Antibody-drug conjugates comprising Anti-cladin-18.2 antibody and their therapeutic

Abstract

The present application relates to an antibody-drug conjugate and uses thereof.

Description

Technical Field

[0001] The present application relates to an antibody-drug conjugate. The antibody-drug conjugate provided by some embodiments of the present application has a therapeutic use for cancer or tumor.

[0002] In addition, the present application provides a pharmaceutical composition comprising an antibody-drug conjugate, a treatment method using the antibody-drug conjugate, a therapeutic use of the antibody-drug conjugate, and a use of the antibody-drug conjugate for manufacturing a drug for treating cancer. Background Art

[0003] Antibodies are biomolecules with the function of recognizing specific molecules and are used in various industrial applications. For example, antibodies can be used to detect or search (screen) for specific substances, identify the path of a specific substance moving in the body or in cells, and can be used for therapeutic purposes by inducing an immune response against a specific substance.

[0004] Attempts have been made to improve such antibodies to expand their functions. Generally, attempts have been made to label or conjugate various substances (such as drugs or radioactive moieties, etc.) to supplement or expand the functions of antibodies. Generally, antibodies can be labeled with fluorescent materials and used for fluorescence assays, or antibodies can be labeled or conjugated with reagents for treating specific diseases to maximize the therapeutic efficacy of the antibodies. These attempts and techniques can be referred to as antibody labeling or antibody-targeting moiety conjugation, and the present application relates to antibody labeling or antibody-targeting moiety conjugation.

[0005] In past studies, antibody-targeting moiety conjugates were prepared using highly reactive amino acid residues (such as amino or sulfhydryl groups) among the amino acid residues constituting the antibody. Specifically, after introducing a reactive group capable of reacting with the residue into the targeting moiety, a targeting moiety (more specifically, a modified targeting moiety into which a reactive group has been introduced) capable of reacting with the reactive residue of the antibody is prepared, and an antibody-targeting moiety conjugate is prepared by reacting the modified targeting moiety with the antibody.

[0006] These past studies were carried out by randomly attaching the targeting moiety substance to the antibody, and these past methods have many problems.

[0007] Basically, the past methods cannot accurately control the site where the targeting moiety binds to the antibody. In addition, it is impossible to accurately control the "quantity" of the targeting moiety bound to the antibody. That is, the antibody-targeting moiety conjugates prepared by the methods in the existing methods have the problem of non-uniformity of the drug structure.

[0008] The problem of the structural heterogeneity of this drug inevitably leads to the problem of the heterogeneity of drug effects caused by the "differences" in the drug structure. These problems have become the main obstacles to the development of antibody-drug conjugate (ADC) technology that requires high safety and reproducibility.

[0009] In addition, the problem of the structural heterogeneity of the drug leads to the problem of the inhibition of antibody function. An antibody contains a Fab region and an Fc region. The Fab region contains an antigen-binding domain that recognizes an antigen, and the Fc region is involved in the crystallization of the antibody. Non-site-specific binding / labeling makes it impossible to precisely control the binding position of the targeting moiety to the antibody, making it impossible to prevent the targeting moiety from binding to the antigen-binding domain of the antibody or to a position adjacent to the antigen-binding domain, thereby interfering with the recognition function of the antibody.

[0010] Therefore, there is a need in the art for a technique to label antibodies in a site-specific manner to ensure the structural homogeneity of the antibody-targeting moiety conjugate. Although some techniques have been developed, most of them lack technical and economic benefits, such as genetic manipulation or antibody modification.

[0011] Therefore, the present inventors have developed a novel antibody-drug conjugate using a technique capable of transferring a click chemistry functional group as a targeting substance to an antibody and using a compound containing an Fc-binding unit. Summary of the Invention

[0012] [Technical Problem]

[0013] In the production of an antibody-targeting moiety conjugate, when the "position" of the binding of the targeting moiety to the antibody and the "number" of the targeting moieties bound to the antibody cannot be precisely controlled, there will be a problem of the non-uniformity of the drug structure. In particular, in drug manufacturing, this problem of the non-uniformity of the drug structure inevitably leads to the problem of the non-uniformity of drug action. In addition, the problem of the non-uniformity of the drug structure leads to the problem of the inhibition of antibody function. Therefore, the present invention provides an antibody-drug conjugate produced by a method of site-specific delivery of a targeting substance (e.g., a drug) to an antibody, and its uses.

[0014] [Technical Solution]

[0015] The present invention provides an antibody-drug conjugate comprising an anti-claudin18.2 antibody. The antibody-drug conjugate of the present invention is characterized in that the drug is site-specifically linked to the antibody.

[0016] [Beneficial Effects]

[0017] The present invention provides a pharmaceutical composition for treating cancer, comprising an antibody-drug conjugate containing an anti-claudin18.2 antibody.

[0018] In addition, the present invention provides a method for treating cancer using an antibody-drug conjugate comprising an anti-claudin18.2 antibody.

[0019] In addition, the present invention provides the use of an antibody-drug conjugate comprising an anti-claudin18.2 antibody for treating cancer.

[0020] In addition, the present invention provides the use of an antibody-drug conjugate comprising an anti-claudin18.2 antibody for preparing a medicament for treating cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 A graph showing the absorbance measured after treating the CHO-K1 cell line transiently transfected with only the NOCK vector (empty vector) (MOCK CHO-K1) with antibody-A and ADC-A, respectively.

[0022] Figure 2 A graph showing the absorbance measured after treating the CHO-K1 cell line transiently transfected with the gene encoding claudin18.1 protein (CLDN18.1) (SEQ ID NO:18) (Claudin18.1CHO-K1) with antibody-A and ADC-A, respectively.

[0023] Figure 3 A graph showing the absorbance measured after treating the CHO-K1 cell line transiently transfected with the gene encoding claudin18.2 protein (CLDN18.2) (SEQ ID NO:19) (Claudin18.2CHO-K1) with antibody-A and ADC-A, respectively.

[0024] Figure 4 A graph showing the absorbance measured after treating CLDN18.2-virus-like particles (VLP) expressing claudin18.2 protein (CLDN18.2) with antibody-A and ADC-A, respectively.

[0025] Figure 5 A graph showing the 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 6 A graph showing the absorbance measured after treating the SNU601 cell line with antibody-A, ADC-A, antibody-B, and ADC-B, respectively.

[0027] Figure 7Shows a graph illustrating the absorbance measured after treating the PATU8988S cell line with antibody-A, ADC-A, antibody-B, and ADC-B, respectively.

[0028] Figure 8 Shows a graph illustrating the absorbance measured after treating the MIA PaCa-2 (CLDN18.2-) cell line with antibody-A, ADC-A, antibody-B, and ADC-B, respectively.

[0029] Figure 9 Shows a graph illustrating the internalization levels measured hourly after treating the MIA PaCa-2~CLDN18.2 cell line with antibody-A and ADC-A, respectively. At this time, the proportion of the red area on the vertical axis refers to the proportion of the area of antibody-A or ADC-A that has penetrated into the cells (measured as the area of the red dots) to the total area of the cells.

[0030] Figure 10 Shows a graph illustrating the internalization levels measured hourly after treating the MIA PaCa-2 (CLDN18.2-) cell line with antibody-A and ADC-A, respectively. At this time, the proportion of the red area on the vertical axis refers to the proportion of the area of antibody-A or ADC-A that has penetrated into the cells (measured as the area of the red dots) to the total area of the cells.

[0031] Figure 11 Shows a graph illustrating the internalization levels measured hourly after treating the SNU601 cell line with antibody-A and ADC-A, respectively. At this time, the proportion of the red area on the vertical axis refers to the proportion of the area of antibody-A or ADC-A that has penetrated into the cells (measured as the area of the red dots) to the total area of the cells.

[0032] Figure 12 Shows a graph illustrating the changes in cell viability measured after treating the MIA PaCa-2~CLDN18.2 cell line with different concentrations of antibody-A, ADC-A, antibody-B, ADC-B, and the combination of antibody-A and MMAE, respectively.

[0033] Figure 13 Shows a graph illustrating the changes in cell viability measured after treating the PATU8988S cell line with different concentrations of antibody-A, ADC-A, antibody-B, ADC-B, and the combination of antibody-A and MMAE, respectively.

[0034] Figure 14 Shows a graph illustrating the changes in cell viability measured after treating the SNU601 cell line with different concentrations of antibody-A, ADC-A, antibody-B, ADC-B, and the combination of antibody-A and MMAE, respectively.

[0035] Figure 15Shows a graph illustrating the changes in cell viability measured after treating the NUGC4 cell line with different concentrations of antibody-A, ADC-A, antibody-B, ADC-B, and the combination of antibody-A and MMAE, respectively.

[0036] Figure 16 Shows a graph illustrating the changes in cell viability measured after treating the MIA PaCa-2 (CLDN18.2-) cell line with different concentrations of antibody-A, ADC-A, antibody-B, ADC-B, and the combination of antibody-A and MMAE, respectively.

[0037] Figure 17 Shows a graph illustrating the changes in cell viability measured after treating the AGS cell line with different concentrations of antibody-A, ADC-A, antibody-B, ADC-B, and the combination of antibody-A and MMAE, respectively.

[0038] Figure 18 Shows a graph illustrating the relative percentages of total antibody or total ADC measured after treating human plasma with antibody-A, ADC-A, and ADC-B, respectively, and extracting the supernatant from samples incubated for different time periods.

[0039] Figure 19 Shows a graph illustrating the relative percentages of total antibody or total ADC measured after treating monkey plasma with antibody-A, ADC-A, and ADC-B, respectively, and extracting the supernatant from samples incubated for different time periods.

[0040] Figure 20 Shows a graph illustrating the relative percentages of total antibody or total ADC measured after treating rat plasma with antibody-A, ADC-A, and ADC-B, respectively, and extracting the supernatant from samples incubated for different time periods.

[0041] Figure 21 Shows a graph illustrating the relative percentages of total antibody or total ADC measured after treating mouse plasma with antibody-A, ADC-A, and ADC-B, respectively, and extracting the supernatant from samples incubated for different time periods.

[0042] Figure 22 Shows a graph illustrating the tumor volumes measured after intravenously injecting antibody-A, ADC-A, and ADC-C into tumor model mice under various conditions (groups G1 to G8).

[0043] Figure 23 Shows a graph illustrating the body weights measured after intravenously injecting antibody-A, ADC-A, and ADC-C into tumor model mice under various conditions (groups G1 to G8).

[0044] Figure 24Shows comparative photographs of tumors at autopsy 28 days after intravenous injection of antibody-A, ADC-A, and ADC-C, respectively, into tumor model mice under various conditions (Groups G1 to G8).

[0045] Figure 25 Shows a graph illustrating the tumor weights at autopsy 28 days after intravenous injection of antibody-A, ADC-A, and ADC-C, respectively, into tumor model mice under various conditions (Groups G1 to G8).

[0046] Figure 26 Shows a graph illustrating the concentrations of total antibody and total ADC measured after a certain period of time following intravenous administration of ADC-A to rats at different concentrations.

[0047] Figure 27 Shows the structure of ADC-A.

[0048] Figure 28 Shows the detailed structures of the linker and drug moieties contained in ADC-A.

[0049] Figure 29 Shows some reaction processes during the manufacture of ADC-A.

[0050] Figure 30 Shows the results of HIC-HPLC analysis of antibody-A.

[0051] Figure 31 Shows the results of HIC-HPLC analysis of Compound 3.

[0052] Figure 32 Shows the results of HIC-HPLC analysis of crude ADC-A. Detailed Description of the Invention

[0053] Some embodiments of the present application provide an antibody-drug conjugate.

[0054] Some embodiments of the present application provide an antibody-drug conjugate having a structure of Formula 1:

[0055] [Formula 1]

[0056]

[0057] Wherein,

[0058] Wherein, Ab is an antibody unit,

[0059] L is a linker unit,

[0060] D is a drug unit,

[0061] n is an integer from 1 to 4,

[0062] The antibody unit is a conjugated anti-claudin18.2 antibody,

[0063] The drug unit is linked to one or more of the lysine residue at position 246 (K246) and the lysine residue at position 248 (K248) of the Fc region of the antibody unit,

[0064] The linker unit has the structure of Formula 2:

[0065] [Formula 2]

[0066]

[0067] wherein b is an integer from 0 to 6,

[0068] X’ is NH-, -C(O)- or -NHC(O)-,

[0069] B’ is a group formed by a click chemical reaction between click chemical functional groups,

[0070] PM 1 and PM 2 are each independently a PEG moiety, the PEG moiety comprising 1 to 10 ethylene glycol units, and the ethylene glycol unit being CH2OCH2-, -OCH2CH2- or -CH2CH2O-,

[0071] 1* represents the attachment site to Ab,

[0072] 2* represents the attachment site to D, and

[0073] The drug unit is monomethyl auristatin E (MMAE).

[0074] In a specific embodiment, B’ may include a structure selected from

[0075]

[0076] wherein R x may be selected from H, halogen and C 1～3 alkyl, and

[0077] A1 and A2 may each represent the attachment site to the remaining structure of the linker unit.

[0078] In a specific embodiment, b may be 2.

[0079] In a specific embodiment, X’ may be -C(O)-.

[0080] In a specific embodiment, PM 1 and PM 2 each independently represents a PEG moiety, the PEG moiety may include 1-10 ethylene glycol units, and the ethylene glycol unit may be -[CH2OCH2]-, -[OCH2CH2]-, or -[CH2CH2O]-. In a particular embodiment, PM 1 and PM 2 may each have the following structure:

[0081]

[0082] In a specific embodiment, the drug unit may have the structure of Formula 5:

[0083] [Formula 5]

[0084]

[0085] wherein, 3* may represent the attachment site to L.

[0086] In a specific embodiment, in addition to the binding portion having the linker unit, the structure of the conjugated anti-claudin18.2 antibody may be the same as that of the anti-claudin18.2 antibody. At this time, the binding portion is K246 or K248 of the heavy chain of the anti-claudin18.2 antibody.

[0087] In a specific embodiment, the anti-claudin18.2 antibody may be an IgG antibody, and the IgG antibody may be selected from the subclasses of IgG1, IgG2, IgG3, and IgG4.

[0088] In a specific embodiment, the heavy chain of the anti-claudin18.2 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-claudin18.2 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.

[0089] In a specific embodiment, the anti-claudin18.2 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.

[0090] In a specific embodiment, n may be 2,

[0091] The antibody unit may include two heavy chains (a first heavy chain and a second heavy chain),

[0092] The antibody-drug conjugate may include two drug units (a first drug unit and a second drug unit),

[0093] The first drug unit may be linked to one of K246 and K248 of the first heavy chain, and

[0094] The second drug unit may be linked to one of K246 and K248 of the second heavy chain.

[0095] In a specific embodiment, the antibody-drug conjugate may have the structure of Formula 7:

[0096] [Formula 7]

[0097]

[0098] Some embodiments of the present application provide a pharmaceutical composition for treating cancer, comprising a therapeutically effective amount of an antibody-drug conjugate.

[0099] At this time, the pharmaceutical composition for treating cancer may further include a pharmaceutically acceptable carrier and / or a pharmaceutically acceptable adjuvant.

[0100] Some embodiments of the present invention provide a method for treating cancer, comprising:

[0101] administering a pharmaceutical composition comprising a therapeutically effective amount of an antibody-drug conjugate.

[0102] Some embodiments of the present invention provide the use of an antibody-drug conjugate for treating cancer.

[0103] Some embodiments of the present invention provide the use of an antibody-drug conjugate for manufacturing a drug for treating cancer.

[0104] [Embodiments of the Invention]

[0105] Hereinafter, the content of the present invention will be described in more detail by way of embodiments and examples. The invention disclosed in the present application can be implemented in various forms and is not limited to the specific embodiments described herein.

[0106] Those of ordinary skill in the art to which the invention disclosed in the present application pertains will be able to conceive of various modifications and other aspects of the content of the invention disclosed in the present application. Therefore, it should be understood that the content of the invention disclosed in this specification is not limited to the specific embodiments or examples described herein, and its modifications and other embodiments are also included within the scope of the invention disclosed in the present application.

[0107] Glossary of Terms

[0108] Unless otherwise indicated, all technical and scientific terms used in this application have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. All publications, patents, and other references mentioned herein are incorporated by reference in their entirety.

[0109] "Halogen" or "halo" refers to a group including fluorine, chlorine, bromine, and iodine, which is included in the halogen group of elements in the periodic table.

[0110] 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 together 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 including one or more heteroatoms in the main chain. As another example, heteroaryl refers to an aryl containing one or more heteroatoms on the ring (e.g., a C6 aryl, wherein one or more carbons on the ring are each independently replaced by a selected heteroatom). 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, etc. Preferably, the term includes polyvalent elements, such as N, O, and S. For example, when a structure contains one or more heteroatoms, each heteroatom can be independently selected from N, O, and S.

