Antibody-drug conjugate comprising heterogeneous drugs and method for preparing same

Abstract

The present invention relates to an antibody-drug conjugate comprising heterogeneous drugs. Particularly, provided is an antibody-drug conjugate that comprises a first drug and a second drug that are bound to one antibody and thus can simultaneously exhibit the effects of the two drugs. To provide an antibody-drug conjugate not only exhibiting strong effects in the prevention and treatment of cancer, but also with minimized toxicity and enhanced safety, the present invention proposes a ratio of heterogeneous drugs bound to an antibody.

Description

Antibody-drug conjugate containing a heterologous drug and method for preparing the same

[0001] The present invention relates to an antibody-drug conjugate comprising a heterologous drug bound to a single antibody, and provides an antibody-drug conjugate having a more potent effect while maintaining the safety of each drug.

[0002] Antibody-drug conjugates (ADCs) have traditionally been considered a novel drug modality that can expand the therapeutic window by maximizing the amount of payload used by improving the safety of the payload by attaching a potent payload to an antibody that selectively recognizes cancer cells, while simultaneously maximizing the efficacy by delivering the payload intensively to cancer cells.

[0003] However, the clinical development results of many ADCs have confirmed that the therapeutic window of ADCs does not improve. Specifically, contrary to conventional belief, ADCs are not delivered only to cancer cells to release their payloads, but rather, the payloads are released after non-selective uptake through pinocytosis in all tissues throughout the body. This problem has emerged where the payload side effects appear in all tissues except cancer cells. As a result, even when converted to an ADC form, the amount of payload used does not increase, but rather, the amount of payload used remains the same, and the therapeutic effect is improved even when the same amount of payload is used.

[0004] However, this has resulted in limitations in the potency and usage of the payload used, and even if a large amount of a strong cytotoxic anticancer drug is used, a very small portion of cancer cells are not completely killed and survive, creating anticancer drug-resistant cancer cells. This has resulted in problems such as incomplete therapeutic effects or development of resistance despite the use of ADCs.

[0005] Traditionally, this problem has been solved through the combination of anticancer drugs with different mechanisms of action. This approach, which minimizes the development of resistant cancer cells by simultaneously treating cancer cells that survive the use of a single anticancer drug with a different mechanism of action, has become the standard in chemotherapy. This approach has also recently been increasing in cancer treatment using ADCs. In particular, the combination of a topoisomerase I inhibitor ADC, which induces DNA damage, with another targeted anticancer drug that can amplify its effect (e.g., PARP inhibitors, ATM / ATR / CHK1 / CHK2 inhibitors), or the combination of a topoisomerase I inhibitor ADC with a cytotoxic anticancer drug with a different mechanism of action (e.g., paclitaxel) has been widely studied recently.

[0006] The efficacy of this combination therapy is maximized when there is a difference between the maximum tolerated dose (MTD) and the maximum efficacious dose (MED) of a specific cytotoxic anticancer agent, especially when the relationship MTD > MED holds. As described above, cytotoxic anticancer agents generally increase efficacy up to a certain dose (MED) when treating specific cancer cells. However, above the MED, cell death is not further promoted and saturation occurs. However, because normal cells in the body can tolerate the effects of anticancer agents up to the MTD, the difference in MTD and MED can be utilized to co-administer anticancer agents with different mechanisms of action, thereby securing even better anticancer efficacy. In this case, the first and second anticancer agents should not have a synergistic mechanism that amplifies toxic effects, and the side effect profiles of the two anticancer agents should be different, if possible, to ensure sufficient safety.

[0007] Accordingly, the inventors of the present invention developed an antibody-drug conjugate that has a more potent effect while maintaining the safety of each drug, with the aim of improving the instability caused by co-administration of drugs.

[0008] To solve the above-mentioned problems, the present invention seeks to provide a carrier-drug conjugate that can contain heterogeneous drugs while maintaining the stability of each drug and enhancing its efficacy.

[0009] Although methods and materials similar or equivalent to those disclosed herein can be used in the practice or testing of the embodiments disclosed herein, certain preferred methods, configurations, devices, and materials are disclosed herein. However, it should be understood that the present invention is not limited to the specific molecules, compositions, methodologies, or protocols disclosed herein, as these may vary based on routine experimentation and optimization. It should be understood that the terminology used herein is for the purpose of describing particular versions or embodiments only and is not intended to limit the scope of the embodiments disclosed herein.

[0010] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. However, in the event of a conflict, the present specification, including its definitions, shall prevail. Accordingly, the following definitions shall apply in the context of the embodiments disclosed herein.

[0011] The overall DAR value used herein does not necessarily mean an integer. The DAR value includes a value with a margin of error of approximately 15% of the defined integer value. For example, an overall DAR of 8 may mean an average value through rounding to include a margin of error, and may mean, but is not limited to, 7.6 to 8.4, 7.7 to 8.3, 7.8 to 8.2, or 7.9 to 8.1. In addition, DAR 1 does not necessarily mean an integer. As non-limiting examples, DAR 1 may mean 0.8 to 1.2, 0.9 to 1.1, 0.92 to 1.08, or 0.95 to 1.05.

[0012] Unless otherwise indicated, all numbers expressing sizes, amounts, and physical properties of features used in this specification and claims are to be understood as being modified in all instances by the term "about." Accordingly, unless indicated to the contrary, the numerical parameters set forth in this specification and attached claims are approximations that may vary depending on the desired properties sought to be obtained by those skilled in the art utilizing the teachings disclosed herein. Preferably, "about" can mean ±15% of the stated value.

[0013] As used herein, the term "comprise" and its linguistic variations mean the presence of the recited feature(s), element(s), method step(s), etc., without excluding the presence of additional feature(s), element(s), method step(s), etc. Conversely, the term "consisting of" and its linguistic variations mean the presence of the recited feature(s), element(s), method step(s), etc., but excludes unrecited feature(s), element(s), method step(s), etc., except for impurities that are generally associated therewith. The phrase "consisting essentially of" refers to the recited feature(s), element(s), method step(s), etc., and feature(s), element(s), method step(s), etc., that do not materially affect the basic characteristics of the composition, system, or method. Many implementations herein are described using open-ended "comprising" language. Such implementations alternatively include a number of closed “consisting of” and / or “consisting essentially of” implementations that could be claimed or described using such language.

[0014] To solve the above problem, a first aspect of the present invention provides an antibody for antibody-drug conjugates to which one or more drugs are bound, wherein the antibody is provided to which the first drug is bound at a ratio of about DAR 1. The antibody of the present invention may have a knob-in-hole structure so that the first drug can be bound at a ratio of about DAR 1 (or a ratio of antibody:drug of about 1:1).

[0015] The present invention can form an antibody-drug conjugate in which drugs are bound at a desired ratio by additionally binding a heterologous drug (e.g., a second drug) to an antibody to which a first drug is bound via DAR1. In particular, when the DAR ratio of the first drug and the second drug is 1:n, the technical task of the present invention can be effectively achieved. Here, the 1:n ratio specifically includes ratios such as 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, and 1:9, and by securing the optimal dosage of each drug according to such specific ratios, an antibody-dual drug conjugate that satisfies both therapeutic efficacy and safety can be provided.

[0016] The present invention also provides a carrier-drug conjugate (antibody-drug conjugate), comprising a carrier (particularly, an antibody); and a first drug and a second drug linked to the carrier. In the present invention, the antibody-drug conjugate in which the first drug and the second drug are linked is also referred to as an 'antibody-dual drug conjugate' or 'AD2C'. In one embodiment of the present invention, the first drug and the second drug are different from each other, the DAR ratio of the first drug and the second drug is 1:5 to 8, and the total DAR of the carrier-drug conjugate is 6 to 9. The first drug and the second drug may each be linked to the carrier via a linker.

[0017] The first drug of the present invention is a microtubule polymerization inhibitory compound, a microtubule inhibitor, and an Auristatin compound (or a derivative thereof) can be used, and any compound having a bystander effect can be used as the first drug of the present invention. Specifically, MMAE (Monomethyl auristatin E), MMAF (Monomethyl auristatin F), MMAU (Monomethyl auristatin U), Auristatin E, DM1 (Emtansine), DM4, Eribulin, SC209, Chemical Formula 1-1, Aur0101, Duostatin 5, etc. can be used without limitation, and compounds having a structure in which the bystander effect is controlled within an optimal range by controlling a specific position of these compounds can also be used in the present invention.

[0018] [Chemical Formula 1-1]

[0019]

[0020] The linker used to attach the first drug of the present invention to the carrier (antibody) may be a linker that is easily cleaved by an enzyme selectively activated within a cancer cell or in the tumor microenvironment to release the payload. Specifically, a GGFG linker, an AAA linker, a Val-Cit linker, a Val-Ala linker, a Glucuronidase-cleaved linker that is cleaved by other enzymes, a Legumarin-cleaved linker, etc. may also be readily used. In addition, a tandem linker that releases the drug only after cleavage by two enzymes rather than one may also be used. Specific examples of linker-drug (payload) combinations that can be used to conjugate the first drug according to the present invention to the carrier (particularly, the antibody) are as shown in the following Chemical Formulas 1 and 2, but are not limited thereto.

[0021] [Chemical Formula 1]

[0022]

[0023] [Chemical Formula 2]

[0024]

[0025]

[0026] The second drug of the present invention is a compound that inhibits topoisomerase I activity, and includes a topoisomerase I inhibitor. Any compound that can strongly inhibit the topoisomerase I enzyme inside cancer cells and bring about a death effect on cancer cells can be used without limitation.

[0027] Specific examples of the second drug of the present invention include DXd, Exatecan, FL118, SN-38, camptothecin series compounds (e.g., compounds of Chemical Formula 3 and Chemical Formula 4), compounds of Chemical Formula 4-1 and Chemical Formula 4-2 and derivatives thereof, Belotecan and derivatives thereof, Topotecan and derivatives thereof, etc. More preferred examples include compounds represented by Chemical Formula 3 or Chemical Formula 4. When the compound represented by Chemical Formula 3 or Chemical Formula 4 is selected as the second drug and is conjugated to a carrier together with the first drug described above, the first drug and the second drug can provide improved anticancer efficacy compared to combined administration of an antibody-drug conjugate each linked to a carrier.

[0028] [Chemical Formula 3]

[0029]

[0030] [Chemical Formula 4]

[0031]

[0032] [Chemical Formula 4-1]

[0033]

[0034] [Chemical Formula 4-2]

[0035]

[0036] The linker used to attach the second drug of the present invention to the carrier (particularly, the antibody) may be a linker that is easily cleaved by an enzyme selectively activated within a cancer cell or in the tumor microenvironment to release the payload. Specifically, a GGFG linker, an AAA linker, a Val-Cit linker, a Val-Ala linker, a Glucuronidase-cleaved linker that is cleaved by other enzymes, a Legumarin-cleaved linker, etc. may be used. In addition, a tandem linker that releases the drug only after cleavage by two enzymes rather than one may also be used. Specific examples of linker-drug (payload) combinations that can be used to conjugate the first drug according to the present invention to the carrier are as shown in the following chemical formulae 5 to 8, but are not limited thereto.

[0037] [Chemical Formula 5]

[0038]

[0039] [Chemical Formula 6]

[0040]

[0041] [Chemical Formula 6-1]

[0042]

[0043] [Chemical Formula 6-2]

[0044]

[0045] [Chemical Formula 7]

[0046]

[0047] [Chemical Formula 7-1]

[0048]

[0049] [Chemical Formula 8]

[0050]

[0051] [Chemical Formula 8-1]

[0052]

[0053] The compounds corresponding to the first and second drugs according to the present invention include not only the above-mentioned compounds or isomers thereof, but also pharmaceutically acceptable salts thereof, solvates thereof, and prodrugs thereof.

[0054] In this specification, pharmaceutically acceptable salts refer to salts commonly used in the pharmaceutical industry, and include, for example, salts of inorganic ions including sodium, potassium, calcium, magnesium, lithium, copper, manganese, zinc, and iron, and salts of inorganic acids such as hydrochloric acid, phosphoric acid, and sulfuric acid, and salts of organic acids such as ascorbic acid, citric acid, tartaric acid, lactic acid, maleic acid, malonic acid, fumaric acid, glycolic acid, succinic acid, propionic acid, acetic acid, orotate acid, and acetylsalicylic acid, and salts of amino acids such as lysine, arginine, and guanidine. In addition, there are salts of organic ions such as tetramethyl ammonium, tetraethyl ammonium, tetrapropyl ammonium, tetrabutyl ammonium, benzyl trimethyl ammonium, and benzethonium that can be used in pharmaceutical reactions, purification, and separation processes, but are not limited thereto.

[0055] "Prodrug" has the meaning used in the relevant technical field. For example, it refers to a drug that is converted from an inactive compound to an active form through drug metabolism in the body. For example, it refers to a compound that is chemically modified from a physiologically active substance or therapeutically active organic compound and designed to liberate or release the parent compound in the body under enzymatic or other conditions. A prodrug is converted into the desired compound in the body after administration. Chemical modifications are applied to drugs that, despite being useful, have unsuitable properties such as side effects, stability, solubility, absorbability, and duration of action, making them clinically viable.

