon atoms in its main chain, whether substituted or unsubstituted.

[0083] The term “heteroalkylene,” used to refer to the molecule itself or to a part of the molecule, means a divalent radical derived from a heteroalkyl group. The term “heteroalkylene” may be used with the terms “substituted” or “unsubstituted,” as needed. If the term “heteroalkylene” is not used with the terms “substituted” or “unsubstituted,” the term “heteroalkylene” is intended to encompass both substituted and unsubstituted heteroalkylene forms. For example, a heteroalkylene may refer to a group having 1 to 100 carbon atoms and heteroatoms in the main chain (e.g., the total number of carbon atoms and heteroatoms in the main chain is between 1 and 100). Examples of heteroalkylene groups include, but are not limited to, -CH2-CH2-O-CH2-CH2- and -CH2-O-CH2-CH2-NH-CH2-. A heteroalkylene group may contain one or more heteroatoms, each of which may be the same or different. For example, a heteroalkylene group may contain one or more heteroatoms at positions other than the ends of a chain or branch, and each heteroatom may be the same or different. For example, a heteroalkylene group may contain one or more heteroatoms at each end of a chain or branch, or at all ends of a chain or branch, and each heteroatom may be the same or different. Exemplarily, "C" in this specification x~y The term "heteroalkylene" is used to refer to a heteroalkylene having a total of x to y atoms in the main chain, whether substituted or unsubstituted (for example, the sum of the number of carbon atoms and heteroatoms in the main chain is x to y). For example, C3 heteroalkylene may be used to mean a heteroalkylene having two carbon atoms and one heteroatom in the main chain. As another example, C5 heteroalkylene may be used to mean a heteroalkylene having three carbon atoms and two heteroatoms in the main chain. C5 heteroalkylenes include structures such as -CH2-CH2-O-CH2-CH2- and -CH2-O-CH2-NH-CH2-.

[0084] The term "cycloalkyl" is used to refer to a fully saturated cyclic hydrocarbon group. "Cycloalkyl" includes monocyclic and polycyclic groups. Unless otherwise defined, monocyclic cycloalkyl groups generally have 3 to about 20, preferably 3 to 10, carbon atoms on the ring. The rings other than the first ring in a polycyclic cycloalkyl group may be selected from saturated, unsaturated, and aromatic rings. Cycloalkyls include bicyclic molecules in which 1, 2, or 3 or more atoms are shared between two rings. The term "fusion cycloalkyl" refers to a polycyclic cycloalkyl group in which each ring shares two adjacent atoms with the other ring. The rings other than the first ring in a fusion polycyclic cycloalkyl group may be selected from saturated, unsaturated, and aromatic rings. Cycloalkyl may also be used with the terms substituted or unsubstituted, where substituted cycloalkyl refers to a group provided when one or more hydrogen atoms linked to carbon atoms on the ring are substituted with one or more independent substituents. Furthermore, the term cycloalkyl may also be used with the term heterocycloalkyl, which refers to a cycloalkyl group containing one or more heteroatoms on the ring.

[0085] The term "cycloalkylene" is used to mean a divalent radical derived from a cycloalkyl group. For example, the term cycloalkylene may be used with the terms substituted or unsubstituted. For example, the term cycloalkylene may be used with the term hetero.

[0086] The terms “alkene” or “alkenyl,” used to refer to a molecule itself or a part of a molecule, include one or more double bonds as a straight-chain or branched non-aromatic hydrocarbon. For example, a straight-chain or branched alkenyl group may have 2 to about 60, 2 to 20, or 2 to 10 carbon atoms.

[0087] The terms "heteroalkene" or "heteroalkenyl" refer to an alkenyl containing one or more heteroatoms. In this case, the heteroatoms are selected independently.

[0088] The term “alkenylene,” used to refer to the molecule itself or to a part of the molecule, means a divalent radical derived from an alkenyl. The term “alkenylene” may be used with the terms “substituted” or “unsubstituted,” as needed. When the term “alkenylene” is not used with the terms “substituted” or “unsubstituted,” the term “alkenylene” is intended to encompass both substituted and unsubstituted forms of alkenylene. For example, an alkenylene may refer to a group having 2 to 100 carbon atoms in the main chain. Examples of alkenylenes may include, but are not limited to, -C=C-, -CCC=CC=C-, or -CCCC=C-, etc. In this specification, “C x~y When used with "Alkenylene", C x~y The term "alkenylene" is used to refer to alkenylenes having x to y carbon atoms in their main chain, whether substituted or unsubstituted.

[0089] The term “heteroalkenylene,” used to refer to the molecule itself or to a part of the molecule, means a divalent radical derived from a heteroalkenyl. For example, the term “heteroalkenylene” may be used to refer to an alkenylene group containing one or more heteroatoms in the main chain. For example, heteroalkenylene may refer to a group having 2 to 100 carbon atoms and heteroatoms in the main chain (e.g., the total number of carbon atoms and heteroatoms is 2 to 100). The term “heteroalkenylene” may be used with the terms “substituted” or “unsubstituted” as needed. In this specification, “C x~y When used with heteroalkenylene, C x~y The term heteroalkenylene is used to mean a heteroalkenylene that has x to y carbon atoms and heteroatoms in its main chain, whether substituted or unsubstituted (for example, the sum of the number of carbon atoms and heteroatoms is x to y).

[0090] The terms "cycloalkene" or "cycloalkenyl" refer to cyclic hydrocarbons containing one or more double bonds on a ring. "Cycloalkenyls" include monocyclic and polycyclic groups. Unless otherwise defined, monocyclic cycloalkenyl groups generally have 3 to about 20, preferably 3 to 10, carbon atoms on the ring. The rings other than the first ring in polycyclic cycloalkenyls may be selected from saturated, unsaturated, and aromatic rings. Cycloalkenyls include bicyclic molecules in which one, two, or three or more atoms are shared between two rings. The term "fusion cycloalkenyl" refers to a polycyclic cycloalkenyl in which each ring shares two adjacent atoms with the other ring. The rings other than the first ring in fusion polycyclic cycloalkenyls may be selected from saturated, unsaturated, and aromatic rings. The term "cycloalkenyl" may also be used with the terms "substituted" or "unsubstituted," where a substituted cycloalkenyl refers to a group provided when one or more hydrogen atoms linked to a carbon atom on the ring are substituted with one or more independent substituents. Furthermore, the term "cycloalkenyl" may also be used with the term "hetero," where a heterocycloalkenyl refers to a cycloalkenyl group containing one or more heteroatoms on the ring.

[0091] The term "cycloalkenylene" is used to mean a divalent radical derived from a cycloalkenyl. For example, the term cycloalkenylene may be used with the terms substituted or unsubstituted. For example, the term cycloalkenylene may be used with the term hetero.

[0092] The terms “alkyne” or “alkynyl,” used to refer to a molecule itself or a part of a molecule, include one or more triple bonds as a linear or branched non-aromatic hydrocarbon. For example, a linear or branched alkynyl group may have 2 to about 60, 2 to 20, or 2 to 10 carbon atoms.

[0093] The terms "heteroalkynyl" or "heteroalkynyl" refer to an alkynyl compound containing one or more heteroatoms. In this case, the heteroatoms are selected independently.

[0094] The term “alkynylene,” used to refer to the molecule itself or to a part of the molecule, means a divalent radical derived from alkynyl. The term “alkynylene” may be used with the terms “substituted” or “unsubstituted,” as needed. If the term “alkynylene” is not used with the terms “substituted” or “unsubstituted,” the term “alkynylene” is intended to encompass both substituted and unsubstituted forms of alkynylene. For example, alkynylene may refer to a group having 2 to 100 carbon atoms in the main chain. In this specification, “C x~y When used with "alkynylene", C x~y The term "alkynylene" is used to refer to alkynylenes having x to y carbon atoms in the main chain, whether substituted or unsubstituted.

[0095] The term “heteroalkylynylene,” used to refer to the molecule itself or to a part of the molecule, means a divalent radical derived from a heteroalkylyl. For example, the term “heteroalkylynylene” may be used to refer to an alkylylene group containing one or more heteroatoms in the main chain. For example, heteroalkylynylene may refer to a group having 2 to 100 carbon atoms and heteroatoms in the main chain (e.g., the total number of carbon atoms and heteroatoms in the main chain is 2 to 100). The term “heteroalkylynylene” may be used with the terms “substituted” or “unsubstituted,” as needed. In this specification, “C x~y When used with heteroalkylene, C x~y The term heteroalkylene is used to refer to a heteroalkylene having x to y carbon atoms and heteroatoms in its main chain, whether substituted or unsubstituted (for example, the sum of the number of carbon atoms and heteroatoms is x to y).

[0096] The term "cycloalkyne" or "cycloalkynyl" refers to a cyclic hydrocarbon containing one or more triple bonds on the ring, also known as a "strained alkyne." "Cycloalkynyls" include monocyclic and polycyclic groups. Unless otherwise defined, monocyclic cycloalkynyls generally have 3 to about 10 carbon atoms on the ring. The rings other than the first ring in a polycyclic cycloalkynyl may be selected from saturated, unsaturated, and aromatic rings. Cycloalkynyls include bicyclic molecules in which 1, 2, or 3 or more atoms are shared between two rings. The term "fusion cycloalkynyl" refers to a polycyclic cycloalkynyl in which each ring shares two adjacent atoms with the other ring. The rings other than the first ring in a fusion polycyclic cycloalkynyl may be selected from saturated, unsaturated, and aromatic rings. The term "cycloalkynyl" may be used with the terms "substituted" or "unsubstituted," where a substituted cycloalkynyl refers to a group provided when one or more hydrogen atoms linked to a carbon atom on the ring are substituted with one or more independent substituents. Furthermore, the term "cycloalkynyl" may be used with the term "hetero," where a heterocycloalkynyl refers to a cycloalkynyl group containing one or more heteroatoms on the ring.

[0097] The term "cycloalkylylene" is used to mean a divalent radical derived from cycloalkylyl. For example, the term cycloalkylylene may be used with the terms substituted or unsubstituted. For example, the term cycloalkylylene may be used with the term hetero.

[0098] The term "aryl" is used to refer to a group containing an aromatic ring, and specifically to a group derived from an aromatic compound, an arene. The term aryl includes monocyclic and polycyclic groups. The term "aryl" may also be used with the term "hetero," where heteroaryl is used to refer to an aryl group having one or more heteroatoms on the ring. The term "aryl" may also be used with the terms "substituted" or "unsubstituted," where substituted aryl refers to an aryl group in which one or more hydrogen atoms bonded to a carbon atom on the ring are substituted with one or more types of substituents. The term "aryl" may also be used to encompass both substituted and unsubstituted aryls, and substituted and unsubstituted heteroaryls. Examples of aryls include, but are not limited to, phenyl, pyridyl, naphthyl, and biphenyl.

[0099] The term "arirene" is used to mean a divalent radical derived from an aryl group. For example, the term arirene may be used with the terms substituted or unsubstituted. For example, the term arirene may be used with the term hetero. The term "arirene" may be used to encompass all substituted and unsubstituted arirenes, and substituted and unsubstituted heteroarirenes.

[0100] As used herein, the term “substituted” means that, if the valence of the atom is normal and the substituted compound is stable, one or more hydrogen atoms on an atom are substituted with substituents including deuterium and hydrogen variants. If the substituent is oxygen (i.e., =O), this means that two hydrogen atoms are substituted. If the substituent is a halogen (e.g., Cl, F, Br, and I), this means that one hydrogen atom is substituted with a halogen. If two or more substituents are present on a single group, the substituents present on that group may be the same or different. Unless otherwise specified, the type and number of substituents may be arbitrary, as long as it is chemically achievable. Exemplary substituents may be selected from -R, =O, =S, -NO2, -CR3, -NR2, =NR, -OR, -SR, -C(=O)R, -C(=O)CR3, -C(=O)OR, and -C(=O)NR2, where R is H, halogen, C 1~6 Alkyl, C 3~10 Cycloalkyl, C 3~10 The substituents may be independently selected from heterocycloalkyl, aryl, heteroaryl, -OH, -NH2, -COOH, =O, =S, and -SH (provided the substituent is not -H). A typical example of a substituent is -C 1~4 This includes, but is not limited to, alkyl, -C(=O)H, -C(=O)CH3, -C(=O)OH, -C(=O)NH2, -NH2, =NH, =O, =S, -OH, -NO2, and -SH. The terms substituted or unsubstituted may be used in conjunction with terms used to refer to the molecule itself or to a part of the molecule. For example, substituted C 10~20 Alkylene can mean that one or more hydrogen atoms linked to the main chain are substituted with substituents, and each substituent may be independently selected.

[0101] In this specification, when representing the structure of a compound, a wavy line drawn in a direction approximately perpendicular to the bond (for example, [ka] ) is used to represent a group and a part to which another group is linked. For example, structure [ka] When expressed as such, it indicates that an X group in a component, molecule, or compound is linked to another part by a bond. For example, structure [ka] When expressed as such, it indicates that the X group in a component, molecule, or compound is linked to other parts by a bond. For example, in a compound having the structure "AX", if only the structure of the X group is shown, it indicates that the structure [ka] It can be expressed as follows. For example, in a compound having the structure "AXB", if only the structure of X is given as an example, it can be expressed as follows: [ka] It can be represented as follows. If necessary, a wavy line drawn nearly perpendicular to the bond may be represented by additional notation. For example, in a compound having the structure AXB, if only the structure of the X group is illustrated, if necessary, the structure of the X group may be represented as follows. [ka] This can also be illustrated as an example, and by mentioning "* is a part connected to A, and ** is a part connected to B," it is possible to provide information about which part each wavy line represents a connection to.

[0102] Furthermore, the wavy lines drawn in a direction nearly perpendicular to the bonds indicate that the "structure illustrated by the wavy lines" is directly covalently bonded to the "groups other than the structure illustrated by the wavy lines." The wavy lines should not be interpreted as meaning that other additional elements may be included between the "structure illustrated by the wavy lines" and the "groups other than the structure illustrated by the wavy lines." If additional elements are included, this will be explained by a separate, relevant explanation.

[0103] Structures used in structures or formulas disclosed herein [ka] " is C x It is used to mean alkylene. For example, structure [ka] This can be used to represent C4 alkylenes such as -CH2-CH2-CH2-CH2-. In this, if x is 0, it means a bond. That is, structure [ka] is structure [ka] It can be represented by:

[0104] The compounds of the present invention may have specific geometric or stereoisomeric forms. When a compound is disclosed without specifying it in this application, isomers such as cis and trans isomers, (-)- and (+)-enantiomers, (R)- and (S)-enantiomers, diastereoisomers, (D)-isomers, (L)-isomers, and racemates of the compound are included in the scope of this application. That is, formulas or structures disclosed herein may be associated with isomers (e.g., *, [ka] , [ka] If it does not have (etc.), it means that the disclosed formula or structure includes all possible isomers.

[0105] As used herein, the term “amino acid” may be used to refer to both amino acids that are not bound to other amino acids and amino acid residues that are bound to other amino acids in proteins or peptides, and may be interpreted as appropriate according to the content or context of the paragraph in which the term “amino acid” is used. As used herein, the term “amino acid” may be used to include both natural and non-natural amino acids. As used herein, natural amino acids refer to the 20 amino acids that are synthesized in the human body through gene transcription and translation. Specifically, natural amino acids include alanine (Ala, A), arginine (Arg, R), asparagine (Asn, N), aspartic acid (Asp, D), cysteine ​​(Cys, C), glutamic acid (Glu, E), glutamine (Gln, Q), glycine (Gly, G), histidine (His, H), isoleucine (Ile, I), leucine (Leu, L), lysine (Lys, K), methionine (Met, M), phenylalanine (Phe, F), proline (Pro, P), serine (Ser, S), threonine (Thr, T), tryptophan (Trp, W), tyrosine (Tyr, Y), and valine (Val, V). As used herein, non-natural amino acids mean amino acids that are not synthesized in the human body by gene transcription and translation, but are synthesized by processes other than transcription and translation, or are artificially synthesized, or can be synthesized by other organisms other than humans. Non-natural amino acids may include, for example, ornithine (Orn), diaminopropionic acid (Dap), diaminobutyric acid (Dab), and naphthylalanine. As stated above, the term “amino acid” as used herein may be used to refer to both amino acids that are not bound to other amino acids, and amino acid residues that are bound to other amino acids contained in proteins or peptides. For example, alanine may be used to refer to alanine and / or an alanine residue. For example, arginine may be used to refer to arginine and / or an arginine residue. As used herein, the term “amino acid” may be used to include both L-type and D-type amino acids.In some embodiments, if there is no reference to L-type or D-type, the amino acid may be interpreted as an L-type amino acid.