[0111] The terms "alkyl" or "alkane" used to refer to the molecule itself or a part of the molecule are used to refer to a fully saturated straight-chain or branched-chain hydrocarbon group. Straight-chain and branched-chain alkyls are, for example, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, isobutyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, etc. An alkyl can include a cyclic structure. The term "C x-y " is intended to include a residue containing x to y carbon atoms in the chain or ring, for example, when used with the term alkyl. For example, the term "C x-y alkyl" can refer to an alkyl including x to y carbon atoms as a substituted or unsubstituted straight-chain alkyl, branched-chain alkyl, or alkyl including a cyclic structure. C0 alkyl refers to hydrogen. C 1-4 Examples of alkyls include methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, and isobutyl, but are not limited thereto. For example, a straight-chain alkyl or branched-chain alkyl can have 1 - about 60, 1 - 20, or 1 - 10 carbon atoms.

[0112] As used herein, the term "heteroalkyl" refers to an alkyl including one or more heteroatoms. In this case, the heteroatoms are each independently selected.

[0113] The term "alkylene", which is used to refer to a molecule itself or a part of a molecule, refers to a divalent group derived from an alkyl group. As needed, the term "alkylene" can be used together with the terms "substituted" or "unsubstituted". When the term "alkylene" is not used together with the terms "substituted" or "unsubstituted", the term "alkylene" is intended to cover aspects of both substituted and unsubstituted alkylene. For example, alkylene can refer to a group having 1 to 100 carbon atoms in the main chain. Examples of alkylene can include, but are not limited to, -CH2-, -CH2CH2-, -CH2CH2CH2-, and -CH2CH2CH2CH2-. For example, alkylene can be used as C2 alkylene, which refers to an alkylene having two carbon atoms in the main chain. Illustratively, "C x-y alkylene" is used herein to refer to a substituted or unsubstituted alkylene having X to Y carbon atoms in the main chain.

[0114] The term "heteroalkylene", which is used to refer to a molecule itself or a part of a molecule, refers to a divalent group derived from a heteroalkyl group. If needed, the term "heteroalkylene" can be used together with the terms "substituted" or "unsubstituted". When the term "heteroalkylene" is not used together with the terms "substituted" or "unsubstituted", the term "heteroalkylene" is intended to cover aspects of both substituted and unsubstituted heteroalkylene. For example, heteroalkylene can refer to a group having 1 - 100 carbon atoms and heteroatoms in the main chain (e.g., the sum of the number of carbon atoms and the number of heteroatoms in the main chain is 1 - 100). Examples of heteroalkylene include, but are not limited to, -CH2-CH2-O-CH2-CH2- and -CH2-O-CH2-CH2-NH-CH2-. Heteroalkylene can contain one or more heteroatoms, and each heteroatom can be the same or different. For example, heteroalkylene can contain one or more heteroatoms at positions that are not at the ends of the chain or branch, and each heteroatom can be the same or different. For example, heteroalkylene can contain one or more heteroatoms at each end or all ends of the chain or branch, and each heteroatom can be the same or different. Illustratively, "C x-y heteroalkylene" in this specification is used to refer to a substituted or unsubstituted heteroalkylene having a total of x to y atoms in the main chain (e.g., the sum of the number of carbon atoms and the number of heteroatoms located in the main chain is x to y). For example, C3 heteroalkylene can be used to refer to a heteroalkylene having two carbon atoms and one heteroatom in the main chain. As another example, C5 heteroalkylene can be used to refer to a heteroalkylene having 3 carbon atoms and 2 heteroatoms in the main chain. C5 heteroalkylene includes structures such as, for example, -CH2-CH2-O-CH2-CH2- and -CH2-O-CH2-NH-CH2-.

[0115] The term "cycloalkyl" is used to refer to a fully saturated cyclic hydrocarbon group. "Cycloalkyl" includes monocyclic groups and polycyclic groups. Unless otherwise defined, monocyclic cycloalkyls generally have 3 to about 20, preferably 3 to 10 carbon atoms in the ring. Rings other than the first ring of polycyclic cycloalkyls can be selected from saturated rings, unsaturated rings, and aromatic rings. Cycloalkyl includes bicyclic molecules in which one, two, three, or more atoms are shared between the two rings. The term "fused cycloalkyl" refers to a polycyclic cycloalkyl in which each ring shares two adjacent atoms with the other rings. Rings other than the first ring of fused polycyclic cycloalkyls can be selected from saturated rings, unsaturated rings, and aromatic rings. Cycloalkyl can be used with the terms substituted or unsubstituted, and substituted cycloalkyl refers to the group provided when one or more hydrogen atoms attached to a carbon atom in the ring are replaced by one or more independent substituents. In addition, cycloalkyl can be used with the term hetero, where heterocycloalkyl refers to a cycloalkyl containing one or more heteroatoms in the ring.

[0116] The term "cycloalkylene" is used to denote a divalent group derived from cycloalkyl. For example, the term cycloalkylene can be used with the terms substituted or unsubstituted. For example, the term cycloalkylene can be used with the term hetero.

[0117] The term "olefin" or "alkenyl" is used to refer to the molecule itself or a part of the molecule that contains one or more double bonds as a straight-chain or branched-chain non-aromatic hydrocarbon. For example, a straight-chain alkenyl or a branched-chain alkenyl can have 2 to about 60, 2 to 20, or 2 to 10 carbon atoms.

[0118] The term "heteroolefin" or "heteroalkenyl" refers to an alkenyl containing one or more heteroatoms. In this case, the heteroatoms are each independently selected.

[0119] The term "alkenylene" used to refer to the molecule itself or a part of the molecule refers to a divalent group derived from alkenyl. As needed, the term "alkenylene" can be used with the terms "substituted" or "unsubstituted". When the term alkenylene is not used with the terms substituted or unsubstituted, the term alkenylene is intended to cover aspects of both substituted and unsubstituted alkenylene. For example, alkenylene can refer to a group having 2 to 100 carbon atoms in the main chain. Examples of alkenylene can include C=C-, -C-C-C=C-C=C-, or -C-C-C-C=C-, etc., but are not limited thereto. In this specification, when used with "C x-y alkenylene", "C x-y alkenylene" is used to denote a substituted or unsubstituted alkenylene having x to y carbon atoms in the main chain.

[0120] The term "heteroalkenylene", which is used to refer to a molecule itself or a part of a molecule, refers to a divalent group derived from an alkenyl group. For example, the term "heteroalkenylene" can be used to refer to an alkenylene group containing one or more heteroatoms in the main chain. For example, heteroalkenylene can refer to a group having 2 - 100 carbon atoms and heteroatoms in the main chain (for example, the sum of the number of carbon atoms and the number of heteroatoms is 2 - 100). As needed, the term "heteroalkenylene" can be used together with the terms "substituted" or "unsubstituted". In this specification, when used with "C x-y heteroalkenylene", C x-y heteroalkenylene is used to represent a substituted or unsubstituted heteroalkenylene having x to y carbon atoms and heteroatoms in the main chain (for example, the sum of the number of carbon atoms and the number of heteroatoms is x to y).

[0121] The term "cycloalkene" or "cycloalkenyl" is a cyclic hydrocarbon containing one or more double bonds in the ring. "Cycloalkenyl" includes monocyclic groups and polycyclic groups. Unless otherwise defined, a monocyclic cycloalkenyl usually has 3 - about 20, preferably 3 - 10 carbon atoms in the ring. The rings other than the first ring of a polycyclic cycloalkenyl can be selected from saturated rings, unsaturated rings, and aromatic rings. Cycloalkenyl includes bicyclic molecules, in which one, two, three, or more atoms are shared between the two rings. The term "fused cycloalkenyl" refers to a polycyclic cycloalkenyl in which each ring shares two adjacent atoms with the other rings. The rings other than the first ring of a fused polycyclic cycloalkenyl can be selected from saturated rings, unsaturated rings, and aromatic rings. The term "cycloalkenyl" can be used together with the terms "substituted" or "unsubstituted", and a substituted cycloalkenyl refers to a group provided when one or more hydrogen atoms attached to the carbon atoms in the ring are replaced by one or more independent substituents. In addition, the term "cycloalkenyl" can be used together with the term "hetero", where a heterocycloalkenyl refers to a cycloalkenyl containing one or more heteroatoms in the ring.

[0122] The term "cycloalkenylene" is used to represent a divalent group derived from a cycloalkenyl group. For example, the term cycloalkenylene can be used together with the terms substituted or unsubstituted. For example, the term cycloalkenylene can be used together with the term hetero.

[0123] The term "alkyne" or "alkynyl", which is used to refer to a molecule itself or a part of a molecule, contains one or more triple bonds as a straight-chain or branched-chain non-aromatic hydrocarbon. For example, a straight-chain alkynyl or a branched-chain alkynyl can have 2 - about 60, 2 - 20, or 2 - 10 carbon atoms.

[0124] The term "heteroalkynyl" or "heteroalkyne" refers to an alkynyl group containing one or more heteroatoms. In this case, the heteroatoms are each independently selected.

[0125] The term "alkynylene", which is used to refer to a molecule itself or a part of a molecule, refers to a divalent group derived from an alkynyl group. As needed, the term "alkynylene" can be used together with the terms "substituted" or "unsubstituted". When the term alkynylene is not used together with the terms substituted or unsubstituted, the term alkynylene is intended to cover aspects of both substituted and unsubstituted alkynylene groups. For example, alkynylene can refer to a group having 2 to 100 carbon atoms in the main chain. In this specification, when used together with "C x-y alkynylene", "C x-y alkynylene" is used to refer to a substituted or unsubstituted alkynylene group having x to y carbon atoms in the main chain.

[0126] The term "heteroalkynylene", which is used to refer to a molecule itself or a part of a molecule, refers to a divalent group derived from a heteroalkynyl group. For example, the term "heteroalkynylene" can be used to refer to an alkynylene group containing one or more heteroatoms in the main chain. For example, heteroalkynylene can refer to a group having 2 to 100 carbon atoms and heteroatoms in the main chain (e.g., the sum of the number of carbon atoms and the number of heteroatoms in the main chain is 2 to 100). As needed, the term "heteroalkynylene" can be used together with the terms "substituted" or "unsubstituted". In this specification, when used together with "C x-y heteroalkynylene", C x-y heteroalkynylene is used to denote a substituted or unsubstituted heteroalkynylene group having x to y carbon atoms and heteroatoms in the main chain (e.g., the sum of the number of carbon atoms and the number of heteroatoms is x to y).

[0127] The term "cycloalkyne" or "cycloalkynyl" refers to a cyclic hydrocarbon containing one or more triple bonds in the ring and is also referred to as "strained alkyne". "Cycloalkynyl" includes monocyclic groups and polycyclic groups. Unless otherwise defined, a monocyclic cycloalkynyl typically has 3 to about 10 carbon atoms in the ring. The rings other than the first ring of a polycyclic cycloalkynyl can be selected from saturated rings, unsaturated rings, and aromatic rings. Cycloalkynyl includes bicyclic molecules in which one, two, three, or more atoms are shared between the two rings. The term "fused cycloalkynyl" refers to a polycyclic alkynyl in which each ring shares two adjacent atoms with the other rings. The rings other than the first ring of a fused polycyclic cycloalkynyl can be selected from saturated rings, unsaturated rings, and aromatic rings. The term "cycloalkynyl" can be used together with the terms "substituted" or "unsubstituted", and a substituted cycloalkynyl refers to a group provided when one or more hydrogen atoms attached to a carbon atom in the ring are replaced by one or more independent substituents. In addition, the term "cycloalkynyl" can be used together with the term "hetero", where a heterocycloalkynyl refers to a cycloalkynyl containing one or more heteroatoms in the ring.

[0128] The term "cycloalkynylene" is used to denote a divalent group derived from a cycloalkynyl group. For example, the term cycloalkynylene can be used together with the terms substituted or unsubstituted. For example, the term cycloalkynylene can be used together with the term hetero.

[0129] The term "aryl" is used to refer to a group containing an aromatic ring and denotes a group derived from an aromatic hydrocarbon (which is an aromatic compound). The term aryl includes monocyclic groups and polycyclic groups. The term "aryl" can be used together with the term "hetero", and heteroaryl is used to refer to an aryl having one or more heteroatoms on the ring. The term "aryl" can be used together with the terms "substituted" or "unsubstituted", and substituted aryl refers to an aryl in which one or more hydrogen atoms attached to the carbon atoms on the ring are replaced by one or more substituents. The term "aryl" can be used to encompass substituted or unsubstituted aryl and substituted or unsubstituted heteroaryl. Examples of aryl include phenyl, pyridyl, naphthyl, biphenyl, etc., but are not limited thereto.

[0130] The term "arylene" is used to denote a divalent group derived from an aryl group. For example, the term arylene can be used together with the terms substituted or unsubstituted. For example, the term arylene can be used together with the term hetero. The term arylene can be used to encompass all substituted or unsubstituted arylene and substituted or unsubstituted heteroarylene.

[0131] As used herein, the term "substituted" means that the atomic valence is normal and the compound being substituted is stable, and one or more hydrogen atoms on the atom are replaced by substituents including deuterium and hydrogen variants. When the substituent is oxygen (i.e., =O), this means that two hydrogen atoms are replaced. When a substituent is a halogen (e.g., Cl, F, Br, and I, etc.), this means that one hydrogen atom is replaced by the halogen. When two or more substituents are present in a group, the substituents present in the group can be the same or different. Unless otherwise specified, the type and number of substituents can be arbitrary as long as it is chemically achievable. Illustratively, the substituents can 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 can each independently be selected from H, halogen, C 1-6 alkyl, C 3-10 cycloalkyl, C 3-10 heterocycloalkyl, aryl, heteroaryl, -OH, -NH2, -COOH, =O, =S, and -SH, but are not limited thereto (provided that the substituent is not -H). Representative examples of substituents include -C 1-4alkyl, -C(=O)H, -C(=O)CH3, -C(=O)OH, -C(=O)NH2, -NH2, =NH, =O, =S, -OH, -NO2, and -SH, but not limited thereto. The term substituted or unsubstituted can be used with a term referring to the molecule itself or a term referring to a part of the molecule. For example, substituted C 10-20 alkylene may mean that one or more hydrogen atoms attached to the main chain are substituted with substituents, where each substituent can be independently selected.

[0132] In this specification, when expressing the structure of a compound, a wavy line drawn in a direction substantially perpendicular to the bond (e.g., ) is used to indicate the part where a group is connected to another group. For example, when represented as the structure , it means that the group X in a substance, molecule, or compound is connected to another part by a bond. For example, when represented as the structure , it means that the group X in a substance, molecule, or compound is connected to other parts by a bond. For example, in a compound having the structure "A-X", when only the structure of the group X is described, it can be represented as the structure For example, in a compound having the structure "A-X-B", when only the X structure is described, it can be expressed as the structure If necessary, a wavy line drawn substantially perpendicular to the bond can be indicated by an additional symbol. For example, in a compound having the A-X-B structure, when only the structure of the group X is described, the structure of the group X can be described as wherein, by mentioning that "* is the part connected to A and ** is the part connected to B", information about what part each wavy line indicates is connected to can be provided.

[0133] In addition, a wavy line drawn in a direction substantially perpendicular to the bond means that the "structure shown by the wavy line" is directly connected to the "group other than the structure shown by the wavy line" by a covalent bond. The wavy line should not be interpreted as that there can be other additional elements between the "structure shown by the wavy line" and the "group other than the structure shown by the wavy line". When additional elements can be included, they will be described by separate relevant descriptions.

[0134] The structure used in the structures or formulas disclosed herein is used to represent C x alkylene. For example, the structure can be used to represent C4 alkylene, such as -CH2-CH2-CH2-CH2-. Here, the case where x is 0 represents a bond. In other words, the structure can be represented by the structure .

[0135] The compounds of the present invention may have specific geometric or stereoisomeric forms. When a compound is disclosed without specifying its form in the present application, isomers of the compound such as cis- and trans-isomers, (-)- and (+)-enantiomers, (R)- and (S)-enantiomers, diastereoisomers, (D)-isomers, (L)-isomers, and racemates are included within the scope of the present application. In other words, when the formula or structure disclosed herein does not have symbols related to isomers (e.g., *, etc.), it means that the disclosed formula or structure includes all possible isomers.

[0136] As used herein, the term "amino acid" can be used to refer to an amino acid that is not bonded to other amino acids and an amino acid residue that is bonded to other amino acids in a protein or peptide, and can be appropriately interpreted according to the context or content of the paragraph in which the term "amino acid" is used. As used herein, the term "amino acid" can be used to include natural and unnatural amino acids. As used herein, natural amino acids refer to 20 types of amino acids synthesized in the human body through the processes of 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, unnatural amino acids refer to amino acids that are not synthesized in the human body through the processes of gene transcription and translation, are amino acids synthesized through other processes than transcription and translation, or are artificially synthesized amino acids, or can be synthesized by other organisms other than humans. Unnatural amino acids may include, for example, ornithine (Orn), diaminopropionic acid (Dap), diaminobutyric acid (Dab), naphthylalanine, etc. As described above, the term "amino acid" used herein can be used to refer to an amino acid that is not bonded to other amino acids and an amino acid residue that is bonded to other amino acids included in a protein or peptide. For example, alanine can be used to refer to alanine and / or alanine residue. For example, arginine can be used to refer to arginine and / or arginine residue. As used herein, the term "amino acid" can be used to include L-type amino acids and D-type amino acids. In some embodiments, when the L-type or D-type is not mentioned, the amino acid can be interpreted as an L-type amino acid.