[0056] "Solvate" means a compound or an isomer thereof and a pharmaceutically acceptable salt thereof, which further comprises a stoichiometric or non-stoichiometric amount of a solvent bound by non-covalent intermolecular forces. If the solvent is water, the solvate is a hydrate.

[0057] In the present invention, the "linker" should be stable in the bloodstream to prevent the drug from being separated from a carrier such as an antibody, and maintain its structure until it reaches a target such as an antigen, thereby minimizing damage to normal tissues. Ideally, the antibody-drug conjugate should be stable when circulated throughout the body, but should be cleaved in the target cell to appropriately release the cytotoxic drug, thereby safely delivering the drug to the target, so that the antibody-drug conjugate has both efficacy and safety.

[0058] In the carrier-drug conjugate of the present invention, the drug can be linked to the linker at an appropriate site as long as its properties, such as anticancer activity, do not change.

[0059] In the present invention, the linker may be in a form that is cleavable under specific intracellular environments and / or conditions, i.e., such that the drug can be released from the antibody through cleavage of the linker in the intracellular environment.

[0060] For example, the linker can be cleaved by a cleavage agent present in the intracellular environment, for example, in a lysosome or an endosome, and can be a peptide linker that can be cleaved by an intracellular peptidase or protease enzyme, for example, a lysosomal or endosomal protease. Typically, the peptide linker has a length of at least two amino acids. The cleavage agent can include cathepsin B and cathepsin D, plasmin, and hydrolyzes the peptide to release the drug into the target cell. The peptide linker can be cleaved by a thiol-dependent protease cathepsin-B, which is highly expressed in cancer tissues, and examples thereof include, but are not limited to, Gly-Gly-Phe-Gly (GGFG), Phe-Leu, or Gly-Phe-Leu-Gly linkers. Additionally, the peptide linker may be cleaved by, for example, an intracellular protease, and may be a Val-Cit linker or a Phe-Lys linker.

[0061] In the present invention, the cleavable linker is pH-sensitive and may be susceptible to hydrolysis at a specific pH value. Generally, a pH-sensitive linker indicates that it can be hydrolyzed under acidic conditions. For example, it may be an acid-labile linker that can be hydrolyzed in lysosomes, such as a hydrazone, a semicarbazone, a thiosemicarbazone, a cis-aconitic amide, an orthoester, an acetal, a ketal, etc.

[0062] Additionally, in the present invention, the linker may be cleaved under reducing conditions, and for example, a disulfide linker may correspond thereto. Various disulfide bonds may be formed using SATA (Nsuccinimidyl-S-acetylthioacetate), SPDP (N-succinimidyl-3-(2-pyridyldithio)propionate), SPDB (Nsuccinimidyl-3-(2-pyridyldithio)butyrate), and SMPT (N-succinimidyl-oxycarbonyl-alpha-methyl-alpha-(2-pyridyl-dithio) toluene).

[0063] The above linker may include a beta-glucuronide linker that is recognized and hydrolyzed by beta-glucuronidase, which is present in large numbers in lysosomes or is overexpressed in some tumor cells. For example, a beta-glucuronide linker disclosed in Korean Patent Publication No. 2015-0137015, such as a beta-glucuronide linker containing a self-immolative group, may be used.

[0064] Additionally, the linker may be, for example, a non-cleavable linker, and the drug is released through an antibody hydrolysis step, producing, for example, an amino acid-linker-drug conjugate. This type of linker may be a thioether group or a maleimidocaproyl group, and may maintain stability in blood.

[0065] According to a preferred specific embodiment, the linker according to the present invention may include GGFG. According to one embodiment of the present invention, a portion of at least one amino acid side chain constituting the linker may be substituted with a hydrophilic functional group capable of separation under specific conditions. The hydrophilic functional group is preferably a monovalent hydrophilic functional group, and examples thereof include, but are not limited to, esters or carbonates having β-glucuronide or PEG (Polyethylene Glycol) groups having 3 to 100 ethylene glycol repeating units.

[0066] More preferably, the linker may have a structure of chemical formula 9 or chemical formula 10, but is not limited thereto.

[0067] [Chemical Formula 9]

[0068]

[0069] [Chemical Formula 9-1]

[0070]

[0071] [Chemical Formula 10]

[0072]

[0073] In the above chemical formula 10, n can be an integer from 3 to 10.

[0074]

[0075] In the present invention, when the carrier in the carrier-drug conjugate is an antibody, the drug and / or drug-linker may be randomly conjugated via lysine within the antibody, or via cysteine ​​exposed upon reduction of the disulfide bond chain. In some cases, the linker-drug may be conjugated via a genetically engineered tag, for example, a lysine or cysteine ​​present in a peptide or protein.

[0076]

[0077] In the present invention, a "carrier" means a substance having the ability to selectively and specifically deliver a drug according to the present invention to a target site, for example, a cancer cell, and may be, but is not limited to, an antibody, a peptide, a lipobody, and / or an aptamer, and is preferably an antibody.

[0078] In the present invention, examples of targets to which the carrier binds, for example, antigens, include, but are not limited to, antigens selectively distributed on the surface of cancer cells, such as Her2, FolR, and PSMA, and antigens overexpressed in cancer cells that are also distributed in small numbers in normal tissues, such as Trop2.