[0106] As used herein, the term “amino acid residue” refers to a structure derived from an amino acid contained in a compound, peptide, and / or protein (e.g., an antibody) that is covalently linked to other parts of the compound, peptide, and / or protein. For example, when alanine, arginine, and glutamic acid are linked by an amide bond to form a peptide having an ARE sequence, the peptide contains three amino acid residues, and A, R, and E may be called the alanine residue, the arginine residue, and the glutamic acid residue, respectively. Furthermore, as described above, in a peptide having an ARE sequence, the peptide may contain three amino acids, and A, R, and E may also be called alanine, arginine, and glutamic acid, respectively. As another example, when aspartic acid, phenylalanine, and lysine are linked by an amide bond to form a peptide having a DFK sequence, the peptide contains three amino acid residues, and D, F, and K may be called the aspartic acid residue, the phenylalanine residue, and the lysine residue, respectively. Furthermore, as described above, in a peptide having a DFK sequence, the peptide may contain three amino acids, where D, F, and K may also be called aspartic acid, phenylalanine, and lysine, respectively.

[0107] Unless otherwise stated, when amino acid sequences are described herein, single-letter or three-letter notation for amino acids is used, and the sequence is described in the direction from the N-terminus to the C-terminus. For example, when represented as RNVP, it refers to a peptide in which arginine, asparagine, valine, and proline are sequentially linked in the direction from the N-terminus to the C-terminus. As another example, when represented as Thr-Leu-Lys, it refers to a peptide in which threonine, leucine, and lysine are sequentially linked in the direction from the N-terminus to the C-terminus. For amino acids that cannot be represented by single-letter notation, other letters are used to describe these amino acids, and additional explanations are provided. Sequences described herein may include sequences having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or greater sequence identity with the described sequence, provided that the desired function is identical.

[0108] As used herein, the term “click chemistry” refers to a chemical concept introduced by K. Barry Sharpless of the Scripps Research Institute to describe complementary chemical functional groups and chemical reactions designed to enable two molecules to rapidly and stably form a covalent bond. As used herein, click chemistry does not imply a specific reaction, but rather the concept of a fast and stable reaction. In one embodiment, several conditions should be satisfied for intermolecular bonding to occur by click chemistry. These conditions include high yield, excellent selectivity to reactive sites, modular work for organically combining molecules, and proceeding in a thermodynamically stabilized direction for fast and accurate product production. The click chemistry described herein involves the reaction of mutually reactive pairs of click chemical functional groups (including, for example, terminal alkynes, azides, strained alkynes, dienes (e.g., Diels-alderdiene), dienophiles (e.g., Diels-alderdienophile), trans-cyclooctene, alkenes, thiols, tetrazines, triazines, dibenzocyclooctin (DBCO), and bicyclononines (including bicyclo[6.1.0]nona-4-yne)). Examples of click chemical reactions include Huisgen 1,3-dipolar cycloaddition (see Tornoe et al., Journal of Organic Chemistry (2002) 67:3075-3064, etc.); Diels-Alder reactions; reverse electron-demanded Diels-Alder reactions; nucleophilic addition to small, strained rings such as epoxides and aziridines; nucleophilic addition to activated carbonyl groups; Staudinger ligation; and addition reactions to carbon-carbon double or triple bonds.

[0109] As used herein, the term “antibody” refers to an immunoglobulin molecule or a fragment thereof. Immunoglobulins are typically well known and possess the ability to specifically bind to one or more antigens. As used herein, the term “antibody” is also used to encompass fragments thereof, and therefore it is not important that the antibody does not possess the ability to bind to a specific antigen, as in the case of the Fc fragment. Unless used with respect to an antibody with specific limitations, the term “antibody” may be interpreted, without specific limitations, to include all monospecific antibodies, bispecific antibodies, trispecific antibodies, monoclonal antibodies, human antibodies, humanized antibodies, recombinant antibodies, and chimeric antibodies. For example, an antibody may consist of two heavy chains and two light chains. For example, in this case, the antibody may have a structure in which two heavy chains are linked by one or more bridges (e.g., disulfide bonds), one heavy chain and one light chain are linked by one or more bridges, and the other heavy chain and light chain are linked by one or more bridges. An antibody may be divided into an Fc region (or Fc domain) and a Fab region, the Fab region containing a site capable of binding to an antigen, and the Fc region containing a portion of the constant region of the heavy chain. The term “antibody” as used herein may be used to include both conjugated and unconjugated antibodies (e.g., free antibodies).

[0110] In this application, when referring to the numbering of amino acid residues in the Fc domain (or Fc region) of an antibody, the numbering of amino acid residues follows the EU numbering system unless otherwise specified. Since the IgG sequence was investigated, as described in Edelman GM, et al., The covalent structure of an entire gamma-G immunoglobulin molecule, Proc Natl Acad Sci USA., 1969 May;63(1):78-85, the EU numbering system has been widely used as a sequencing system for the Fc region. For example, at lysine 246 in the Fc region, the number 246 is a number assigned according to the EU numbering system. As another example, at lysine 248 in the Fc region, the number 248 is a number assigned according to the EU numbering system.

[0111] The terms “linked” or “connected” as used herein mean that two or more elements present in a conceptualizable structure are directly or indirectly linked (e.g., by other elements such as linkers), and are not intended to mean that no other additional elements can exist between the two or more elements. For example, a description such as “element B linked to element A” is intended to include both cases where one or more other elements are present between elements A and B (i.e., element A is linked to element B by one or more other elements) and cases where one or more other elements are not present between elements A and B (i.e., elements A and B are directly linked), and is not intended to be interpreted restrictively.

[0112] As used herein, the term “sequence identity” is a term used in relation to the degree of similarity between two or more sequences. For example, the term “sequence identity” is used in conjunction with a term referring to a reference sequence and a term expressing a percentage. For example, the term “sequence identity” may be used to describe a sequence that is similar to or substantially identical to a reference amino acid sequence. When a description such as “a sequence having 90% or more sequence identity with sequence A” is used herein, the reference sequence is sequence A. For example, percent sequence identity may be calculated by aligning the reference sequence and the sequence that is the target of the measurement of the percentage of sequence identity. The method for calculating and / or determining percent sequence identity is not particularly limited, and percent sequence identity may be calculated and / or determined by any reasonable method or algorithm available to those skilled in the art.

[0113] In some embodiments, the term “unit” as used herein is used to distinguish the conjugated substance from the free substance. The term “unit” as used in some embodiments is illustrated by illustrating antibody units and free antibodies. A free antibody refers to an antibody molecule that is not covalently bonded to another molecule or group. An antibody unit refers to a group derived from the free antibody that is covalently bonded to another molecule or group. For example, an antibody-functional group conjugate can be prepared when a free antibody and a functional substance are combined by the reaction of the reactive group of the functional group with the amine group of a lysine residue of the free antibody. In this case, the portion derived from the free antibody may be called an antibody unit. In an antibody-functional group conjugate, the antibody unit may be understood to be structurally identical to the free antibody from which the antibody unit originates, except for the portion that is conjugated with the non-antibody portion of the antibody-functional group conjugate. For example, if the amine group of the lysine residue involved in the reaction in the free antibody is illustrated separately, the structure of the free antibody is: [ka] It can be expressed as ". When the junction between the antibody unit and a part other than the antibody unit is illustrated separately in an antibody unit, the structure of the antibody unit is " [ka] It can be expressed as "[...]." Therefore, the antibody unit and the free antibody can be understood to be structurally identical except for the amine group of the lysine residue used in the reaction. Thus, in some embodiments, the antibody unit and the free antibody cannot be distinguished separately and may be called "antibody," and these terms may be interpreted appropriately according to the context. In some embodiments, when a statement such as "the antibody unit is derived from the antibody" is used, the antibody unit and the antibody can be understood to have the relationship described above. In some embodiments, when a statement such as "the Fc-binding unit is derived from the Fc-binding substance" is used, the "Fc-binding unit and the Fc-binding substance" can be understood to have a similar relationship to the one described above. That is, the Fc-binding unit can be understood to have the same structure as the Fc-binding substance from which it originates, except for the junction to which the Fc-binding unit is attached to a part other than the Fc-binding unit. As in the case of the antibody unit, in some embodiments, the Fc-binding unit may be called the Fc-binding substance from which it originates, and these terms may be interpreted appropriately according to the context.

[0114] The terms “comprise” or “include” as used herein may be used to mean that, in addition to the object of the term “comprise” or “include” (e.g., the object), other elements may be present or equivalent to the object of the term. For example, when a statement such as “A includes B” is used, it should be interpreted that the statement “A includes B” does not exclude the fact that A includes additional components other than B. That is, “A includes B” is intended to include cases in which additional elements other than B are present in A (e.g., B and C are present in A), cases in which A is B, cases in which A consists of B, and cases in which A is represented by B. Accordingly, when a statement such as “A includes B” is used, the statement may be changed to “A is B,” “A consists of B,” or “A is represented by B.”

[0115] The term “having” as used herein may be used to mean that, in addition to the object of the term “having” (e.g., the object), other elements may be present or equivalent to the object of the term. For example, when a description such as “A has B” is used, the description includes cases where additional elements other than B are present in A (e.g., B and C are present in A), cases where A is B, cases where A consists of B, and cases where A is represented by B. Accordingly, when a description such as “A has B” is used, the description may be changed to “A is B,” “A consists of B,” or “A is represented by B.”

[0116] Where compounds (e.g., small compounds, peptides, antibodies, and conjugates) are disclosed herein, it should be understood that their salt forms are also disclosed. Examples of ions that form salts of compounds include ammonium, calcium, sodium, potassium, and acetic acid (CH3COO). - ), carbon dioxide (CO3 2- ), chloride (Cl- ), citric acid, cyanide, fluoride (F - ), nitric acid (NO3 - ), nitrite (NO2 - ), phosphoric acid (PO3 - ), and sulfuric acid (SO4 2- ) and others, but are not particularly limited thereto. If necessary, salt-forming ions commonly used in the art may be used for the formation of salts of the compounds. The salt may be, for example, a pharmaceutically acceptable salt, in which case a pharmaceutically acceptable salt means a salt that has the potency of the parent and is not biologically undesirable (e.g., has little or no toxicity). Preferred salts include, for example, salts that can be formed by mixing a solution of a pharmaceutically acceptable acid such as hydrochloric acid, phosphoric acid, sulfuric acid, or acetic acid with a solution of the parent. For example, if the compound contains an acidic substructure, its pharmaceutically acceptable salt may include salts formed using alkali metal ions (sodium or potassium), alkaline earth metal ions (calcium or magnesium), and preferred organic ligands such as ammonium ions.

[0117] Hereafter, for better understanding, antibody structures will be specifically described based on what is generally known in the art, and the scope of this application is not limited by the following description.

[0118] The structure of an antibody can be divided into a heavy chain region and a light chain region depending on the type of chain. The structure of an antibody can be divided into a fragment antigen-binding region (Fab region) and a fragment crystallizable region (Fc region) according to its antigen-binding function. The structure of an antibody can be divided into a variable region and a constant region according to the variability of its amino acid sequence. Other parts of the antibody structure include a hinge region and a tail region. The heavy chain region and light chain region can be described as functionally divided into a fragment antigen-binding region (Fab region) and a fragment crystallizable region (Fc region). The Fab region is a portion containing a part that binds to the antigen (antigen-binding site). The Fc region is a portion capable of binding to the fc receptor. The heavy chain region can be described as having both a Fab region and an Fc region, and the light chain region can be described as having a Fab region.

[0119] The Fab region of the heavy chain includes the variable heavy chain region (VH) and the constant heavy chain region 1 (CH1). For example, in IgG1, the Fc region is known to include the constant heavy chain region 2 (CH2) and the constant heavy chain region 3 (CH3). In this case, the entire constant heavy chain region of the antibody can be called CH. For example, the entire region combining CH1, CH2, and CH3 of IgG1 can be represented as CH.

[0120] The Fab region of the light chain includes the variable light chain region (VL) and the constant light chain region (CL). The light chain region can be described as lacking an Fc region.

[0121] The aforementioned VH, CH1, CH2, CH3, VL, and CL can each be called immunoglobulin domains.

[0122] The immunoglobulin domains in the heavy chain region are known to be located in the order of VH, CH1, CH2, and CH3 or VH, CH1, CH2, CH3, and CH4, from the N-terminus to the C-terminus. The immunoglobulin domains in the light chain region are known to be located in the order of VL and CL, from the N-terminus to the C-terminus. In general, the heavy chain region and the light chain region are linked by disulfide bonds, and the Fab region and the Fc region are linked by hinges. Specifically, the C-terminus of CH1 and the N-terminus of CH2 in the heavy chain region are known to be linked by hinges.

[0123] The variable regions (VH and VL) are regions containing the antigen-binding site. Within the variable regions, there is a portion with the greatest variability (hypervariable region), and the corresponding portion is called the complementarity-determining region (CDR). VH contains three CDRs, which are generally referred to as CDRH1, CDRH2, or CDRH3. The CDRs in VH can be understood to be located in the order CDRH1, CDRH2, and CDRH3 from the N-terminus to the C-terminus. VL also contains three CDRs, which are generally referred to as CDRL1, CDRL2, or CDRL3. The CDRs in VH can be understood to be located in the order CDRL1, CDRL2, and CDRL3 from the N-terminus to the C-terminus.

[0124] The constant region of an antibody is a region distinct from the antigen-binding region, and it is known that the constant region can interact with cells or molecules of the immune system. For example, the constant region can interact with (bind to or ligate to) the cell membrane of immune cells (e.g., lymphocytes, neutrophils, dendritic cells, and / or macrophages). Specifically, the hinge region and / or the CH2 portion of the constant region can bind to receptors on the cell membrane of immune cells (e.g., FcεRIII). In another embodiment, the constant region can bind to FcRn.

[0125] The constant regions of the heavy chain domains mentioned above (hereinafter referred to as "heavy chain constant regions") can be broadly divided into five types (classes or isotypes): alpha (α), gamma (γ), delta (δ), epsilon (ε), and mu (μ). In this case, the type of heavy chain constant region mentioned above is not determined individually for CH1, CH2, CH3, and CH4, but is determined by considering all heavy chain constant regions contained in the antibody (CH1, CH2, and CH3; or CH1, CH2, CH3, and CH4).

[0126] There are two types of steady-state regions in the light chain (hereinafter referred to as the "light chain steady-state region"), and these two types are lambda (λ) and kappa (κ).

[0127] Antibody types are known to be broadly classified into five types (classes or isotypes). These five types are determined by the type of heavy chain constant region.

[0128] The five antibody types mentioned above are immunoglobulin M (IgM), immunoglobulin D (IgD), immunoglobulin G (IgG), immunoglobulin A (IgA), immunoglobulin A (IgA), and immunoglobulin E (IgE). If the type of the constant region of the heavy chain of an antibody is classified as alpha, the antibody type may be recognized as IgA. If the type of the constant region of the heavy chain of an antibody is classified as gamma, the antibody type may be recognized as IgG. If the type of the constant region of the heavy chain of an antibody is classified as delta, the antibody type may be recognized as IgD. If the type of the constant region of the heavy chain of an antibody is classified as epsilon, the antibody type may be recognized as IgE. If the type of the constant region of the heavy chain of an antibody is classified as mu, the antibody type may be recognized as IgM. For example, each heavy chain of IgG is known to contain four immunoglobulin domains (VH, CH1, CH2, and CH3).