[0137] As used herein, the term "amino acid residue" refers to a structure derived from an amino acid included in a compound, peptide, and / or protein (e.g., an antibody, etc.) 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 amide bonds to form a peptide with the ARE sequence, the peptide contains three amino acid residues, where A, R, and E can be referred to as alanine residue, arginine residue, and glutamic acid residue, respectively. In addition, as described above, in the peptide with the ARE sequence, the peptide can contain three amino acids, and A, R, and E can also be referred to as alanine, arginine, and glutamic acid, respectively. As another example, when aspartic acid, phenylalanine, and lysine are linked by amide bonds to form a peptide with the DFK sequence, the peptide contains three amino acid residues, where D, F, and K can be referred to as aspartic acid residue, phenylalanine residue, and lysine residue, respectively. In addition, as described above, in the peptide with the DFK sequence, the peptide can contain three amino acids, and D, F, and K can also be referred to as aspartic acid, phenylalanine, and lysine, respectively.

[0138] Unless otherwise specified, when describing an amino acid sequence herein, the single-letter symbol or three-letter symbol of the amino acid is used, and the description is made in the direction from the N-terminus to the C-terminus. For example, when expressed 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 expressed 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. In the case of an amino acid that cannot be represented by a single-letter symbol, other letters are used to describe these amino acids, and an additional description is provided. The sequences described herein may include sequences having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more sequence identity with the described sequence, provided that the required functions are the same.

[0139] 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 that are designed such that two molecules can rapidly and stably form a covalent bond. The click chemistry of this specification does not mean a specific reaction, but rather the concept of rapid and stable reactions. In one embodiment, several conditions should be met in order to form a bond between molecules by click chemistry. The above conditions include high yield, excellent selectivity for reactive sites, operation in a modular manner to combinatorially assemble molecules, and proceeding in a thermodynamically stable direction to rapidly and accurately produce products. The click chemistry of this specification includes reactions of mutually reactive pairs in click chemistry functional groups (e.g., including terminal alkynes, azides, strained alkynes, dienes (e.g., Diels - Alder dienes), dienophiles (e.g., Diels - Alder dienophiles), trans - cyclooctene, alkenes, thiols, tetrazines, triazines, dibenzocyclooctynes (DBCO), and bicyclononynes (including bicyclo[6.1.0]non - 4 - yne)). Examples of click chemical reactions include the Huisgen 1,3 - dipolar cycloaddition (see Tornoe et al. Journal of Organic Chemistry (2002) 67:3075 - 3064, etc.); the Diels - Alder reaction; the inverse electron demand Diels - Alder reaction; nucleophilic addition to small strained rings such as epoxides and aziridines; nucleophilic addition to activated carbonyls; the Staudinger ligation; and addition reactions to carbon - carbon double or triple bonds.

[0140] The term "antibody" as used herein is used to refer to an immunoglobulin molecule or a fragment thereof. Immunoglobulins are generally well - known and have the ability to specifically bind one or more antigens. As used herein, the term "antibody" is also used to encompass its fragments, so it is immaterial that an antibody does not have the ability to bind a specific antigen, as in the case of the Fc fragment. Unless there are specific restrictions regarding the use of the antibody, the term "antibody" can be interpreted without particular limitation to include all monospecific antibodies, bispecific antibodies, trispecific antibodies, monoclonal antibodies, human antibodies, humanized antibodies, recombinant antibodies, chimeric antibodies, etc. For example, an antibody can comprise two heavy chains and two light chains. For example, in such a case, a known antibody can have the following structure: two heavy chains are connected by one or more bridge bonds (e.g., disulfide bonds), one heavy chain and one light chain are connected by one or more bridge bonds, and the other heavy chain and light chain are connected by one or more bridge bonds. An antibody can be divided into an Fc region (or Fc domain) and an Fab region, the Fab region contains the site capable of binding an antigen, and the Fc region contains a part of the heavy - chain constant region. As used herein, the term "antibody" can be used to include both bound and unbound antibodies (e.g., free antibodies).

[0141] In the present application, when referring to the numbering of amino acid residues in the Fc domain (or Fc region) of an antibody, unless otherwise specified, the numbering of amino acid residues follows the EU numbering system. Since the IgG sequence was studied, the EU numbering system has been widely used as a sequencing system for the Fc region, as described by Edelman GM et al. in "The covalent structure of an entire gammaG immunoglobulin molecule" (Proc Natl Acad Sci U S A., 1969 May; 63(1): 78-85). For example, in the lysine at position 246 in the Fc region, the number 246 is the number assigned according to the EU numbering system. For another example, in the lysine at position 248 in the Fc region, the number 248 is the number assigned according to the EU numbering system.

[0142] As used herein, the term "linked" or "linking" means that two or more elements present within a conceptualizable structure are directly or indirectly connected (e.g., through other elements such as a linker), and is not intended to mean that no other additional elements can exist between the two or more elements. For example, the description of "element B is linked to element A" is intended to include both cases between element A and element B: the case of one or more other elements (i.e., when element A is linked to element B through one or more other elements), and the case where there are no one or more other elements between element A and element B (i.e., when element A and element B are directly linked), and should not be construed in a limiting manner.

[0143] As used herein, the term "sequence identity" is a term used to refer to the similarity between two or more sequences. For example, the term "sequence identity" is used together with a term referring to a reference sequence and a term representing a ratio (e.g., percentage). For example, the term "sequence identity" can be used to describe a sequence that is similar or substantially identical to a reference amino acid sequence. When using a description such as "a sequence having 90% or higher sequence identity with sequence A", the reference sequence herein is sequence A. For example, the sequence identity percentage can be calculated by aligning the reference sequence and the sequence that is the target of measurement of the sequence identity percentage. The method for calculating and / or determining the sequence identity percentage is not particularly limited, and the sequence identity percentage can be calculated and / or determined by any reasonable method or algorithm available to those of ordinary skill in the art.

[0144] As used herein, the term "unit" is used in some embodiments to distinguish between conjugated substances and free substances. The term "unit" as used in some embodiments will be described by way of example with antibody units and free antibodies. A free antibody refers to an antibody molecule that is not covalently bound to other molecules or groups. An antibody unit refers to a group derived from a free antibody that is covalently linked to other molecules or groups. For example, when a free antibody and a functional substance are combined through the reaction of a reactive group of a functional group with the amine group of a lysine residue of the free antibody, an antibody-functional group conjugate can be prepared. At this time, the portion derived from the free antibody can be referred to as an antibody unit. In the antibody-functional group conjugate, the antibody unit can be understood to have the same structure as the free antibody from which the antibody unit is derived, except for the portion that binds to the non-antibody portion of the antibody-functional group conjugate. For example, when the amine group of the lysine residue participating in the reaction in the free antibody is described separately, the structure of the free antibody can be expressed as "Ab-NH2". In the antibody unit, when the connection between the antibody unit and the portion other than the antibody unit is described separately, the structure of the antibody unit can be represented as Therefore, it can be understood that the antibody unit and the free antibody have the same structure except for the amine group of the lysine residue used in the reaction. Thus, in some embodiments, the antibody unit and the free antibody may not be distinguished separately and may be referred to as "antibody", and these terms can be appropriately interpreted according to the context. In some embodiments, when a description such as "the antibody unit is derived from an antibody" is used, it can be understood that the antibody unit and the antibody have the relationship described above. In some embodiments, when a description such as "the Fc-binding unit is derived from an Fc-binding substance" is used, it can be understood that "the Fc-binding unit and the Fc-binding substance" have a relationship similar to the relationship described above. That is, it can be understood that the Fc-binding unit has the same structure as the Fc-binding substance from which it is derived, except for the connection where the Fc-binding unit is linked to the portion other than the Fc-binding unit. As in the case of the antibody unit, in some embodiments, the Fc-binding unit can be referred to as the Fc-binding substance from which it is derived, and these terms can be appropriately interpreted according to the context.

[0145] As used herein, the term "comprise" or "include" may be used to mean that there may additionally exist other elements in addition to the object of the term "comprise" or "include" (e.g., an object), or they are equivalent to the object of the term. For example, when a description such as "A includes B" is used, the description of "A includes B" should be interpreted as not excluding that A may also include additional elements other than B. In other words, "A includes B" is intended to cover cases such as the case where there are additional elements other than B in A (e.g., the case where B and C exist in A), the case where A is B, the case where A consists of B, and the case where A is represented by B, etc. Therefore, when a description such as "A includes B" is used, the description can be modified to "A is B", "A consists of B", or "A is represented by B".

[0146] As used herein, the term "has" may be used to mean that there may exist other elements in addition to the object of the term "has" (e.g., an object), or they are equivalent to the object of the term. For example, when a description such as "A has B" is used, the description covers cases such as the case where there are additional elements other than B in A (e.g., the case where B and C exist in A), the case where A is B, the case where A consists of B, and the case where A is represented by B, etc. Therefore, when a description such as "A has B" is used, the description can be modified to "A is B", "A consists of B", or "A is represented by B".

[0147] When a compound (e.g., a small molecule compound, a peptide, an antibody, a conjugate, etc.) is disclosed herein, it should be understood that its salt forms are also disclosed. Examples of the ions forming the salt of the compound include ammonium ion, calcium ion, sodium ion, potassium ion, acetate (CH3COO - ), carbonate (CO3 2- ), chloride ion (Cl - ), citrate, cyanide ion, fluoride ion (F - ), nitrate (NO3 - ), nitrite (NO2 - ), phosphate (PO3 - ), sulfate (SO4 2-) and the like, but not particularly limited thereto. Depending on the need, salt-forming ions commonly used in the art can be used to form salts of the compound. The salt can be, for example, a pharmaceutically acceptable salt. In this case, a pharmaceutically acceptable salt refers to a salt having the efficacy of the parent drug and no biological adverse reactions (for example, less toxic or non-toxic). Suitable salts include, for example, salts that can be formed by mixing a solution of the parent drug with a solution of a pharmaceutically acceptable acid such as hydrochloric acid, phosphoric acid, sulfuric acid, or acetic acid. For example, when the compound includes an acidic moiety, its pharmaceutically acceptable salts can include salts formed with suitable organic ligands such as alkali metal ions (sodium or potassium), alkaline earth metal ions (calcium or magnesium), and ammonium ions.

[0148] Hereinafter, for better understanding, the structure of the antibody will be specifically explained based on what is generally known in the art, and the scope of the present application is not limited by the following description.

[0149] According to the type of chain, the structure of the antibody is divided into a heavy chain region and a light chain region. According to the antigen-binding function of the antibody, the structure of the antibody is divided into a fragment antigen-binding region (Fab region) and a fragment crystallizable region (Fc region). According to the variability of the amino acid sequence, the structure of the antibody is divided into a variable region and a constant region. Other structures of the antibody include a hinge portion and a tail portion. The heavy chain region and the light chain region can be functionally interpreted as being roughly divided into a fragment antigen-binding region (Fab region) and a fragment crystallizable region (Fc region). The Fab region is the part that contains the part that binds to the antigen (antigen-binding part). The Fc region is the part that can bind to the fc receptor. The heavy chain region can be interpreted as having both a Fab region and an Fc region, and the light chain region can be interpreted as having a Fab region.

[0150] The Fab region of the heavy chain region contains a heavy chain variable region (VH) and a heavy chain constant region 1 (CH1). For example, in IgG1, it is known that the Fc region contains a heavy chain constant region 2 (CH2) and a heavy chain constant region 3 (CH3). In this case, the entire heavy chain constant region of the antibody can be referred to as CH. For example, the entire region combining CH1, CH2, and CH3 of IgG1 can be represented as CH.

[0151] The Fab region of the light chain region contains a light chain variable region (VL) and a light chain constant region (CL). The light chain region can be interpreted as not having an Fc region.

[0152] The above VH, CH1, CH2, CH3, VL, CL, etc. can each be referred to as an immunoglobulin domain.

[0153] The immunoglobulin domains contained in the heavy chain region are positioned in the order of VH, CH1, CH2, and CH3 or VH, CH1, CH2, CH3, and CH4 in the direction from the N-terminus to the C-terminus. The immunoglobulin domains contained in the light chain region are positioned in the order of VL and CL in the direction from the N-terminus to the C-terminus. Generally, the heavy chain region and the light chain region are known to be linked by disulfide bonds, and the Fab region and the Fc region are linked by a hinge portion. Specifically, the C-terminal portion of CH1 and the N-terminal portion of CH2 in the heavy chain region are linked by the hinge portion.

[0154] The variable regions (VH and VL) are regions containing the antigen-binding portion. Even within the variable regions, there are portions with the greatest variability (hypervariable regions), and the corresponding portions are called complementarity-determining regions (CDRs). VH includes three CDRs, and the three CDRs included in VH are generally called CDRH1, CDRH2, or CDRH3, respectively. The CDRs in VH can be understood to be positioned in the order of CDRH1, CDRH2, and CDRH3 in the direction from the N-terminus to the C-terminus. VL includes three CDRs, and the three CDRs included in VL are generally called CDRL1, CDRL2, or CDRL3, respectively. The CDRs in VL can be understood to be positioned in the order of CDRL1, CDRL2, and CDRL3 in the direction from the N-terminus to the C-terminus.

[0155] The constant region of an antibody is a region separated from the antigen-binding portion, and the constant region is known to be able to interact with cells or molecules of the immune system. For example, the constant region can interact (can bind or can be linked) with the cell membrane of immune cells (such as lymphocytes, neutrophils, dendritic cells, and / or macrophages, etc.). Specifically, the hinge region and / or the CH2 portion of the constant region can bind to receptors (such as FcεRⅢ, etc.) on the cell membrane of immune cells. As another embodiment, the constant region can bind to FcRn.

[0156] The constant region of the above heavy chain region (hereinafter referred to as "heavy chain constant region") is roughly divided into five types (classes or isotypes): alpha (α), gamma (γ), delta (δ), epsilon (ε), and mu (μ). In this case, the type of the above heavy chain constant region is not determined individually for CH1, CH2, CH3, and CH4, but is determined considering all the heavy chain constant regions (CH1, CH2, and CH3; or CH1, CH2, CH3, and CH4) contained in the antibody.

[0157] There are two types of constant regions in the light chain region (hereinafter referred to as "light chain constant regions"), and these two types are lambda (λ) and kappa (κ).

[0158] Known antibody types can be roughly divided into five types (classes or isotypes). These five types are determined by the type of the heavy chain constant region.

[0159] The above five types of antibodies are immunoglobulin M (IgM), immunoglobulin D (IgD), immunoglobulin G (IgG), immunoglobulin A (IgA), immunoglobulin A (IgA), and immunoglobulin E (IgE). When the type of the constant region of the heavy chain of an antibody is classified as α, the type of the antibody can be identified as IgA. When the type of the constant region of the heavy chain of an antibody is classified as γ, the type of the antibody can be identified as IgG. When the type of the constant region of the heavy chain of an antibody is classified as δ, the type of the antibody can be identified as IgD. When the type of the constant region of the heavy chain of an antibody is classified as ε, the type of the antibody can be identified as IgE. When the type of the constant region of the heavy chain of an antibody is classified as μ, the type of the antibody can be identified as IgM. For example, it is known that each heavy chain of IgG contains four immunoglobulin domains (VH, CH1, CH2, and CH3).

[0160] Among the 5 antibody types, IgG and IgA are known to be further classified into more detailed subclasses. For example, when describing the case where the antibody is a human antibody, when the type of the constant region of the heavy chain of the antibody is gamma 1 (γ1), the type of the antibody is IgG1; when the type of the constant region of the heavy chain of the antibody is gamma 2 (γ2), the type of the antibody is IgG2; when the type of the constant region of the heavy chain of the antibody is gamma 3 (γ3), the type of the antibody is IgG3; and when the type of the constant region of the heavy chain of the antibody is gamma 4 (γ4), the type of the antibody is IgG4. When the constant region of the heavy chain of a human antibody is alpha 1 (α1), the type of the antibody is IgA1; when the constant region of the heavy chain of a human antibody is alpha 2 (α2), the type of the antibody is IgA2.

[0161] Antibody-drug Conjugates of the Present Disclosure

[0162] Overview of Antibody-Drug Conjugates

[0163] According to one aspect of the present disclosure, an antibody-drug conjugate is disclosed.

[0164] An antibody-drug conjugate refers to the connection of an antibody and a drug. At this time, the antibody is an anti-claudin18.2 antibody, and the drug is monomethyl auristatin E (MMAE). At this time, the antibody-drug conjugate has a structure in which the drug is linked to one or more of the lysine residue at position 246 (K246) and the lysine residue at position 248 (K248) located in the Fc region of the antibody.

[0165] In some embodiments, the antibody-drug conjugate has the structure of Formula 1:

[0166] [Formula 1]

[0167]

[0168] In Formula 1, Ab is an antibody unit.

[0169] In Formula 1, L is a linker unit.

[0170] In Formula 1, D is a drug unit.

[0171] In Formula 1, n is an integer from 1 to 4.

[0172] At this time, in Formula 1, the drug unit can be linked to one or more of the lysine residue at position 246 (K246) and the lysine residue at position 248 (K248) of the Fc region of the antibody unit. Specifically, in Formula 1, the linker unit can be covalently linked to the N atom of the lysine amino group (ε-amino group) derived from the antibody.

[0173] Hereinafter, each element of the compound of Formula 1 will be described in detail.