[0079] 예시적인 암 세포 표적 항원은 5T4, ABL, ABCF1, ACVR1, ACVR1B, ACVR2, ACVR2B, ACVRL1, ADORA2A, AFP, Aggrecan, AGR2, AICDA, AIF1, AIGI, AKAP1, AKAP2, ALCAM, ALK, AMH, AMHR2, ANGPT1, ANGPT2, ANGPTL3, ANGPTL4, ANPEP, APC, APOCl, AR, 아로마타제 (aromatase), ASPH, ATX, AX1, AXL, AZGP1 (zinc-a-glycoprotein), B4GALNT1, B7, B7.1, B7.2, B7-H1, B7-H3, B7-H4, B7-H6, BAD, BAFF, BAG1, BAI1, BCR, BCL2, BCL6, BCMA, BDNF, BLNK, BLR1 (MDR15), BIyS, BMP1, BMP2, BMP3B (GDFIO), BMP4, BMP6, BMP8, BMP10, BMPR1A, BMPR1B, BMPR2, BPAG1 (플렉틴), BRCA1, C19orflO (IL27w), C3, C4A, C5, C5R1, CA6, CA9, CANT1, CAPRIN-1, CASP1, CASP4, CAV1, CCBP2 (D6 / JAB61), CCL1 (1-309), CCLI1 (에오탁신), CCL13 (MCP-4), CCL15 (MIP-Id), CCL16 (HCC-4), CCL17 (TARC), CCL18 (PARC), CCL19 (MIP-3b), CCL2 (MCP-1), MCAF, CCL20 (MIP-3a), CCL21 (MEP-2), SLC, exodus-2, CCL22(MDC / STC-I), CCL23 (MPIF-I), CCL24 (MPIF-2 / 에오탁신-2), CCL25 (TECK), CCL26(에오탁신-3), CCL27 (CTACK / ILC), CCL28, CCL3 (MIP-Ia), CCL4 (MIPIb), CCL5(RANTES), CCL7 (MCP-3), CCL8 (mcp-2), CCNA1, CCNA2, CCND1,CCNE1, CCNE2, CCR1 (CKR1 / HM145), CCR2 (mcp-IRB / RA), CCR3 (CKR3 / CMKBR3), CCR4, CCR5(CMKBR5 / ChemR13), CCR6 (CMKBR6 / CKR-L3 / STRL22 / DRY6), CCR7 (CKR7 / EBI1), CCR8 또는 CDw198 (CMKBR8 / TERI / CKR-L1), CCR9 (GPR-9-6), CCRL1 (VSHK1), CCRL2 (L-CCR), CD13, CD164, CD19, CDH6, CDIC, CD2, CD20, CD21, CD200, CD22, CD23, CD24, CD27, CD28, CD29, CD3, CD33, CD35, CD37, CD38, CD3E, CD3G, CD3Z, CD4, CD40, CD40L, CD44, CD45RB, CD47, CD52, CD56, CD69, CD70, CD72, CD74, CD79A, CD79B, CD8, CD80, CD81, CD83, CD86, CD97, CD99, CD117, CD125, CD137, CD147, CD179b, CD223, CD279, CD152, CD274, CDH1 (E-카드헤린), CDH1O, CDH12, CDH13, CDH18, CDH19, CDH2O, CDH3, CDH5, CDH7, CDH8, CDH9, CDH17, CDK2, CDK3, CDK4, CDK5, CDK6, CDK7, CDK9, CDKN1A (p21Wap1 / Cip1), CDKN1B (p27Kip1), CDKN1C, CDKN2A (p16INK4a), CDKN2B, CDKN2C, CDKN3, CEA, CEACAM5, CEACAM6, CEBPB, CERI, CFC1B, CHGA, CHGB, 키티나제 (Chitinase), CHST1O, CIK, CKLFSF2, CKLFSF3, CKLFSF4, CKLFSF5, CKLFSF6, CKLFSF7, CKLFSF8, CLDN3, CLDN6, CLDN7 (클라우딘-7), CLDN18, CLEC5A, CLEC6A,CLEC11A, CLEC14A, CLN3, CLU (클러스테린), CMKLR1, CMKOR1 (RDC1), CNR1, C-MET, COL18A1, COLIA1, COL4A3, COL6A1, CR2, Cripto, CRP, CSF1 (M-CSF), CSF2 (GM-CSF), CSF3 (GCSF), CTAG1B (NY-ESO-1), CTLA4, CTL8, CTNNB1 (b-카테닌), CTSB (카텝신 B), CX3CL1 (SCYD1), CX3CR1 (V28), CXCL1 (GRO1), CXCL1O (IP-IO), CXCLI1 (1-TAC / IP-9), CXCL12 (SDF1), CXCL13, CXCL14, CXCL16, CXCL2 (GRO2), CXCL3 (GRO3), CXCL5 (ENA-78 / LIX), CXCL6 (GCP-2), CXCL9 (MIG), CXCR3 (GPR9 / CKR-L2), CXCR4, CXCR6 (TYMSTR / STRL33 / Bonzo), CYB5, CYC1, CYSLTR1, DAB2IP, DES, DKFZp451J0118, DLK1, DNCL1, DPP4, E2F1, Engel, Edge, Fennel, EFNA3, EFNB2, EGF, EGFR, ELAC2, ENG, Enola, ENO2, ENO3, EpCAM, EPHA1, EPHA2, EPHA3, EPHA4, EPHA5, EPHA6, EPHA7, EPHA8, EPHA9, EPHA10, EPHB1, EPHB2, EPHB3, EPHB4, EPHB5, EPHB6, EPHRIN-A1, EPHRIN-A2, EPHRINA3, EPHRIN-A4, EPHRIN-A5, EPHRIN-A6, EPHRIN-B1, EPHRIN-B2, EPHRIN-B3, EPHB4, EPG, ERBB2 (HER-2), ERBB3, ERBB4, EREG, ERK8, 에스트로겐 수용체, Earl, ESR2, F3 (TF), FADD, FAP, 파르네실트란스퍼라제, FasL, FASNf FCER1A, FCER2, FCGR3A, FGF,FGF1 (aFGF), FGF10, FGF1 1, FGF12, FGF12B, FGF13, FGF14, FGF16, FGF17, FGF18, FGF19, FGF2 (bFGF), FGF20, FGF21, FGF22, FGF23, FGF3 (int-2), FGF4 (HST), FGF5, FGF6 (HST-2), FGF7 (KGF), FGF8, FGF9, FGFR1, FGFR2, FGFR3, FGFR4, FIGF (VEGFD), FIL1 (EPSILON), FBL1 (ZETA), FLJ12584, FLJ25530, FLRT1 (fibronectin), FLT1, FLT-3, FOLR1, FOS, FOSL1(FRA-1), FR-alpha, FY (DARC), GABRP (GABAa), GAGEB1, GAGEC1, GALNAC4S-6ST, GATA3, GD2, GD3, GDF5, GFI1, GFRA1, GGT1, GM-CSF, GNAS1, GNRH1, GPC1, GPC3, GPNB, GPR2 (CCR10), GPR31, GPR44, GPR81 (FKSG80), GRCC1O (C1O), GRP, GSN (Gelsolin), GSTP1, GUCY2C, HAVCR1, HAVCR2, HDAC, HDAC4, HDAC5, HDAC7A, HDAC9, Hedgehog, HER3, HGF, HIF1A, HIP1, histamine and histamine receptor, HLA-A, HLA-DR, HLA-DRA, HLA-E, HM74, HMOXI, HSP90, HUMCYT2A, ICEBERG, ICOSL, ID2,IFN-a,IFNA1,IFNA2,IFNA4,IFNA5,EFNA6,BFNA7,IFNB1,IFNgamma,IFNW1,IGBP1,IGF1,IGFIR,IGF2,IGFBP2,IGFBP3,IGFBP6,DL-1,ILIO,ILIORA,ILIORB,IL-1,IL1R1 (CD121a), IL1R2 (CD121b), IL-IRA, IL-2, IL2RA (CD25), IL2RB (CD122),IL2RG(CD132), IL-4, IL-4R(CD123), IL-5, IL5RA(CD125), IL3RB(CD131), IL-6, IL6RA, (CD126), IR6RB(CD130), IL-7, IL7RA(CD127), IL-8, CXCR1 (IL8RA), CXCR2, (IL8RB / CD128), IL-9, IL9R(CD129), IL-10, IL10RA(CD210), IL10RB(CDW210B), IL-11, IL11RA, IL-12, IL-12A, IL-12B, IL-12RB1, IL-12RB2, IL-13, IL13RA1, IL13RA2, IL14, IL15, IL15RA, IL16, IL17, IL17A, IL17B, IL17C, IL17R, IL18, IL18BP, IL18R1, IL18RAP, IL19, ILIA, ILIB, ILIF10, ILIF5, IL1F6, ILIF7, IL1F8, DL1F9, ILIHYI, ILIR1, IL1R2, ILIRAP, ILIRAPLI, ILIRAPL2, ILIRL1, IL1RL2, ILIRN, IL20, IL20RA. IL21R, IL22, IL22R, IL22RA2, IL23, DL24, IL25, IL26, IL27, IL28A, IL28B, IL29, IL2RA, IL2RB, IL2RG, IL3, IL30, IL3RA, IL4, 1L4, IL6ST (당단백자 130), ILK, INHA, INHBA, INSL3, INSL4. IRAK1, IRAK2, ITGA1, ITGA2, ITGA3, ITGA6 (α6 인하그린), ITGAV, ITGB3, ITGB4 (β4 인하그린), JAG1, JAK1, JAK3, JTB, JUN, K6HF, KAI1, KDR, KIT, KITLG, KLF5 (GC Box BP), KLF6, KLK10, KLK12, KLK13, KLK14, KLK15, KLK3, KLK4. KLK5, KLK6, KLK9, KRT1, KRT19 (キ라틴 19), KRT2A,KRTHB6 (hair-specific type II keratin), L1CAM, LAG3, LAMA5, LAMP1, LEP (leptin), Lewis Y antigen ("LeY"), LILRB1, Lingo-p75, Lingo-Troy, LGALS3BP, LRRC15, LPS, LTA (TNF-b), LTB, LTB4R (GPR16), LTB4R2, LTBR, ​​LY75, LYPD3, MACMARCKS, MAG or OMgp, MAGEA3, MAGEA6, MAP2K7 (c-Jun), MCP-1, MDK, MIB1, midkine, MIF, MISRII, MJP-2, MLSN, MK, MKI67 (Ki-67), MMP2, MMP9, MS4A1, MSMB, MT3 (metallothionectin-UI), mTOR, MTSS1, MUC1 (mucin), MUC16, MYC, MYD88, NCK2, NCR3LG1, neurocan, NFKBI, NFKB2, NGFB (NGF), NGFR, NgR-Lingo, NgRNogo66, (Nogo), NgR-p75, NgR-Troy, NMEI (NM23A), NOTCH, NOTCH1, NOTCH3, NOX5, NPPB, NROB1, NROB2, NRID1, NR1D2, NR1H2, NR1H3, NR1H4, NR112, NR113, NR2C1, NR2C2, NR2E1, NR2E3, NR2F1, NR2F2, NR2F6, NR3C1, NR3C2, NR4A1, NR4A2, NR4A3, NR5A1, NR5A2, NR6A1, NRP1, NRP2, NT5E, NTN4, NY-ESO1, ODZI, OPRDI, P2RX7, PAP, PART1, PATE, PAWR, P-cadherin, PCA3, PCD1, PD-L1, PCDGF, PCNA, PDGFA, PDGFB, PDGFRA, PDGFRB, PECAMI, L1-CAM, peg-asparaginase, PF4 (CXCL4), PGF, PGR, phosphacan, PIAS2, PI3 kinase, PIK3CG,PLAU (uPA), PLG, PLXDCI, PKC, PKC-beta, PPBP (CXCL7), PPID, PR1, PRAME, PRKCQ, PRKD1, PRL, PROC, PROK2, PSAP, PSCA, PSMA, PTAFR, PTEN, PTHR2, PTGS2 (COX-2), PTN, PVRIG, RAC2 (P21Rac2), RANK, RANK ligand, RARB, RGS1, RGS13, RGS3, RNFI1O (ZNF144), Ron, ROBO2, ROR1, RXR, S100A2, SCGB 1D2 (lipophilin B), SCGB2A1 (mammaglobin 2), SCGB2A2 (mammaglobin 1), SCYE1 (endothelial monocyte-activating cytokine), SDF2, SERPENA1, SERPINA3, SERPINB5 (MASPIN), SERPINEI (PAI-I), SERPINFI, SHIP-1, SHIP-2, SHB1, SHB2, SHBG, SfcAZ, SLAMF7, SLC2A2, SLC33A1, SLC43A1, SLC44A4, SLC34A2, SLIT2, SPP1, SPRR1B (Spr1), ST6GAL1, ST8SIA1, STAB1, STATE, STEAP, STEAP2, TB4R2, TBX21, TCP1O, TDGF1, TEK, TGFA, TGFB1, TGFB1I1, TGFB2, TGFB3, TGFBI, TGFBR1, TGFBR2, TGFBR3, THIL, THBS1 (thrombospondin-1), THBS2, THBS4, THPO, TIE (Tie-1), TIMP3, tissue factor, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TLR11, TNF, TNF-a, TNFAIP2 (B94), TNFAIP3, TNFRSFI1A, TNFRSF1A, TNFRSF1B, TNFRSF21, TNFRSF5, TNFRSF6 (Fas), TNFRSF7, TNFRSF8, TNFRSF9, TNFSF1O (TRAIL),TNFRSF10A, TNFRSF10B, TNFRSF12A, TNFRSF17, TNFSF1 1 (TRANCE), TNFSF12 (APO3L), TNFSF13 (April), TNFSF13B, TNFSF14 (HVEM-L), TNFRSF14 (HVEM), TNFSF15 (VEGI), TNFSF18, TNFSF4 (OX40 ligand), TNFSF5 (CD40 ligand), TNFSF6 (FasL), TNFSF7 (CD27 ligand), TNFSF8 (CD30 ligand), TNFSF9 (4-1BB ligand), TOLLIP, Toll-like receptor, TOP2A (topoisomerase Iia), TP53, TPM1, TPM2, TRADD, TRAF1, TRAF2, TRAF3, TRAF4, TRAF5, TRAF6, TRKA, TREM1, TREM2, TROP2, TRPC6, TSLP, TWEAK, Tyrosinase, uPAR, VEGF, VEGFB, VEGFC, versican, VHL C5, VLA-4, WT1, Wnt-1, XCL1 (lymphotactin), XCL2 (SCM-Ib), XCRI (GPR5 / CCXCR1), YY1, ZFPM2, CLEC4C (BDCA-2, DLEC, CD303, CDH6, CLECSF7), CLEC4D (MCL, CLECSF8), CLEC4E (Mincle), CLEC6A (Dectin-2), CLEC5A (MDL-1, CLECSF5), CLEC1B (CLEC-2), CLEC9A (DNGR-1), CLEC7A (Dectin-1), CLEC11A, PDGFRa, SLAMF7, GP6 (GPVI), LILRA1 (CD85I), LILRA2 (CD85H, ILT1), LILRA4 (CD85G, ILT7), LILRA5 (CD85F, ILT11), LILRA6 (CD85b, ILT8), LILRB1, NCR1 (CD335, LY94, NKp46), NCR3 (CD335, LY94, NKp46), NCR3 (CD337, NKp30), OSCAR, TARM1,CD30, CD300C, CD300E, CD300LB (CD300B), CD300LD (CD300D), KIR2DL4 (CD158D), KIR2DS, KLRC2 (CD159C, NKG2C), KLRK1 (CD314, NKG2D), NCR2 (CD336, NKp44), PILRB, SIGLEC1 (CD169, SN), SIGLEC5, SIGLEC6, SIGLEC7, SIGLEC8, SIGLEC9, SIGLEC10, SIGLEC11, SIGLEC12, SIGLEC14, SIGLEC15 (CD33L3), SIGLEC16, SIRPA, SIRPB1 (CD172B), TREM1 (CD354), TREM2, KLRF1 (NKp80), 17-1A, SLAM7, MSLN, CTAG1B / NY-ESO-1, MAGEA3 / A6, ATP5I (Q06185), OAT (P29758), AIFM1 (Q9Z0X1), AOFA (Q64133), MTDC (P18155), CMC1 (Q8BH59), PREP (Q8K411), YMEL1 (O88967), LPPRC (Q6PB66), LONM (Q8CGK3), ACON (Q99KI0), ODO1 (Q60597), IDHP (P54071), ALDH2 (P47738), ATPB (P56480), AATM (P05202), TMM93 (Q9CQW0), ERGI3 (Q9CQE7), RTN4 (Q99P72), CL041 (Q8BQR4), ERLN2 (Q8BFZ9), TERA (Q01853), DAD1 (P61804), CALX (P35564), CALU (O35887), VAPA (Q9WV55), MOGS (Q80UM7), GANAB (Q8BHN3), ERO1A (Q8R180), UGGG1 (Q6P5E4), P4HA1 (Q60715), HYEP (Q9D379), CALR (P14211), AT2A2 (O55143), PDIA4 (P08003), PDIA1 (P09103), PDIA3 (P27773), PDIA6 (Q922R8),CLH (Q68FD5), PPIB (P24369), TCPG (P80318), MOT4 (P57787), NICA (P57716), BASI (P18572), VAPA (Q9WV55), ENV2 (P11370), VAT1 (Q62465), 4F2 (P10852), ENOA (P17182), ILK (O55222), GPNMB (Q99P91), ENV1 (P10404), ERO1A (Q8R180), CLH (Q68FD5), DSG1A (Q61495), AT1A1 (Q8VDN2), HYOU1 (Q9JKR6), TRAP1 (Q9CQN1), GRP75 (P38647), ENPL (P08113), CH60 (P63038), or CH10 (Q64433), but is not limited thereto. In addition, the target antigen may be an antigen that is distributed more than 10 times more abundantly in cancer cells than in normal cells.

[0080] Specifically, when the carrier of the present disclosure is an antibody, "antibody-drug conjugate" means a linkage of the antibody or an antigen-binding fragment thereof with another anti-tumor compound, such as a chemotherapeutic agent, toxin, immunotherapeutic agent, imaging probe, or the like. The linkage may be a covalent bond.

[0081] The term "antibody," as used herein, refers to an immunoglobulin molecule that specifically binds to an antigen. An antibody may be an intact immunoglobulin derived from a natural or recombinant source, or may be an immunoreactive portion of an intact immunoglobulin. Antibodies are typically tetramers of immunoglobulin molecules. The antibodies of the present invention may exist in various forms, including, for example, polyclonal antibodies, monoclonal antibodies, single-chain antibodies, and humanized antibodies.

[0082] The term "antigen-binding fragment thereof" as used herein means a portion of a full-length antibody, generally an antigen-binding or variable region thereof. Examples of antibody fragments include Fab fragments, Fab' fragments, Fab'-SH, Fv fragments, scFv fragments, F(ab')2 fragments, VL fragments, VH fragments, ScFv-Fc fragments, (ScFv)2-Fc fragments, diabodies, linear antibodies, fragments generated from Fab expression libraries, anti-idiotypic (anti-Id) antibodies, CDRs (complementarity determining regions), and epitope-binding fragments of any of the above that immunospecifically bind to cancer cell antigens, viral antigens, or microbial antigens, single-chain antibody molecules, and multispecific antibodies formed from antibody fragments.