[0129] Among the five antibody types, IgG and IgA are known to be further classified into more detailed subclasses. For example, when describing an antibody that is a human antibody, if the type of the heavy chain constant region of the antibody is gamma 1 (γ1), the antibody type is IgG1; if the type of the heavy chain constant region of the antibody is gamma 2 (γ2), the antibody type is IgG2; if the type of the heavy chain constant region of the antibody is gamma 3 (γ3), the antibody type is IgG3; and if the type of the heavy chain constant region of the antibody is gamma 4 (γ4), the antibody type is IgG4. If the heavy chain constant region of a human antibody is alpha 1 (α1), the antibody type is IgA1; and if the heavy chain constant region of a human antibody is alpha 2 (α2), the antibody type is IgA2.

[0130] Antibody-drug conjugates of this disclosure

[0131] Overview of Antibody-Drug Conjugates

[0132] An antibody-drug conjugate is disclosed in accordance with one aspect of this disclosure.

[0133] An antibody-drug conjugate means that an antibody and a drug are linked together. In this case, the antibody is an anti-claudin 18.2 antibody, and the drug is monomethyl auristatin E (MMAE). The antibody-drug conjugate has a structure in which the drug is linked to one or more of the lysine residues 246 (K246) and 248 (K248) located in the Fc region of the antibody.

[0134] In some embodiments, the antibody-drug conjugate is expressed by formula 1: [Formula 1] [ka] It has the structure of [the object].

[0135] In Equation 1, Ab is an antibody unit.

[0136] In Equation 1, L is the linker unit.

[0137] In Equation 1, D is a drug unit.

[0138] In Equation 1, n is an integer between 1 and 4.

[0139] In this case, in Formula 1, the drug unit may be linked to one or more of the lysine residues 246 (K246) and 248 (K248) of the Fc region of the antibody unit. Specifically, in Formula 1, the linker unit may be covalently linked to an N atom derived from the lysine amino group (ε-amino group) of the antibody.

[0140] From here on, each element of the compound in Equation 1 will be explained in detail.

[0141] Antibody unit

[0142] The antibody unit may be derived from an anti-claudin 18.2 antibody (anti-CLDN18.2 antibody) and may be called a conjugated anti-CLDN18.2 antibody. Furthermore, since the structure of the antibody unit is identical to that of the anti-CLDN18.2 antibody from which it is derived, except for the conjugated portion, the antibody unit may also be called an anti-CLDN18.2 antibody. In this case, the conjugated portion may be either lysine residue 246 (K246) or lysine residue 248 (K248) of the heavy chain of the anti-CLDN18.2 antibody.

[0143] In some embodiments, the anti-CLDN18.2 antibody may include the Fc region of IgG. In some embodiments, the Fc region of the anti-CLDN18.2 antibody may be the Fc region of IgG.

[0144] In some embodiments, the anti-CLDN18.2 antibody may be an IgG antibody. The IgG antibody includes human IgG antibodies, humanized IgG antibodies, and chimeric IgG antibodies.

[0145] IgG may be selected from any one of the subclasses IgG1, IgG2, IgG3, and IgG4.

[0146] In some embodiments, the anti-CLDN18.2 antibody may be an IgG1 antibody. The IgG1 antibody includes human IgG1 antibody, humanized IgG1 antibody, and chimeric IgG1 antibody.

[0147] In some embodiments, the anti-CLDN18.2 antibody may include the Fc region of IgG1. The Fc region of the anti-CLDN18.2 antibody may be the Fc region of IgG1.

[0148] In some embodiments, the anti-CLDN18.2 antibody may be an IgG2 antibody. The IgG2 antibody includes human IgG2 antibody, humanized IgG2 antibody, and chimeric IgG2 antibody.

[0149] In some embodiments, the anti-CLDN18.2 antibody may include the Fc region of IgG2. The Fc region of the anti-CLDN18.2 antibody may be the Fc region of IgG2.

[0150] In some embodiments, the anti-CLDN18.2 antibody may be an IgG3 antibody. The IgG3 antibody includes human IgG3 antibody, humanized IgG3 antibody, and chimeric IgG3 antibody.

[0151] In some embodiments, the anti-CLDN18.2 antibody may include the Fc region of IgG3. The Fc region of the anti-CLDN18.2 antibody may be the Fc region of IgG3.

[0152] In some embodiments, the anti-CLDN18.2 antibody may be an IgG4 antibody. The IgG4 antibody includes human IgG4 antibody, humanized IgG4 antibody, and chimeric IgG4 antibody.

[0153] In some embodiments, the anti-CLDN18.2 antibody may include the Fc region of IgG4. The Fc region of the anti-CLDN18.2 antibody may be the Fc region of IgG4.

[0154] In some embodiments, the anti-CLDN18.2 antibody may have an amino acid sequence selected from SEQ ID NOs: 1 to SEQ ID NOs: 5. Alternatively, the anti-CLDN18.2 antibody may have an amino acid sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or greater sequence identity with respect to the selected amino acid sequence. In certain embodiments, the anti-CLDN18.2 antibody may have an amino acid sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more sequence identity thereto.

[0155] In some embodiments, the anti-CLDN18.2 antibody includes an IgG Fc region, in which case the IgG Fc region may have an amino acid sequence selected from SEQ ID NOs: 1 to 5. The IgG Fc region may have an amino acid sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or greater sequence identity with respect to the selected amino acid sequence. In certain embodiments, the anti-CLDN18.2 antibody may include an IgG Fc region, in which case the IgG Fc region may have an amino acid sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or greater sequence identity with respect to the selected amino acid sequence.

[0156] In some embodiments, the anti-CLDN18.2 antibody or its Fc region may include the amino acid sequence of KPKDTLM (SEQ ID NO: 6) and the amino acid sequence of MHEALHNH (SEQ ID NO: 7).

[0157] In some embodiments, the anti-CLDN18.2 antibody or its Fc region may include the amino acid sequence of KPKDTLM (SEQ ID NO: 6) and the amino acid sequence of MHEALHNHY (SEQ ID NO: 8).

[0158] In some embodiments, the anti-CLDN18.2 antibody or its Fc region may include the amino acid sequence GPSVFLFPPKPKDTLM (SEQ ID NO: 9).

[0159] In some embodiments, the anti-CLDN18.2 antibody may have an amino acid sequence selected from sequence numbers 1-5, or amino acid sequences having 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or greater sequence identity with respect to them, and may basically include the amino acid sequence of KPKDTLM (sequence number 6) and the amino acid sequence of MHEALHNH (sequence number 7). In some embodiments, the anti-CLDN18.2 antibody may also include an IgG Fc region, in which case the IgG Fc region may have any one amino acid sequence selected from sequence numbers 1-5, or amino acid sequences having 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or greater sequence identity with respect to them, and may basically include the amino acid sequence of KPKDTLM (sequence number 6) and the amino acid sequence of MHEALHNH (sequence number 7).

[0160] In some embodiments, the anti-CLDN18.2 antibody may have an amino acid sequence selected from amino acid sequences having 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to SEQ ID NOs: 1-4, and may essentially include the amino acid sequence of KPKDTLM (SEQ ID NO: 6) and the amino acid sequence of MHEALHNHY (SEQ ID NO: 8). In some embodiments, the anti-CLDN18.2 antibody may include an IgG Fc region (for example, the antibody's Fc region may be the IgG's Fc region), in which case the IgG Fc region may have an amino acid sequence selected from amino acid sequences having 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more sequence identity with respect to SEQ ID NOs: 1-4, in which case the amino acid sequence may basically include the amino acid sequence of KPKDTLM (SEQ ID NO: 6) and the amino acid sequence of MHEALHNHY (SEQ ID NO: 8).

[0161] In some embodiments, the anti-CLDN18.2 antibody may have an amino acid sequence selected from amino acid sequences having 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or greater sequence identity with respect to SEQ ID NOs: 1-5, and may basically include the amino acid sequence of GPSVFLFPPKPKDTLM (SEQ ID NO: 9). In some embodiments, the anti-CLDN18.2 antibody may also include an IgG Fc region, in which case the IgG Fc region may have an amino acid sequence selected from amino acid sequences having 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or greater sequence identity with respect to

[0162] In some embodiments, the antibody having binding affinity to the Fc-binding substance or peptide of the present invention may be an IgG isotype antibody. In some embodiments, the antibody having binding affinity to the Fc-binding substance or peptide of the present invention may be an IgG1 isotype antibody, an IgG2 isotype antibody, an IgG3 isotype antibody, or an IgG4 isotype antibody. In some embodiments, the antibody having binding affinity to the Fc-binding substance or peptide of the present invention may be an IgG1 isotype antibody, an IgG2 isotype antibody, or an IgG4 isotype antibody.

[0163] In some embodiments, the anti-CLDN18.2 antibody may be any known anti-CLDN18.2 antibody (see reference [Korean Patent Application No. 10-2021-7023724 (Publication No. 10-2021-0110339)]).

[0164] In some embodiments, the heavy chain of the anti-CLDN18.2 antibody may include CDRH1 having the amino acid sequence of SEQ ID NO: 10 (TYGVH) or an amino acid sequence having 90% or more sequence identity thereto; CDRH2 having the amino acid sequence of SEQ ID NO: 11 (VIWAGGSTNYNSALMS) or an amino acid sequence having 90% or more sequence identity thereto; and CDRH3 having the amino acid sequence of SEQ ID NO: 12 (AAYYGNGLDY) or an amino acid sequence having 90% or more sequence identity thereto. In certain embodiments, the anti-CLDN18.2 antibody may have two heavy chains comprising CDRH1 having the amino acid sequence of SEQ ID NO: 10, CDRH2 having the amino acid sequence of SEQ ID NO: 11, and CDRH3 having the amino acid sequence of SEQ ID NO: 12.

[0165] In some embodiments, the light chain of the anti-CLDN18.2 antibody may include CDRL1 having the amino acid sequence of SEQ ID NO: 13 (KSSQTLLNSGNQKNYLT) or an amino acid sequence having 90% or more sequence identity thereto; CDRL2 having the amino acid sequence of SEQ ID NO: 14 (WASTGES) or an amino acid sequence having 90% or more sequence identity thereto; and CDRL3 having the amino acid sequence of SEQ ID NO: 15 (QNAYFYPFT) or an amino acid sequence having 90% or more sequence identity thereto. In certain embodiments, the anti-CLDN18.2 antibody may have two light chains comprising CDRL1 having the amino acid sequence of SEQ ID NO: 13, CDRL2 having the amino acid sequence of SEQ ID NO: 14, and CDRL3 having the amino acid sequence of SEQ ID NO: 15.

[0166] In some embodiments, the heavy chain of the anti-CLDN18.2 antibody may include CDRH1 having the amino acid sequence of SEQ ID NO: 10 or an amino acid sequence having 90% or more sequence identity thereto, CDRH2 having the amino acid sequence of SEQ ID NO: 11 or an amino acid sequence having 90% or more sequence identity thereto, and CDRH3 having the amino acid sequence of SEQ ID NO: 12 or an amino acid sequence having 90% or more sequence identity thereto. The light chain of the anti-CLDN18.2 antibody may include CDRL1 having the amino acid sequence of SEQ ID NO: 13 or an amino acid sequence having 90% or more sequence identity thereto, CDRL2 having the amino acid sequence of SEQ ID NO: 14 or an amino acid sequence having 90% or more sequence identity thereto, and CDRL3 having the amino acid sequence of SEQ ID NO: 15 or an amino acid sequence having 90% or more sequence identity thereto. In certain embodiments, the anti-CLDN18.2 antibody may have two heavy chains comprising CDRH1 having the amino acid sequence of SEQ ID NO: 10, CDRH2 having the amino acid sequence of SEQ ID NO: 11, and CDRH3 having the amino acid sequence of SEQ ID NO: 12, and two light chains comprising CDRL1 having the amino acid sequence of SEQ ID NO: 13, CDRL2 having the amino acid sequence of SEQ ID NO: 14, and CDRL3 having the amino acid sequence of SEQ ID NO: 15.

[0167] In some embodiments, the anti-CLDN18.2 antibody may comprise a heavy chain having the amino acid sequence of SEQ ID NO: 16 or an amino acid sequence having 90% or greater sequence identity thereto, and a light chain having the amino acid sequence of SEQ ID NO: 17 or an amino acid sequence having 90% or greater sequence identity thereto. In certain embodiments, the anti-CLDN18.2 antibody may comprise two heavy chains having the amino acid sequence of SEQ ID NO: 16 and two light chains having the amino acid sequence of SEQ ID NO: 17.

[0168] Linker unit

[0169] The linker unit is a structure of a region that connects an anti-claudin 18.2 antibody (anti-CLDN18.2 antibody) and a drug.

[0170] Overview of the linker unit

[0171] L (linker unit) in Formula 1 is of Formula 2: [Formula 2]

Chemical formula

[0172] At this time, In Formula 2, b is an integer from 0 to 6. In some embodiments, b may be 0, 1, 2, 3, 4, 5, or 6, or an integer between two of these numbers. For example, b may be an integer from 1 to 3. In a specific embodiment, b may be 2.

[0173] In Formula 2, X' is -NH-, -C(O)-, or -NHC(O)-.

[0174] In Formula 2, B' is a group formed by a click chemical reaction between click chemical functional groups.

[0175] In Formula 2, PM 1 and PM 2 are each an independent PEG partial structure.

[0176] In Formula 2, 1* represents the attachment site to Ab.

[0177] In Formula 2, 2* represents the attachment site to D.

[0178] B' of Formula 2

[0179] B' of Formula 2 is

Chemical formula

[0180] At this time, R x is H, halogen, and C 1~3Alkyl can be selected, A1 and A2 represent attachment sites of the linker unit to the rest of the structure, respectively. In a particular embodiment, A1 may represent an attachment site to X'. In another particular embodiment, A2 may represent an attachment site to X'.

[0181] PM of Equation 2 1 and PM 2

[0182] In Equation 2, PM 1 and PM 2 Each of these independently represents a PEG substructure.

[0183] In this case, the PEG substructure may contain 1 to 30, 1 to 20, or 1 to 10 ethylene glycol units (e.g., -CH2OCH2-, -OCH2CH2-, or -CH2CH2O-).

[0184] For example, the PEG substructure may have the structure of formula 3.

[0185] [Formula 3]

[0186] [ka]

[0187] In Equation 3, D PEG This is a spacer in the PEG substructure.

[0188] For example, D PEG This may be a group with a main chain length of 0 to 6 (i.e., a group in which the total number of atoms located in the main chain is between 0 and 6).

[0189] For example, D PEG is a combined, or substituted or unsubstituted C 1~6 Alkylene, substituted or unsubstituted C 1~6 Heteroalkylenes, substituted or unsubstituted C 1~6Alkenylene, substituted or unsubstituted C 1~6 The heteroalkenylene, substituted or unsubstituted alkylene, or substituted or unsubstituted heteroalkylene may contain one or more substituents, selected from -R, =O, =S, -NO2, -CR3, -NR2, -OR, -SR, -C(=O)R, -C(=O)CR3, -C(=O)OR, and -C(=O)NR2, where R is independently H, halogen, or C 1~6 Alkyl, C 3~10 Cycloalkyl, C 3~10 The substituents are heterocycloalkyl, aryl, heteroaryl, -OH, -NH2, =O, =S, and -SH. In certain embodiments, the substituent is -C 1~4 The heteroalkylene, heteroalkenylene, and heteroalkynylene each independently contain one or more heteroatoms, and each heteroatom may be independently selected from N, O, and S.

[0190] In a particular embodiment, D PEG is a combined, or substituted or unsubstituted C 1~3 Alkylene, or substituted or unsubstituted C 1~3 The heteroalkylene may be a substituted alkylene or a substituted heteroalkylene may contain one or more types of substituents, the substituent being =O, and the heteroalkylene may contain one or more heteroatoms, each of which may be independently selected from N, O, and S.