[0174] Antibody unit

[0175] The antibody unit is derived from an anti-claudin 18.2 antibody (anti-CLDN18.2 antibody) and can be referred to as a conjugated anti-CLDN18.2 antibody. In addition, since the structure of the antibody unit is the same as that of the anti-CLDN18.2 antibody from which it is derived except for the conjugated part, the antibody unit can be referred to as an anti-CLDN18.2 antibody. In this case, the conjugated part can be the lysine residue at position 246 (K246) or the lysine residue at position 248 (K248) of the heavy chain of the anti-CLDN18.2 antibody.

[0176] 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.

[0177] In some embodiments, the anti-CLDN18.2 antibody may be an IgG antibody. IgG antibodies include human IgG antibodies, humanized IgG antibodies, and chimeric IgG antibodies.

[0178] IgG can be selected from any one of the IgG1, IgG2, IgG3, and IgG4 subclasses.

[0179] In some embodiments, the anti-CLDN18.2 antibody may be an IgG1 antibody. IgG1 antibodies encompass human IgG1 antibodies, humanized IgG1 antibodies, and chimeric IgG1 antibodies.

[0180] 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.

[0181] In some embodiments, the anti-CLDN18.2 antibody can be an IgG2 antibody. IgG2 antibodies encompass human IgG2 antibodies, humanized IgG2 antibodies, and chimeric IgG2 antibodies.

[0182] In some embodiments, the anti-CLDN18.2 antibody can include the Fc region of IgG2. The Fc region of the anti-CLDN18.2 antibody can be the Fc region of IgG2.

[0183] In some embodiments, the anti-CLDN18.2 antibody can be an IgG3 antibody. IgG3 antibodies encompass human IgG3 antibodies, humanized IgG3 antibodies, and chimeric IgG3 antibodies.

[0184] In some embodiments, the anti-CLDN18.2 antibody can include the Fc region of IgG3. The Fc region of the anti-CLDN18.2 antibody can be the Fc region of IgG3.

[0185] In some embodiments, the anti-CLDN18.2 antibody can be an IgG4 antibody. IgG4 antibodies encompass human IgG4 antibodies, humanized IgG4 antibodies, and chimeric IgG4 antibodies.

[0186] In some embodiments, the anti-CLDN18.2 antibody can include the Fc region of IgG4. The Fc region of the anti-CLDN18.2 antibody can be the Fc region of IgG4.

[0187] In some embodiments, the anti-CLDN18.2 antibody can have an amino acid sequence selected from SEQ ID NO:1 to SEQ ID NO:5. Alternatively, the anti-CLDN18.2 antibody can 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 higher sequence identity with the selected amino acid sequence. In a specific embodiment, the anti-CLDN18.2 antibody can have the amino acid sequence of SEQ ID NO:1 or 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 higher sequence identity therewith.

[0188] In some embodiments, the anti-CLDN18.2 antibody comprises an IgG Fc region, and in this case, the IgG Fc region may have an amino acid sequence selected from SEQ ID NO: 1 to SEQ ID NO: 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 higher sequence identity with the selected amino acid sequence. In a specific embodiment, the anti-CLDN18.2 antibody may comprise the Fc region of IgG, and in this case, the Fc region of IgG may have the amino acid sequence of SEQ ID NO: 1 or 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 higher sequence identity therewith.

[0189] In some embodiments, the anti-CLDN18.2 antibody or its Fc region may comprise the amino acid sequence of KPKDTLM (SEQ ID NO: 6) and the amino acid sequence of MHEALHNH (SEQ ID NO: 7).

[0190] In some embodiments, the anti-CLDN18.2 antibody or its Fc region may comprise the amino acid sequence of KPKDTLM (SEQ ID NO: 6) and the amino acid sequence of MHEALHNHY (SEQ ID NO: 8).

[0191] In some embodiments, the anti-CLDN18.2 antibody or its Fc region may comprise the amino acid sequence of GPSVFLFPPKPKDTLM (SEQ ID NO: 9).

[0192] In some embodiments, the anti-CLDN18.2 antibody may have an amino acid sequence selected from SEQ ID NOs: 1 to 5 or an amino acid sequence having 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or higher sequence identity thereto, and may substantially comprise the amino acid sequences of KPKDTLM (SEQ ID NO: 6) and MHEALHNH (SEQ ID NO: 7). In some embodiments, the anti-CLDN18.2 antibody may comprise an IgG Fc region, and in this case, the IgG Fc region may have an amino acid sequence selected from SEQ ID NOs: 1 to 5 or an amino acid sequence having 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or higher sequence identity thereto, and may substantially comprise the amino acid sequences of KPKDTLM (SEQ ID NO: 6) and MHEALHNH (SEQ ID NO: 7).

[0193] In some embodiments, the anti-CLDN18.2 antibody may have an amino acid sequence selected from SEQ ID NOs: 1 to 4 or an amino acid sequence having 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or higher sequence identity thereto, and may substantially comprise the amino acid sequences of KPKDTLM (SEQ ID NO: 6) and MHEALHNHY (SEQ ID NO: 8). In some embodiments, the anti-CLDN18.2 antibody may comprise an IgG Fc region (e.g., the Fc region of the antibody may be the Fc region of IgG), and in this case, the IgG Fc region may have an amino acid sequence selected from SEQ ID NOs: 1 to 4 or an amino acid sequence having 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or higher sequence identity thereto, and in this case, the amino acid sequence may substantially comprise the amino acid sequences of KPKDTLM (SEQ ID NO: 6) and MHEALHNHY (SEQ ID NO: 8).

[0194] In some embodiments, the anti-CLDN18.2 antibody may have an amino acid sequence selected from SEQ ID NOs: 1 to 5 or an amino acid sequence having 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or higher sequence identity thereto, and may substantially comprise the amino acid sequence of GPSVFLFPPKPKDTLM (SEQ ID NO: 9). In some embodiments, the anti-CLDN18.2 antibody may comprise an IgG Fc region, wherein the IgG Fc region may have an amino acid sequence selected from SEQ ID NOs: 1 to 5 or an amino acid sequence having 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or higher sequence identity thereto, and wherein the amino acid sequence may substantially comprise the amino acid sequence of GPSVFLFPPKPKDTLM (SEQ ID NO: 9).

[0195] In some embodiments, the antibody of the present application having binding affinity for an Fc-binding substance or peptide may be an antibody of the IgG isotype. In some embodiments, the antibody of the present application having binding affinity for an Fc-binding substance or peptide may be an IgG1 isotype antibody, an IgG2 isotype antibody, an IgG3 isotype antibody, or an IgG4 isotype antibody. In some embodiments, the antibody of the present application having binding affinity for an Fc-binding substance or peptide may be an IgG1 isotype antibody, an IgG2 isotype antibody, or an IgG4 isotype antibody.

[0196] In some embodiments, the anti-CLDN18.2 antibody may be a known anti-CLDN18.2 antibody (see Document [Korean Patent Application No. 10-2021-7023724 (Publication No. 10-2021-0110339)]).

[0197] In some embodiments, the heavy chain of the anti-CLDN18.2 antibody may comprise CDRH1 having an amino acid sequence of SEQ ID NO: 10 (TYGVH) or an amino acid sequence having 90% or higher sequence identity thereto, CDRH2 having an amino acid sequence of SEQ ID NO: 11 (VIWAGGSTNYNSALMS) or an amino acid sequence having 90% or higher sequence identity thereto, and CDRH3 having an amino acid sequence of SEQ ID NO: 12 (AAYYGNGLDY) or an amino acid sequence having 90% or higher sequence identity thereto. In a specific embodiment, 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.

[0198] 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 higher sequence identity thereto, CDRL2 having the amino acid sequence of SEQ ID NO: 14 (WASTGES) or an amino acid sequence having 90% or higher sequence identity thereto, and CDRL3 having the amino acid sequence of SEQ ID NO: 15 (QNAYFYPFT) or an amino acid sequence having 90% or higher sequence identity thereto. In a specific embodiment, the anti-CLDN18.2 antibody may have two light chains, including 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.

[0199] 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 higher sequence identity thereto, CDRH2 having the amino acid sequence of SEQ ID NO: 11 or an amino acid sequence having 90% or higher sequence identity thereto, and CDRH3 having the amino acid sequence of SEQ ID NO: 12 or an amino acid sequence having 90% or higher sequence identity thereto, and 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 higher sequence identity thereto, CDRL2 having the amino acid sequence of SEQ ID NO: 14 or an amino acid sequence having 90% or higher sequence identity thereto, and CDRL3 having the amino acid sequence of SEQ ID NO: 15 or an amino acid sequence having 90% or higher sequence identity thereto. In a specific embodiment, the anti-CLDN18.2 antibody may have two heavy chains and two light chains, the 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 the 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.

[0200] In some embodiments, the anti-CLDN18.2 antibody can be an antibody comprising a heavy chain having the amino acid sequence of SEQ ID NO:16 or an amino acid sequence having 90% or higher 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 higher sequence identity thereto. In a specific embodiment, the anti-CLDN18.2 antibody can 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.

[0201] Linker Unit

[0202] The linker unit is a structure in the region connecting the anti-claudin18.2 antibody (anti-CLDN18.2 antibody) and the drug.

[0203] Overview of Linker Unit

[0204] L (linker unit) of Formula 1 can have the structure of Formula 2:

[0205] [Formula 2]

[0206]

[0207] At this time,

[0208] In Formula 2, b is an integer from 0 to 6. In some embodiments, b can be 0, 1, 2, 3, 4, 5 or 6, or an integer between two of these numbers. For example, b can be an integer from 1 to 3. In a specific embodiment, b can be 2.

[0209] In Formula 2, X’ is -NH-, -C(O)- or -NHC(O)-.

[0210] In Formula 2, B’ is a group formed by a click chemical reaction between click chemical functional groups.

[0211] In Formula 2, PM 1 and PM 2 are each independent PEG moieties.

[0212] In Formula 2, 1* represents the attachment site to Ab.

[0213] In Formula 2, 2* represents the attachment site to D.

[0214] B' of Formula 2

[0215] B' of Formula 2 can include any one of the following structures:

[0216]

[0217]

[0218] At this time,

[0219] R x may be selected from H, halogen, and C 1-3 alkyl,

[0220] A1 and A2 each represent a connection site to the remaining structure of the linker unit. In a specific embodiment, A1 may represent the connection site to X'. In another specific embodiment, A2 may represent the connection site to X'.

[0221] PM of Formula 2 1 and PM 2

[0222] In Formula 2, PM 1 and PM 2 each independently represent a PEG moiety.

[0223] At this time, the PEG moiety may contain 1 - 30, 1 - 20, or 1 - 10 ethylene glycol units (e.g., -CH2OCH2-, -OCH2CH2-, or -CH2CH2O-).

[0224] For example, the PEG moiety may have the structure of Formula 3.

[0225] [Formula 3]

[0226]

[0227] In Formula 3, D PEG is a spacer of the PEG moiety.

[0228] For example, D PEG may be a group with a main chain length of 0 - 6 (i.e., a group with the sum of the number of atoms in the main chain being 0 - 6).

[0229] For example, D PEG may be a bond, or a substituted or unsubstituted C 1-6 alkylene, a substituted or unsubstituted C 1-6 heteroalkylene, a substituted or unsubstituted C 1-6 alkenylene, a substituted or unsubstituted C 1-6Heteroalkenyl, substituted or unsubstituted alkynyl, or substituted or unsubstituted heteroalkynyl, wherein the substituted alkyl, substituted heteroalkyl, substituted alkenyl, or substituted heteroalkenyl may comprise one or more substituents, which may be selected from -R, =O, =S, -NO2, -CR3, -NR2, -OR, -SR, -C(=O)R, -C(=O)CR3, -C(=O)OR, and -C(=O)NR2, wherein each R is independently selected from H, halogen, C 1-6 alkyl, C 3-10 cycloalkyl, C 3-10 heterocycloalkyl, aryl, heteroaryl, -OH, -NH2, =O, =S, and -SH. In a specific embodiment, the substituents may be selected from -C 1-4 alkyl, -C(=O)H, -C(=O)CH3, -C(=O)OH, -C(=O)NH2, -NH2, =O, =S, -OH, -NO2, and -SH. As used herein, heteroalkyl, heteroalkenyl, and heteroalkynyl each independently comprise one or more heteroatoms, wherein the heteroatoms may each independently be selected from N, O, and S.

[0230] In a specific embodiment, D PEG may be a bond, or a substituted or unsubstituted C 1-3 alkylene, or a substituted or unsubstituted C 1-3 heteroalkylene, wherein the substituted alkylene or substituted heteroalkylene may include one or more substituents, wherein the substituent is =O, and the heteroalkylene may include one or more heteroatoms, wherein the heteroatoms may each independently be selected from N, O, and S.

[0231] In Formula 3, R PEGis a PEG capping group. At this time, the PEG capping group may be absent, or may be -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)CH2CH3, -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, but not limited thereto. In a specific embodiment, 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 a specific embodiment, the total atomic mass of the atoms belonging to the PEG capping group may be 300 daltons or less, 200 daltons or less, 150 daltons or less, 100 daltons or less, or 50 daltons or less, but not limited thereto.

[0232] In formula 3, p may be an integer from 1 to 30, preferably an integer from 1 to 10.

[0233] In formula 3, [EG] is an ethylene glycol unit. At this time, the ethylene glycol unit is -[CH2OCH2]-, -[OCH2CH2]- or -[CH2CH2O]-.

[0234] In a specific embodiment, in formula 3, D PEG is -C(=O)-, [EG] is -[OCH2CH2]-, p is 8, R PEG is -CH3.

[0235] In a specific embodiment, in formula 2, PM 1 and PM 2 are each:

[0236]

[0237] Specific Embodiments of Linker Unit

[0238] In a specific embodiment, the linker unit may have the structure of formula 4:

[0239] [Formula 4]

[0240]

[0241] In Formula 4, PM 1 and PM 2 are each independently a PEG moiety and are described as in the "PM of Formula 2 1 and PM 2 " section regarding the description of PM 1 and PM 2 .

[0242] In Formula 4, 1* represents the attachment site to Ab,

[0243] In Formula 4, 2* represents the attachment site to D.

[0244] Drug unit

[0245] A drug unit refers to a drug that binds to an antibody or a structure derived from a drug. The drug can be monomethyl auristatin E (MMAE).

[0246] In a specific embodiment, the drug unit can have the structure of Formula 5:

[0247] [Formula 5]

[0248]

[0249] In Formula 5, 3* represents the attachment site to L of Formula 1.

[0250] Position where the drug unit is attached

[0251] In Formula 1, n is an integer from 1 to 4. In some embodiments, n can be an integer from 1 to 2. In a specific embodiment, n can be 2.

[0252] The drug unit can be attached (through L or a linker unit) to any one or more of the lysine residues at position 246 (K246) and position 248 (K248) of the Fc region of the antibody unit. More specifically, the antibody unit can comprise two heavy chains (a first heavy chain and a second heavy chain), and in this case, "-L" can be attached to any one or more of K246 of the first heavy chain, K248 of the first heavy chain, K246 of the second heavy chain, and K248 of the second heavy chain.

[0253] For example, when n is 1,

[0254] one drug unit can be attached to K246 of the first heavy chain of the antibody unit (through L or a linker unit). Alternatively, one drug unit can be attached to K248 of the first heavy chain of the antibody unit.

[0255] For example, when n is 2,

[0256] One of the two drug units can be linked to K246 of the first heavy chain of the antibody unit, and the other drug unit can be linked to K246 of the second heavy chain. Alternatively, one of the two drug units can be linked to K248 of the first heavy chain of the antibody unit, and the other drug unit can be linked to K248 of the second heavy chain. Alternatively, one of the two drug units can be linked to K246 of the first heavy chain of the antibody unit, and the other drug unit can be linked to K248 of the second heavy chain. Alternatively, one of the two drug units can be linked to K246 of the first heavy chain of the antibody unit, and the other drug unit can be linked to K248 of the first heavy chain.

[0257] For example, when n is 3,

[0258] The three drug units can be independently linked to K246 of the first heavy chain, K248 of the first heavy chain, and K246 of the second heavy chain, respectively.

[0259] Alternatively, the drug units of this generation can be independently linked to K246 of the first heavy chain, K248 of the first heavy chain, and K248 of the second heavy chain, respectively.

[0260] More specifically, when n is 3, the antibody-drug conjugate includes three drug units (the first drug unit, the second drug unit, and the third drug unit). At this time, the first drug unit can be linked to K246 of the first heavy chain, the second drug unit can be linked to K248 of the first heavy chain, and the third drug unit can be linked to K246 of the second heavy chain. Alternatively, the first drug unit can be linked to K246 of the first heavy chain, the second drug unit can be linked to K248 of the first heavy chain, and the third drug unit can be linked to K248 of the second heavy chain.

[0261] For example, when n is 4,

[0262] The four drug units can be independently linked to K246 of the first heavy chain of the antibody unit, K248 of the first heavy chain, K246 of the second heavy chain, and K248 of the second heavy chain, respectively.

[0263] More specifically, when n is 4, the antibody-drug conjugate includes four drug units (the first drug unit, the second drug unit, the third drug unit, and the fourth drug unit). At this time, the first drug unit can be linked to K246 of the first heavy chain, the second drug unit can be linked to K248 of the first heavy chain, the third drug unit can be linked to K246 of the second heavy chain, and the fourth drug unit can be linked to K248 of the second heavy chain.