[0083] Non-limiting examples of antibodies in the present invention include Urelumab, Utomilumab, Bebtelovimab, Aducanumab, Bapinezumab, Crenezumab, Donanemab, Gantenerumab, Lecanemab, Solanezumab, Nesvacumab, Evinacumab, Enoblituzumab, Omburtamab, Belimumab, Ianalumab, Tabalumab, Bertilimumab, Mogamulizumab, Leronlimab (Leronlimab), Siplizumab, Foralumab, Muromonab-CD3, Otelixizumab, Teplizumab, Ibalizumab, Tregalizumab, Zanolimumab, Itolizumab, Efalizumab, Inebilizumab, Tafasitamab, Tositumomab, Ocrelizumab, Ofatumumab, Rituximab, Ublituximab, Veltuzumab, Epratuzumab Basiliximab, Daclizumab, Varlilumab, Lulizumab, Iratumumab, Lintuzumab, Daratumumab, Felzartamab, Isatuximab,Mezagitamab, Bleselumab, Dacetuzumab, Iscalimab, Lucatumumab, Mitazalimab, Sotigalimab, Dapirolizumab, Apamistamab, Ligufalimab, Magrolimab, Alemtuzumab, Crizanlizumab, Inclacumab, Cusatuzumab, Oleclumab, Milatuzumab, Galiximab, Carotuximab, Adecatumumab Adecatumumab, Eptinezumab, Erenumab, Fremanezumab, Galcanezumab, Zolbetuximab, Onartuzumab, Eculizumab, Pozelimab, Ravulizumab, Lacnotuzumab, Axatilimab, Cabiralizumab, Emactuzumab, Ipilimumab, Quavonlimab, Tremelimumab, Zalifrelimab, Cetuximab, Depatuxizumab (Depatuxizumab), Futuximab, Imgatuzumab, Matuzumab, Modotuximab, Necitumumab, Nimotuzumab, Panitumumab, Tomuzotuximab,Zalutumumab, Batoclimab, Nipocalimab, Rozanolixizumab, Burosumab, Farletuzumab, Dinutuximab, Naxitamab, Ragifilimab, Gimsilumab, Lenzilumab, Mavrilimumab, Namilumab, Otilimab, Plonmarlimab, Codrituzumab, Margetuximab, Pertuzumab, Trastuzumab, Datopotamab Patritumab, Seribantumab, Duligotuzumab, Ficlatuzumab, Rilotumumab, Alomfilimab, Anifrolumab, Emapalumab, Ligelizumab, Omalizumab, Cixutumumab, Dalotuzumab, Figitumumab, Ganitumab, Teprotumumab, Bermekimab, Canakinumab, Gevokizumab, Briakinumab, Ustekinumab Anrukinzumab, Cendakimab, Lebrikizumab, Tralokinumab, Brodalumab, Bimekizumab, Ixekizumab,Secukinumab, Brazikumab, Guselkumab, Mirikizumab, Risankizumab, Tildrakizumab, Nemolizumab, Imsidolimab, Spesolimab, Pascolizumab, Dupilumab, Defemokimab, Mepolizumab, Reslizumab, Benralizumab, Clazakizumab, Olokizumab, Siltuximab, Sirukumab, Ziltivekimab, Revilimab Levilimab, Sarilumab, Satralizumab, Tocilizumab, Abituzumab, Fabezelimab, Fianlimab, Ieramilimab, Relatlimab, Simtuzumab, Abagovomab, Oregovomab, Tanezumab, Ivuxolimab, Rocatinlimab, Tavolimab, Telazorlimab, Vonlerolizumab, Alirocumab, Bococizumab, Ebronuximab (Ebronucimab), Evolocumab, Frovocimab, Ongericimab, Tafolecimab, Dostarlimab, Balstilimab, Camrelizumab,Cemiplimab, Geptanolimab, Nivolumab, Pembrolizumab, Penpulimab, Pidilizumab, Prolgolimab, Retifanlimab, Sasanlimab, Serplulimab, Sintilimab, Spartalizumab, Tislelizumab, Toripalimab, Ezabenlimab, Zimberelimab, Atezolizumab, Avelumab, Cosibelimab, Sugemalimab (Sugemalimab), Durvalumab, Envafolimab, Subratoxumab, Denosumab, Zilovertamab, Elotuzumab, Domvanalimab, Etigilimab, Ociferlimab, Tiragolumab, Vibostolimab, Surzebiclimab, Cobolimab, Sabatolimab, Concizumab, Marstacimab, Adalimumab, Golimumab, Infliximab, Certolizumab (Certolizumab), Conatumumab, Tigatuzumab, Tezepelumab, Gatipotuzumab, Cabiralizumab, Bevacizumab, Brolucizumab,Ranibizumab, Olinvacimab, Icrucumab, Ramucirumab, Caplacizumab, Abrilumab, Etrolizumab, Vedolizumab, Intetumumab, Natalizumab, Obrindatamab, Elranatamab, Linvoseltamab, Teclistamab, Epcoritamab, Glofitamab, Mosunetuzumab, Odronextamab, Flotetuzumab, Bivecotamab (Vibecotamab), Catumaxomab, Cibisatamab, Talquetamab, Ubamatamab, Emfizatamab, Blinatumomab, Amivantamab, Emicizumab, Zenocutuzumab, Zanidatamab, Tibulizumab, Naptumomab, Belantamab, Pivekimab, Praluzatamab, Coltuximab, Denintuzumab, Loncastuximab, Ibritumomab, Inotuzumab (Inotuzumab), Epratuzumab, Moxetumomab, Brentuximab, Gemtuzumab, Vadastuximab, Lorvotuzumab, Polatuzumab,Tusamitamab, Telisotuzumab, Rovalpituzumab, Depatuxizumab, Farletuzumab, Mirvetuximab, Disitamab, Anetumab, Enfortumab, Sacituzumab, Vobarilizumab, Cadonilimab, Vudalimab, Tebotelimab, Ivonescimab, Erfonrilimab, Ozoralizumab, Faricimab, Vanucizumab (Vanucizumab) or navicixizumab, but is not limited thereto, and most preferably trastuzumab.

[0084] The antibody of the present invention may have a knob-in-hole structure. The knob-in-hole technology induces mutations in the CH3 domains of two different Ig heavy chains through genetic manipulation, thereby creating a hole structure in the CH3 domain of one Ig heavy chain and a knob structure in the CH3 domain of another Ig heavy chain, thereby inducing the two Ig heavy chains to form a heterodimer. For a technology for producing an antibody having the knob-in-hole structure, reference may be made to, for example, U.S. Patent No. US 7695936 B2, and the structure, embodiments, and production method of an antibody having a knob-in-hole structure disclosed in the U.S. Patent No. 7695936 B2 may be referred to as a description in the present specification.

[0085] Since the carrier (antibody)-drug conjugate of the present invention includes heterologous drugs, it provides a binding ratio (DAR) that can simultaneously increase the efficacy and stability of the drugs by considering the cytotoxicity and / or properties of the two drugs.

[0086] The DAR of the first drug of the present invention against the antibody is 1 to 2, most preferably 1. The DAR of the second drug of the present invention against the antibody is 4 to 8, preferably 5 to 7. The total DAR of the antibody-drug conjugate of the present invention is 5 to 10, preferably 6 to 9.

[0087] In one embodiment of the present invention, a carrier-drug conjugate is provided, wherein the DAR of the first drug is 1, the DAR of the second drug is 4 to 8 (preferably 5 to 8), and the total DAR is 5 to 9 (preferably 6 to 9). If the total DAR in the carrier-drug conjugate of the present invention is less than 5 or more than 9, the selectivity for target cells is low, the efficacy of each drug is reduced, and toxicity to non-target cells may occur. Furthermore, since different drugs are used, when the total DAR is 5 to 9, the DAR of the first drug should be 1 and the DAR of the second drug should be 4 to 8 in a ratio to increase the efficacy and stability of both drugs simultaneously and produce a synergistic effect. However, if the ratio is exceeded, the therapeutic index will be lowered, and overlapping toxicity of the first or second drug may occur due to the different toxicity mechanisms (non-overlapping toxicity) of each drug.

[0088] The present invention also provides a method for preparing a carrier (antibody)-drug conjugate. The method for preparing an antibody-drug conjugate of the present invention may include the steps of: a) preparing an antibody having a knob-in-hole structure and a free cysteine ​​in a heavy chain having a hole; b) conjugating a first drug to the antibody having the knob-in-hole structure at a ratio of DAR 1; and additionally, c) conjugating a second drug to the antibody to which the first drug is bound by DAR 1. The step b) may include: b-1) a first reduction step of reducing at least some of the disulfide bonds of the antibody having the knob-in-hole structure to form a free thiol group (-SH); and b-2) a first oxidation step of oxidizing the antibody so that only one free thiol group (-SH) at a selected position is maintained; and b-3) a step of conjugating the first drug to the free thiol group (-SH) of the antibody. The contents of the antibody-dual drug conjugate mentioned in this specification can be equally applied to the present manufacturing method, and for the specific manufacturing method, refer to the contents described in FIG. 13.

[0089] A second aspect of the present invention relates to a pharmaceutical composition for preventing or treating cancer, comprising a carrier-drug conjugate comprising an antibody; and a first drug and a second drug linked to the antibody.

[0090] The description of the above carrier-drug conjugate applies equally to the pharmaceutical composition, to the extent that it does not limit its inherent properties.

[0091] The cancer of the present invention includes all cancers that can be treated with a topoisomerase I inhibitor and / or a microtubule-targeting therapeutic agent (inhibitor), and may be a solid cancer or a blood cancer. Specifically, pseudomyxoma, intrahepatic cholangiocarcinoma, hepatoblastoma, liver cancer, thyroid cancer, colon cancer, testicular cancer, myelodysplastic syndrome, glioblastoma, oral cancer, lip cancer, mycosis fungoides, acute myeloid leukemia, acute lymphoblastic leukemia, basal cell carcinoma, ovarian epithelial cancer, ovarian germ cell cancer, ovarian cancer, male breast cancer, brain cancer, pituitary adenoma, multiple myeloma, gallbladder cancer, biliary tract cancer, colon cancer, chronic myeloid leukemia, chronic lymphocytic leukemia, retinoblastoma, choroidal melanoma, ampulla of Vater cancer, bladder cancer, peritoneal cancer, parathyroid cancer, adrenal cancer, paranasal sinus cancer, non-small cell lung cancer, tongue cancer, astrocytoma, small cell lung cancer, pediatric brain cancer, pediatric lymphoma, pediatric leukemia, small intestine cancer, meningioma, esophageal cancer, glioma, renal pelvis cancer, kidney cancer, Including, but not limited to, heart cancer, duodenal cancer, malignant soft tissue cancer, malignant bone cancer, malignant lymphoma, malignant mesothelioma, malignant melanoma, eye cancer, vulvar cancer, ureteral cancer, urethral cancer, cancer of unknown primary site, gastric lymphoma, stomach cancer, gastric carcinoid, gastrointestinal stromal cancer, Wilms' cancer, breast cancer, triple-negative breast cancer (TNBC), sarcoma, penile cancer, pharyngeal cancer, gestational trophoblastic disease, cervical cancer, endometrial cancer, uterine sarcoma, prostate cancer, metastatic bone cancer, metastatic brain cancer, mediastinal cancer, rectal cancer, rectal carcinoid, vaginal cancer, spinal cancer, acoustic neuroma, pancreatic cancer, salivary gland cancer, Kaposi's sarcoma, Paget's disease, tonsil cancer, squamous cell carcinoma, lung adenocarcinoma, lung cancer, lung squamous cell carcinoma, skin cancer, anal cancer, rhabdomyosarcoma, laryngeal cancer, pleural cancer, blood cancer, and thymic cancer. Additionally, the above cancer includes not only primary cancer but also metastatic cancer.

[0092] The carrier-drug conjugate of the present invention or a pharmaceutical composition comprising the same can be administered to a subject in need thereof in a therapeutically effective amount.

[0093] As used herein, "patient," "subject," and "subject" refer to an animal, such as a mammal. In certain embodiments, the patient is a human. In other embodiments, the patient is a non-human animal, such as a dog, cat, domestic animal (e.g., a horse, pig, or donkey), chimpanzee, or monkey.

[0094] The term "therapeutically effective amount" as used herein refers to the amount of the carrier-drug conjugate or pharmaceutical composition containing the same that is effective in treating or preventing cancer. Specifically, a "therapeutically effective amount" means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment, and the effective dosage level can be determined according to factors including the type and severity of the individual, age, sex, type of disease, activity of the drug, sensitivity to the drug, time of administration, route of administration and excretion rate, duration of treatment, concurrently used drugs, and other factors well known in the medical field. The pharmaceutical composition of the present invention can be administered as an individual therapeutic agent or in combination with other therapeutic agents, and can be administered sequentially or simultaneously with commercially available therapeutic agents. And it can be administered singly or in multiple doses. It is important to take all of the above factors into consideration and administer an amount that can achieve the maximum effect with the minimum amount without side effects. Since the carrier-drug conjugate of the present invention exhibits a dose-dependent effect, the administration dosage can be easily determined by those skilled in the art according to various factors such as the patient's condition, age, sex, and complications. Since the effective ingredient of the pharmaceutical composition of the present invention has excellent safety, it can be used even in amounts exceeding the determined dosage.

[0095] The present invention also provides a use of the carrier-drug conjugate for use in the manufacture of a medicament for use in the treatment or prevention of cancer.

[0096] The carrier-drug conjugate for manufacturing a drug can be mixed with pharmaceutically acceptable excipients, diluents, carriers, etc., and can be manufactured into a complex preparation together with other active agents to have a synergistic effect of the active ingredients.

[0097] In this specification, the anticancer effect or therapeutic effect of an anticancer agent may refer to an action that occurs while a patient is suffering from a specific cancer, such as reducing the severity of the cancer, reducing the tumor size, or delaying or slowing the progression of the cancer.