[0191] In Equation 3, R PEGis a PEG capping group. In this case, the PEG capping group may be absent, or -CH3, C2 alkyl, C3 alkyl, -NH2, -CH2NH2, -SC(=O)CH3, -SC(=O)CH2CH3, -CH2SC(=O)CH3, -CH2SC(=O)CH2CH3, -OH, -CH2OH, -SH, -CH2SH, -OCH3, -CH2OCH3, -CH2OCH2CH3, -C(=O)CH3, -C(=O)C H2CH3, -CH2C(=O)CH3, -CH2C(=O)CH2CH3, -NHC(=O)CH3, -NHC(=O)CH2CH3, -CH2NHC(=O)CH3, -CH2CH2NHC(=O)CH3, -CH2CH2NHC(=O)CH2CH3, -CH2NHC(=O)CH2CH3, -CH2CH2COOH, glucose, or -O-glucose. In certain embodiments, the PEG capping group may be absent or may be -CH3, -OCH3, -CH2OCH3, -C(=O)CH3, -CH2C(=O)CH3, -NHC(=O)CH3, -CH2NHC(=O)CH3, or -CH2CH2COOH. In certain embodiments, the total atomic mass of atoms belonging to the PEG capping group may be 300 doltons or less, 200 doltons or less, 150 doltons or less, 100 doltons or less, or 50 doltons or less, but is not limited to these.

[0192] In Equation 3, p may be an integer between 1 and 30, preferably between 1 and 10.

[0193] In Equation 3, [EG] is an ethylene glycol unit. In this case, the ethylene glycol unit is -[CH2OCH2]-, -[OCH2CH2]-, or -[CH2CH2O]-.

[0194] In a particular embodiment, in formula 3, D PEG is -C(=O)-, [EG] is -[OCH2CH2]-, p is 8, R PEG It is -CH3.

[0195] In a particular embodiment, in formula 2, PM 1 and PM 2 Each of them is, [ka] That is the case.

[0196] Specific Embodiments of Linker Units

[0197] In a particular embodiment, the linker unit is defined by Equation 4: [Formula 4] [ka] It may have a structure.

[0198] In Equation 4, PM 1 and PM 2 These are each independent PEG substructures, and their description is as follows: <<PM of Equation 2 above 1 and PM 2 >>PM in the section 1 and PM 2 This is explained in detail.

[0199] In Equation 4, 1* represents the attachment site with Ab. In Equation 4, 2* represents the attachment site with D.

[0200] Drug Unit

[0201] A drug unit refers to a drug conjugated to an antibody or a structure derived from a drug. The drug may be monomethyl auristatin E (MMAE).

[0202] In a particular embodiment, the drug unit is defined by formula 5: [Formula 5] [ka] It may have a structure.

[0203] In Equation 5, 3* represents the attachment site to L in Equation 1.

[0204] Location where the drug unit is connected

[0205] In Equation 1, n is an integer between 1 and 4. In some embodiments, n may be an integer between 1 and 2. In certain embodiments, n may be 2.

[0206] The drug unit may be linked to one or more of the lysine residues 246 (K246) and 248 (K248) of the Fc region of the antibody unit (via L or a linker unit). More specifically, the antibody unit may contain two heavy chains (a first heavy chain and a second heavy chain), in which case "-L-" may be linked to one or more of the K246, K248 of the first heavy chain, K246 of the second heavy chain, and K248 of the second heavy chain.

[0207] For example, if n is 1, One drug unit may be linked to K246 of the first heavy chain of the antibody unit (via L or a linker unit). Alternatively, one drug unit may be linked to K248 of the first heavy chain of the antibody unit.

[0208] For example, if n is 2, One of the two drug units may be linked to K246 of the first heavy chain of the antibody unit, and the other drug unit may be linked to K246 of the second heavy chain. Alternatively, one of the two drug units may be linked to K248 of the first heavy chain of the antibody unit, and the other drug unit may be linked to K248 of the second heavy chain. Alternatively, one of the two drug units may be linked to K246 of the first heavy chain of the antibody unit, and the other drug unit may be linked to K248 of the second heavy chain. Alternatively, one of the two drug units may be linked to K246 of the first heavy chain of the antibody unit, and the other drug unit may be linked to K248 of the first heavy chain.

[0209] For example, if n is 3, The three drug units may be independently linked to K246 of the first heavy chain, K248 of the first heavy chain, and K246 of the second heavy chain, respectively.

[0210] Alternatively, the drug units of that generation may be independently linked to K246 of the first heavy chain, K248 of the first heavy chain, and K248 of the second heavy chain, respectively.

[0211] More specifically, when n is 3, the antibody-drug conjugate contains three drug units (a first drug unit, a second drug unit, and a third drug unit). In this case, the first drug unit may be linked to K246 of the first heavy chain, the second drug unit may be linked to K248 of the first heavy chain, and the third drug unit may be linked to K246 of the second heavy chain. Alternatively, the first drug unit may be linked to K246 of the first heavy chain, the second drug unit may be linked to K248 of the first heavy chain, and the third drug unit may be linked to K248 of the second heavy chain.

[0212] For example, if n is 4, The four drug units may be independently linked to K246, K248, K246, and K248 of the first heavy chain of the antibody unit.

[0213] More specifically, when n is 4, the antibody-drug conjugate contains 4 drug units (a first drug unit, a second drug unit, a third drug unit, and a fourth drug unit). In this case, the first drug unit may be linked to K246 of the first heavy chain, the second drug unit may be linked to K248 of the first heavy chain, the third drug unit may be linked to K246 of the second heavy chain, and the fourth drug unit may be linked to K248 of the second heavy chain.

[0214] Specific Embodiments of Antibody-Drug Conjugates

[0215] In certain embodiments, formula 1 may be represented by formula 6. That is, the antibody-drug conjugate is given by the following formula 6: [Formula 6] [ka] It may have a structure.

[0216] In Equation 6, Ab is an antibody unit, and its description is as described in relation to the drug unit in the <<Antibody Unit>> section above.

[0217] In Equation 6, PM 1 and PM 2 These are each independent PEG substructures, and their explanation is as follows: <<PM in Equation 2 above 1 and PM 2 >>PM in the section 1 and PM 2 This is explained in detail.

[0218] In Equation 6, D is a drug unit, and its description is as described in the section above concerning drug units.

[0219] In equation 6, n is an integer between 1 and 4.

[0220] In Equation 6, the position where the drug unit is attached to the antibody unit is described in the section above titled "<<Position where the drug unit is attached>>".

[0221] In certain embodiments, formula 1 may be represented by formula 7. That is, the antibody-drug conjugate is given by the following formula 7: [Formula 7] [ka] It may have a structure.

[0222] In Equation 7, Ab is an antibody unit, and its description is as described above in the section on "Antibody Units" with respect to the drug unit.

[0223] In equation 7, n is an integer between 1 and 4.

[0224] In Equation 7, the location where the antibody unit is bound is described in the section above titled "<<Location where the drug unit is bound>>".

[0225] Characteristics of antibody-drug conjugates in this disclosure

[0226] The binding of the antibody-drug conjugate of this disclosure to the antigen

[0227] The antibody-drug conjugates disclosed herein may be conjugated to an antigen (claudin 18.2 protein). Specifically, the antibody unit contained in the antibody-drug conjugate may be conjugated to an antigen (claudin 18.2 protein).

[0228] In some embodiments, the antibody-drug conjugate may bind to an antigen (claudin 18.2 protein) expressed on the surface (or membrane) of a cell. Specifically, the antibody unit contained in the antibody-drug conjugate may bind to an antigen (claudin 18.2 protein) expressed on the surface (or membrane) of a cell.

[0229] In some embodiments, the antibody-drug conjugate may bind to claudin 18.2-positive cells (or cells expressing claudin 18.2). Specifically, the antibody unit contained in the antibody-drug conjugate may bind to claudin 18.2-positive cells (or cells expressing claudin 18.2).

[0230] In some embodiments, the binding affinity of the antibody-drug conjugate disclosed herein to the antigen (claudin 18.2 protein) is the same as and / or the same as the binding affinity of the antibody (anti-claudin 18.2 antibody) before conjugation to the antigen (claudin 18.2 protein).

[0231] Internalization of the antibody-drug conjugate of this disclosure into cells

[0232] The antibody-drug conjugate disclosed herein may bind to a claudin 18.2 protein expressed in a cell and subsequently enter (or be internalized) the cell. In this process, the portion of the antibody-drug conjugate corresponding to the beta-glucuronide linker may be cleaved by an intracellular beta-glucuronidase, which may be produced in the lysosome of the cell.

[0233] Therefore, the drug units separated from the antibody-drug conjugate may be released into the cells by the cleavage process described above.

[0234] The structure of the beta-glucuronide linker contained in the antibody-drug conjugate is shown in Equation 8.

[0235] [Formula 8]

[0236] [ka]

[0237] In Equation 8, 4* represents the attachment site to the linker unit.

[0238] In Equation 8, 5* represents the attachment site to the drug unit.

[0239] Anticancer effects of antibody-drug conjugates in this disclosure

[0240] The antibody-drug conjugates disclosed herein have anticancer effects. In this context, anticancer effects refer to the inhibition of the development, proliferation, and / or metastasis of cancer cells. The anticancer effects are exerted by the synergistic effect between the function of the drug released after the antibody-drug conjugate enters cancer cells, and / or the function of the antibody itself.

[0241] In some embodiments, antibody-drug conjugates can attack cancer cells.

[0242] In some embodiments, antibody-drug conjugates can kill cancer cells.

[0243] Half-life of antibody-drug conjugates in this disclosure

[0244] The in vivo half-life of the antibody-drug conjugate disclosed herein may be in the range of 3 to 7 days. In some embodiments, the in vivo half-life of the antibody-drug conjugate is in the range of 4 to 6 days. In certain embodiments, the in vivo half-life of the antibody-drug conjugate is in the range of 4.5 to 5.2 days.

[0245] In some embodiments, the in vivo half-life of the antibody-drug conjugate is the same as and / or similar to the in vivo half-life of the pre-conjugation antibody (anti-claudin 18.2 antibody).

[0246] Use of antibody-drug conjugates in this disclosure

[0247] Summary of Use of Antibody-Drug Conjugates in This Disclosure

[0248] The antibody-drug conjugates of this disclosure have use in the treatment of cancer.

[0249] This disclosure discloses a pharmaceutical composition for treating cancer, comprising the antibody-drug conjugate described above.

[0250] This disclosure discloses a method for treating cancer using the antibody-drug conjugate described above.

[0251] Various embodiments of antibody-drug conjugates used in cancer treatment will be described later.

[0252] Target diseases

[0253] The target diseases for which the antibody-drug conjugates of this disclosure may be used for therapeutic purposes are cancer, tumors, cancer-related diseases, or tumor-related diseases.

[0254] In some embodiments, the cancer or tumor may be any one selected from bladder cancer, bone cancer, brain cancer, breast cancer, heart cancer, cervical cancer, colon cancer, rectal cancer, esophageal cancer, fibrosarcoma, gastric cancer, digestive tract cancer, biliary tract 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, genitourinary cancer, testicular germ cell carcinoma, thymoma, and thymic carcinoma. In certain embodiments, the cancer or tumor may be any one selected from gastric cancer, pancreatic cancer, esophageal cancer, ovarian cancer, lung cancer, liver cancer, bladder cancer, colon cancer, bile duct cancer, and genitourinary cancer.

[0255] In some embodiments, the cancer or tumor is a claudin 18.2-positive cancer or tumor. In this case, the cancer or tumor may express the claudin 18.2 protein, and specifically, cancer cells or tumor cells may express the claudin 18.2 protein.

[0256] In some embodiments, the disease to be treated by the pharmaceutical composition may be gastric cancer. In certain embodiments, the disease to be treated by the pharmaceutical composition is claudin 18.2-positive gastric cancer. In this case, claudin 18.2-positive gastric cancer may refer to gastric cancer caused by cancer cells expressing the claudin 18.2 protein.

[0257] In some embodiments, the disease to be treated by the pharmaceutical composition may be pancreatic cancer. In certain embodiments, the disease to be treated by the pharmaceutical composition is claudin 18.2-positive pancreatic cancer. In this case, claudin 18.2-positive pancreatic cancer may refer to pancreatic cancer caused by cancer cells expressing the claudin 18.2 protein.

[0258] In some embodiments, the disease to be treated by the pharmaceutical composition may be esophageal cancer. In certain embodiments, the disease to be treated by the pharmaceutical composition is claudin 18.2-positive esophageal cancer. In this case, claudin 18.2-positive esophageal cancer may refer to esophageal cancer caused by cancer cells expressing the claudin 18.2 protein.

[0259] In some embodiments, the disease to be treated by the pharmaceutical composition may be ovarian cancer. In certain embodiments, the disease to be treated by the pharmaceutical composition is claudin 18.2-positive ovarian cancer. In this case, claudin 18.2-positive ovarian cancer may refer to ovarian cancer caused by cancer cells expressing the claudin 18.2 protein.

[0260] In some embodiments, the disease to be treated by the pharmaceutical composition may be lung cancer. In certain embodiments, the disease to be treated by the pharmaceutical composition is claudin 18.2-positive lung cancer. In this case, claudin 18.2-positive lung cancer may refer to lung cancer caused by cancer cells expressing the claudin 18.2 protein.

[0261] In some embodiments, the disease to be treated by the pharmaceutical composition may be liver cancer. In certain embodiments, the disease to be treated by the pharmaceutical composition is claudin 18.2-positive liver cancer. In this case, claudin 18.2-positive liver cancer may refer to liver cancer caused by cancer cells expressing the claudin 18.2 protein.

[0262] In some embodiments, the disease to be treated by the pharmaceutical composition may be bladder cancer. In certain embodiments, the disease to be treated by the pharmaceutical composition is claudin 18.2-positive bladder cancer. In this case, claudin 18.2-positive bladder cancer may refer to bladder cancer caused by cancer cells expressing the claudin 18.2 protein.

[0263] In some embodiments, the disease to be treated by the pharmaceutical composition may be colon cancer. In certain embodiments, the disease to be treated by the pharmaceutical composition is claudin 18.2-positive colon cancer. In this case, claudin 18.2-positive colon cancer may refer to colon cancer caused by cancer cells expressing the claudin 18.2 protein.

[0264] In some embodiments, the disease to be treated by the pharmaceutical composition may be cholangiocarcinoma. In certain embodiments, the disease to be treated by the pharmaceutical composition is claudin 18.2-positive cholangiocarcinoma. In this case, claudin 18.2-positive cholangiocarcinoma may refer to cholangiocarcinoma caused by cancer cells expressing the claudin 18.2 protein.

[0265] In some embodiments, the disease to be treated by the pharmaceutical composition may be genitourinary cancer. In certain embodiments, the disease to be treated by the pharmaceutical composition is claudin 18.2-positive genitourinary cancer. In this case, claudin 18.2-positive genitourinary cancer may refer to genitourinary cancer caused by cancer cells expressing the claudin 18.2 protein.

[0266] Pharmaceutical compositions containing antibody-drug conjugates

[0267] Overview of Pharmaceutical Compositions

[0268] In accordance with one aspect of this disclosure, a pharmaceutical composition comprising an antibody-drug conjugate for treating cancer, tumors, cancer-related diseases, or tumor-related diseases is disclosed. Herein, the antibody-drug conjugate refers to the antibody-drug conjugate disclosed in this disclosure. The pharmaceutical composition comprises a therapeutically effective amount of the antibody-drug conjugate. The pharmaceutical composition may further comprise a pharmaceutically acceptable carrier and / or a pharmaceutically acceptable adjuvant, wherein the pharmaceutically acceptable carrier may be used for appropriate formulation of the pharmaceutical composition.

[0269] Target diseases of pharmaceutical compositions

[0270] The target disease of the pharmaceutical composition is cancer, tumor, cancer-related disease, or tumor-related disease, as described in the <<Target Disease>> subsection of the <<Use of Antibody-Drug Conjugates of the Disclosure>> section.

[0271] Examples of pharmaceutical composition formulations

[0272] In some embodiments, the pharmaceutical composition may be formulated as lozenges, medicinal drops, tablets, aqueous suspensions, oily suspensions, prepared powders, granules, emulsions, hard capsules, soft capsules, syrups, or elixirs.

[0273] In some embodiments, the pharmaceutical composition may be formulated as an injectable, suppository, respiratory inhalation powder, spray aerosol, ointment, topical powder, oil, or cream.