[0264] Specific Embodiments of Antibody-Drug Conjugates

[0265] In a specific embodiment, Formula 1 can be represented by Formula 6. In other words, the antibody-drug conjugate can have the structure of the following Formula 6:

[0266] [Formula 6]

[0267]

[0268] In Formula 6, Ab is an antibody unit, which is described as in the "antibody unit" part above regarding the antibody unit.

[0269] In Formula 6, PM 1 and PM 2 are each independently a PEG moiety, which is described as in the part of "PM 1 and PM 2 " above regarding PM 1 and PM 2 .

[0270] In Formula 6, D is a drug unit, which is described as in the "drug unit" part above regarding the drug unit.

[0271] In Formula 6, n is an integer from 1 to 4.

[0272] In Formula 6, the description of the position where the drug unit is linked to the antibody unit is as described in the part of "the position where the drug unit is linked" above.

[0273] In a specific embodiment, Formula 1 can be represented by Formula 7. In other words, the antibody-drug conjugate can have the structure of the following Formula 7:

[0274] [Formula 7]

[0275]

[0276] In Formula 7, Ab is an antibody unit, which is described as in the "antibody unit" part above regarding the antibody unit.

[0277] In Formula 7, n is an integer from 1 to 4.

[0278] In Formula 7, the description of the bonding position of the antibody unit is as described in the part of "the position where the drug unit is linked" above.

[0279] Characteristics of Antibody-drug Conjugates of the Present Disclosure

[0280] Binding of the Antibody-Drug Conjugate of the Present Disclosure to an Antigen

[0281] The antibody-drug conjugate disclosed in the present disclosure can bind to an antigen (claudin18.2 protein). Specifically, the antibody unit included in the antibody-drug conjugate can bind to the antigen (claudin18.2 protein).

[0282] In some embodiments, the antibody-drug conjugate can bind to an antigen (claudin18.2 protein) expressed on the surface (or membrane) of a cell. Specifically, the antibody unit included in the antibody-drug conjugate can bind to the antigen (claudin18.2 protein) expressed on the surface (or membrane) of a cell.

[0283] In some embodiments, the antibody-drug conjugate can bind to claudin18.2 positive cells (or cells expressing claudin18.2). Specifically, the antibody unit included in the antibody-drug conjugate can bind to claudin18.2 positive cells (or cells expressing claudin18.2).

[0284] In some embodiments, the binding affinity of the antibody-drug conjugate disclosed in the present disclosure for the antigen (claudin18.2 protein) is the same as and / or similar to the binding affinity of the antibody (anti-claudin18.2 antibody) before binding to the antigen (claudin18.2 protein).

[0285] The antibody-drug conjugate of the present disclosure internalizes into cells

[0286] The antibody-drug conjugate disclosed in the present disclosure can bind to the claudin18.2 protein expressed in a cell and then enter the cell (or internalize into the cell). At this time, the portion corresponding to the β-glucuronide linker of the antibody-drug conjugate can be cleaved by β-glucuronidase in the cell. At this time, β-glucuronidase can be produced in the lysosome of the cell.

[0287] Therefore, the drug unit separated from the antibody-drug conjugate can be released into the cell through the above cleavage process.

[0288] The structure of the β-glucuronide linker included in the antibody-drug conjugate is shown in Formula 8.

[0289] [Formula 8]

[0290]

[0291] In Formula 8, 4* represents the connection site with a part of the linker unit.

[0292] In Formula 8, 5* represents the connection site with the drug unit.

[0293] The anti-cancer effect of the antibody-drug conjugate of the present disclosure

[0294] The antibody-drug conjugate disclosed by the present disclosure has an anti-cancer effect. At this time, the anti-cancer effect refers to inhibiting the occurrence, growth, and / or metastasis of cancer cells. The anti-cancer effect is exerted through the synergistic effect between the function of the drug released after the antibody-drug conjugate enters the cancer cells and / or the function of the antibody itself.

[0295] In some embodiments, the antibody-drug conjugate can attack cancer cells.

[0296] In some embodiments, the antibody-drug conjugate can kill cancer cells.

[0297] The half-life of the antibody-drug conjugate of the present disclosure

[0298] The in vivo half-life of the antibody-drug conjugate disclosed by the present disclosure can be 3 to 7 days. In some embodiments, the in vivo half-life range of the antibody-drug conjugate is 4 to 6 days. In a specific embodiment, the in vivo half-life of the antibody-drug conjugate is 4.5 days to 5.2 days.

[0299] 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 antibody before conjugation (anti-claudin18.2 antibody).

[0300] Uses of Antibody-drug Conjugates of the Present Disclosure

[0301] Overview of the uses of the antibody-drug conjugate of the present disclosure

[0302] The antibody-drug conjugate of the present disclosure has the use for treating cancer.

[0303] The present disclosure discloses a pharmaceutical composition for treating cancer, comprising the above-mentioned antibody-drug conjugate.

[0304] The present disclosure discloses a method for treating cancer using the above-mentioned antibody-drug conjugate.

[0305] The following describes various embodiments of the antibody-drug conjugate for cancer treatment.

[0306] Target disease

[0307] The target diseases for which the antibody-drug conjugate of the present disclosure can be used for treatment purposes are cancer, tumor, diseases related to cancer, or diseases related to tumor.

[0308] In a specific embodiment, the cancer or tumor can 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, gastrointestinal cancer, cholangiocarcinoma, 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 cancer, thymoma, and thymic carcinoma. In a specific embodiment, the cancer or tumor can be any one selected from gastric cancer, pancreatic cancer, esophageal cancer, ovarian cancer, lung cancer, liver cancer, bladder cancer, colon cancer, cholangiocarcinoma, and genitourinary cancer.

[0309] In some embodiments, the cancer or tumor is a claudin18.2-positive cancer or tumor. At this time, the cancer or tumor can be a cancer or tumor that expresses the claudin18.2 protein. Specifically, the cancer cells or tumor cells can be cancer cells or tumor cells that express the claudin18.2 protein.

[0310] In some embodiments, the disease to be treated by the pharmaceutical composition can be gastric cancer. In a specific embodiment, the disease to be treated by the pharmaceutical composition is claudin18.2-positive gastric cancer. At this time, claudin18.2-positive gastric cancer can refer to gastric cancer caused by cancer cells that express the claudin18.2 protein.

[0311] In some embodiments, the disease to be treated by the pharmaceutical composition can be pancreatic cancer. In a specific embodiment, the disease to be treated by the pharmaceutical composition is claudin18.2-positive pancreatic cancer. At this time, claudin18.2-positive pancreatic cancer can refer to pancreatic cancer caused by cancer cells that express the claudin18.2 protein.

[0312] In some embodiments, the disease to be treated by the pharmaceutical composition can be esophageal cancer. In a specific embodiment, the disease to be treated by the pharmaceutical composition is claudin18.2-positive esophageal cancer. At this time, claudin18.2-positive esophageal cancer can refer to esophageal cancer caused by cancer cells that express the claudin18.2 protein.

[0313] In some embodiments, the disease to be treated by the pharmaceutical composition can be ovarian cancer. In a specific embodiment, the disease to be treated by the pharmaceutical composition is claudin18.2-positive ovarian cancer. At this time, claudin18.2-positive ovarian cancer can refer to ovarian cancer caused by cancer cells that express the claudin18.2 protein.

[0314] In some embodiments, the disease to be treated by the pharmaceutical composition can be lung cancer. In a specific embodiment, the disease to be treated by the pharmaceutical composition is claudin18.2-positive lung cancer. At this time, claudin18.2-positive lung cancer can refer to lung cancer caused by cancer cells expressing the claudin18.2 protein.

[0315] In some embodiments, the disease to be treated by the pharmaceutical composition can be liver cancer. In a specific embodiment, the disease to be treated by the pharmaceutical composition is claudin18.2-positive liver cancer. At this time, claudin18.2-positive liver cancer can refer to liver cancer caused by cancer cells expressing the claudin18.2 protein.

[0316] In some embodiments, the disease to be treated by the pharmaceutical composition can be bladder cancer. In a specific embodiment, the disease to be treated by the pharmaceutical composition is claudin18.2-positive bladder cancer. At this time, claudin18.2-positive bladder cancer can refer to bladder cancer caused by cancer cells expressing the claudin18.2 protein.

[0317] In some embodiments, the disease to be treated by the pharmaceutical composition can be colon cancer. In a specific embodiment, the disease to be treated by the pharmaceutical composition is claudin18.2-positive colon cancer. At this time, claudin18.2-positive colon cancer can refer to colon cancer caused by cancer cells expressing the claudin18.2 protein.

[0318] In some embodiments, the disease to be treated by the pharmaceutical composition can be cholangiocarcinoma. In a specific embodiment, the disease to be treated by the pharmaceutical composition is claudin18.2-positive cholangiocarcinoma. At this time, claudin18.2-positive cholangiocarcinoma can refer to cholangiocarcinoma caused by cancer cells expressing the claudin18.2 protein.

[0319] In some embodiments, the disease to be treated by the pharmaceutical composition can be genitourinary cancer. In a specific embodiment, the disease to be treated by the pharmaceutical composition is claudin18.2-positive genitourinary cancer. At this time, claudin18.2-positive genitourinary cancer can refer to genitourinary cancer caused by cancer cells expressing the claudin18.2 protein.

[0320] A pharmaceutical composition comprising an antibody-drug conjugate

[0321] Overview of Pharmaceutical Compositions

[0322] According to one aspect of the present disclosure, a pharmaceutical composition comprising an antibody-drug conjugate for treating cancer, tumor, cancer-related diseases or tumor-related diseases is disclosed. Herein, the antibody-drug conjugate refers to the antibody-drug conjugate disclosed in the present disclosure. The pharmaceutical composition comprises a therapeutically effective amount of the antibody-drug conjugate. The pharmaceutical composition may further include a pharmaceutically acceptable carrier and / or a pharmaceutically acceptable adjuvant. Herein, the pharmaceutically acceptable carrier can be used for the proper formulation of the pharmaceutical composition.

[0323] Target Diseases of Pharmaceutical Compositions

[0324] The target diseases of the pharmaceutical composition are cancer, tumor, cancer-related diseases or tumor-related diseases, as described in the "Target Diseases" section of the "Use of the Antibody-Drug Conjugate of the Present Disclosure" section.

[0325] Examples of Formulations of Pharmaceutical Compositions

[0326] In some embodiments, the pharmaceutical composition can be formulated as lozenges, troches, tablets, aqueous suspensions, oily suspensions, prefabricated powders, granules, emulsions, hard capsules, soft capsules, syrups or elixirs.

[0327] In some embodiments, the pharmaceutical composition can be formulated as injections, suppositories, powders for respiratory inhalation, aerosols for spraying, ointments, powders for topical application, oils or creams.

[0328] In some embodiments, the pharmaceutical composition can be formulated as an injection. Specifically, a therapeutically effective amount of the antibody-drug conjugate can be mixed with a stabilizer or buffer in water to prepare a solution or suspension, which can be formulated as a unit dose in an ampoule or vial.

[0329] In some embodiments, the pharmaceutical composition can be formulated as an aerosol, which is prepared by mixing a propellant, etc. with additives to prepare a water-dispersible concentrate or wet powder.

[0330] In some embodiments, when the pharmaceutical composition is formulated for transdermal use, an ointment, cream, powder for topical application, oil, topical skin preparation, etc. can be prepared by adding a therapeutically effective amount of the antibody-drug conjugate to a carrier (such as animal fat, vegetable fat, wax, paraffin, starch, tragacanth, cellulose derivatives, polyethylene glycol, silicone, bentonite, silica, talc, zinc oxide, etc.).

[0331] Examples of Pharmaceutically Acceptable Carriers

[0332] Pharmaceutically acceptable carriers can be those commonly used in formulations and can include saline solutions, sterile water, Ringer's solution, buffered saline, cyclodextrin, glucose solution, maltodextrin solution, glycerol, ethanol, liposomes, etc., but are not limited thereto, and can further include other common additives as needed, such as antioxidants and buffers. In addition, diluents, dispersants, surfactants, binders, lubricants, etc. can be additionally added to formulate injectable preparations, such as aqueous solutions, suspensions and emulsions, pills, capsules, granules or tablets. Regarding suitable pharmaceutically acceptable carriers and formulations, each component can preferably be formulated using the methods disclosed in Remington's Pharmaceutical Sciences (19th Edition, 1995).

[0333] In some embodiments, the pharmaceutical composition can include binders as pharmaceutically acceptable carriers, such as lactose, sucrose, sorbitol, mannitol, starch, amylopectin, cellulose or gelatin; excipients, such as dibasic calcium 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; syrups; liquid carriers, such as fatty oils; sterile aqueous solutions; propylene glycol; polyethylene glycol; injectable esters, such as ethyl oleate; suspending agents; emulsifying agents; lyophilized preparations; topical preparations; stabilizers; buffers; animal oils; vegetable oils; waxes; paraffin wax; starch; tragacanth; cellulose derivatives; polyethylene glycol; silicone resins; bentonite; silica; talc; zinc oxide or suitable combinations thereof.

[0334] In some embodiments, to prepare the pharmaceutical composition of the present disclosure, the antibody-drug conjugate can be mixed with a pharmaceutically acceptable carrier. In other words, the pharmaceutical composition can include a pharmaceutically acceptable carrier. In addition, the pharmaceutical composition can further contain one or more other elements suitable for treating or preventing cancer. The term "pharmaceutically acceptable carrier" can be used to include excipients, diluents or adjuvants. The carrier can be one or more selected from, for example, lactose, glucose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum arabic, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, polyvinylpyrrolidone, water, normal saline, buffers such as phosphate buffered saline (PBS), methyl paraben, propyl paraben, talc, magnesium stearate and mineral oil. The carrier can contain fillers, anticoagulants, lubricants, wetting agents, flavoring agents, emulsifying agents, preservatives or combinations thereof.

[0335] Methods of treatment using antibody-drug conjugates

[0336] Overview of Treatment Methods

[0337] According to one aspect of the present disclosure, a method for treating cancer, tumor, 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 in the present disclosure. The treatment method includes administering a suitable preparation of a therapeutically effective antibody-drug conjugate, i.e., a pharmaceutical composition, to a subject using a suitable administration method, a suitable dosing regimen, and a suitable dose.

[0338] Target Diseases of Treatment Methods

[0339] The target diseases of the treatment method are cancer, tumor, cancer-related diseases or tumor-related diseases, as described in the "Target Diseases" section of the "Use of the Antibody-Drug Conjugate of the Present Disclosure" section.

[0340] Treatment of Subjects

[0341] The subject of the treatment method can be a human or a non-human animal.

[0342] Examples of Administration Methods

[0343] The treatment method can be administering a therapeutically effective antibody-drug conjugate of a suitable preparation to a subject by a suitable administration method. In one embodiment, the treatment method can be administering a therapeutically effective antibody-drug conjugate of a suitable preparation to a subject by a method selected from oral administration, parenteral administration, intravenous administration, intraperitoneal administration, intramuscular administration, transdermal administration, and subcutaneous administration. In a specific embodiment, the treatment method can be administering a therapeutically effective antibody-drug conjugate of a suitable preparation to a subject by intravenous administration.

[0344] Examples of Dosages

[0345] The treatment method can be administering a therapeutically effective antibody-drug conjugate of a suitable dose and a suitable preparation to a subject. In one embodiment, based on the antibody-drug conjugate, the dose can be about 0.01 mg - 1000 mg / kg of the subject's body weight. In a specific embodiment, based on the antibody-drug conjugate, the dose can be about 0.01 mg / kg of the subject's body weight - 100 mg / kg of the subject's body weight.

[0346] Examples of Administration Cycles

[0347] The treatment method can be administering a therapeutically effective antibody-drug conjugate to a subject in a suitable dosing cycle with a suitable formulation. In one embodiment, the pharmaceutical composition containing a suitable dose of the antibody-drug conjugate can be administered once a day. In another example, the dosing cycle can be administering the pharmaceutical composition containing a suitable dose of the antibody-drug conjugate two or more times a day. In another example, the administration cycle can be administering the pharmaceutical composition containing a suitable dose of the 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.

[0348] Therapeutic use of antibody-drug conjugate

[0349] According to one aspect of the present disclosure, the use of an antibody-drug conjugate for treating cancer, tumor, cancer-related diseases or tumor-related diseases is disclosed. Herein, the antibody-drug conjugate refers to the antibody-drug conjugate disclosed in the present disclosure. Herein, cancer, tumor, cancer-related diseases or tumor-related diseases are as described in the "Target Diseases" section of the "Use of the Antibody-Drug Conjugate of the Present Disclosure" section.

[0350] Use of antibody-drug conjugate in the preparation of cancer therapeutic agents

[0351] According to one aspect of the present disclosure, the use of an antibody-drug conjugate in the manufacture of a medicament for treating cancer, tumor, cancer-related diseases or tumor-related diseases is disclosed. Herein, the antibody-drug conjugate refers to the antibody-drug conjugate disclosed in the present disclosure. Herein, cancer, tumor, cancer-related diseases or tumor-related diseases are as described in the "Target Diseases" section of the "Use of the Antibody-Drug Conjugate of the Present Disclosure" section.