[0098] For example, the anticancer effect of an anticancer drug can be measured in vitro and / or in vivo by measuring the cell viability (the degree of cytotoxicity or changes in cell number) of cancer cells treated with the drug. For example, this can be indirectly confirmed through drug response testing using cell lines or non-clinical animal models (xenografts). Furthermore, the anticancer effect of an anticancer drug can be directly confirmed in cancer patients, and related data can be derived and used as a database. Furthermore, animal model PK parameters and / or toxicity profiles can be considered in parallel when designing dosing guidelines for anticancer drugs.

[0099] The anticancer effect of an anticancer drug is expressed as the % maximum effect of the anticancer drug, which is in vitro data, for example, IC 50 , IC 60 , IC 70 , IC 80 and IC 90 It can be inferred from, and confirmed in nonclinical animal models and clinical cancer patients through in-vivo data such as the drug's peak blood concentration (Cmax) and / or the area under the blood drug concentration-time curve (AUC).

[0100] The reactivity of an anticancer drug refers to the clinical sensitivity in terms of anticancer effect.

[0101] When referring to treatment with anticancer drugs, "sensitivity" and "sensitive" are relative terms referring to the extent to which a compound is effective in alleviating or reducing the progression of the tumor or disease being treated.

[0102] An "effective patient anticancer effect / response" can be, for example, a 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, or greater inhibition of patient response, as measured by any suitable means, such as gene expression, cell counts, assay results, etc.

[0103] In this specification, the dosage is the dose at which the medicinal effect is expected. In the present invention, the medicinal effect may be an anticancer effect. The reactivity (anticancer effect) of an anticancer agent is the degree of response, and the % maximum effect (Maximum effect) of the anticancer agent, for example, IC 50 , IC 60 , IC 70 , IC 80 and IC 90 , the value that exhibits toxicity to normal cells (LC 50 ) may be.

[0104] For example, oral dosage forms can be formulated using various formulation techniques known in the art. For example, they may include a biodegradable (hydrolyzable) polymeric carrier that adheres to the oral mucosa. They are designed to slowly erode over a predetermined period of time, thereby providing essentially total drug delivery.

[0105] Drug delivery in oral dosage forms avoids the disadvantages of oral drug administration, such as slow absorption, degradation of the active agent by fluids present in the gastrointestinal tract, and / or first-pass inactivation in the liver. For biodegradable (hydrolyzable) polymeric carriers, virtually any such carrier may be used as long as the desired drug release profile is not compromised, and the carrier is compatible with any other ingredients present in the oral dosage unit. Typically, polymeric carriers comprise hydrophilic (water-soluble and water-swellable) polymers that adhere to the wet surface of the oral mucosa. An example of a polymeric carrier useful herein is an acrylic acid polymer (e.g., a carbomer). In some embodiments, non-limiting examples of other ingredients that may be incorporated into the oral dosage form include disintegrants, diluents, binders, lubricants, flavoring agents, coloring agents, preservatives, and the like. In some embodiments, the oral dosage form may be in the form of a conventionally formulated tablet, lozenge, or gel for oral or sublingual administration.

[0106] In some embodiments, administration of the compound is continued at the physician's discretion if the patient's condition improves; alternatively, the dose of the drug to be administered may be temporarily reduced or temporarily discontinued for a length of time (i.e., a "drug holiday"). The length of a drug holiday may vary from 2 days to 1 year, and includes, by way of example only, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 12 days, 15 days, 20 days, 28 days, 35 days, 50 days, 70 days, 100 days, 120 days, 150 days, 180 days, 200 days, 250 days, 280 days, 300 days, 320 days, 350 days, or 365 days. In some embodiments, the dose reduction during a drug-free period is 10%-100%, including, by way of example only, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%.

[0107] Once improvement occurs in the patient's condition, a maintenance dose is administered, if necessary. Subsequently, the dosage, frequency, or both may be reduced as a function of symptoms, to a level that maintains the improved disease, disorder, or condition. However, patients require intermittent treatment over a long period of time if any symptoms recur.

[0108] The amount of a given formulation that would correspond to such a quantity will vary depending on factors such as the specific compound, the severity of the disease, the identity (e.g., body weight) of the subject to be treated, but can nevertheless be routinely determined in a manner known in the art, for example, depending on the formulation to be administered, the route of administration, and the particular circumstances surrounding the subject to be treated. In general, however, the dosage employed in adult human treatment will typically range from 0.02 to 5000 mg / day, or from about 1 to 1500 mg / day.

[0109] A single dose herein may be provided as a single dose or as divided doses administered simultaneously, for example as two, three, four or more sub-doses.

[0110] In some embodiments, the oral dosage form is a unit dosage form suitable for single administration of precise dosages. In a unit dosage form, the dosage form is divided into unit doses containing appropriate amounts of one or more compounds. In some embodiments, the unit dose is in the form of a tablet containing discrete quantities of the dosage form. Non-limiting examples include packaged tablets or capsules, and powder vials or ampoules. Aqueous suspension compositions may be packaged in single-dose, non-reclosable containers. Alternatively, multi-dose, reclosable containers may be used, in which case it is typical to include a preservative in the composition.

[0111] In some embodiments, the parenteral injection formulation is provided in unit dosage form, including but not limited to ampoules, or in multi-dose containers, with an added preservative.

[0112] Typically, it is prepared in unit dose injectable form for parenteral administration, i.e., bolus, intravenous, and intratumoral injection, together with a pharmaceutically acceptable parenteral vehicle. It is optionally mixed with pharmaceutically acceptable diluents, carriers, excipients, or stabilizers (Remington's Pharmaceutical Sciences (1980) 16th edition, Osol, A. Ed.) as a lyophilized preparation or aqueous solution.

[0113] The matters mentioned in the carrier-drug conjugate, pharmaceutical composition containing the same, use, manufacturing method and treatment method presented in the present invention are equally applicable unless they are contradictory.

[0114] The present invention provides an antibody-drug conjugate comprising heterologous drugs as a method for co-administering drugs that exhibit a strong synergistic effect in the prevention and treatment of cancer. The present invention comprises a single antibody and a heterologous drug, enabling the efficacy of both drugs to be achieved with a single administration.

[0115] The antibody-drug conjugate of the present invention has the advantage of minimizing toxicity and improving safety due to combination of heterologous drugs, while exhibiting high therapeutic efficacy against target cancers, specifically cancers that overexpress HER2 (preferably breast cancer, ovarian cancer, bladder cancer, pancreatic cancer, or stomach cancer).

[0116] In addition, the present invention provides an antibody structure designed to implement a drug-to-antibody ratio (DAR) of 1 in an antibody-drug conjugate. The antibody of the present invention has a knob-in-hole structure, enabling formation of an antibody-drug conjugate with a DAR of 1. Furthermore, even when the antibody is used as an antibody-dual drug conjugate in which different drugs are bound, the DAR ratio can be precisely controlled so as to satisfy a dosage range suitable for administration of each drug.

[0117] Figure 1 illustrates the overlapping interval (yellow) between MMAE as the first drug and Dxd as the second drug when manufacturing an ADC according to the present invention. When the ratio and DAR of drugs expected to exhibit superior efficacy are derived, Cases 2, 3, 4, and 5 (DARs 6 to 9) demonstrate superior results.

[0118] Figure 2 shows the results of cytotoxicity testing on KPL-4 cell lines (HER2+). Each cell was treated with ADCs manufactured with a total DAR of 8 and different ratios of heterologous drugs bound to the carrier (first drug:second drug = 1:4, 1:5, 1:6, 1:7, 1:8, 0:8).

[0119] Figure 3 shows the results of cytotoxicity testing on MDA-MB-468 cell lines (HER2-). Each cell was treated with ADCs manufactured with a total DAR of 8 but with different ratios of heterologous drugs bound to the carrier (first drug:second drug = 1:4, 1:5, 1:6, 1:7, 1:8, 0:8).

[0120] Figure 4 shows the results of analyzing cell viability after treating ADCs manufactured with a total DAR of 8 but different ratios of heterologous drugs bound to the carrier (first drug:second drug = 1:4, 1:5, 1:6, 1:7, 1:8, 0:8) to HER2 positive cell line (KPL4) and negative cell line (MDA-MB-468) (IC50 values).

[0121] Figure 5 shows the results of cytotoxicity testing on KPL-4 cell lines (HER2+). Each cell was treated with ADCs manufactured with different total DARs (5 to 8) and different ratios of heterologous drugs bound to the carrier (first drug:second drug = 1:4, 1:5, 1:6, 1:7, 1:8, 0:8).

[0122] Figure 6 shows the results of cytotoxicity testing on MDA-MB-468 cell lines (HER2-). Each cell was treated with ADCs manufactured with different total DARs (5 to 8) and different ratios of heterologous drugs bound to the carrier (first drug:second drug = 1:4, 1:5, 1:6, 1:7, 1:8, 0:8).

[0123] Figure 7 shows the results of analyzing cell viability according to treatment of ADC samples containing heterologous drugs after treating ADCs with different total DARs (5 to 8) and ratios of heterologous drugs bound to carriers (first drug:second drug = 1:4, 1:5, 1:6, 1:7, 1:8, 0:8) on HER2 positive cell lines (KPL4) and negative cell lines (MDA-MB-468) (IC 50 values).

[0124] Figure 8 shows the changes in tumor volume after ADC treatment in NOD SCID mice (all groups) bearing NCI-N87 tumors. Data in Figure 8 are expressed as mean ± SEM. (* p<0.05, ** p<0.01)

[0125] Figure 9 shows the results of examining body weight changes after ADC treatment in NOD SCID mice (all groups) bearing NCI-N87 tumors. Data are expressed as mean ± SEM. (* p < 0.05)

[0126] Figure 10 shows the results of confirming the effect of ADC containing a heterologous drug manufactured according to the present invention on the tumor growth inhibition rate in NOD SCID mice (all groups) bearing NCI-N87 tumors. (TGI (%) = [1-(Ti-T0) / (Vi-V0)] Х100, among the treatment groups, Ti: average tumor volume on the corresponding day, T0: average tumor volume on the first day of treatment, Vehicle control group, Vi: average tumor volume on the same day as Ti, V0: average tumor volume on the first day of treatment.)

[0127] Figure 11 is a graph comparing the tumor growth inhibition effect of an ADC manufactured according to one embodiment of the present invention with that of an isotype control.

[0128] Figure 12 shows the structure of the compound (SN-38, Exatecan, Dxd, FL118) mentioned in the present invention.

[0129] FIG. 13 is a schematic diagram illustrating a method for producing an antibody-dual drug conjugate in which a first drug and a second drug have a binding ratio of DAR 1:6 using an antibody having a knob-in-hole structure according to one embodiment of the present invention.

[0130] Figures 14a to 14d show the SEC and HIC analysis results of antibody-dual drug conjugates produced using an antibody with a knob-in-hole structure according to the present invention. Figures 14a and 14c show the SEC and HIC analysis results of the trastuzumab antibody before modification, respectively, and Figures 14b and 14d show the SEC and HIC analysis results of the antibody-dual drug conjugate using the trastuzumab antibody with a knob-in-hole structure.

[0131] FIG. 15 is a schematic diagram illustrating a method for preparing an antibody-dual drug conjugate in which a first drug and a second drug have a binding ratio of DAR 1:7 using a trastuzumab antibody of a general structure according to one embodiment of the present invention.

[0132] Figure 16 shows the results of HIC purification after manufacturing an antibody-dual drug conjugate having a first drug and a second drug having binding ratios of DAR 0:8, 1:7, and 2:6 using a trastuzumab antibody of a general structure according to one embodiment of the present invention.

[0133] Figures 17a and 17d show the SEC and HIC analysis results of antibody-dual drug conjugates manufactured using a trastuzumab antibody of a general structure according to the present invention. Figures 17a and 17c show the SEC and HIC analysis results of the trastuzumab antibody before modification, respectively, and Figures 17b and 17d show the SEC and HIC analysis results of the antibody-dual drug conjugate using the trastuzumab antibody.

[0134] Hereinafter, the present invention will be described in more detail through examples. However, the following examples are intended only to clearly illustrate the technical features of the present invention and do not limit the scope of protection of the present invention.

[0135]

[0136] [Manufacturing example]

[0137] Preparation of materials

[0138] The KPL-4, MDA-MB-468, and NCI-N87 cell lines used in the present invention were purchased from ATCC and the Korea Cell Line Bank (KCLB). Trastuzumab was purchased from Wuxi Biologics, and compounds 6 and 7 were prepared using the following methods. Compound 1 (mc-vc-PAB-MMAE) was purchased from MedChemExpress.

[0139]

[0140] Preparation of compound 6

[0141] (1) Manufacturing method

[0142]

[0143] (2) Step 1

[0144]

[0145] Fmoc-Gly-Gly-OH (5.0 g, 14.1 mmol) was partially dissolved in tetrahydrofuran (125 mL), toluene (42.6 mL), and pyridine (2.15 mL). Then, lead(IV) acetate (7.8 g, 17.6 mmol) was added, and the reaction mixture turned orange. The mixture was heated to reflux. After stirring for 3 h, the reaction mixture was cooled to room temperature, filtered through a layer of Celite, and washed with ethyl acetate. The residue was then concentrated under reduced pressure. The residue was purified by flash chromatography (silica, 10-100% ethyl acetate in heptane) to give (2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetamido)-methyl acetate (3.0 g, 57% yield) as a white solid.