[0274] In some embodiments, the pharmaceutical composition may be formulated as an injectable. Specifically, a therapeutically effective amount of antibody-drug conjugate may be mixed with a stabilizer or buffer in water to prepare a solution or suspension, which may be formulated for unit doses in ampoules or vials.

[0275] In some embodiments, the pharmaceutical composition may be formulated as an aerosol by mixing it with an additive such as a spray to prepare a water-dispersed concentrate or wet powder.

[0276] In some embodiments, when a pharmaceutical composition is formulated for transdermal use, ointments, creams, topical powders, oils, and other topical skin preparations may be prepared by adding a therapeutically effective amount of antibody-drug conjugate to a carrier such as animal fat, vegetable fat, wax, paraffin, starch, tragacanth, cellulose derivatives, polyethylene glycol, silicone, bentonite, silica, talc, or zinc oxide.

[0277] Examples of pharmaceutically acceptable carriers

[0278] A pharmaceutically acceptable carrier may be one commonly used in formulations and may include, but is not limited to, physiological saline, sterile water, Ringer's solution, buffered physiological saline, cyclodextrin, glucose solution, maltodextrin solution, glycerol, ethanol, and liposomes, and may further include other common additives such as antioxidants and buffers as needed. In addition, diluents, dispersants, surfactants, binders, and lubricants may be added to formulate the mixture into injectable formulations such as aqueous solutions, suspensions, and emulsions, pills, capsules, granules, or tablets. With regard to preferred pharmaceutically acceptable carriers and formulations, each mixture component may preferably be formulated using the methods disclosed in Remington's Pharmaceutical Sciences (19th edition, 1995).

[0279] In some embodiments, the pharmaceutical composition may include, as a pharmaceutically acceptable carrier, binders such as lactose, saccharose, sorbitol, mannitol, starch, amylopectin, cellulose, or gelatin; excipients such as dicalcium phosphate; disintegrants such as corn starch or sweet potato starch; lubricants such as magnesium stearate, calcium stearate, sodium stearyl fumarate, or polyethylene glycol wax; sweeteners; flavoring agents; liquid carriers such as syrups or fatty oils; sterile aqueous solutions; injectable esters such as propylene glycol, polyethylene glycol, or ethyl oleate; suspending agents; emulsifiers; freeze-drying agents; topical agents; stabilizers; buffers; animal oils; vegetable oils; waxes; paraffin; starch; tragacanth; cellulose derivatives; polyethylene glycol; silicones; bentonite; silica; talc; zinc oxide; or suitable combinations thereof.

[0280] In some embodiments, the antibody-drug conjugate may be mixed with a pharmaceutically acceptable carrier to prepare the pharmaceutical compositions of this disclosure. That is, the pharmaceutical composition may contain a pharmaceutically acceptable carrier. Furthermore, the pharmaceutical composition may further contain one or more other elements suitable for the treatment or prevention of cancer. The term pharmaceutically acceptable carrier may be used to include excipients, diluents, or adjuvants. The carrier may be one or more selected from, for example, lactose, glucose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, polyvinylpyrrolidone, water, physiological saline, buffers such as phosphate-buffered saline (PBS), methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil. The carrier may also contain fillers, anti-agglutinants, lubricants, wetting agents, flavoring agents, emulsifiers, preservatives, or combinations thereof.

[0281] Treatment methods using antibody-drug conjugates

[0282] Summary of treatment methods

[0283] In accordance with one aspect of this disclosure, a method for treating cancer, tumors, cancer-related diseases, or tumor-related diseases using an antibody-drug conjugate is disclosed. Herein, the antibody-drug conjugate refers to the antibody-drug conjugate disclosed herein. The treatment method includes administering a suitable formulation of a therapeutically effective antibody-drug conjugate, i.e., a pharmaceutical composition, to a target using a suitable administration method, a suitable administration regimen, and a suitable dose.

[0284] Target diseases of treatment methods

[0285] The target disease of the treatment method is cancer, tumor, cancer-related disease, or tumor-related disease, as described in the <<Target Disease>> subsection of the <<Use of Antibody-Drug Conjugates of This Disclosure>> section.

[0286] Subject to treatment

[0287] The treatment method may be applied to humans or non-human animals.

[0288] Examples of administration methods

[0289] The treatment method may involve administering an appropriate formulation of a therapeutically effective antibody-drug conjugate to the target by an appropriate administration method. In one embodiment, the treatment method may involve administering an appropriate formulation of a therapeutically effective antibody-drug conjugate to the target by one method selected from oral administration, parenteral administration, intravenous administration, intraperitoneal administration, intramuscular administration, transdermal administration, and subcutaneous administration. In a particular embodiment, the treatment method may involve administering an appropriate formulation of a therapeutically effective antibody-drug conjugate to the target by intravenous administration.

[0290] Dosage examples

[0291] The treatment method may involve administering an appropriate dosage of an appropriate formulation of an effective antibody-drug conjugate to the target using an appropriate administration method. In one embodiment, the dosage may be approximately 0.01 mg to 1000 mg per kg of the target's body weight, based on the antibody-drug conjugate. In a specific embodiment, the dosage may be approximately 0.01 mg / kg to 100 mg / kg of the target's body weight, based on the antibody-drug conjugate.

[0292] Example of an administration cycle

[0293] The treatment method may involve administering an appropriate formulation of an effective antibody-drug conjugate to the target in an appropriate administration cycle. In one embodiment, the pharmaceutical composition containing an appropriate dose of antibody-drug conjugate may be administered once daily. In another example, the administration cycle may involve administering the pharmaceutical composition containing an appropriate dose of antibody-drug conjugate twice or more times daily. In yet another example, the administration cycle may involve administering the pharmaceutical composition containing an appropriate dose of antibody-drug conjugate at intervals of 1 hour, 2 hours, 6 hours, 12 hours, 24 hours, 2 days, 3 days, 1 week, 2 weeks, 1 month, 2 months, and / or 3 months.

[0294] Therapeutic applications of antibody-drug conjugates

[0295] In accordance with one aspect of this disclosure, the use of antibody-drug conjugates for treating cancer, tumors, cancer-related diseases, or tumor-related diseases is disclosed. Herein, antibody-drug conjugates refer to the antibody-drug conjugates disclosed by this disclosure. Herein, cancer, tumors, cancer-related diseases, or tumor-related diseases are as described in the <<Target Diseases>> subsection of the <<Uses of Antibody-Drug Conjugates in this Disclosure>> section.

[0296] Use of antibody-drug conjugates in the manufacture of cancer treatment drugs

[0297] In accordance with one aspect of this disclosure, the use of an antibody-drug conjugate for treating cancer, tumors, cancer-related diseases, or tumor-related diseases in the manufacture of a drug for treating cancer is disclosed. Herein, antibody-drug conjugate means the antibody-drug conjugate disclosed by this disclosure. Herein, cancer, tumors, cancer-related diseases, or tumor-related diseases are as described in the <<Target Diseases>> subsection of the <<Uses of Antibody-Drug Conjugates in this Disclosure>> section.

[0298] Method for preparing antibody-drug conjugates

[0299] The antibody-drug conjugates disclosed herein may be prepared, for example, by the methods disclosed in Examples 1 and 2.

[0300] Hereafter, the invention provided in this application will be described in more detail by experimental examples and embodiments. These examples are intended to illustrate the content disclosed in this application, and the scope of the content disclosed in this application is not limited by these examples. [Examples]

[0301] Ingredients and experimental methods

[0302] compound

[0303] The compounds were purchased from commercial suppliers and used without further purification. 5-exo-norbornenecarboxylic acid, ammonium sulfate ((NH4)2SO4), N-Boc-ethylenediamine, triisopropylsilane (TIS), and 1,2-ethanedithiol (EDT) were purchased from Sigma-Aldrich CO., LTD. (St. Louis, Missouri, USA).

[0304] N-methylmaleimide, N-ethylmaleimide, N-cyclohexylmaleimide, N-phenylmaleimide, N-benzylmaleimide, N-tert-butylmaleimide, and 3-maleimidopropionic acid were purchased from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). Tert-butylthioglycolic acid was purchased from Angene Chemical PVT., LTD. (Telangana, India). 4-methyltetrazine NHS ester was purchased from Broadpharm Inc. (San Diego, California, USA). Fmoc-PEG8-OH was purchased from Quanta Biodesign (Plain City, Ohio, USA). DM1-SMCC was purchased from eNovation Chemicals LLC. (Bridgewater Township, New Jersey, USA). DBCO-C6-NHS was purchased from Lumiprobe (Hong Kong). All amino acids, Rink amide resins, and coupling reagents were purchased from AAPPtec LLC. (Louisville, Kentucky, USA), GL Biochem LTD. (Shanghai, China), and Combi-Blocks Inc. (San Diego, California, USA). Solvents were used without distillation. Trifluoroacetic acid (TFA), N,N-diisopropylethylamine (DIPEA), ammonium hydroxide (ammonia solution), diethylamine, Na2HPO4, N,N-dimethylformamide (DMF), dichloromethane (DCM), methanol (MeOH), hexane (Hex), ethyl acetate (EA), and diethyl ether were obtained from Daejung Chemicals & Metals CO., LTD. (Siheung, South Korea). High-performance liquid chromatography (HPLC) grade acetonitrile (ACN), isopropanol, and water were purchased from Thermo Fisher Scientific Inc. (Waltham, Massachusetts, USA).

[0305] Antibodies and ADCs

[0306] Two types of anti-claudin 18.2 antibodies (anti-CLDN18.2 antibodies) were prepared (hereinafter referred to as antibody-A and antibody-B).

[0307] Antibody-A is an antibody comprising a heavy chain having the amino acid sequence of SEQ ID NO: 16 and a light chain having the amino acid sequence of SEQ ID NO: 17 (in this respect, as in the structure of a commonly known antibody, antibody-A has two light chains and two heavy chains). Information regarding the antibody is disclosed in the literature [Korean Patent Application No. 10-2021-7023724 (Publication No. 10-2021-0110339)]. Antibody-A is obtained from GenScript ProBio Inc., and a method for preparing antibody-A is described in detail in "Example 3" of the literature [Korean Patent Application No. 10-2021-7023724 (Publication No. 10-2021-0110339)], which is incorporated herein by reference.

[0308] Antibody-B is a known antibody (solbetuximab antibody) and was purchased from Biointron.

[0309] Antibody-C, human IgG antibody (Sigma-Aldrich, I4506), was purchased from Sigma-Aldrich CO., LTD. (St. Louis, Missouri, USA).

[0310] ADC-A is antibody-A conjugated with MMAE and was produced by the processes of Example 1 and Example 2.

[0311] ADC-B is antibody-B conjugated with MMAE, and its production was ordered from Abzena. The method for producing ADC-B was carried out by referring to the information disclosed in the literature [US2018 / 0117174 (application number US15 / 565,848)].

[0312] ADC-C was produced by conjugating MMAE to antibody-C using the processes of Example 1 and Example 2. The only difference is that in Example 2, antibody-C was used, not antibody-A.

[0313] Cells and plasma

[0314] The MIA PaCa-2~CLDN18.2 cell line (C3002) was purchased from Accurus Biosciences Inc. The PATU8988S cell line (ACC204) was purchased from German Collection of Microorganisms and Cell Cultures GmbH (DSMZ). The MIA PaCa-2 (CLDN18.2-) cell line (CRL-1420) was purchased from American Type Culture Collection (ATCC). The SNU601 cell line (00601) was purchased from Korean Cell Line Bank (KCLB). The NUGC-4 cell line (ABC-TC0862) was purchased from AcceGen. The AGS cell line (21739) was purchased from KCLB.

[0315] Human plasma was purchased from BIO IVT. Monkey plasma was purchased from GENIA. Rat plasma was purchased from QuBest Bio CO.,LTD. Mouse plasma was purchased from QuBest Bio CO.,LTD.

[0316] Equipment and conditions

[0317] device

[0318] All peptides and compounds, including the payload, were characterized using HPLC (Waters, XBridge®, C18, 4.6 × 250 mm, 5 μm).

[0319] For HPLC, we used the HPLC Alliance system manufactured by Waters (2996 PAD detector and 2695 separation module).

[0320] For the analysis of all low molecular weight substances, including peptides and compounds, an LC / MS system equipped with a Quatro Premier XE instrument manufactured by Waters and an Acquity Waters LC system was used as the mass spectrometer.

[0321] All antibodies, including those containing antibody conjugates (e.g., ADCs), were characterized using hydrophobic interaction chromatography (HIC)-HPLC (Thermo Fisher Scientific, MAbPac®, HIC butyl, 4.6 × 100 mm, 5 μm), size exclusion chromatography (SEC)-HPLC (Thermo Fisher Scientific, MAbPac®, SEC-1, 300 Å 4 × 300 mm, 5 μm), and UV spectrophotometer (Thermo Fisher Spectrophotometer, using MULTISKAN GO / MicroDrop® plate, N12391).

[0322] A Fleta4 centrifuge (Hanil, South Korea) was used for centrifugation in the spin desalination process.

[0323] In all enzyme-linked immunosorbent assay (ELISA) experiments, OD values ​​were measured using a UV-Vis spectrometer (Thermo Fisher, 4661030N).

[0324] In cytotoxicity experiments, cell viability was measured using GloMax® Discover (Promega, GM3000).

[0325] For real-time internalization analysis, we used the S3 Live Cell Analysis Instrument (SARTORIUS, IncuCyte® S3).

[0326] A Smart R17 Plus (Hanil, SM-R17PL) was used as the centrifugal separator.

[0327] Characterization of peptides and compounds using C18-HPLC

[0328] C18-HPLC was performed at a flow rate of 1 mL / min. All compounds were analyzed using the same elution conditions [initial 80% mobile phase A (0.1% TFA in H2O) for 1 minute, followed by a 20-80% gradient mobile phase B (0.075% TFA in ACN) in phase A for 15 minutes]. Chromatograms of peptides and compounds were acquired at 280 nm, and chromatograms of the payload were acquired at 254 nm.

[0329] Antibody characterization using HIC-HPLC

[0330] HIC-HPLC was performed at a flow rate of 1 mL / min. HIC-HPLC was performed for all antibodies under the same conditions [1 minute, initial 100% mobile phase A (1.5 M (NH4)2SO4, 50 mM Na2HPO4, 5% isopropanol) at pH 7.0, followed by 15 minutes, 0-100% gradient mobile phase B (50 mM sodium phosphate, 20% isopropanol) in A]. All chromatograms were acquired at 280 nm.

[0331] Characterization of antibodies and other substances using SEC-HPLC

[0332] SEC-HPLC was performed at a flow rate of 0.2 mL / min. All antibodies were analyzed under the same conditions [homogeneous solvent mobile phase D (1×PBS) for 20 minutes]. All chromatograms were acquired at 280 nm.

[0333] Measurement of optical density (OD) values ​​in ELISA experiments using a UV-Vis spectrometer.

[0334] OD values ​​were measured by ELISA by measuring absorbance at 450 nm using a UV-Vis spectrometer.

[0335] Measuring cell viability to confirm cytotoxicity

[0336] To measure cell viability in cytotoxicity experiments, luminescence measurements using the GloMax® Discover were employed, and the measurement method followed the instrument's instruction manual.

[0337] Real-time internalization analysis

[0338] Real-time internalization measurements were performed according to the instructions for use of reagents and equipment.

[0339] Process for pre-treating plasma and PK samples using centrifugation.

[0340] Centrifugation was performed in the pretreatment process at 13,000 rpm for 10 minutes at 4°C.

[0341] Example 1. Preparation of Fc-binding substances and compounds, etc.

[0342] Example 1.1 Preparation of Fc-binding material

[0343] The inventors prepared an Fc-binding substance represented by FcBP(Orn) as described below, with reference to the information disclosed in the document [WO2020 / 184944 (Application No. PCT / KR2020 / 003282)]. FcBP(Orn) can be represented by Ac-PEG8-DCAWHOrnGELVWCT-NH2, whose structure is as follows: [ka] That is correct.

[0344] The inventors prepared FcBP(Orn) using solid-phase peptide synthesis (SPPS).