[0352] Method for preparing antibody-drug conjugate

[0353] The antibody-drug conjugate disclosed in the present disclosure can be prepared, for example, by the methods disclosed in Example 1 and Example 2.

[0354] Hereinafter, the invention provided in the present application will be described in more detail through experimental examples and examples. These examples are intended to illustrate the content disclosed in the present application, and the scope of the content disclosed in the present application is not limited by these examples.

[0355] Examples

[0356] Materials and Experimental Methods

[0357] Compound

[0358] The compounds were purchased from commercial suppliers and used without further purification. 5-exo-norbornene carboxylic acid, ammonium sulfate ((NH4)2SO4), N-Boc-ethylenediamine, triisopropylsilane (TIS), and 1,2-ethanedithiol (EDT) were purchased from Sigma-Aldrich CO., LTD (St. Louis, MO, USA).

[0359] N-methylmaleimide, N-ethylmaleimide, N-cyclohexylmaleimide, N-phenylmaleimide, N-benzylmaleimide, N-tert-butylmaleimide, and 3-maleimidopropionic acid were purchased from Tokyo Chemical Industry (Tokyo, Japan). tert-Butyl thioglycolate was purchased from Angene Chemical PVT., LTD (Telangana, India). 4-Methyltetrazine NHS ester was purchased from Broadpharm (San Diego, CA, USA). Fmoc-PEG8-OH was purchased from Quanta Biodesign (Plain City, OH, USA). DM1-SMCC was purchased from eNovation Chemicals LLC (Bridgewater Township, NJ, USA). DBCO-C6-NHS was purchased from (Lumiprobe, Hong Kong, China). All amino acids, rink amide resin, and coupling reagents were purchased from AAPPtec LLC (Louisville, KY, USA), GL Biochem LTD (Shanghai, China), and Combi-Blocks (San Diego, CA, 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 purchased from Daejung Chemicals & Metals CO., LTD (Siheung, Korea). High-performance liquid chromatography (HPLC)-grade acetonitrile (ACN), isopropanol, and water were purchased from Thermo Fisher Scientific (Waltham, MA, USA).

[0360] Antibodies and ADCs

[0361] Two types of anti-claudin18.2 antibodies (anti-CLDN18.2 antibodies) (hereinafter referred to as Antibody-A and Antibody-B) were prepared.

[0362] 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. (Herein, Antibody-A has two light chains and two heavy chains, having the same structure as a known general antibody.) Information on the antibody is disclosed in the literature [Korean Patent Application No. 10-2021-7023724 (Publication No. 10-2021-0110339)]. Antibody-A was obtained from GenScript ProBio, and the 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.

[0363] Antibody-B is a known antibody (zolbetuximab antibody) and was purchased from Biointron.

[0364] Antibody-C is a human IgG antibody (Sigma-Aldrich, I4506) and was purchased from Sigma-Aldrich CO., LTD (St. Louis, MO, USA).

[0365] ADC-A is Antibody-A conjugated with MMAE and was produced by the methods of Example 1 and Example 2.

[0366] ADC-B is Antibody-B conjugated with MMAE and was produced by commissioning Abzena. At this time, the method for producing ADC-B was carried out according to the content disclosed in the reference [US 2018 / 0117174 (Application No. US 15 / 565,848)].

[0367] MMAE was conjugated with Antibody-C by the methods of Example 1 and Example 2 to produce ADC-C. At this time, the only difference is that the antibody used in Example 2 was Antibody-C instead of Antibody-A.

[0368] Cells and Plasma

[0369] The MIA PaCa-2~CLDN18.2 cell line (C3002) was purchased from Accurus Biosciences. The PATU8988S cell line (ACC 204) was purchased from the German Collection of Microorganisms and Cell Cultures GmbH (DSMZ). The MIA PaCa-2 (CLDN18.2-) cell line (CRL-1420) was purchased from the American Type Culture Collection (ATCC). The SNU601 cell line (00601) was purchased from the 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.

[0370] 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.

[0371] Equipment and Conditions

[0372] Equipment

[0373] All peptides and compounds containing payloads were characterized by HPLC (Waters, C18, 4.6×250 mm, 5 μm).

[0374] For HPLC, an HPLC Alliance system (2996 PAD detector and 2695 separation module) manufactured by Waters was used.

[0375] As the mass spectrometer for analyzing all small molecular weight substances such as peptides and compounds, an LC / MS equipped with a Quatro Premier XE device and an Acquity Waters LC system manufactured by Waters was used.

[0376] All antibodies containing antibody conjugates (e.g., ADC, etc.) were analyzed using hydrophobic interaction chromatography (HIC)-HPLC (Thermo Fisher Scientific, MAbPac TM , HIC-butyl, 4.6×100 mm, 5 μm), size exclusion chromatography (SEC)-HPLC (Thermo Fisher Scientific, MAbPacTM, SEC- 4 × 300 mm, 5 μm) and a UV spectrophotometer (Thermo Fisher spectrophotometer, MULTISKAN GO / MicroDrop TM plates were used, N12391) for characterization.

[0377] Centrifugal desalting was performed using a Fleta 4 centrifuge (Hanil, Korea).

[0378] The OD values in all enzyme-linked immunosorbent assay (ELISA) experiments were measured using a UV-Vis spectrophotometer (Thermo Fisher, 4661030N).

[0379] In the cytotoxicity experiment, Discover (Promega, GM3000) was used to measure cell viability.

[0380] A S3 live cell analysis instrument (SARTORIUS, S3) was used for real-time internalization analysis.

[0381] As a centrifuge, Smart R17 Plus (Hanil, SM-R17PL) was used.

[0382] Characterization of Peptides and Compounds Using C18-HPLC

[0383] 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% to 80% gradient of mobile phase B (0.075% TFA in CAN) in A for 15 minutes]. Chromatograms of peptides and compounds were obtained at 280 nm, and chromatograms of payloads were obtained at 254 nm.

[0384] Characterization of Antibodies Using HIC-HPLC

[0385] HIC-HPLC was performed at a flow rate of 1 mL / min. HIC-HPLC was performed on all antibodies under the same conditions [initial 100% mobile phase A (1.5 M (NH4)2SO4, 50 mM Na2HPO4, pH 7.0, 5% isopropanol) for 1 minute, followed by a 0% to 100% gradient of mobile phase B (50 mM sodium phosphate, pH 7.0, 20% isopropanol) in A for 15 minutes]. All chromatograms were obtained at 280 nm.

[0386] Characterization of Antibodies, etc. Using SEC-HPLC

[0387] SEC-HPLC was performed at a flow rate of 0.2 mL / min. All antibodies etc. were analyzed under the same conditions [isocratic mobile phase D (1×PBS), for 20 minutes]. All chromatograms were obtained at 280 nm.

[0388] Measurement of Optical Density (OD) Values in ELISA Experiments Using a UV-Vis Spectrophotometer

[0389] ELISA was performed to measure the OD value by measuring the absorbance at 450 nm using a UV-Vis spectrophotometer.

[0390] Measurement of Cell Viability to Confirm Cytotoxicity

[0391] In the cytotoxicity experiment, to measure cell viability, Discover was used to measure luminescence, and the measurement method was carried out according to the equipment manual.

[0392] Real-time Internalization Analysis

[0393] Real-time internalization measurement was carried out according to the manuals of the reagents and equipment.

[0394] Method for Pretreating Plasma Samples and PK Samples Using a Centrifuge

[0395] Centrifugation during the pretreatment process was carried out at 13,000 rpm and 4 °C for 10 minutes.

[0396] Example 1. Preparation of Fc-binding substances, compounds, etc.

[0397] Example 1.1 Preparation of Fc-binding substances

[0398] The present inventors prepared an Fc-binding substance represented by FcBP(Orn) by referring to the content disclosed in the reference [WO2020 / 184944 (application number PCT / KR2020 / 003282)]. FcBP(Orn) can be represented by Ac-PEG8-DCAWHOrnGELVWCT-NH2, and its structure is as follows:

[0399]

[0400] The present inventors prepared FcBP(Orn) using solid-phase peptide synthesis (SPPS).

[0401]

[0402] Coupling:

[0403] I) Coupling amino acids, HoBt, HBTU, DIPEA / DMF, r.t. 2 h;

[0404] Washing:

[0405] II) DCM (and DMF), 10 V (v / w), 3 times

[0406] Fmoc deprotection:

[0407] III) Piperidine in 20% DMF (v / v); 2 times

[0408] Complete cleavage:

[0409] IV) TFA: EDT: TIS: DW = 94:1:2.5:2.5 (v / v / v / v), 10 V (v / w)

[0410] Acetylation:

[0411] V) Acetic anhydride, DCM, DIPEA

[0412] List of Fmoc Amino Acids and Introduction Order Used in Example 1.1.1

[0413] Fmoc-L-Thr(tBu)-OH, Fmoc-Cys(Trt)-OH, Fmoc-L-Trp(Boc)-OH, Fmoc-L-Val-OH, Fmoc-L-Leu-OH, Fmoc-L-Glu(OtBu)-OH, Fmoc-Gly-OH, Fmoc-Orn(Boc)-OH, Fmoc-L-His(Trt)-OH, Fmoc-L-Trp(Boc)-OH Fmoc-Ala-OH, Fmoc-Cys(Trt)-OH, and Fmoc-Asp(tBu)-OH.

[0414] The specific preparation method is described in detail in the literature [WO 2020 / 184944 (application number PCT / KR2020 / 003282)], which is incorporated herein by reference.

[0415] Introduction of Amino Acids in Example 1.1.2

[0416] The amounts of reagents used in the following methods 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.

[0417] The reactions of activating Fmoc-amino acids and connecting the activated residues to the rink amide resin were carried out sequentially from the C-terminal amino acid.

[0418] Fmoc deprotection was carried out in DMF containing 20% piperidine. To activate and introduce residues, the amino acids prepared in this order were mixed with 2 mL of a DMF solution containing 0.5 M hydroxybenzotriazole (HOBt), 2 mL of a DMF solution containing 0.5 M O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU), and 174 μL of DIPEA for 5 minutes. Then the resulting mixture was poured into a reactor containing the resin and mixed for 2 hours.

[0419] The introduction reaction was confirmed by Kaiser test. When unreacted amines remained, the introduction reaction was repeated once, or capping was carried out with a DMF solution containing 20% Ac2O. The resin was washed thoroughly with DMF and DCM and then moved to the next step in each introduction reaction and Fmoc deprotection process. This step was repeated until the target peptide sequence was completed.

[0420] Introduction of H-PEG8-OH in Example 1.1.3

[0421] To introduce H-PEG8-OH at the N-terminus after all amino acids were introduced, 1 mL of a DMF solution of 0.5 M Fmoc-N-amide-dPEG8-acid, 1 mL of a DMF solution containing 0.5 M HBTU, 1 mL of a DMF solution containing 0.5 M HOBt, and 87 μL of DIPEA were mixed for 5 minutes. Then the resulting mixture was poured into a reactor containing the resin and mixed for 2 hours.

[0422] The reaction progress was confirmed by Kaiser test. When unreacted amines remained, the reaction time was extended by an additional 1 - 3 hours, or the reaction solution was emptied and the above reaction steps were repeated. After removing the N-terminal Fmoc protecting group with DMF containing 20% piperidine, the resin was washed thoroughly with a solution of DMF and DCM and then capped with a DMF solution containing 20% Ac2O. After capping, the resin linked to the peptide was dried with a solution of DCM and ether and weighed.

[0423] The peptide was cleaved from the resin by stirring 250 mg of the resin (to which the peptide prepared during the amino acid introduction was linked) with 2 mL of a mixed solution of TFA, TIS, water, and EDT (94:1.0:2.5:2.5) at room temperature for 120 minutes. The cleavage mixture was filtered, and the resulting filtrate was concentrated to about half with nitrogen and then poured into ether to precipitate the peptide. The precipitated peptide was further washed three times with ether and dried with nitrogen. After dissolving the dried precipitate in water containing 0.1% TFA - 30% ACN, the resulting solution was stirred for 6 hours and then concentrated.

[0424] After dissolving the concentrate in a 0.01 M ammonium acetate buffer (pH 6.5) solution containing 5%-DMSO-20%-ACN at a concentration of 0.1 mg / mL, the resulting solution was stirred for 3 days while being exposed to air. The progress of the disulfide bond formation reaction was observed by HPLC, and when it was determined that the reaction no longer progressed, the reaction solution was lyophilized to obtain a peptide precipitate.

[0425] Purification in Example 1.1.4

[0426] The peptide precipitate obtained by lyophilization during the above H-PEG8-OH introduction process was purified by preparative LC and then lyophilized. The obtained peptide was confirmed to have a purity of 90% or higher by analytical HPLC, and the molecular weight of the synthetic peptide was confirmed by LC / MS.

[0427] -LC / MS analysis result: [M / 3+H]=666.26; [M / 2+H]=999.38

[0428] -HPLC analysis result: 9.004 minutes, purity: 99.9%

[0429] Example 1.2 Preparation of Compound 1

[0430] The present inventors prepared the following Compound 1 using the obtained peptide.

[0431] (Compound 1)

[0432]

[0433] At this time, FcBU is PEG8-DCAWHOrn’GELVWCT-NH2 derived from FcBP(Orn), and Orn’ is the bound ornithine.

[0434] Specifically, the structure of Compound 1 is as follows:

[0435]

[0436] (Exact mass: 2265.0082)

[0437] The preparation method of Compound 1 is described with reference to the following reaction scheme:

[0438]

[0439] 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, and O-benzylhydroxylamine (6.5 g, 52.7 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hexafluorophosphate (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 h. Ethyl acetate (EtOAc) and distilled water were added, and the organic layer was extracted. The collected organic layer was dried over anhydrous magnesium sulfate. After filtering, concentrating, and purifying the organic layer by column chromatography, compound 1-1 (11.4 g, 95%) was obtained.

[0440] After dissolving compound 1-1 (5.4 g, 21.5 mmol) in 180 mL of N,N-dimethylformamide, potassium carbonate (K2CO3, 6.5 g, 47.3 mmol) and methyl iodide (5.4 mL, 86.0 mmol) were added, and the resulting mixture was stirred at room temperature for 3 h. Ethyl acetate and distilled water were added to extract the organic layer. The collected organic layer was dried over anhydrous magnesium sulfate (MgSO4). After filtering, concentrating, and purifying the organic layer by column chromatography, compound 1-2 (5.0 g, 87.7%) was obtained.

[0441] After dissolving compound 1-2 (1 g, 3.7 mmol) in 24 mL of tetrahydrofuran, lithium hydroxide (LiOH, 0.23 g in 8 mL of water) was added, and the resulting mixture was reacted at room temperature for 3 h. Water and ethyl acetate were added, and the water was extracted. The aqueous layer was acidified to pH 3 with 4N HCl. Ethyl acetate was added to extract the organic layer. The collected organic layer was dried over magnesium sulfate (MgSO4). After filtering, concentrating, and purifying the organic layer by column chromatography, compound 1-3 (0.7 g, 74.7%) was obtained.

[0442] After dissolving compound 1-3 (0.85 g, 3.4 mmol) in 13 mL of methanol, Pd / C (0.05 g) was added, and the resulting mixture was stirred under a hydrogen atmosphere. The reaction solution was filtered through diatomaceous earth and concentrated under vacuum to obtain compound 1-4 (0.46 g, quantitative yield) without purification.

[0443] After dissolving Compound 1-4 (1 g, 6.208 mmol) in 4 mL of dichloromethane, 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 for 3 hours at room temperature under a nitrogen atmosphere. After concentration, ethyl acetate and 10% citric acid solution were added to extract the organic layer. After concentration, the organic layer was purified by column chromatography to obtain Compound 1-5 (1.65 mg, 92.2%).

[0444] After dissolving Compound 1-5 (200 mg, 0.694 mmol) in 6.8 mL of dichloromethane, 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 allowed to react for 3 hours at room temperature under an argon atmosphere. After concentration, the mixture was purified by column chromatography to obtain Compound 1-6.

[0445] (221.8 mg, 83%)

[0446] After dissolving FcBP(Orn) (200 mg, 0.1002 mmol) in 0.5 mL of N,N-dimethylformamide, N,N-diisopropylethylamine (0.07 mL) and Compound 1-6 (40.51 mg) were added, and the resulting mixture was stirred for 2 hours at room temperature under an argon atmosphere. After concentration, the mixture was purified by reverse-phase column chromatography to obtain Compound 1-7 (Compound 1) (226.9 mg, 64.8%).

[0447] Thereafter, the obtained Compound 1 was analyzed by mass spectrometry and HPLC, and the results were as follows:

[0448] - LC / MS analysis result: [M / 2 + H] = 1134.35; [M + H] = 2268.37

[0449] - HPLC analysis result: 13.19 minutes, purity 99.9%

[0450] Example 1.3 Preparation of Compound 2 (Payload 1)

[0451] Preparation of Compound 2-1 in Example 1.3.1

[0452] 40 mL of dichloromethane was added to 2-chlorotrityl chloride resin (1.4 mmol / g, 1 g) (1 equivalent), and the resulting mixture was stirred for over 30 minutes. Thereafter, the solution was removed, and ethylenediamine (5.6 mmol, 4 equivalents) and N,N'-diisopropylethylamine (5.6 mmol, 4 equivalents) were mixed with dichloromethane and then with the resin. After stirring at room temperature for over 2 hours, the reaction solution was removed. Compound 2-1 was prepared by washing the resin with sufficient dichloromethane and dimethylformamide three or more times each to remove the residual reaction solution.