[0146] 1 H NMR (400 MHz, CDCl3) δ 7.77 (d,J= 7.4 Hz, 2H), 7.59 (d,J= 7.5 Hz, 2H), 7.45 - 7.37 (m, 2H), 7.36 - 7.29 (m, 2H), 6.98 (s, 1H), 5.34 (s, 1H), 5.26 (d,J= 7.3 Hz, 2H), 4.46 (d,J= 6.8 Hz, 2H), 4.23 (t,J= 6.8 Hz, 1H), 3.94 - 3.84 (m, 2H), 2.06 (s, 3H). m / z 391.2 [M+Na] +

[0147]

[0148] (3) Step 2

[0149]

[0150] (2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetamido)-methyl acetate (0.3 g, 0.814 mmol) was dissolved in dichloromethane (4.35 mL), and benzyl (R)-2-hydroxypropanoate (1.46 g, 8.14 mmol) was added. Pyridinium p-toluenesulfonate (0.020 g, 0.081 mmol) was then added, and the mixture was stirred at reflux overnight. The reaction mixture was cooled to room temperature, diluted with ethyl acetate (20 mL), washed with water (3X20 mL), and dried over Na2SO4. It was then filtered and concentrated under reduced pressure. The residue was purified by flash chromatography (silica, ethyl acetate in heptane 10-70%). Yield: 0.35 g, 88%.

[0151] 1 H NMR (400 MHz, CDCl3) δ 7.77 (d,J= 7.5 Hz, 2H), 7.59 (d,J= 7.5 Hz, 2H), 7.44 - 7.37 (m, 2H), 7.37 - 7.28 (m, 7H), 6.66 - 6.55 (m, 1H), 5.21 - 5.12 (m, 3H), 4.93 - 4.73 (m, 2H), 4.46 (d,J= 6.7 Hz, 2H), 4.26 - 4.19 (m, 2H), 3.85 - 3.69 (m, 2H), 1.42 (d,J= 6.8 Hz, 3H). m / z 511.2 [M+Na] +

[0152]

[0153] (4) Step 3

[0154]

[0155] To a suspension of benzyl (R)-1-(9H-fluoren-9-yl)-10-methyl-3,6-dioxo-2,9-dioxa-4,7-diazaundecan-11-oate (0.35 g, 0.716 mmol) in dichloromethane (3.4 mL) was added diethylamine (1.7 mL) at room temperature. After stirring for 2 h, the reaction mixture was concentrated under reduced pressure and co-evaporated three times with dichloromethane to obtain benzyl (R)-2-((2-aminoacetamido)-methoxy)propanoate as a white solid. The product was used as is. m / z 267.0 [M+H] +

[0156]

[0157] (5) Step 4

[0158]

[0159] To a solution of benzyl (R)-2-((2-aminoacetamido)methoxy)propanoate (0.191 g, 0.716 mmol) in N,N-dimethylformamide (dry) (3.35 mL) was added Fmoc-Phe-OSu (1.041 g, 2.148 mmol) under an argon atmosphere at room temperature. After stirring for 50 min, the reaction mixture was diluted with ethyl acetate, washed with water and brine, dried over Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by flash chromatography (silica, ethyl acetate in heptane 10-100%) to give benzyl (5S,13R)-5-benzyl-1-(9H-fluoren-9-yl)-13-methyl-3,6,9-trioxo-2,12-dioxa-4,7,10-triazatetradecane-14-oate (0.295 g, 64% yield) as a colorless oil.

[0160] 1H NMR (400 MHz, CDCl3) δ 7.76 (d,J= 7.6 Hz, 2H), 7.56 - 7.49 (m, 2H), 7.44 - 7.37 (m, 2H), 7.37 - 7.27 (m, 10H), 7.21 - 7.14 (m, 2H), 6.93 - 6.73 (m, 1H), 6.30 - 6.09 (m, 1H), 5.25 - 5.16 (m, 1H), 5.16 - 5.08 (m, 2H), 4.81 - 4.77 (m, 1H), 4.71 - 4.67 (m, 1H), 4.51 - 4.25 (m, 3H), 4.22 - 4.15 (m, 2H), 3.89 - 3.69 (m, 2H), 3.19 - 2.98 (m, 2H), 1.37 (d,J= 6.9 Hz, 3H). m / z 658.4 [M+Na] +

[0161]

[0162] (6) Step 5

[0163]

[0164] To a suspension of benzyl (5S,13R)-5-benzyl-1-(9H-fluoren-9-yl)-13-methyl-3,6,9-trioxo-2,12-dioxa-4,7,10-triazatetradecan-14-oate (0.295 g, 0.464 mmol) in dichloromethane (2.2 mL) was added diethylamine (1.1 mL) at room temperature. After stirring for 2 h, the reaction mixture was concentrated under reduced pressure and co-evaporated three times with dichloromethane to obtain benzyl (R)-2-((2-((S)-2-amino-3-phenylpropanamido) acetamido) methoxy) propanoate as a colorless residue, which solidified upon standing. The product was used as is. m / z 414.2 [M+H] +

[0165]

[0166] (7) Step 6

[0167]

[0168] To a solution of benzyl (R)-2-((2-((S)-2-amino-3-phenylpropanamido)acetamido)methoxy)propanoate (0.192 g, 0.464 mmol) in N,N-dimethylformamide (dry) (1.482 mL) was added DIPEA (0.324 mL, 1.856 mmol), followed by the sequential addition of Fmoc-Gly-Gly-OH (0.329 g, 0.928 mmol) and HATU (0.353 g, 0.928 mmol). The reaction mixture was stirred at room temperature for 45 min. The reaction mixture was then diluted with ethyl acetate (20 mL) and washed with 10% aqueous citric acid (10 mL). The aqueous layer was extracted with ethyl acetate (2X10 mL). The combined organic layers were washed with water (5 mL), saturated aqueous NaHCO3 (5 mL), and brine (5 mL), respectively, and dried over Na2SO4. The residue was filtered and concentrated under reduced pressure. The residue was purified by flash chromatography (silica, 0-8% methanol in dichloromethane) to give benzyl (11S,19R)-11-benzyl-1-(9H-fluoren-9-yl)-19-methyl-3,6,9,12,15-pentaoxo-2,18-dioxa-4,7,10,13,16-pentaazaicosan-20-oate (0.259 g, 74% yield) as a white solid.

[0169] 1H NMR (400 MHz, DMSO-d6) δ 8.63 - 8.54 (m, 1H), 8.39 - 8.29 (m, 1H), 8.16 (d,J= 8.2 Hz, 1H), 8.07 - 7.97 (m, 1H), 7.89 (d,J= 7.4 Hz, 2H), 7.70 (d,J= 7.6 Hz, 2H), 7.64 - 7.55 (m, 1H), 7.44 - 7.29 (m, 9H), 7.26 - 7.13 (m, 5H), 5.21 - 5.07 (m, 2H), 4.66 - 4.45 (m, 3H), 4.33 - 4.17 (m, 4H), 3.82 - 3.56 (m, 6H), 3.05 (dd,J= 13.7, 4.5 Hz, 1H), 2.84 - 2.73 (m, 1H), 1.26 (d,J= 6.8 Hz, 3H). m / z 772.4 [M+Na] +

[0170]

[0171] (8) Step 7

[0172]

[0173] A solution of benzyl (11S,19R)-11-benzyl-1-(9H-fluoren-9-yl)-19-methyl-3,6,9,12,15-pentaoxo-2,18-dioxa-4,7,10,13,16-pentaazaicosan-20-oate (259 mg, 0.345 mmol) in ethanol (4.1 mL) and ethyl acetate (2.0 mL) was purged with argon for 10 min. Then, Pd / C (10%, 50% wet) (73.5 mg, 0.035 mmol) was added and stirred under a hydrogen atmosphere (balloon) for 2 h. The reaction mixture was filtered through a layer of celite, washed with methanol, and concentrated under reduced pressure to obtain (11S,19R)-11-benzyl-1-(9H-fluoren-9-yl)-19-methyl-3,6,9,12,15-pentaoxo-2,18-dioxa-4,7,10,13,16-pentaazaicosan-20-oic acid (0.204 g, 90% yield) as a pale white solid.

[0174] 1 H NMR (400 MHz, DMSO-d6) δ 8.31 (s, 1H), 7.89 (d,J= 7.6 Hz, 2H), 7.72 (d,J= 7.4 Hz, 2H), 7.59 (d,J= 7.2 Hz, 0H), 7.46 - 7.28 (m, 4H), 7.24 (d,J= 4.4 Hz, 4H), 7.18 (t,J= 4.6 Hz, 1H), 4.69 (s, 1H), 4.58 - 4.46 (m, 2H), 4.35 (s, 1H), 4.31 - 4.18 (m, 3H), 3.97 (d,J= 7.7 Hz, 1H), 3.84 - 3.70 (m, 2H), 3.67 - 3.54 (m, 4H), 3.07 (dd,J= 13.9, 4.5 Hz, 1H), 2.89 - 2.76 (m, 1H), 1.23 (d,J= 6.8 Hz, 3H). m / z 682.2 [M+Na] +

[0175]

[0176] (9) Step 8

[0177]

[0178] To a suspension of (11S,19R)-11-benzyl-1-(9H-fluoren-9-yl)-19-methyl-3,6,9,12,15-pentaoxo-2,18-dioxa-4,7,10,13,16-pentaazaicosan-20-oic acid (50 mg, 0.076 mmol) in dichloromethane (0.5 mL) was added diethylamine (0.25 mL) at room temperature. After stirring for 3 h, the reaction mixture was concentrated under reduced pressure and co-evaporated three times with dichloromethane to obtain (2R,10S)-16-amino-10-benzyl-2-methyl-6,9,12,15-tetraoxo-3-oxa-5,8,11,14-tetraazahexadecanoic acid as a white solid. The product was used as is. m / z 438.2 [M+H] +

[0179]

[0180] (10) Step 9

[0181]

[0182] (2R,10S)-16-Amino-10-benzyl-2-methyl-6,9,12,15-tetraoxo-3-oxa-5,8,11,14-tetraazahexadecanoic acid (33.2 mg, 0.076 mmol) was dissolved in N,N-dimethylformamide (dry) (1 mL), and DIPEA (0.086 mL, 0.494 mmol) was added at room temperature under an argon atmosphere. 6-Maleimidohexanoic acid N-hydroxysucciimide ester (35.1 mg, 0.114 mmol) was then added. The reaction mixture was stirred at room temperature for 1 h. The reaction mixture was diluted with 1 mL of DMF and purified by acidic preparative MPLC (Luna5-40). Afterwards, lyophilization was performed to obtain (2R,10S)-10-benzyl-23-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-2-methyl-6,9,12,15,18-pentaoxo-3-oxa-5,8,11,14,17-pentaazatrichosanoic acid (24 mg, 50% yield) as a white solid.

[0183] 1 H NMR (400 MHz, DMSO-d6) δ 12.60 (s, 1H), 8.61 (s, 1H), 8.32 - 7.93 (m, 4H), 7.28 - 7.21 (m, 4H), 7.20 - 7.15 (m, 1H), 7.00 (s, 2H), 4.68 - 4.60 (m, 1H), 4.59 - 4.44 (m, 2H), 4.04 - 3.96 (m, 1H), 3.80 - 3.55 (m, 6H), 3.40 - 3.34 (m, 2H), 3.06 (dd,J= 13.9, 4.6 Hz, 1H), 2.81 (dd,J= 13.9, 9.7 Hz, 1H), 2.10 (t,J= 7.3 Hz, 2H), 1.52 - 1.41 (m, 4H), 1.26 - 1.14 (m, 5H). m / z 653.4 [M+Na] +

[0184]

[0185] (11) Step 10

[0186]

[0187] (1S,10S)-1-Amino-10-ethyl-10-hydroxy-1,2,3,10,13,16-hexahydro-11H,14H-benzo[de][1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-11,14-dione methanesulfonate (410 mg, 1 eq, 754 μmol) and (2R,10S)-10-benzyl-23-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-2-methyl-6,9,12,15,18-pentaoxo-3-oxa-5,8,11,14,17-pentaazatrichosanoic acid (466 mg, 0.98 eq, 739 μmol) was suspended in N,N-dimethylformamide (10.0 mL). Then, DIPEA (439 mg, 591 μL, 4.5 eq, 3.39 mmol) was added, followed by HATU (430 mg, 1.5 eq, 1.13 mmol). The reaction mixture was stirred at room temperature for 30 minutes. The reaction mixture was purified three times by acidic preparative MPLC (Luna10-50). The obtained product fractions were combined and lyophilized to obtain a pale yellow solid. Yield: 510 mg, 63%.