[0345] [ka] [ka]

[0346] Example 1.1.1 List of Fmoc amino acids used and order of introduction

[0347] Fmoc-L-Thr(tBu)-OH, Fmoc-Cys(Trt)-OH, Fmoc-L-Trp(Boc)-OH, Fmoc-L-Val-OH, Fmoc-L-Leu-OH, F moc-L-Glu(OtBu)-OH, Fmoc-Gly-OH, Fmoc-Orn(Boc)-OH, Fmoc-L-His(Trt)-OH, Fmoc-L-Trp(Boc)-OH Fmoc-Ala-OH, Fmoc-Cys(Trt)-OH, and Fmoc-Asp(tBu)-OH.

[0348] A specific preparation method is described in detail in the document [WO2020 / 184944 (Application No. PCT / KR2020 / 003282)], which is incorporated herein by reference.

[0349] Example 1.1.2 Introduction of amino acids

[0350] The amounts of reagents used in the following process are based on 0.25 mmol. 0.5 g of Rink 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 the C-terminus to the N-terminus.

[0351] The reaction to activate the Fmoc-amino acid and the reaction to attach the activated residue to the CLEAR amide resin were carried out sequentially starting from the C-terminal amino acid.

[0352] Fmoc removal was performed in 20% piperidine-containing DMF. For residue activation and introduction, amino acids prepared according to the sequence were mixed for 5 minutes with 2 mL of 0.5 M hydroxybenzotriazole (HOBt)-containing DMF solution, 2 mL of 0.5 M hexafluorophosphorate azabenzotriazole tetramethyluronium (HBTU)-containing DMF solution, and 174 μL of DIPEA. The resulting mixture was then poured into a reactor containing resin and mixed for 2 hours.

[0353] The introduction reaction was confirmed by a Kaiser test, and if unreacted amines were found to be present, the introduction reaction was either repeated or capping was performed using a 20% Ac2O-containing DMF solution. In each introduction reaction and Fmoc removal process, the resin was thoroughly washed with DMF and DCM before proceeding to the next step. This process was repeated until the target peptide sequence was complete.

[0354] Example 1.1.3 Introduction of H-PEG8-OH

[0355] After all amino acids had been introduced, in order to introduce H-PEG8-OH at the N-terminus, 1 mL of 0.5 M Fmoc-N-amide-dPEG8-acid in DMF solution, 1 mL of 0.5 M HBTU-containing DMF solution, 1 mL of 0.5 M HOBt-containing DMF solution, and 87 μL of DIPEA were mixed for 5 minutes, and then the resulting mixture was poured into a reactor containing resin and mixed for 2 hours.

[0356] The reaction was confirmed by a Kaiser test, and if unreacted amines were found to be present, the reaction time was extended by another 1-3 hours, or the reaction solution was emptied and the reaction process described above was repeated. After removing the N-terminal Fmoc protecting group using 20% ​​piperidine-containing DMF, the resin was thoroughly washed with a solution of DMF and DCM, and then capping was performed using a 20% Ac2O-containing DMF solution. After capping, the resin with the attached peptides was dried with a solution of DCM and diethyl ether and weighed.

[0357] 250 mg of resin to which the peptide prepared during the amino acid introduction process was attached was stirred with 2 mL of a mixed solution of TFA, TIS, water, and EDT (94:1.0:2.5:2.5) at room temperature for 120 minutes to cleave the peptide from the resin. The cleaved mixture was filtered, and the resulting filtrate was concentrated to about half using nitrogen gas, and then diethyl ether was added to precipitate the peptide. The precipitated peptide was further washed three times with diethyl ether and dried using nitrogen gas. The dried precipitate was dissolved in a 0.1% TFA-30% ACN aqueous solution, and the resulting solution was stirred for 6 hours and then concentrated.

[0358] The concentrate was dissolved at a concentration of 0.1 mg / mL in a 0.01 M ammonium acetate buffer (pH 6.5) solution containing 5% DMSO and 20% ACN. The resulting solution was then stirred for 3 days while exposed to air. The progress of the disulfide bond formation reaction was observed by HPLC. If it was determined that the reaction did not proceed further, the reaction solution was freeze-dried to obtain a peptide precipitate.

[0359] Example 1.1.4 Purification

[0360] The peptide precipitate obtained by lyophilization in the H-PEG8-OH introduction process described above was purified by prep-LC and then lyophilized. Analytical HPLC confirmed that the obtained peptide had a purity of 90% or higher, and the molecular weight of the synthesized peptide was confirmed by LC / MS.

[0361] -LC / MS analysis results: [M / 3+H]=666.26;[M / 2+H]=999.38 -HPLC analysis result: 9.004 minutes, purity: 99.9%

[0362] Example 1.2 Preparation of Compound 1

[0363] The inventors prepared compound 1 below using the obtained peptide.

[0364] (Compound 1)

[0365] [ka]

[0366] In this case, FcBU is PEG8-DCAWHOrn'GELVWCT-NH2 derived from FcBP(Orn), and Orn' is conjugated ornithine.

[0367] Specifically, the structure of compound 1 is as follows: [ka] That is correct.

[0368] (Exact mass: 2265.0082)

[0369] The preparation process for compound 1 follows the reaction scheme below: [ka] See the reference for further explanation.

[0370] 5-Ethoxy-5-oxopentanoic acid (10 g, 68.4 mmol) was dissolved in 240 mL of dichloromethane and 9.6 mL of N,N-dimethylformamide. Then, O-benzylhydroxylamine (6.5 g, 52.7 mmol), hexafluorophosphorate azabenzotriazoletetramethyluronium (HATU) (23.7 g, 62.3 mmol), and DIPEA (17.6 mL, 100.6 mmol) were added, and the resulting mixture was stirred at room temperature for 18 hours. Ethyl acetate (RINKAN) and distilled water were added, and the organic layer was extracted. The recovered organic layer was dried on anhydrous magnesium sulfate. After filtration, the organic layer was concentrated and purified by column chromatography to obtain compound 1-1 (11.4 g, 95%).

[0371] Compound 1-1 (5.4 g, 21.5 mmol) was dissolved in 180 mL of N,N-dimethylformamide, then potassium carbonate (K2CO3, 6.5 g, 47.3 mmol) and iodomethane (5.4 mL, 86.0 mmol) were added, and the resulting mixture was stirred at room temperature for 3 hours. Ethyl acetate and distilled water were added, and the organic layer was extracted. The recovered organic layer was dried on anhydrous magnesium sulfate (MgSO4). After filtration, the organic layer was concentrated and purified by column chromatography to obtain compound 1-2 (5.0 g, 87.7%).

[0372] Compound 1-2 (1 g, 3.7 mmol) was dissolved in 24 mL of tetrahydrofuran, then lithium hydroxide (LiOH, 0.23 g in 8 mL of water) was added, and the resulting mixture was reacted at room temperature for 3 hours. Water and ethyl acetate were added, and the water was extracted. The aqueous layer was acidified to pH 3 using 4N HCl. Ethyl acetate was added, and the organic layer was extracted. The recovered organic layer was dried over magnesium sulfate (MgSO4). After filtration, the organic layer was concentrated and purified by column chromatography to obtain compound 1-3 (0.7 g, 74.7%).

[0373] Compound 1-3 (0.85 g, 3.4 mmol) was dissolved in 13 mL of methanol, then Pd / C (0.05 g) was added, and the resulting mixture was stirred under a hydrogen atmosphere. The reaction solution was filtered through Celite and concentrated under reduced pressure to obtain compound 1-4 (0.46 g, quantitative yield) without purification.

[0374] Compound 1-4 (1 g, 6.208 mmol) was dissolved in 4 mL of dichloromethane, then triethylamine (1.77 mL, 13.0368 mmol) and 2-(2-azidoethoxy)acetyl chloride* (1.13 g, 6.828 mmol) were added, and the resulting mixture was stirred at room temperature under a nitrogen atmosphere for 3 hours. After concentration, ethyl acetate and 10% citric acid solution were added, and the organic layer was extracted. After concentration, the organic layer was purified by column chromatography to obtain compound 1-5 (1.65 mg, 92.2%).

[0375] Compound 1-5 (200 mg, 0.694 mmol) was dissolved in 6.8 mL of dichloromethane, then triethylamine (0.1015 mL, 0.728 mmol) and N,N,N',N'-tetramethyl-O-(N-succinimidyl)uronium tetrafluoroborate (TSTU, 229.8 mg, 0.8634 mmol) were added, and the mixture was reacted under an argon atmosphere at room temperature for 3 hours. After concentration, the mixture was purified by column chromatography to obtain compound 1-6 (221.8 mg, 83%).

[0376] FcBP(Orn) (200 mg, 0.1002 mmol) was dissolved in 0.5 mL of N,N-dimethylformamide, then N,N-diisopropylethylamine (0.07 mL) and compound 1-6 (40.51 mg) were added. The resulting mixture was stirred at room temperature under an argon atmosphere for 2 hours. After concentration, the mixture was purified by reverse-phase column chromatography to obtain compound 1-7 (compound 1) (226.9 mg, 64.8%).

[0377] Subsequently, the obtained compound 1 was analyzed by mass spectrometry and HPLC, and the results are as follows: -LC / MS analysis results: [M / 2+H]=1134.35;[M+H]=2268.37 -HPLC analysis result: 13.19 minutes, purity 99.9% That is correct.

[0378] Example 1.3 Preparation of Compound 2 (Payload 1)

[0379] Example 1.3.1 Preparation of Compound 2-1

[0380] 2-chlorotrityl chloride resin (1.4 mmol / g, 1 g) (1 equiv) was mixed with 40 mL of dichloromethane, and the resulting mixture was stirred for longer than 30 minutes. The solution was then removed, and ethylenediamine (5.6 mmol, 4 equiv) and N,N'-diisopropylethylamine (5.6 mmol, 4 equiv) were mixed with the dichloromethane, and then mixed with the resin. After stirring at room temperature for longer than 2 hours, the reaction solution was removed. Compound 2-1 was prepared by washing the resin three or more times with sufficient amounts of dichloromethane and dimethylformamide, respectively, to remove any remaining reaction solution.

[0381] (Compound 2-1)

[0382] [ka]

[0383] Example 1.3.2 Preparation of Compound 2-2

[0384] N-alpha-fluorenylmethoxycarbonyl-N-epsilon-allyl-oxycarbonyl-l-lysine (Fmoc-Lys(alloc)-OH) (2.8 mmol, 2 equivs), N,N'-diisopropylcarbodiimide (2.8 mmol, 2 equivs), hydroxybenzotriazole (5.6 mmol, 4 equivs), and dimethylformamide were thoroughly mixed, and the resulting mixture was added to the resin. After stirring at room temperature for longer than 2 hours, the reaction solution was removed. To remove any remaining reaction solution, the resin was washed at least three times each with sufficient amounts of dichloromethane and dimethylformamide, thereby preparing compound 2-2. The preparation of compound 2-2 was confirmed by a Kaiser test.

[0385] (Compound 2-2)

[0386] [ka]

[0387] Example 1.3.3 Preparation of Compounds 2-3

[0388] A 20% piperidine solution was prepared by dissolving it in dimethylformamide, stirring at room temperature for 10 minutes, and the reaction solution was removed. The above process was repeated a total of two times. To remove any remaining reaction solution, the resin was washed at least three times each with sufficient amounts of dichloromethane and dimethylformamide, thereby preparing compounds 2 and 3.

[0389] (Compound 2-3)

[0390] [ka]

[0391] Example 1.3.4 Preparation of Compounds 2-4

[0392] Mono-tert-butyl succinate (2.8 mmol, 2 equivs), N,N'-diisopropylcarbodiimide (2.8 mmol, 2 equivs), hydroxybenzotriazole (5.6 mmol, 4 equivs), and dimethylformamide were thoroughly mixed, and the resulting mixture was added to the resin. After stirring at room temperature for longer than 2 hours, the reaction solution was removed. To remove any remaining reaction solution, the resin was washed at least three times each with sufficient amounts of dichloromethane and dimethylformamide, thereby preparing compounds 2-4.

[0393] (Compound 2-4)

[0394] [ka]

[0395] Example 1.3.5 Preparation of Compounds 2-5

[0396] The remaining resin was washed three or more times with a sufficient amount of dichloromethane. Tetrakis(triphenylphosphine palladium) (0.7 mmol, 0.5 equiv), 1,3-dimethylbarbitulic acid (14 mmol, 10 equiv), and dichloromethane were thoroughly mixed, and the resulting mixture was added to the resin. After stirring at room temperature for about 1 hour, the reaction solution was removed. To remove any remaining reaction solution, the resin was washed at least three times each with a sufficient amount of dichloromethane and dimethylformamide, thereby preparing compounds 2-5.

[0397] (Compound 2-5)

[0398] [ka]

[0399] Example 1.3.6 Preparation of Compounds 2-6

[0400] N-alpha-N-epsilon-bis(9-fluorenylmethyloxycarbonyl)-l-lysine (Fmoc-Lys(fmoc)-OH) (2.8 mmol, 2 equiv), N,N'-diisopropylcarbodiimide (2.8 mmol, 2 equiv), Hydroxybenzotriazole (5.6 mmol, 4 equiv), and Dimethylformamide was thoroughly mixed, and the resulting mixture was added to the resin. After stirring at room temperature for more than 2 hours, the reaction solution was removed. To remove any remaining reaction solution, the resin was washed at least three times each with sufficient amounts of dichloromethane and dimethylformamide, thereby preparing compound 2-6. The preparation of compound 2-6 was confirmed by a Kaiser test.

[0401] (Compound 2-6)

[0402] [ka]

[0403] Example 1.3.7 Preparation of Compounds 2-7

[0404] A 20% piperidine solution was prepared by dissolving it in dimethylformamide, stirring at room temperature for 10 minutes, and the reaction solution was removed. The above process was repeated a total of two times. To remove any remaining reaction solution, the resin was washed at least three times each with sufficient amounts of dichloromethane and dimethylformamide, thereby preparing compounds 2-7.

[0405] (Compound 2-7)

[0406] [ka]

[0407] Example 1.3.8 Preparation of Compounds 2-8

[0408] 2,5,8,11,14,17,20,23-Octaoxahacosacosan-26-Eucic Acid (m-PEG8 Acid) (2.8 mmol, equiv), N,N'-Diisopropylcarbodiimide (2.8 mmol, 2 equiv), Hydroxybenzotriazole (5.6 mmol, 4 equiv), and dimethylformamide were thoroughly mixed, and the resulting mixture was added to the resin. After stirring at room temperature for longer than 2 hours, the reaction solution was removed. To remove any remaining reaction solution, the resin was washed at least three times each with sufficient amounts of dichloromethane and dimethylformamide, thereby preparing compound 2-8.

[0409] (Compound 2-8)

[0410] [ka]

[0411] Example 1.3.9 Preparation of Compounds 2-9

[0412] A mixture of trifluoroacetic acid and distilled water in a 95:5 ratio was prepared, and a sufficient amount of this mixture was added to the resin. The resulting mixture was stirred at room temperature for 2 hours. After stirring, the solution and resin were separated. A sufficient amount of 0°C diethyl ether was added to the separated solution and mixed. The resulting mixture was left at 0°C for longer than 2 hours to ensure sufficient precipitation. Compound 2-9 was prepared by centrifugation and drying. The preparation of compound 2-9 was confirmed by mass and HPLC analysis ([M / 2+H]=603.38; [M+H]=1206.21 (accurate mass: 1204.72)).

[0413] (Compound 2-9)

[0414] [ka]

[0415] Example 1.3.10 Preparation of Compounds 2-10

[0416] 1 g (0.83 mmol) of the product obtained from the previous process was dissolved in dimethylformamide, and then DBCO-C6-NHS (0.995 mmol, 1.2 equiv) and N,N'-diisopropylethylamine (1.659 mmol, 2 equiv) were added. The resulting mixture was stirred at room temperature for more than 1 hour. After the reaction was complete, the reaction solution was purified by Prep-HPLC (C18) and lyophilized to obtain product (compound 2-10) (0.394 mmol, 47.5%). The preparation of compound 2-10 was confirmed by mass and HPLC analysis ([M / 2+H] = 761.09 (accurate mass: 1519.84)).