[0453] (Compound 2-1)

[0454]

[0455] Preparation of Compound 2-2 in Example 1.3.2

[0456] N-α-Fmoc-N-ε-allyloxycarbonyl-L-lysine (Fmoc-Lys(alloc)-OH) (2.8 mmol, 2 equivalents), N,N'-diisopropylcarbodiimide (2.8 mmol, 2 equivalents), hydroxybenzotriazole (5.6 mmol, 4 equivalents) and dimethylformamide were thoroughly mixed, and the resulting mixture was added to the resin. After stirring at room temperature for over 2 hours, the reaction solution was removed. To remove the residual reaction solution, the resin was washed with sufficient dichloromethane and dimethylformamide at least 3 times each, thereby preparing Compound 2-2. The preparation of Compound 2-2 was confirmed by the Kaiser test.

[0457] (Compound 2-2)

[0458]

[0459] Preparation of Compound 2-3 in Example 1.3.3

[0460] A 20% piperidine solution dissolved in dimethylformamide was prepared, stirred at room temperature for 10 minutes, and the reaction solution was removed. The above process was carried out 2 times in total. To remove the residual reaction solution, the resin was washed with sufficient dichloromethane and dimethylformamide at least three times each, thereby preparing Compound 2-3.

[0461] (Compound 2-3)

[0462]

[0463] Example 1.3.4 Preparation of Compound 2-4

[0464] Mix succinic acid monoter-butyl ester (2.8 mmol, 2 equivalents), N,N'-diisopropylcarbodiimide (2.8 mmol, 2 equivalents), hydroxybenzotriazole (5.6 mmol, 4 equivalents) and dimethylformamide thoroughly, and add the resulting mixture to the resin. After stirring at room temperature for more than 2 hours, remove the reaction solution. To remove the residual reaction solution, the resin is washed with sufficient dichloromethane and dimethylformamide at least 3 times respectively, thereby preparing Compound 2-4.

[0465] (Compound 2-4)

[0466]

[0467] Preparation of Compound 2-5 in Example 1.3.5

[0468] The remaining resin is washed with sufficient dichloromethane three or more times. Mix tetrakis(triphenylphosphine)palladium(0) (0.7 mmol, 0.5 equivalent), 1,3-dimethylbarbituric acid (14 mmol, 10 equivalents) and dichloromethane thoroughly, and add the resulting mixture to the resin. After stirring at room temperature for about 1 hour, remove the reaction solution. To remove the residual reaction solution, the resin is washed with sufficient dichloromethane and dimethylformamide at least three times respectively, thereby preparing Compound 2-5.

[0469] (Compound 2-5)

[0470]

[0471] Preparation of Compound 2-6 in Example 1.3.6

[0472] Mix N-α-N-ε-bis(9-fluorenylmethoxycarbonyl)-L-lysine (Fmoc-Lys(fmoc)-OH) (2.8 mmol, 2 equivalents), N,N’-diisopropylcarbodiimide (2.8 mmol, 2 equivalents), hydroxybenzotriazole (5.6 mmol, 4 equivalents), and dimethylformamide thoroughly, and add the resulting mixture to the resin. After stirring at room temperature for more than 2 hours, remove the reaction solution. To remove the residual reaction solution, the resin is washed with sufficient dichloromethane and dimethylformamide at least 3 times respectively, thereby preparing Compound 2-6. The preparation of Compound 2-6 is confirmed by Kaiser test.

[0473] (Compound 2-6)

[0474]

[0475] Preparation of Compound 2-7 in Example 1.3.7

[0476] Prepare a 20% piperidine solution dissolved in dimethylformamide, stir it at room temperature for 10 minutes, and remove the reaction solution. The above steps are carried out twice in total. To remove the residual reaction solution, the resin is washed at least three times with sufficient dichloromethane and dimethylformamide respectively, thereby preparing Compound 2-7.

[0477] (Compound 2-7)

[0478]

[0479] Preparation of Compound 2-8 in Example 1.3.8

[0480] Fully mix 2,5,8,11,14,17,20,23-octaoxahacosacosan-26-oic acid (m-PEG8-acid) (2.8 mmol, equivalent), N,N′-diisopropylcarbodiimide (2.8 mmol, 2 equivalents), hydroxybenzotriazole (5.6 mmol, 4 equivalents) and dimethylformamide, and add the resulting mixture to the resin. After stirring at room temperature for more than 2 hours, remove the reaction solution. To remove the residual reaction solution, the resin is washed at least three times with sufficient dichloromethane and dimethylformamide respectively, thereby preparing Compound 2-8.

[0481] (Compound 2-8)

[0482]

[0483] Preparation of Compound 2-9 in Example 1.3.9

[0484] Prepare a mixture of trifluoroacetic acid and distilled water in a ratio of 95:5, add a sufficient amount of the mixture to the resin, and stir the resulting mixture at room temperature for 2 hours. After stirring, separate the solution and the resin. Add a sufficient amount of 0 °C ether to the separated solution, mix, and let the resulting mixture stand at 0 °C for more than 2 hours to ensure sufficient precipitation. Compound 2-9 is prepared by centrifugation and drying. The preparation of Compound 2-9 is confirmed by mass spectrometry and HPLC analysis ([M / 2 + H] = 603.38; [M + H] = 1206.21 (accurate mass: 1204.72)).

[0485] (Compound 2-9)

[0486]

[0487] Preparation of Compound 2-10 in Example 1.3.10

[0488] Dissolve 1 g (0.83 mmol) of the product obtained from the previous process in dimethylformamide, then add DBCO-C6-NHS (0.995 mmol, 1.2 equivalents) and N,N'-diisopropylethylamine (1.659 mmol, 2 equivalents), and stir the resulting mixture at room temperature for more than 1 hour. After completion of the reaction, the reaction solution was purified by preparative-HPLC (C18) and lyophilized to obtain the product (Compound 2-10) (0.394 mmol, 47.5%). The preparation of Compound 2-10 was confirmed by mass spectrometry and HPLC analysis ([M / 2 + H] = 761.09 (Exact mass: 1519.84)).

[0489] (Compound 2-10)

[0490]

[0491] Preparation of Compound 2-11 in Example 1.3.11

[0492] Dissolve the product obtained from the previous process (0.394 mmol) in dimethylformamide, then add tetramethyl-O-(N-succinimidyloxy)uronium tetrafluoroborate (0.788 mmol, 2 equivalents) and N,N'-diisopropylethylamine (0.788 mmol, 2 equivalents), and stir the resulting mixture at room temperature for more than 1 hour. After completion of the reaction, the reaction solution was purified by preparative-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 spectrometry and HPLC analysis ([M / 2 + H] = 809.67 (Exact mass: 1616.86)).

[0493] (Compound 2-11)

[0494]

[0495] Preparation of Compound 2-12 (Compound 2, Payload 1) in Example 1.3.12

[0496] Dissolve the product obtained from the previous process (0.138 mmol) in dimethylformamide, then add the pre-prepared H2N-BG-MMAE (0.152 mmol, 1.1 equivalents) and N,N'-diisopropylethylamine (0.276 mmol, 2 equivalents), and stir the resulting mixture at room temperature for more than 1 hour. After completion of the reaction, the reaction solution was purified by preparative-HPLC (C18) and lyophilized to obtain the product (Compound 2, Payload 1) (0.065 mmol, 47.5%).

[0497] (Compound 2, Payload 1)

[0498]

[0499] Thereafter, Compound 2 obtained by mass spectrometry and HPLC analysis gave the following results:

[0500] - LC / MS analysis result: [M / 2 + H] = 1318.06; [M + H] = 2635.38 (Exact mass: 2631.45)

[0501] - HPLC analysis result: 16.721 minutes, purity 100%

[0502] Preparation of ADC-A in Example 2

[0503] The present inventors prepared ADC-A by conjugating a drug (MMAE) with an anti-claudin18.2 antibody (Antibody-A). The structure of ADC-A is as Figure 27 shown.

[0504] At this time, ADC-A includes an antibody containing two heavy chains, and the payload is linked to the lysine residue at position 246 or 248 of one of the heavy chains, and the payload is linked to the lysine residue at position 246 or 248 of the other heavy chain. At this time, the structure of the payload linked to each heavy chain is the same.

[0505] Specifically, the structure of the payload linked to the lysine residue at position 246 or 248 of one of the heavy chains of ADC-A is as Figure 28 shown.

[0506] Reference Figure 29 describes the preparation process of ADC-A according to the reaction process.

[0507] Example 2.1 Preparation of Compound 3

[0508] The present inventors prepared a conjugate (Compound 3) in which an azide was linked to Antibody-A.

[0509] (Compound 3)

[0510]

[0511] The specific method is as follows.

[0512] Compound 1 was prepared at a concentration of 10 mM in DMSO solvent. The prepared Compound 1 (277.8 μL, 2.778 nmol, 4.0 equivalents) 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 at 1×PBS (pH 7.4) and reacted for 3 hours. The final DMSO product was fixed at 10%. The reaction (crosslinking of Compound 1 with Antibody-A) was monitored by HIC-HPLC analysis, and the monitoring results are as Figure 30 and Figure 31 shown.

[0513] After the reaction was completed, unreacted Compound 1 with Antibody-A was removed using spin desalting (Zeba TM spin desalting column, 40K molecular weight cut-off, 10 mL, 1000 g force) and SEC. As a result, Compound 3 was obtained.

[0514] Example 2.2 Preparation of ADC-A

[0515] The present inventors prepared a conjugate (ADC-A) using the obtained Compounds 2 and 3, in which the payload of Compound 2 was linked to Antibody-A. The specific method is as follows.

[0516] Compound 2 was prepared at a concentration of 10 mM in DMSO solvent. The prepared Compound 2 (416.7 μL, 4.167 nmol, 6 equivalents) 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 at 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 as Figure 32 shown.

[0517] After the reaction was completed, unreacted Compound 2 with Compound 3 was removed using spin desalting (Zeba TM spin desalting column, 40K molecular weight cut-off, 10 mL, 1000 g force) and dialysis (pH 7.4, 1×PBS, 4 h, 4 h, 12 h, 3 times). Thus, crude ADC-A (DAR 2) was obtained.

[0518] Verification of the Efficacy of ADC-A in Example 3

[0519] Example 3.1 Target Specificity Verification 1 - Confirmation of Binding to Antigen-Expressing Cells

[0520] The present inventors conducted an experiment according to Example 3.1.1 to confirm whether antibody-A and ADC-A could specifically bind to the claudin18.2 protein (CLDN18.2), and the results are as described in Example 3.1.2.

[0521] Experimental Method in Example 3.1.1

[0522] Prepare a CHO-K1 cell line transiently transfected with the gene encoding claudin18.1 protein (CLDN18.1) (SEQ ID NO:18) (hereinafter referred to as "Claudin18.1 CHO-K1"), a CHO-K1 cell line transiently transfected with the gene encoding claudin18.2 protein (CLDN18.2) (SEQ ID NO:19) (hereinafter referred to as "Claudin18.2 CHO-K1"), and a CHO-K1 cell line transiently transfected only with the NOCK vector (empty vector) (hereinafter referred to as "MOCK CHO-K1"). At this time, the pcDNA3.1 vector is used for Claudin18.1 CHO-K1, and the pcDNA3.1(+) vector is used for Claudin18.2 CHO-K1.

[0523] The CHO-K1 cell line was prepared by the following method.

[0524] In an incubator at 37°C and 5% CO2, the CHO-K1 cell line was cultured in RPMI1640 containing 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin.

[0525] Transient transfection was performed according to the TransIT2020 manual. The transfected cells were inoculated on a 96-well plate and cultured, and then treated with antibody-A and ADC-A respectively. Thereafter, a cell-based enzyme-linked immunosorbent assay (ELISA) was performed, and the optical density (OD) value was measured. At this time, the measured values are as Figures 1 to 3 shown.

[0526] Using GraphPad Prism 9.0 software, the measured results were used for regression analysis to calculate EC50 and Bmax. Based on the calculated values, the cell binding affinities of antibody-A and ADC-A were evaluated. At this time, the calculated values are listed in Table 1.

[0527] Experimental Results in Example 3.1.2

[0528] By Figures 1 to 3, it was confirmed that antibody-A and ADC-A did not bind to the negative control MOCK CHO-K1 cell line and the Claudin18.1 CHO-K1 cell line, and specifically bound only to the Claudin18.2 CHO-K1 cell line expressing CLDN18.2.

[0529] From Table 1, it was confirmed that the EC50 and binding maximum (Bmax) (representing the maximum binding affinity) of ADC-A were almost the same as those of antibody-A. In other words, it was found that although the linker and payload were conjugated to antibody-A, the binding affinity of the antibody to the antigen was not significantly affected.

[0530] [Table 1]

[0531]

[0532] Example 3.2. Target Specificity Verification 2 - Confirmation of Binding to Antigen Protein

[0533] The present inventors conducted the experiment according to Example 3.2.1 to confirm whether antibody-A and ADC-A could bind to the claudin18.2 protein (CLDN18.2), and the results are as described in Example 3.2.2.

[0534] Experimental Method in Example 3.2.1

[0535] To confirm the binding strength to the antigen, CLDN18.2-VLPs (virus-like particles) expressing the claudin18.2 protein (CLDN18.2) were prepared. At this time, the CLDN18.2-VLPs were CSB-MP005498HU (A5) from CUSABIO TECHNOLOGY LLC.

[0536] After coating a 96-well plate with CLDN18.2 VLPs by ELISA and treating each with antibody-A and ADC-A, indirect ELISA was performed to measure the OD value. At this time, the measured values are as Figure 4 shown.

[0537] The measured results were used for regression analysis in GraphPad Prism 9.0 software to calculate the EC50 and Bmax. Based on the calculated values, the antigen binding affinities of antibody-A, ADC-A, antibody-B, and ADC-B were evaluated. At this time, the calculated values are listed in Table 2.

[0538] Experimental Results in Example 3.2.2

[0539] By Figure 4As shown in Table 2, it can be confirmed that the measured binding affinities of each test substance for CLDN18.2 VLP increase 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 affinities for the antigen than ADC-B and antibody-B.

[0540] [Table 2]

[0541]

[0542] Example 3.3. Target Specificity Verification 3 - Confirmation of Binding to Cancer Cells Expressing the Antigen

[0543] The present inventors conducted the experiment according to Example 3.3.1 to confirm whether antibody-A and ADC-A can bind to cancer cells expressing the claudin18.2 protein (CLDN18.2-positive cancer cells), and the results are as described in Example 3.3.2.

[0544] Experimental Method in Example 3.3.1

[0545] Four types of cell lines (MIA PaCa-2~CLDN18.2, PATU8988S, parental MIA PaCa-2 (CLDN18.2-), and SNU601) were prepared. At this time, MIA PaCa-2~CLDN18.2 and PATU8988S are CLDN18.2-positive pancreatic cancer cell lines. MIAPaCa-2 (CLDN18.2-) is a CLDN18.2-negative pancreatic cancer cell line. SNU601 is a CLDN18.2-positive gastric cancer cell line.

[0546] In an incubator under the conditions of 37°C and 5% CO2, the prepared cell lines were cultured 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), and DMEM containing 5% FBS, 5% serum, 1% penicillin / streptomycin, and 2 mM L-glutamine (PATU8988S).

[0547] The prepared cell lines were inoculated on a 96-well plate for culture, and then treated with antibody-A, ADC-A, antibody-B, and ADC-B, respectively. Thereafter, cell-based ELISA was performed to measure the OD value. At this time, the measured values are as Figures 5 to 8 shown.

[0548] The measurement results were used for GraphPad Regression analysis in the 9.0 software was performed to calculate the EC50 and Bmax. Based on the calculated values, the cellular binding affinities of antibody-A, ADC-A, antibody-B, and ADC-B to each cell line were evaluated. At this time, the calculated values are shown in Table 3.

[0549] Experimental Results in Example 3.3.2

[0550] By Figures 5 to 8 and Table 3, it was confirmed that the measured binding affinities to pancreatic cancer and gastric cancer cell lines increased in the order of antibody-B, ADC-B, ADC-A, and antibody-A.

[0551] Furthermore, it was confirmed that the binding affinities to all test substances (antibody-A, ADC-A, ADC-B, and antibody-B) increased in the order of PATU8988S, SNU601, and MIA PaCa-2 to CLDN18.2, and it was confirmed that no test substance bound to the parental MIA PaCa-2 (which is a CLDN18.2-negative cell line). These experimental results imply that the expression level of CLDN18.2 increased in the order of PATU8988S, SNU601, and MIA PaCa-2 to CLDN18.2.

[0552] [Table 3]

[0553]

[0554] Example 3.4 Verification of Cellular Internalization

[0555] The present inventors conducted the experiment according to Example 3.4.1 to confirm the cellular internalization of ADC-A, and the results are as described in Example 3.4.2.