[0188] 1H NMR (400 MHz, DMSO-d6) δ 8.62 (t,J= 6.6 Hz, 1H), 8.50 (d,J= 9.1 Hz, 1H), 8.29 (t,J= 5.9 Hz, 1H), 8.13 - 7.96 (m, 3H), 7.39 (s, 1H), 7.27 - 7.12 (m, 6H), 6.99 (s, 2H), 6.48 (s, 1H), 6.26 (d,J= 6.0 Hz, 2H), 5.61 - 5.51 (m, 1H), 5.45 - 5.33 (m, 2H), 5.20 - 4.99 (m, 2H), 4.72 - 4.63 (m, 1H), 4.57 - 4.41 (m, 2H), 4.11 (q,J= 6.7 Hz, 1H), 3.78 - 3.53 (m, 6H), 3.40 - 3.34 (m, 2H), 3.16 - 2.95 (m, 3H), 2.74 (dd,J= 13.8, 9.5 Hz, 1H), 2.17 - 2.03 (m, 4H), 1.91 - 1.78 (m, 2H), 1.52 - 1.35 (m, 7H), 1.25 - 1.12 (m, 2H), 0.87 (t,J= 7.3 Hz, 3H). m / z 1060.02 [M+H] +

[0189]

[0190] Preparation of compound 7

[0191] (1) Manufacturing method

[0192]

[0193] (2) Step 1

[0194]

[0195] To a solution of benzyl (R)-1-(9H-fluoren-9-yl)-10-methyl-3,6-dioxo-2,9-dioxa-4,7-diazaundecan-11-oate (211 mg, 432 μmol) was added diethylamine (1.0 mL, 9.7 mmol) using dichloromethane (2.0 mL). The reaction mixture was stirred at room temperature for 1.5 h, concentrated under reduced pressure, and co-evaporated six times with DCM to obtain an opaque oil. This was used in the next step.

[0196] SC_ACID: m / z 267.2 [M+H]+

[0197]

[0198] (3) Step 2

[0199]

[0200] Benzyl (R)-2-((2-aminoacetamido)methoxy)propanoate (115 mg, 432 μmol) was dissolved in DMF (5 mL), and DIPEA (226 μL, 1.30 mmol) and (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(4-(((2S, 3R, 4S, 5S,6S))-3,4,5-triacetoxy-6-(methoxycarbonyl)tetrahydro-2H-pyran-2-yl)oxy)phenyl)propanoic acid (466 mg, 648 μmol) and HATU (246 mg, 648 μmol) were added, and the reaction mixture was stirred at room temperature for 1 h. The reaction mixture was diluted with ethyl acetate and washed with water (2x) and brine (2x). The organic layer was dried over Na2SO4, filtered, and concentrated to give a thick yellow oil (656 mg). The product was purified by flash column chromatography (24 g Si, 0-100% EtOAc in heptane), and the product fractions were collected and concentrated to give the product as a white solid. Yield: 317 mg, 71%

[0201] SC_ACID: m / z 990.2 [M+Na]+

[0202] 1H NMR (400 MHz, CDCl3) δ 7.76 (d, J = 7.5 Hz, 2H), 7.56 - 7.48 (m, 2H), 7.40 (t, J = 7.5 Hz, 2H), 7.37 - 7.27 (m, 7H), 7.14 - 7.05 (m, 2H), 6.94 (d, J = 8.1 Hz, 2H), 6.87 (s, 1H), 6.29 (s, 1H), 5.36 - 5.19 (m, 4H), 5.18 - 5.05 (m, 3H), 4.86 - 4.78 (m, 1H), 4.72 - 4.64 (m, 1H), 4.53 - 4.44 (m, 1H), 4.43 - 4.26 (m, 2H), 4.24 - 4.08 (m, 3H), 3.79 (m, 2H), 3.70 (s, 3H), 3.15 - 2.91 (m, 2H), 2.08 - 2.00 (m, 9H), 1.61 - 1.53 (m, 2H), 1.38 (d, J = 7.0 Hz, 3H).

[0203]

[0204] (4) Step 3

[0205]

[0206] Piperidine (71.2 μL, 720 μmol) was added to a solution of (2S,3R,4S,5S,6S)-2-(4-((4R,12S)-12-(((9H-fluoren-9-yl)methoxy)carbonyl)amino)-4-methyl-3,8,11-trioxo-1-phenyl-2,5-dioxa-7,10-diazatridecan-13-yl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate (317 mg, 327 μmol) in DMF (4.5 ml), and the reaction mixture was stirred at room temperature for 30 min. The reaction mixture was quenched with acetic acid (41.2 μL, 720 μmol), purified by acid preparative MPLC (Luna 10-50), and the product fractions were combined and lyophilized to obtain a white solid. Yield: 200 mg, 82%

[0207] SC_ACID: m / z 746.4 [M+H]+

[0208] 1H NMR (400 MHz, CDCl3) δ 7.81 (t, J = 5.8 Hz, 1H), 7.38 - 7.30 (m, 5H), 7.17 - 7.11 (m, 2H), 6.99 - 6.92 (m, 2H), 6.83 (t, J = 6.8 Hz, 1H), 5.39 - 5.25 (m, 3H), 5.18 (d, J = 4.7 Hz, 2H), 5.16 - 5.12 (m, 1H), 4.88 - 4.73 (m, 2H), 4.29 - 4.14 (m, 2H), 3.89 - 3.85 (m, 2H), 3.73 (s, 3H), 3.63 (dd, J = 8.9, 4.2 Hz, 1H), 3.17 (dd, J = 13.7, 4.2 Hz, 1H), 2.80 - 2.70 (m, 1H), 2.10 - 2.01 (m, 9H), 1.42 (d, J = 6.9 Hz, 3H).

[0209]

[0210] (5) Step 4

[0211]

[0212] To a solution of (2S,3R,4S,5S,6S)-2-(4-((4R,12S)-12-amino-4-methyl-3,8,11-trioxo-1-phenyl-2,5-dioxa-7,10-diazatridecan-13-yl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate (197 mg, 264 μmol) in DMF (3.0 ml) were added DIPEA (138 μL, 793 μmol), Fmoc-Gly-Gly-OH (140 mg, 396 μmol), and HATU (151 mg, 396 μmol), and the reaction mixture was stirred at room temperature for 45 min. The reaction mixture was purified directly by acidic preparative MPLC (Luna30-70). The product fractions were combined and lyophilized to obtain a white solid. Yield: 215 mg, 75%

[0213] SC_ACID: m / z 1104.2 [M+Na]+

[0214] 1H NMR (400 MHz, CDCl3) δ 7.75 (d, J = 7.6 Hz, 2H), 7.57 (t, J = 6.7 Hz, 2H), 7.49 - 7.27 (m, 10H), 7.20 - 7.04 (m, 5H), 6.92 - 6.85 (m, 2H), 5.97 (t, J = 5.6 Hz, 1H), 5.39 - 5.27 (m, 2H), 5.27 - 5.20 (m, 1H), 5.18 - 5.06 (m, 3H), 4.80 - 4.72 (m, 1H), 4.71 - 4.63 (m, 1H), 4.56 (q, J = 7.1 Hz, 1H), 4.44 (d, J = 6.7 Hz, 2H), 4.27 - 4.15 (m, 3H), 3.94 - 3.72 (m, 6H), 3.68 (s, 3H), 3.21 - 3.12 (m, 1H), 2.99 - 2.89 (m, 1H), 2.08 - 2.01 (m, 9H), 1.36 (d, J = 6.9 Hz, 3H).

[0215]

[0216] (6) 단계 5

[0217]

[0218] A suspension of (2S,3R,4S,5S,6S)-2-(4-((4R,12S)-12-(2-(2-(((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetamido)acetamido)-4-methyl-3,8,11-trioxo-1-phenyl-2,5-dioxa-7,10-diazatridecan-13-yl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate (193 mg, 178 μmol) dissolved in methanol (8 ml) was purged with nitrogen for 10 min. Then, Pd / C (10%, 50% wet) (38.0 mg, 5% Wt, 17.8 μmol) was added and the mixture was stirred under hydrogen for 30 minutes. The reaction mixture was flushed with nitrogen for 10 minutes, filtered through Celite, eluted with methanol, and concentrated to obtain a white solid (155 mg). The product was purified by acidic preparative MPLC (Luna20-60), and the product fraction was lyophilized to obtain a white solid. Yield: 117 mg, 66%

[0219] SC_ACID: m / z 1014.0 [M+Na]+

[0220] 1H NMR (400 MHz, DMSO) δ 12.60 (s, 1H), 8.64 - 8.57 (m, 1H), 8.31 (t, J = 5.8 Hz, 1H), 8.22 - 8.04 (m, 2H), 7.89 (d, J = 7.5 Hz, 2H), 7.71 (d, J = 7.4 Hz, 2H), 7.61 (t, J = 6.1 Hz, 1H), 7.41 (t, J = 7.4 Hz, 2H), 7.32 (t, J = 7.4 Hz, 2H), 7.22 - 7.15 (m, 2H), 6.91 - 6.85 (m, 2H), 5.61 (d, J = 8.0 Hz, 1H), 5.45 (t, J = 9.6 Hz, 1H), 5.11 - 5.01 (m, 2H), 4.72 - 4.52 (m, 3H), 4.52 - 4.42 (m, 1H), 4.32 - 4.26 (m, 2H), 4.26 - 4.15 (m, 1H), 4.03 (q, J = 6.9 Hz, 1H), 3.83 - 3.55 (m, 9H), 3.04 - 2.95 (m, 1H), 2.81 - 2.71 (m, 1H), 2.04 - 1.96 (m, 9H), 1.23 (d, J = 6.9 Hz, 3H).

[0221]

[0222] (7) 단계 6

[0223]

[0224] (1S,10S)-1-Amino-10-ethyl-10-hydroxy-1,2,3,10,13,16-hexahydro-11H,14H-benzo[de][1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-11,14-dione methanesulfonate (55 mg, 1 Eq, 0.10 mmol) was suspended in DMF (2.5 mL). (11S,19R)-1-(9H-fluoren-9-yl)-19-methyl-3,6,9,12,15-pentaoxo-11-(4-(((2S,3R,4S,5S,6S)-3,4,5-triacetosan-6-(methoxycarbonyl)tetrahydro-2H-pyran-2-yl)oxy)benzyl)-2,18-dioxa-4,7,10,13,16-pentaazaicosane-20-acid (0.10 g, 1 Eq, 0.10 mmol) and DIPEA (39 mg, 53 μL, 3 Eq, 0.30 mmol) were added, followed by addition of HATU (48 mg, 1.25 Eq, 0.13 mmol) and stirring was performed at room temperature for 15 min. The reaction mixture was purified directly by acid preparative MPLC (Luna20-60), the product fractions were combined and lyophilized to obtain a yellow solid (100 mg, 70%).

[0225] SC_ACID: m / z 1421.8 [M+H]+

[0226] 1H NMR (400 MHz, DMSO-d6) δ 8.65 (t, J = 6.6 Hz, 1H), 8.49 (d, J = 9.1 Hz, 1H), 8.31 (t, J = 5.8 Hz, 1H), 8.10 (d, J = 7.9 Hz, 1H), 8.03 (t, J = 5.7 Hz, 1H), 7.87 (d, J = 7.6 Hz, 2H), 7.69 (d, J = 7.4 Hz, 2H), 7.57 (t, J = 6.0 Hz, 1H), 7.43 - 7.26 (m, 5H), 7.23 (s, 1H), 7.19 - 7.11 (m, 2H), 6.91 - 6.84 (m, 2H), 6.47 (s, 1H), 6.26 (d, J = 7.2 Hz, 2H), 5.64 - 5.51 (m, 2H), 5.45 (t, J = 9.7 Hz, 1H), 5.42 - 5.32 (m, 2H), 5.17 - 4.99 (m, 4H), 4.72 - 4.63 (m, 2H), 4.54 (m, 1H), 4.48 - 4.38 (m, 1H), 4.31 - 4.07 (m, 4H), 3.81 - 3.55 (m, 9H), 3.15 - 2.88 (m, 3H), 2.75 - 2.67 (m, 1H), 2.16 - 2.05 (m, 2H), 2.05 - 1.95 (m, 9H), 1.92 - 1.77 (m, 2H), 1.39 (d, J = 6.8 Hz, 3H), 0.86 (t, J = 7.3 Hz, 3H).