[0417] (Compound 2-10)

[0418] [ka]

[0419] Example 1.3.11 Preparation of Compound 2-11

[0420] The product obtained from the previous process (0.394 mmol) was dissolved in dimethylformamide, and then tetramethyl-O-(N-succinimidyl)uronium tetrafluoroborate (0.788 mmol, 2 equiv) and N,N'-diisopropylethylamine (0.788 mmol, 2 equiv) were added. The resulting mixture was stirred at room temperature for longer than 1 hour. After the reaction was complete, the reaction solution was purified by Prep-HPLC (C18) and lyophilized to obtain the product (compound 2-11) (0.138 mmol, 35%). The preparation of compound 2-11 was confirmed by mass and HPLC analysis ([M / 2+H] = 809.67 (accurate mass: 1616.86)).

[0421] (Compound 2-11)

[0422] [ka]

[0423] Example 1.3.12 Preparation of Compound 2-12 (Compound 2, Payload 1)

[0424] The product obtained from the previous process (0.138 mmol) was dissolved in dimethylformamide, and then the pre-prepared H2N-BG-MMAE (0.152 mmol, 1.1 equiv) and N,N'-diisopropylethylamine (0.276 mmol, 2 equiv) were added. The resulting mixture was stirred at room temperature for longer than 1 hour. After the reaction was complete, the reaction solution was purified by Prep-HPLC (C18) and lyophilized to obtain the product (compound 2, payload 1) (0.065 mmol, 47.5%).

[0425] (Compound 2, Payload 1)

[0426] [ka]

[0427] Subsequently, the obtained compound 2 was analyzed by mass spectrometry and HPLC, and the results are as follows: -LC / MS analysis result: [M / 2+H]=1318.06;[M+H]=2635.38 (Accurate mass: 2631.45) -HPLC analysis result: 16.721 minutes, purity 100% That is correct.

[0428] Example 2. Preparation of ADC-A

[0429] The inventors prepared ADC-A by conjugating a drug (MMAE) with an anti-claudin 18.2 antibody (antibody-A). The structure of ADC-A is shown in Figure 27.

[0430] In this configuration, ADC-A has an antibody containing two heavy chains, with the payload ligated to lysine residue 246 or 248 of one of the heavy chains, and the other payload ligated to lysine residue 246 or 248 of the other heavy chain. The structure of the payloads ligated to each heavy chain is identical.

[0431] Specifically, Figure 28 shows a structure in which the payload is linked to one lysine residue 246 or 248 of the antibody heavy chain in ADC-A.

[0432] The preparation process for ADC-A is described with reference to the reaction process shown in Figure 29.

[0433] Example 2.1 Preparation of Compound 3

[0434] The inventors prepared a conjugate (compound 3) in which azide is linked to antibody-A.

[0435] (Compound 3)

[0436] [ka]

[0437] The specific method is as follows:

[0438] Compound 1 was prepared in DMSO solvent at a concentration of 10 mM. The prepared compound 1 (277.8 μL, 2,778 nmol, 4.0 equiv) was mixed with 101.4 mg of antibody-A (146 kDa, 5.2 mg / mL, 19.5 mL, 694.5 nmol). The resulting mixture was stirred under 1×PBS (pH 7.4) and the reaction was carried out for 3 hours. The final DMSO product was fixed with 10%. The reaction (crosslinking of compound 1 with antibody-A) was monitored by HIC-HPLC analysis, and the monitoring results are shown in Figures 30 and 31.

[0439] After the reaction was complete, compound 1, which did not react with antibody-A, was removed using spin desalting (Zeba® spin desalting column, 40K molecular weight cutoff, 10 mL, 1000 gravitational acceleration) and SEC. As a result, compound 3 was obtained.

[0440] Example 2.2 Preparation of ADC-A

[0441] The inventors used the obtained compounds 2 and 3 to prepare a conjugate (ADC-A) in which the payload of compound 2 was linked to antibody-A. The specific method is as follows.

[0442] Compound 2 was prepared in DMSO solvent at a concentration of 10 mM. The prepared compound 2 (416.7 μL, 4,167 nmol, 6 equiv) was mixed with 101.4 mg of compound 3 (146 kDa, 5.2 mg / mL, 19.5 mL, 694.5 nmol). The resulting mixture was mixed under 1×PBS (pH 7.4) and incubated at 25°C for 24 hours. The reaction was monitored by HIC-HPLC, and the monitoring results are shown in Figure 32.

[0443] After the reaction was complete, compound 2, which did not react with compound 3, was removed using spin desalting (Zeba® spin desalting column, 40K molecular weight cutoff, 10 mL, 1000 gravitational acceleration) and dialysis (pH 7.4, 1 × PBS, 4 hours, 4 hours, 12 hours, 3 times). As a result, crude ADC-A(DAR2) was obtained.

[0444] Example 3. Verification of the effectiveness of ADC-A.

[0445] Example 3.1 Verification of Target Specificity 1 - Confirmation of Binding to Antigen-Expressing Cells

[0446] The inventors conducted experiments according to Example 3.1.1 to confirm whether antibody-A and ADC-A can specifically bind to claudin 18.2 protein (CLDN18.2), and the results are described in Example 3.1.2.

[0447] Example 3.1.1 Experimental Method

[0448] We prepared CHO-K1 cell lines transiently transfected with the gene encoding claudin 18.1 protein (CLDN18.1) (sequence number: 18) (hereinafter referred to as "Claudin 18.1CHO-K1"), CHO-K1 cell lines transiently transfected with the gene encoding claudin 18.2 protein (CLDN18.2) (sequence number: 19) (hereinafter referred to as "Claudin 18.2CHO-K1"), and CHO-K1 cell lines transiently transfected with only a MOCK vector (empty vector) (hereinafter referred to as "MOCK CHO-K1"). In this process, we used the pcDNA3.1 vector for claudin 18.1CHO-K1 and the pcDNA3.1(+) vector for claudin 18.2CHO-K1.

[0449] The CHO-K1 cell line was prepared using the following process.

[0450] CHO-K1 cell lines were cultured in an incubator at 37°C and 5% CO2 in RPMI1640 containing 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin.

[0451] Transient transfection was performed according to the TransIT2020 instruction manual, and the transfected cells were spread into 96-well plates for culture and then treated with antibody-A and ADC-A, respectively. Subsequently, cell-based enzyme-linked immunosorbent assay (ELISA) was performed and the optical density (OD) values ​​were measured. The measured values ​​are shown in Figures 1-3.

[0452] The measurement results were used for regression analysis using GraphPad PRISM® 9.0 software to calculate EC50 and Bmax. Based on the calculated values, the cell binding affinity of antibody-A and ADC-A was evaluated. The calculated values ​​are listed in Table 1.

[0453] Example 3.1.2 Experimental Results

[0454] Figures 1-3 confirm that antibody-A and ADC-A do not bind to the negative control MOCK CHO-K1 cell line or the claudin 18.1CHO-K1 cell line, but specifically bind only to the claudin 18.2CHO-K1 cell line expressing CLDN18.2.

[0455] Table 1 confirms that the EC50 and maximum binding amount (Bmax), which represent maximum binding affinity, of ADC-A are almost the same as those of antibody-A. In other words, although the linker and payload are conjugated to antibody-A, the binding affinity of the antibody to the antigen was not significantly affected.

[0456] [Table 1] [Table 1]

[0457] Example 3.2. Verification of target specificity 2 - Confirmation of binding to antigen protein

[0458] The inventors conducted experiments according to Example 3.2.1 to confirm whether antibody-A and ADC-A can bind to claudin 18.2 protein (CLDN18.2), and the results are described in Example 3.2.2.

[0459] Example 3.2.1 Experimental Method

[0460] To confirm its binding activity to the antigen, CLDN18.2-VLPs (virus-like particles) expressing claudin 18.2 protein (CLDN18.2) were prepared. The CLDN18.2-VLP used was CSB-MP005498HU(A5) from CUSABIO TECHNOLOGY LLC.

[0461] For ELISA, 96-well plates were coated with CLDN18.2VLP and treated with antibody-A and ADC-A respectively. Indirect ELISA was then performed to measure the OD values. The measured values ​​are shown in Figure 4.

[0462] The measurement results were used for regression analysis in GraphPad PRISM® 9.0 software to calculate EC50 and Bmax. Based on the calculated values, the antigen-binding affinity of antibody-A, ADC-A, antibody-B, and ADC-B was evaluated. The calculated values ​​are listed in Table 2.

[0463] Example 3.2.2 Experimental Results

[0464] Figure 4 and Table 2 confirm that the measured binding affinity of each test substance to CLDN18.2VLP was highest in the order of antibody-B, ADC-B, ADC-A, and antibody-A. These results indicate that antibody-A and ADC-A have better binding affinity to the antigen than ADC-B and antibody-B.

[0465] [Table 2] [Table 2]

[0466] Example 3.3. Verification of target specificity 3 - Confirmation of binding to antigen-expressing cancer cells

[0467] The inventors conducted experiments according to Example 3.3.1 to confirm whether antibody-A and ADC-A can bind to cancer cells expressing claudin 18.2 protein (CLDN18.2-positive cancer cells), and the results are described in Example 3.3.2.

[0468] Example 3.3.1 Experimental Method

[0469] Four types of cell lines were prepared: MIA PaCa-2~CLDN18.2, PATU8988S, parent MIA PaCa-2(CLDN18.2-), and SNU601. MIA PaCa-2~CLDN18.2 and PATU8988S are CLDN18.2-positive pancreatic cancer cell lines. MIA PaCa-2(CLDN18.2-) is a CLDN18.2-negative pancreatic cancer cell line. SNU601 is a CLDN18.2-positive gastric cancer cell line.

[0470] The prepared cell lines were cultured in an incubator at 37°C and 5% CO2 in DMEM (MIA PaCa-2~CLDN18.2, MIA PaCa-2(CLDN18.2-)) containing 10% FBS and 1% penicillin / streptomycin, RPMI1640 (SNU601) containing 10% FBS and 1% penicillin / streptomycin, and DMEM (PATU8988S) containing 5% FBS, 5% serum, 1% penicillin / streptomycin, and 2 mM L-glutamine.

[0471] The prepared cell lines were spread onto 96-well culture plates and then treated with antibody-A, ADC-A, antibody-B, and ADC-B, respectively. Cell-based ELISA was then performed, and the OD values ​​were measured. The measured values ​​are shown in Figures 5-8.

[0472] The measurement results were used for regression analysis in GraphPad PRISM® 9.0 software to calculate EC50 and Bmax. Based on the calculated values, the cell binding affinity of antibody-A, ADC-A, antibody-B, and ADC-B to each cell line was evaluated. The calculated values ​​are listed in Table 3.

[0473] Example 3.3.2 Experimental Results

[0474] Figures 5-8 and Table 3 confirm that the measured binding affinity to pancreatic cancer cell lines and gastric cancer cell lines increased in the order of antibody-B, ADC-B, ADC-A, and antibody-A.

[0475] In addition, binding affinity was confirmed to be higher for all test substances (antibody-A, ADC-A, ADC-B, and antibody-B) in the order of PATU8988S, SNU601, and MIA PaCa-2 to CLDN18.2, and none of the test substances were confirmed to bind to the CLDN18.2-negative cell line, parent MIA PaCa-2. These experimental results mean that the expression level of CLDN18.2 is higher in the order of PATU8988S, SNU601, and MIA PaCa-2 to CLDN18.2.

[0476] [Table 3] [Table 3]

[0477] Example 3.4 Verification of intracellular relocation

[0478] The inventors conducted an experiment according to Example 3.4.1 to confirm the intracellular localization effect of ADC-A, and the results are as described in Example 3.4.2.

[0479] Example 3.4.1 Experimental Method

[0480] To confirm the intracellular uptake (internalization) of antibody-A and ADC-A depending on the presence or absence of CLDN18.2 expression, MIA PaCa (CLDN18.2-) cell lines that do not express CLDN18.2, and MIA PaCa-2~CLDN18.2 and SNU601 cell lines that express CLDN18.2 were prepared.

[0481] The prepared cell lines were cultured in an incubator at 37°C and 5% CO2 in DMEM (MIA PaCa-2~CLDN18.2, MIA PaCa-2(CLDN18.2-)) containing 10% FBS and 1% penicillin / streptomycin, and in RPMI1640 (SNU601) containing 10% FBS and 1% penicillin / streptomycin. MIA PaCa(CLDN18.2-), MIA PaCa-2~CLDN18.2, and SNU601 cell lines were plated in 96-well plates at concentrations of 3,000 to 5,000 cells / well.

[0482] Samples for verifying the internalization of antibody-A and ADC-A were prepared according to the instructions for the IncuCyte® Fabfluor-pH reagent.

[0483] Finally, antibody-A and ADC-A were diluted to 8, 4, and 2 μg / mL, respectively, and 50 μL of each was used to treat the specified wells. The internalization level of the plate mixed with the sample was measured hourly using the IncuCyte® Live-Cell Analysis System. The measured values ​​are shown in Figures 9-11.

[0484] Example 3.4.2 Experimental Results

[0485] Figures 9-11 confirm that antibody-A and ADC-A were rapidly internalized within 20 hours in MIA PaCa-2 to CLDN18.2 and SNU601 cell lines expressing CLDN18.2. On the other hand, antibody-A and ADC-A were not internalized in MIA PaCa-2 (CLDN18.2-) cells that do not express CLDN18.2. In addition, antibody-A and ADC-A showed similar internalization efficiency at almost all concentrations. These results confirm that the linker and drug (payload) constituting ADC-A do not affect intracellular internalization compared to antibody-A, and that internalization is determined by the expression of the antigen CLDN18.2 in the cell line.

[0486] Example 3.5 Cytotoxicity Evaluation

[0487] The inventors conducted experiments according to Example 3.5.1 to evaluate the cytotoxicity of ADC-A, and the results are described in Example 3.5.2.

[0488] Example 3.5.1 Experimental Method

[0489] Four types of cell lines were prepared: MIA PaCa-2~CLDN18.2, PATU8988S, parent MIA PaCa-2(CLDN18.2-), SNU601, NUGC-4, and AGS. MIA PaCa-2~CLDN18.2 and PATU8988S are CLDN18.2-positive pancreatic cancer cell lines. MIA PaCa-2(CLDN18.2-) is a CLDN18.2-negative pancreatic cancer cell line. SNU601 and NUGC-4 are CLDN18.2-positive gastric cancer cell lines. AGS is a CLDN18.2-negative gastric cancer cell line.

[0490] The prepared cell lines were cultured in an incubator at 37°C and 5% CO2 in DMEM containing 10% FBS and 1% penicillin / streptomycin (MIA PaCa-2~CLDN18.2, MIA PaCa-2(CLDN18.2-)), RPMI1640 containing 10% FBS and 1% penicillin / streptomycin (SNU601, NUGC-4, and AGS), and DMEM containing 5% FBS, 5% horse serum, 1% penicillin / streptomycin, and 2 mL of L-glutamine (PATU8988S).

[0491] The prepared cell lines were spread into 96-well culture plates at concentrations of 500 to 5,000 cells / well, and then treated with each test substance (antibody-A, ADC-C, ADC-A, antibody-B, ADC-C, and a combination of antibody-A and MMAE) that had been serially diluted 10-fold (10,000, 1,000, 10, 1, 0.1, 0.01, 0.001, and 0 ng / mL). In this case, the combination of antibody-A and MMAE means that the cells were treated with a combination of antibody-A and MMAE, and the concentration of MMAE was the same molar concentration as the MMAE contained in other ADCs (e.g., ADC-A).

[0492] The change in cell viability following treatment with each test substance was measured using the same method as in the CellTiter-Glo instruction manual. The measured values ​​are shown in Figures 12-17.

[0493] The measurement results were used for regression analysis in GraphPad PRISM® 9.0 software to calculate the IC50. Based on the calculated values, the cytotoxicity of each test substance was evaluated. The calculated values ​​are listed in Table 4.