[0556] Experimental Method in Example 3.4.1

[0557] To confirm the cellular uptake (internalization) of antibody-A and ADC-A based on the presence or absence of CLDN18.2 expression, the MIAPaCa (CLDN18.2-) cell line that does not express CLDN18.2 and the MIAPaCa-2 to CLDN18.2 and SNU601 cell lines that express CLDN18.2 were prepared.

[0558] In an incubator at 37 °C and 5% CO2, the prepared cell lines were cultured in DMEM containing 10% FBS and 1% penicillin / streptomycin (MIA PaCa-2~CLDN18.2, MIA PaCa-2 (CLDN18.2-)) and RPMI1640 containing 10% FBS and 1% penicillin / streptomycin (SNU601). The MIA PaCa (CLDN18.2-), MIA PaCa-2~CLDN18.2, and SNU601 cell lines were seeded at 3,000 - 5,000 cells / well on a 96-well plate.

[0559] According to the Fabfluor-pH reagent manual, antibody-A and ADC-A samples for internalization verification were prepared.

[0560] Finally, antibody-A and ADC-A were each diluted to 8 μg / mL, 4 μg / mL, and 2 μg / mL, and the designated wells were each treated with 50 μL. Using a live cell analysis system, the internalization level of the well plate mixed with the samples was measured hourly. The measured values are as Figures 9 to 11 shown.

[0561] Experimental Results in Example 3.4.2

[0562] By Figures 9 to 11 , it was confirmed that antibody-A and ADC-A were rapidly internalized within 20 hours in the MIA PaCa-2~CLDN18.2 and SNU601 cell lines expressing CLDN18.2. On the other hand, it was confirmed that antibody-A and ADC-A were not internalized in MIA PaCa-2 (CLDN18.2-) that does not express CLDN18.2. In addition, antibody-A and ADC-A showed similar internalization efficiencies at almost all concentrations. These results confirmed that, compared with antibody-A, the linker and drug (payload) constituting ADC-A do not affect cell internalization, and internalization depends on the expression of the antigen CLDN18.2 in the cell line.

[0563] Example 3.5 Cytotoxicity Evaluation

[0564] The present inventors conducted an experiment according to Example 3.5.1 to evaluate the cytotoxicity of ADC-A, and the results are as described in Example 3.5.2.

[0565] Example 3.5.1 Experimental Method

[0566] Four types of cell lines (MIA PaCa-2~CLDN18.2, PATU8988S, parental MIA PaCa-2 (CLDN18.2-), SNU601, NUGC-4, and AGS) were prepared. At this time, 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.

[0567] In an incubator under the conditions of 37 °C and 5% CO2, the prepared cell lines were cultured 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 L-glutamine (PATU8988S).

[0568] The prepared cell lines were seeded on 96-well plates and cultured at 500 to 5000 cells / well, and then treated with each test substance (antibody-A, ADC-C, ADC-A, antibody-B, ADC-C, and the combination of antibody-A and MMAE) diluted in a 10-fold gradient (10000, 1000, 100, 10, 1, 0.1, 0.01, 0.001, and 0 ng / mL). At this time, the combination of antibody-A and MMAE means that the cells were treated with the combination of antibody-A and MMAE, and the concentration of MMAE was the same molar concentration as that of MMAE contained in other ADCs (e.g., ADC-A).

[0569] Using the same method as the CellTiter-Glo manual, the changes in cell viability according to each test substance treatment were measured. At this time, the measured values are as Figures 12 to 17 shown.

[0570] The results of the measurement 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. At this time, the calculated values are listed in Table 4.

[0571] Experimental Results in Example 3.5.2

[0572] By Figures 12 to 17As shown in Table 4, it was determined that ADC-A exhibited anti-cancer activity in CLDN18.2-positive cancer cell lines regardless of the type of cancer. In addition, it was confirmed that the IC50 value of ADC-A was 10 - 35 times lower than that of ADC-B used as a control. In particular, in the MIA PaCa-2 to CLDN18.2 cell lines, ADC-A exhibited an IC50 value lower than that of the co-administration group of antibody-A and MAE, and also exhibited an IC50 value of 1 nM or less in PATU8988S and SNU601. In addition, in CLDN18.2-negative cell lines, it was confirmed that ADC-A had almost no toxicity at the highest treatment concentration, while ADC-B used as a control exhibited toxicity.

[0573] [Table 4]

[0574]

[0575] *: Unable to derive the IC 50 value.

[0576] Example 3.6 Plasma Stability Assessment

[0577] The present inventors conducted the experiment 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.

[0578] Experimental Method in Example 3.6.1

[0579] Human, monkey, rat, and mouse plasma were prepared. After treating the prepared plasma with each of the test substances (antibody-A, ADC-A, and ADC-B), the plasma was incubated at a temperature of 37 °C for 5 minutes, 1 day, 5 days, 8 days, 12 days, and 15 days (a total of 108 samples were prepared).

[0580] At each time point, only the supernatant was taken out from the prepared samples using a centrifuge and used for analysis. The plasma stability of the test substances over time was confirmed by ELISA analysis. The ELISA plate was coated with CLDN18.2 VLP and treated with plasma diluted 5000-fold, and then treated with each of antibody-A, ADC-A, and ADC-B. The amount of total antibody (total Ab) was analyzed using an anti-human IgG antibody (Promega, W4031), and the amount of total ADC was analyzed using an anti-MMAE antibody (an antibody generated in-house by ABFRONTIER). At this time, total Ab includes not only antibodies not bound to the linker, such as antibody-A, but also substances bound to the linker, such as ADC-A. Total ADC only includes substances bound to the linker, such as ADC-A or ADC-B, and does not include antibodies not bound to the linker. For example, when the linker is cleaved in ADC-A, it is not included in total ADC.

[0581] Convert the amount of total antibody or total ADC over time to a relative percentage based on the remaining amount on Day 0 (5 minutes). The conversion results are as Figures 18 to 21 shown.

[0582] Experimental Results in Example 3.6.2

[0583] By Figures 18 to 21 , the following results can be seen.

[0584] In the case of 15 days in human plasma, the total antibody of Antibody-A remains at 85.2%, the total antibody of ADC-A remains at 74.8%, the total ADC of ADC-A remains at 75.1%, the total antibody of ADC-B remains at 36.9%, and the total ADC of ADC-B remains at 16.6%.

[0585] In the case of 15 days in monkey plasma, the total antibody of Antibody-A remains at 97.3%, the total antibody of ADC-A remains at 90.3%, the total ADC of ADC-A remains at 77.5%, the total antibody of ADC-B remains at 24.3%, and the total ADC of ADC-B remains at 7.4%.

[0586] In the case of 15 days in rat plasma, the total antibody of Antibody-A remains at 68.2%, the total antibody of ADC-A remains at 64.0%, the total ADC of ADC-A remains at 50.3%, the total antibody of ADC-B remains at 17.8%, and the total ADC of ADC-B remains at 15.3%.

[0587] In the case of 15 days in mouse plasma, the total antibody of Antibody-A remains at 72.0%, the total antibody of ADC-A remains at 78.7%, the total ADC of ADC-A remains at 77.0%, the total antibody of ADC-B remains at 25.5%, and the total ADC of ADC-B remains at 17.7%.

[0588] Within 15 days, the stability of Antibody-A and ADC-A in plasma shows similar levels and trends in all types of plasma. In contrast, the total antibody and total ADC of ADC-B show a pattern of rapid decrease over time. Since ADC-A shows the same value as Antibody-A in plasma over time, it can be interpreted that no aggregation or degradation occurs in ADC-A due to the linker and drug binding to the antibody. Therefore, it can be seen that ADC-A shows comparable stability to the antibody in in vitro plasma, and the linker and drug bound to the antibody in ADC-A do not affect the stability of the ADC.

[0589] Example 3.7 Evaluation of in vivo drug efficacy

[0590] The present 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 as described in Example 3.7.5.

[0591] Cell Culture in Example 3.7.1

[0592] In an incubator at 37 °C and 5% CO2, the SNU601 cell line (which is a CLDN18.2-positive gastric cancer cell line) was cultured in RPMI culture solution containing 10% FBS and 1% penicillin / streptomycin. The SNU601 cell line was treated with trypsin-ethylenediaminetetraacetic acid (EDTA) and passaged regularly once a week.

[0593] Tumor Inoculation and Drug Administration in Example 3.7.2

[0594] For tumor development, the SNU601 cell line (1×10 7 ) was mixed with serum-free medium and Matrigel, and the resulting mixture was subcutaneously inoculated into the left ventral side of each BALB / c nude mouse (purchased from Charles River Laboratories Japan Inc.). Based on the average tumor volume in each group reaching approximately 155 mm 3 , the mice were randomly assigned to 8 groups (G1 to G8). Thereafter, the drugs were administered to each group as shown in Table 5.

[0595] [Table 5]

[0596]

[0597] Observation and Measurement of Tumors in Example 3.7.3

[0598] All animals were observed twice a day for mortality, abnormal symptoms, pain, and signs of stress, and clinical signs were observed once a day. The changes in body weight and tumor volume were measured three times a week, and the tumor weight was determined after autopsy.

[0599] Tumor volume, tumor difference, and tumor growth inhibition were calculated as follows:

[0600] Tumor volume (mm 3 ) = (major axis length × minor axis length 2 ) / 2

[0601] Tumor difference (%) = ((tumor weight 测试组 - tumor weight 对照(G1) ) / tumor weight 对照(G1) × 100

[0602] Tumor growth inhibition (%) = 100 - ((T f / T i ) 测试组) / (((T f / T i )) 对照组 )×100

[0603] T f is the last tumor volume measured before autopsy, and T i is the first tumor volume measured.

[0604] At this time, the measured tumor volumes are as Figure 22 shown. The measured body weights are as Figure 23 shown. The shape of the tumor after autopsy is as Figure 24 shown. The tumor weight measured after autopsy is as Figure 25 shown.

[0605] Statistical Analysis in Example 3.7.4

[0606] The numerical data of the study were calculated as mean and standard deviation. The Bartlett's test was used to compare the variance between groups for each parameter, with a significance level of 0.05. When the difference between the variances between groups was not significant, one-way analysis of variance (ANOVA) of the parameters was performed. When ANOVA indicated a significant difference between the means (p ≤ 0.05), Dunnett's test was used to compare the group means between the control group and each treatment group. Whenever the Bartlett's test showed that the variances between groups were not homogeneous (p ≤ 0.05), the non-parametric Kruskal-Wallis test was used to compare all the groups considered. When significance (p ≤ 0.05) was found by the Kruskal-Wallis test, Dunn's test was used to evaluate the significance of the difference between the control group and each treatment group. For the comparison of groups, the significance levels of 0.05, 0.01, and 0.001 were reported. All statistical analyses were performed using GraphPad Prism version 5.0.

[0607] Experimental Results in Example 3.7.5

[0608] No unplanned deaths were observed during the study.

[0609] During the study, the test substances (Antibody-A, ADC-A, and ADC-C) had no effect on body weight change. The average change in body weight for each group is shown in Table 6 and Figure 23 as follows.

[0610] [Table 6]

[0611]

[0612]

[0613] On day 28, it was confirmed that the average tumor volume in groups G7 and G8 was significantly lower than that in the control. The average tumor volume and tumor growth inhibition in each group during the study are shown in Table 7 and Figure 22 as shown. The average tumor weight after autopsy in each group is shown in Table 8 and Figure 25 as shown.

[0614] [Table 7]

[0615]

[0616] *TGI: Tumor growth inhibition

[0617] Compared with G1 P < 0.05; P < 0.01; P < 0.001.

[0618] Significance was calculated by the Kruskal-Wallis test and Dunn's multiple comparison test.

[0619] [Table 8]

[0620]

[0621] P < 0.05; P < 0.01; P < 0.001 vs G1.

[0622] Significance was calculated by the Kruskal-Wallis test and Dunn's multiple comparison test.

[0623] In this example, a SNU601 gastric cancer tumor model was created using BALB / c nude mice, and the anti-cancer 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) was 60.1% - 98.3%. In particular, when ADC-A was administered at 1.75 mg / kg (G7), the TGI was 98.3%, indicating a very strong anti-cancer effect.

[0624] No unplanned deaths occurred in any of the animals, and no significant changes in body weight were observed. Even in the highest concentration dosing group G8, no histological gastric damage related to the antibody-A and ADC-A dosing groups was observed. In summary, ADC-A exhibited high anti-cancer efficacy in the gastric cancer model without weight loss or histological gastric damage.

[0625] Example 3.8 Pharmacokinetic evaluation

[0626] The present inventors conducted experiments according to Example 3.8.1 to evaluate the pharmacokinetics of ADC-A, and the results are as described in Example 3.8.2.

[0627] Experimental Method in Example 3.8.1

[0628] After single administration of 1 mg / kg and 3 mg / kg doses of ADC-A to rats (purchased from SAMTAKO Bio Korea CO., LTD.) via the tail vein, blood was collected over a period of time and analyzed to compare and evaluate the pharmacokinetic curves. The supernatant was obtained from the plasma using a centrifuge, and the concentration of ADC-A in the plasma was analyzed using the ELISA method. By using software, pharmacokinetic analysis was performed in a non-compartmental manner to calculate the parameters. The calculated values are as Figure 26 shown.

[0629] Experimental Results in Example 3.8.2

[0630] As a result of single intravenous administration of the test substance to rats, it was confirmed that all animals were exposed to the test substance, and the pharmacokinetic curves according to the administered dose were evaluated. After single intravenous administration of ADC-A to rats at doses of 1 mg / kg and 3 mg / kg, as a result of measuring the concentration of total antibody (total Ab) in the blood, the half-lives (t1 / 2) were determined to be 4.5 days and 4.7 days, and the total clearance rates were determined to be 2.9 mL / hr / kg and 2.8 mL / hr / kg, respectively. As a result of measuring the total ADC concentration, the half-lives (t1 / 2) were determined to be 5.2 and 4.9 days, and the total clearance rates were determined to be 3.0 mL / hr / kg and 2.5 mL / hr / kg, respectively. Through analysis, it was confirmed that the changes over time in the blood concentration of total antibody and the total ADC of ADC-A were similar.

Claims

1. An antibody-drug conjugate having the structure of Formula 1: [Formula 1] Among them, Ab is an antibody unit, L is a linker unit, D is a drug unit, n is an integer from 1 to 4, The antibody unit is a bound anti-claudin18.2 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 has the structure of Formula 2: [Formula 2] Wherein, b is an integer from 0 to 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 independently is a PEG moiety, wherein the PEG moiety contains 1 to 10 ethylene glycol units, and wherein the ethylene glycol unit is CH2OCH2-, -OCH2CH2- or -CH2CH2O- 1* represents the attachment site to Ab, 2* represents the attachment site to D, The drug unit is monomethyl auristatin E (MMAE).

2. The antibody-drug conjugate according to claim 1, B comprises a selection from a structure in Among them, R x selected from H, halogen, and C 1-3 alkyl, and A1 and A2 each represent a connection site to the remaining structure of the linker unit.

3. The antibody-drug conjugate according to claim 1, B’ is Among them, A2 represents the attachment site to X'.

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

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

6. The antibody-drug conjugate according to claim 1, PM 1 and PM 2 respectively represent the PEG part, The PEG moiety contains 1 to 10 ethylene glycol units, The ethylene glycol unit is -[CH2OCH2]-, -[OCH2CH2]- or -[CH2CH2O]-.

7. The antibody-drug conjugate according to claim 1, PM 1 and PM 2 respectively have the following structures, 8. The antibody-drug conjugate according to claim 1, The drug unit has the structure of Formula 5: [Formula 5] Among them, 3* represents the attachment site to L.

9. The antibody-drug conjugate according to claim 1, Among them, Except for the part bound to the linker unit, the structure of the bound anti-claudin18.2 antibody is the same as the structure of the anti-claudin18.2 antibody.

10. The antibody-drug conjugate according to claim 9, The bound part is K246 or K248 of the anti-claudin18.2 antibody heavy chain.

11. The antibody-drug conjugate according to claim 9, The anti-claudin18.2 antibody is an IgG antibody, The IgG antibody is selected from the subclasses of IgG1, IgG2, IgG3 and IgG4.

12. The antibody-drug conjugate according to claim 9, Among them, The heavy chain of the anti-claudin18.2 antibody contains 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 wherein, the light chain of the anti-claudin18.2 antibody contains 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.

13. The antibody-drug conjugate according to claim 9, The anti-claudin18.2 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.

14. The antibody-drug conjugate according to claim 1, 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), Among them, 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.

15. The antibody-drug conjugate according to claim 14, 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.

16. The antibody-drug conjugate according to claim 1, The antibody-drug conjugate has the structure of Formula 7: [Formula 7] 17. A pharmaceutical composition for treating cancer, comprising a therapeutically effective amount of an antibody-drug conjugate, The antibody-drug conjugate is the antibody-drug conjugate according to any one of claims 1-16.

18. The pharmaceutical composition for treating cancer according to claim 17, The pharmaceutical composition for treating cancer further comprises a pharmaceutically acceptable carrier and / or a pharmaceutically acceptable adjuvant.

19. A method for treating cancer, comprising: administering a pharmaceutical composition comprising a therapeutically effective amount of an antibody-drug conjugate, wherein the antibody-drug conjugate is the antibody-drug conjugate according to any one of claims 1-16.

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

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

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