[0494] Example 3.5.2 Experimental Results

[0495] Figures 12-17 and Table 4 confirm that ADC-A exhibits anticancer activity in CLDN18.2-positive cancer cell lines, regardless of cancer type. In addition, the IC50 value of ADC-A was confirmed to be 10 to 35 times lower than that of the control ADC-B. In particular, in MIA PaCa-2~CLDN18.2 cell lines, ADC-A showed a lower IC50 than that of the antibody-A and MMAE co-administration group, and in PATU8988S and SNU601, it showed an IC50 of 1 nM or lower. Furthermore, in CLDN18.2-negative cell lines, ADC-A was confirmed to have almost no toxicity up to the highest treatment concentration, while the control group ADC-B showed toxicity.

[0496] [Table 4] [Table 4]

[0497] Example 3.6 Plasma Stability Evaluation

[0498] The inventors conducted experiments according to Example 3.6.1 to evaluate the plasma stability of ADC-A, and the results are as described in Example 3.6.2.

[0499] Example 3.6.1 Experimental Method

[0500] Human, monkey, rat, and mouse plasma was prepared. The prepared plasma was treated with each of the test substances (antibody-A, ADC-A, and ADC-B), and then incubated at 37°C for 5 minutes, 1 day, 5 days, 8 days, 12 days, and 15 days (a total of 108 samples were prepared).

[0501] Centrifugation was used at each time point to extract only the supernatant from the prepared samples, which was then used for analysis. ELISA analysis confirmed the plasma stability of the test substance over time. ELISA plates were coated with CLDN18.2VLP and treated with 5000-fold diluted plasma, followed by treatment with antibody-A, ADC-A, and ADC-B, respectively. The total amount of antibody (total Ab) was analyzed using anti-human IgG antibody (Promega, W4031), and the total amount of ADC was analyzed using anti-MMAE antibody (antibody produced in-house by ABFRONTIER). Total Ab included not only antibodies without linker conjugates, such as antibody-A, but also substances with linker conjugates, such as ADC-A. Total ADC included only substances with linker conjugates, such as ADC-A or ADC-B, and did not include antibodies without linker conjugates. For example, if the linker in ADC-A is cleaved, it is not included in total ADC.

[0502] The total amount of antibody or total ADC over time was converted to a relative percentage based on the remaining amount at day 0 (5 minutes). The conversion results are shown in Figures 18-21.

[0503] Example 3.6.2 Experimental Results

[0504] Figures 18-21 show the following results.

[0505] In human plasma over 15 days, total antibodies against antibody-A were maintained at 85.2%, total antibodies against ADC-A were maintained at 74.8%, total ADCs against ADC-A were maintained at 75.1%, total antibodies against ADC-B were maintained at 36.9%, and total ADCs against ADC-B were maintained at 16.6%.

[0506] In monkey plasma over 15 days, total antibodies against antibody-A were maintained at 97.3%, total antibodies against ADC-A were maintained at 90.3%, total ADCs against ADC-A were maintained at 77.5%, total antibodies against ADC-B were maintained at 24.3%, and total ADCs against ADC-B were maintained at 7.4%.

[0507] In rat plasma over 15 days, total antibodies against antibody-A were maintained at 68.2%, total antibodies against ADC-A were maintained at 64.0%, total ADCs against ADC-A were maintained at 50.3%, total antibodies against ADC-B were maintained at 17.8%, and total ADCs against ADC-B were maintained at 15.3%.

[0508] In mouse plasma over 15 days, total antibodies against antibody-A were maintained at 72.0%, total antibodies against ADC-A were maintained at 78.7%, total ADCs against ADC-A were maintained at 77.0%, total antibodies against ADC-B were maintained at 25.5%, and total ADCs against ADC-B were maintained at 17.7%.

[0509] The stability of antibody-A and ADC-A in plasma showed similar levels and trends across all plasma types over 15 days. In contrast, the total antibody and total ADCs related to ADC-B showed a pattern of rapid decrease over time. Since ADC-A showed the same values ​​over time as antibody-A in plasma, it can be interpreted that aggregation or degradation caused by the linker and drug conjugated to the antibody does not occur in ADC-A. Therefore, it can be concluded that ADC-A exhibits stability equivalent to that of the antibody in in vitro plasma, and that the linker and drug conjugated to the antibody in ADC-A do not affect the stability of the ADC.

[0510] Example 3.7 Evaluation of in vivo drug efficacy

[0511] The inventors conducted experiments according to Examples 3.7.1 to 3.7.4 to evaluate the in vivo efficacy of ADC-A, and the results are described in Example 3.7.5.

[0512] Example 3.7.1 Cell Culture

[0513] SNU601 cells, a CLDN18.2-positive gastric cancer cell line, were cultured in an incubator at 37°C and 5% CO2 in an RPMI culture medium containing 10% FBS and 1% penicillin / streptomycin. The SNU601 cells were regularly subcultured once a week by treatment with trypsin-ethylenediaminetetraacetic acid (EDTA).

[0514] Example 3.7.2 Tumor Inoculation and Drug Administration

[0515] For tumor development, the SNU601 cell line (1 × 10 7 The mixture was mixed with serum-free medium and Matrigel, and the resulting mixture was subcutaneously inoculated into the left flank of each BALB / c nude mouse (purchased from Charles River Laboratories Japan Inc.). The mice were divided into eight groups (G1-G8), with an average tumor volume of approximately 155 mm² in each group. 3 Participants were randomly assigned based on reaching a certain threshold. Subsequently, each group was administered the drug as shown in Table 5.

[0516] [Table 5] [Table 5]

[0517] Example 3.7.3 Observation and Measurement of Tumors

[0518] All animals were observed twice daily for mortality, abnormal symptoms, pain, and signs of stress, and once daily for clinical signs. Changes in body weight and tumor volume were measured three times a week, and tumor weight was determined after autopsy.

[0519] Tumor volume, tumor differences, and inhibition of tumor growth are as follows: Tumor volume (mm 3 ) = (length of major axis × length of minor axis) 2 ) / 2 Tumor difference, % = ((tumor weight) 試験群 - Tumor weight 対照(G1) ) / tumor weight 対照(G1) ) × 100 Tumor growth inhibition (%)=100-((T f / T i ) 試験群 ) / (((T f / T i )) 対照 ) × 100 The calculation was performed as follows.

[0520] T f This is the final tumor volume measured before autopsy, and T i This is the tumor volume measured initially.

[0521] The tumor volume measured at this time is shown in Figure 22. The measured body weight is shown in Figure 23. The shape of the tumor after autopsy is as shown in Figure 24. The tumor weight determined after autopsy is shown in Figure 25.

[0522] Example 3.7.4 Statistical Analysis

[0523] Numerical data related to the survey were calculated as mean and standard deviation. Bartlett's test was used to compare group variances for each parameter at a significance level of 0.05. If the difference between group variances was not significant, a parametric one-way analysis of variance (ANOVA) was performed. If a significant difference between means was expressed by ANOVA (p ≤ 0.05), Dunnett's test was used to compare the group means between the control and each treatment group. Whenever Bartlett's test showed heterogeneous group variances (p ≤ 0.05), a non-parametric Kruskal-Wallis test was used to compare all examined groups. If significance was found by the Kruskal-Wallis test (p ≤ 0.05), Dunn's test was used to assess the significance of the difference between the control and each treatment group. Significance was reported at levels of 0.05, 0.01, and 0.001 for each group comparison. All statistical analyses were performed using GraphPad PRISM® Version 5.0.

[0524] Example 3.7.5 Experimental Results

[0525] No unplanned deaths were observed during the clinical trial period.

[0526] During the clinical trial period, the test substances (antibodies-A, ADC-A, and ADC-C) had no effect on changes in body weight. The mean changes in body weight for each group are shown in Table 6 and Figure 23.

[0527] [Table 6] [Table 6]

[0528] On day 28, the average tumor volume in groups G7 and G8 was confirmed to be significantly lower than that in the control group. The average tumor volume and tumor growth inhibition for each group during the trial period are shown in Table 7 and Figure 22. The average tumor weight after autopsy for each group is shown in Table 8 and Figure 25.

[0529] [Table 7] [Table 7]

[0530] Significance was calculated using the Kruskal-Wallis test and the Dunn multiple comparison test.

[0531] [Table 8] [Table 8]

[0532] Significance was calculated using the Kruskal-Wallis test and the Dunn multiple comparison test.

[0533] In this example, a SNU601 gastric cancer tumor model was created using BALB / c nude mice, and the anticancer efficacy of antibody-A, ADC-A, and ADC-C was evaluated. In all ADC-A treatment groups, the measured tumor volume was lower than that of the control, and the measured tumor growth inhibition (TGI) ranged from 60.1% to 98.3%. In particular, when ADC-A was administered at 1.75 mg / kg (G7), the TGI was 98.3%, indicating a very strong anticancer effect.

[0534] No unplanned deaths occurred in any of the animals, and no significant changes in body weight were observed. Histological gastric lesions associated with antibody-A and ADC-A administration were not observed, even in the highest concentration group (G8). In conclusion, ADC-A demonstrated high anticancer efficacy in a gastric cancer model without weight loss or histological gastric lesions.

[0535] Example 3.8 Pharmacokinetic evaluation

[0536] The inventors conducted experiments according to Example 3.8.1 to evaluate the pharmacokinetics of ADC-A, and the results are described in Example 3.8.2.

[0537] Example 3.8.1 Experimental Method

[0538] Rats (purchased from SAMTAKO Bio Korea CO.,LTD.) were administered a single dose of ADC-A at doses of 1 and 3 mg / kg via tail vein. Blood was collected and analyzed for a set period of time to compare and evaluate pharmacokinetic profiles. The supernatant was obtained from the plasma using a centrifuge, and the concentration of ADC-A in the plasma was analyzed using ELISA. Parameters were calculated by performing non-compartmental pharmacokinetic analysis using Phoenix® WinNonlin® software. The calculated values ​​are shown in Figure 26.

[0539] Example 3.8.2 Experimental Results

[0540] Following a single intravenous administration of the test substance to rats, all animals were confirmed to have been exposed to the test substance, and the pharmacokinetic profiles were evaluated according to the administered dose. After single intravenous administration of ADC-A at doses of 1 and 3 mg / kg to rats, the total antibody (total Ab) concentrations in the blood were measured, resulting in half-lives (t1 / 2) of 4.5 and 4.7 days, and total clearances of 2.9 and 2.8 mL / hr / kg, respectively. The total ADC concentrations were measured, resulting in half-lives (t1 / 2) of 5.2 and 4.9 days, and total clearances of 3.0 and 2.5 mL / hr / kg, respectively. Analysis confirmed that the time-course blood concentrations of total antibody and total ADC related to ADC-A were similar.

Claims

1. The following formula 1: [Formula 1] 【Chemistry 1】 Antibody-drug conjugate having the structure [In the formula, Ab is an antibody unit, L is the linker unit, D is a drug unit, n is an integer between 1 and 4. The antibody unit is a conjugated anti-claudin 18.2 antibody. The drug unit is linked to one or more of K246 and K248 in the Fc region of the antibody unit. The linker unit is given by the following equation 2: [Formula 2] 【Chemistry 2】 Having a structure, [In the formula, b is an integer between 0 and 6, X' is NH-, -C(O)-, or -NHC(O)-, B' is a group formed by a click chemical reaction between click chemical functional groups. PM １ and PM ２ Each of these is independently a PEG substructure, and the PEG substructure contains 1 to 10 ethylene glycol units, and the ethylene glycol units are CH ２ OCH ２ -, -OCH ２ CH ２ -, or -CH ２ CH ２ It is O-, 1* represents the attachment site with Ab, 2* indicates the attachment site with D. The drug unit is monomethyl auristatin E (MMAE).

2. B is, 【Transformation 3】 、 【Chemistry 4】 、 【Transformation 5】 、 【Transformation 6】 、 【Transformation 7】 、 【Transformation 8】 、 【Chemistry 9】 、 【Chemistry 10】 、 【Chemistry 11】 、 【Chemistry 12】 、 【Chemistry 13】 、 【Chemistry 14】 、 【Chemistry 15】 and 【Chemistry 16】 、 Includes one structure selected from, The antibody-drug conjugate according to claim 1 [wherein, R ｘ is selected from H, halogen, and C １～３ alkyl] A １ and A ２ [Each of these points represents the attachment point to the rest of the linker unit's structure.]

3. B' is 【Chemistry 17】 That is, The antibody-drug conjugate according to claim 1 [In the formula, A2 represents the attachment site with X'.]

4. b is 2. The antibody-drug conjugate according to claim 1.

5. X' is -C(O)- The antibody-drug conjugate according to claim 1.

6. PM １ and PM ２ Each of these represents the aforementioned PEG substructure, The PEG substructure comprises 1 to 10 ethylene glycol units. The aforementioned ethylene glycol unit is -[CH ２ OCH ２ ]-,-[OCH ２ CH ２ ]-, or-[CH ２ CH ２ O] - is The antibody-drug conjugate according to claim 1.

7. PM １ and PM ２ Each has the following structure [Chemistry 18] Having, The antibody-drug conjugate according to claim 1.

8. The drug unit is represented by the following formula 5: [Formula 5] 【Chemistry 19】 Having a structure The antibody-drug conjugate according to claim 1 [In the formula, 3* represents the attachment site with L.]

9. The structure of the conjugated anti-claudin 18.2 antibody is the same as that of the anti-claudin 18.2 antibody, except for the portion conjugated with the linker unit. The antibody-drug conjugate according to claim 1.

10. The conjugated portion is K246 or K248 of the heavy chain of the anti-claudin 18.2 antibody. The antibody-drug conjugate according to claim 9.

11. The aforementioned anti-claudin 18.2 antibody is an IgG antibody, The IgG antibody is selected from the subclasses IgG1, IgG2, IgG3, and IgG4. The antibody-drug conjugate according to claim 9.

12. The heavy chain of the anti-claudin 18.2 antibody comprises CDRH1 represented by the amino acid sequence of SEQ ID NO: 10, CDRH2 represented by the amino acid sequence of SEQ ID NO: 11, and CDRH3 represented by the amino acid sequence of SEQ ID NO: 12, and the light chain of the anti-claudin 18.2 antibody comprises CDRL1 represented by the amino acid sequence of SEQ ID NO: 13, CDRL2 represented by the amino acid sequence of SEQ ID NO: 14, and CDRL3 represented by the amino acid sequence of SEQ ID NO:

15. The antibody-drug conjugate according to claim 9.

13. The anti-claudin 18.2 antibody comprises a heavy chain represented by the amino acid sequence of SEQ ID NO: 16 and a light chain represented by the amino acid sequence of SEQ ID NO:

17. The antibody-drug conjugate according to claim 9.

14. n is 2, The antibody unit comprises two heavy chains (a first heavy chain and a second heavy chain), The antibody-drug conjugate comprises two drug units (a first drug unit and a second drug unit), The first drug unit is linked to one of K246 and K248 of the first heavy chain, The second drug unit is linked to one of K246 and K248 of the second heavy chain. The antibody-drug conjugate according to claim 1.

15. The first drug unit is linked to K246 of the first heavy chain, The second drug unit is linked to K248 of the second heavy chain. The antibody-drug conjugate according to claim 14.

16. The following formula 7: [Formula 7] 【Chemistry 20】 Having a structure The antibody-drug conjugate according to claim 1.

17. A pharmaceutical composition for treating cancer, comprising a therapeutically effective amount of antibody-drug conjugate, The antibody-drug conjugate is the antibody-drug conjugate according to any one of claims 1 to 16, in the pharmaceutical composition.

18. Further comprising a pharmaceutically acceptable carrier and / or a pharmaceutically acceptable adjuvant, A pharmaceutical composition for treating cancer according to claim 17.

19. A step of administering a pharmaceutical composition containing a therapeutically effective amount of antibody-drug conjugate, Here, the antibody-drug conjugate is the antibody-drug conjugate described in any one of claims 1 to 16. A method for treating cancer, comprising [a specific feature / feature].

20. Use of an antibody-drug conjugate according to any one of claims 1 to 16 for treating cancer.

21. Use of an antibody-drug conjugate according to any one of claims 1 to 16 for the manufacture of a drug for treating cancer.

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