[0227]

[0228] (8) 단계 7

[0229]

[0230] (2S,3R,4S,5S,6S)-2-(4-((S)-11-((2-((((R)-1-(((1S,10S)-10-ethyl-10-hydroxy-11,14-dioxo-2,3,10,11,14,16-hexahydro-1H,13H-benzo[de][1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizide [1,2-b]quinolin-1-yl)amino)-1-oxopropan-2-yl)oxy)methyl)amino)-2-oxoethyl)carbamoyl)-1-(9H-fluoren-9-yl)-3,6,9-trioxo-2-oxa-4,7,10-triazadodecan-12-yl)phenooxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate (50 mg, 1 Eq, 35 μmol) was dissolved in methanol / THF (1:1, 6 mL) and cooled to 0°C. Lithium hydroxide monohydrate (15 mg, 10 Eq, 0.35 mmol) dissolved in water (600 μL) was added. The reaction mixture was stirred at 0°C for 2 h, acetic acid (0.11 g, 0.10 mL, 50 Eq, 1.8 mmol) was added, and the organic solvent was removed under reduced pressure. DMSO (2 mL) was added to the aqueous residue. The solution was purified by acid preparative MPLC (Luna5-40), and the product fractions were combined and lyophilized to obtain a white solid (27 mg, 72%).

[0231] SC_ACID: m / z 1059.6 [M+H]+

[0232] 1H NMR (400 MHz, DMSO-d6) δ 8.71 (t, J = 6.6 Hz, 1H), 8.54 (d, J = 9.2 Hz, 1H), 8.38 (t, J = 5.9 Hz, 1H), 8.29 - 8.13 (m, 1H), 7.93 (d, J = 8.2 Hz, 1H), 7.40 (s, 1H), 7.23 (s, 1H), 7.08 (d, J = 8.3 Hz, 2H), 6.86 (d, J = 8.2 Hz, 2H), 6.48 (s, 1H), 6.26 (d, J = 5.0 Hz, 2H), 5.61 - 5.51 (m, 1H), 5.45 - 5.34 (m, 2H), 5.29 - 4.98 (m, 4H), 4.84 (d, J = 7.5 Hz, 1H), 4.74 - 4.64 (m, 1H), 4.58 - 4.41 (m, 2H), 4.12 (q, J = 6.7 Hz, 1H), 3.82 - 3.48 (m, 9H, coincides with H2O peak), 3.24 - 2.92 (m, 13H, coincides with H2O peak), 2.64 - 2.53 (m, 1H), 2.17 - 2.08 (m, 2H), 1.92 - 1.78 (m, 2H), 1.39 (d, J = 6.8 Hz, 3H), 0.87 (t, J = 7.4 Hz, 3H).

[0233]

[0234] (9) 단계 8

[0235]

[0236] (2S,3S,4S,5R,6S)-6-(4-((S)-2-(2-(2-aminoacetamido)acetamido)-3-((2-((((R)-1-(((1S,10S)-10-ethyl-10-hydroxy-11,14-dioxo-2,3,10,11,14,16-hexahydro-1H,13H-benzo[de] [1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-1-oxopropan-2-yl)oxy)methyl)amino)-2-oxoethyl)amino)-3-oxopropyl)phenooxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (60 mg, 1 Eq, 57 μmol) and N-succinimidyl 6-maleimidohexanoate (35 mg, 2 Eq, 0.11 mmol) were dissolved in DMF (2.5 mL). DIPEA (15 mg, 20 μL, 2 Eq, 0.11 mmol) was added and the reaction mixture was stirred at room temperature for 45 min. The reaction mixture was purified directly by acid preparative MPLC (Luna5-50), the product fractions were combined and lyophilized to obtain a pale yellow solid (49 mg, 69%).

[0237] U_AN_ACID: m / z 1252.2 [M+H]+

[0238] 1H NMR (400 MHz, DMSO-d6) δ 8.62 (t, J = 6.6 Hz, 1H), 8.50 (d, J = 9.1 Hz, 1H), 8.27 (t, J = 5.9 Hz, 1H), 8.11 - 7.98 (m, 3H), 7.39 (s, 1H), 7.23 (s, 1H), 7.15 - 7.07 (m, 2H), 6.98 (s, 2H), 6.92 - 6.84 (m, 2H), 6.47 (s, 1H), 6.26 (d, J = 4.3 Hz, 2H), 5.61 - 5.52 (m, 1H), 5.46 - 5.32 (m, 3H), 5.24 - 4.93 (m, 4H), 4.67 (dd, J = 10.1, 6.6 Hz, 1H), 4.53 (dd, J = 10.1, 6.6 Hz, 1H), 4.46 - 4.36 (m, 1H), 4.11 (q, J = 6.7 Hz, 1H), 3.86 (d, J = 9.5 Hz, 1H), 3.80 - 3.53 (m, 7H), 3.43 - 3.36 (m, 2H), 3.29 - 3.19 (m, 2H), 3.16 - 2.89 (m, 3H), 2.69 (dd, J = 13.9, 9.1 Hz, 1H), 2.19 - 2.03 (m, 4H), 1.94 - 1.76 (m, 2H), 1.52 - 1.37 (m, 7H), 1.23 - 1.11 (m, 2H), 0.87 (t, J = 7.3 Hz, 3H).

[0239]

[0240]

[0241] (10) 단계 9

[0242]

[0243] Benzylamine (1.18 mL, 10.8 mmol) was added to a THF (40 mL) suspension of methyl 1,2,3,4-tetra-O-acetyl-beta-D-glucuronate (3.39 g, 9.01 mmol). The reaction mixture was stirred at room temperature overnight. The reaction mixture was concentrated in vacuo to give a red oil. The product was purified by flash column chromatography (80 g Si, 0–50% EtOAc in heptane) to give a red oil (3.12 g). The material was coated on a separator and purified by silica flash column chromatography (80 g Si, 0–50% EtOAc in heptane) to give the product as a yellow oil. Yield: 2.35 g, 76%

[0244] SC_ACID: m / z 691.0 [2M+Na]+

[0245] 1H NMR (400 MHz, CDCl3) mixture of anomers: δ 5.62 - 5.53 (m, 2H), 5.35 - 5.15 (m, 1.5H), 4.96 - 4.89 (m, 1.25H), 4.83 - 4.78 (m, 0.25H), 4.59 (d, J = 10.0 Hz, 1H), 4.17 - 4.07 (m, 1H), 3.79 - 3.72 (m, 4H), 3.72 - 3.53 (m, 1H), 2.13 - 2.07 (m, 4H), 2.07 - 1.99 (m, 8H).

[0246]

[0247] (11) Step 10

[0248]

[0249] (3R,4S,5S,6S)-2-Hydroxy-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate (2.35 g, 7.03 mmol) and DCM (40 mL) were cooled to 0°C, and 2,2,2-trichloroacetonitrile (3.52 mL, 35.2 mmol) and DBU (210 μL, 1.41 mmol) were subsequently added. The reaction mixture was warmed to room temperature and stirred for 3 h. The reaction mixture was concentrated under reduced pressure to obtain a red oil. The product was coated on a separator and purified by flash column chromatography (80 g Si, 0–35% EtOAc in heptane). The product fractions were collected and concentrated under reduced pressure to obtain the product as a beige solid. Yield: 1.86 g, 55%

[0250] SC_ACID: m / z 499.8 [M+Na]+

[0251] 1H NMR (400 MHz, CDCl3) δ 8.74 (s, 1H), 6.64 (d, J = 3.6 Hz, 1H), 5.63 (t, J = 9.9 Hz, 1H), 5.31 - 5.23 (m, 1H), 5.15 (dd, J = 10.2, 3.6 Hz, 1H), 4.50 (d, J = 10.2 Hz, 1H), 3.75 (s, 3H), 2.07 - 2.04 (m, 6H), 2.02 (s, 3H).

[0252]

[0253] (12) Step 11

[0254]

[0255] H-Tyr-OBzl (4.0 g, 15 mmol) was dissolved in chloroform (100 mL), and Fmoc-OSu (5.0 g, 15 mmol) was added. The reaction mixture was stirred at room temperature overnight. The reaction mixture was diluted with DCM (100 mL) and washed with water (2 x 100 mL) and brine (100 mL). The organic layer was concentrated under reduced pressure to obtain an off-white solid (7.22 g). The solid was filtered and rinsed with DCM (3 x 10 mL). The solid was collected and dried in a vacuum oven at 40 °C for 2 days to obtain the product as a white solid. Yield: 5.77 g, 79%

[0256] SC_ACID: m / z 494.4 [M+H]+ , 516.4 [M+Na]+

[0257] 1H NMR (400 MHz, DMSO) δ 9.25 (s, 1H), 7.95 - 7.81 (m, 3H), 7.66 (t, J = 7.4 Hz, 1H), 7.53 - 7.20 (m, 9H), 7.08 - 6.89 (m, 2H), 6.70 - 6.60 (m, 2H), 5.15 - 5.01 (m, 2H), 4.48 - 3.97 (m, 4H), 3.02 - 2.61 (m, 2H).

[0258]

[0259] (13) Step 12

[0260]

[0261] A suspension of (2S,3S,4S,5R)-2-(methoxycarbonyl)-6-(2,2,2-trichloro-1-iminoethoxy)tetrahydro-2H-pyran-3,4,5-triyl triacetate (51 mg, 0.11 mmol) and benzyl(((9H-fluoren-9-yl)methoxy)carbonyl)-L-tyrosinate (53 mg, 0.11 mmol) in DCM (dry) (1.5 mL) was added to 4A molecular sieves and cooled to 0°C. BF3.OEt2 (ca. 48% BF3) (15 μL, 0.12 mmol) was added and the reaction mixture was stirred at room temperature for 2 h. The reaction mixture was quenched with trimethylamine (2 M in THF) (59 μL, 0.12 mmol) and purified directly by flash column chromatography (12 g Si, 0–60% EtOAc in heptane). The product fractions were collected and concentrated under reduced pressure to give the product as a white solid. Yield: 32 mg, 37%

[0262] SC_ACID: m / z 810.6 [M+H]+ , 832.6 [M+Na]+

[0263] 1H NMR (400 MHz, CDCl3) δ 7.78 (d, J = 7.6 Hz, 2H), 7.59 - 7.51 (m, 2H), 7.45 - 7.28 (m, 9H), 6.93 - 6.75 (m, 4H), 5.37 - 5.09 (m, 6H), 5.02 (d, J = 7.1 Hz, 1H), 4.73 - 4.65 (m, 1H), 4.49 - 4.42 (m, 1H), 4.37 - 4.29 (m, 1H), 4.23 - 4.18 (m, 1H), 4.16 - 4.06 (m, 1H), 3.71 (s, 3H), 3.15 - 2.99 (m, 2H), 2.09 - 2.02 (m, 9H).

[0264]

[0265] (14) Step 13

[0266]

[0267] A suspension of (2S,3R,4S,5S,6S)-2-(4-((S)-2-(((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(benzyloxy)-3-oxopropyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate (527 mg, 651 μmol) in EtOAc (17 mL) was purged with nitrogen for 10 min. Then, Pd / C (10%, 50% wet) (139 mg, 5% Wt, 65.1 μmol) was added and the mixture was stirred under hydrogen for 5 h. The reaction mixture was purged with nitrogen, filtered through Celite, rinsed with ethyl acetate, concentrated under reduced pressure, and concentrated under reduced pressure with DCM (2x) to give the product as a white solid (468 mg, quantitative yield).

[0268] SC_ACID: m / z 742.0 [M+Na]+

[0269] 1H NMR (400 MHz, CDCl3) δ 7.77 (d, J = 7.6 Hz, 2H), 7.59 - 7.49 (m, 2H), 7.41 (t, J = 7.5 Hz, 2H), 7.35 - 7.28 (m, 2H), 7.07 (d, J = 8.1 Hz, 2H), 6.91 (d, J = 8.0 Hz, 2H), 5.36 - 5.29 (m, 2H), 5.29 - 5.18 (m, 2H), 5.06 (d, J = 7.3 Hz, 1H), 4.70 - 4.61 (m, 1H), 4.52 - 4.43 (m, 1H), 4.39 - 4.30 (m, 1H), 4.23 - 4.06 (m, 2H), 3.68 (s, 3H), 3.21 - 3.11 (m, 1H), 3.11 - 3.02 (m, 1H), 2.09 - 1.98 (m, 9H).

[0270]

[0271] [Example 1]

[0272] Manufacturing of an ADC with a total DAR of 8

[0273] In this Example 1, the effects of varying DAR of each drug are compared when the overall DAR is constant at 8. Trastuzumab was used as the antibody.

[0274]

[0275] Preparation of carrier-drug conjugates

[0276] 1-1. Manufacturing of ADC 1 (Total DAR 8, Drug DAR Ratio 0:8)

[0277] Trastuzumab antibody (4 mg / ml) and 20-fold molar equivalents of TCEP were reacted in reduction buffer (150 mM NaCl, 50 mM Histidine pH 6.0) at 25°C for 2 hours. Residual TCEP was removed using a PD-10 column, and 2 mg / ml antibody and 12-fold molar equivalents of compound of formula 7 (hereinafter referred to as compound 7) were added in reaction buffer (25 mM Histidine pH 6.0) containing 10% DMSO, and the reaction was performed at 25°C for 1 hour. Residual linker-payload was removed using a PD-10 column. When DAR was analyzed using LC-MS, the DAR of compound 7 was confirmed to be 7.74.

[0278]

[0279] 1-2. Preparation of ADC 2 (Total DAR 8, Drug DAR Ratio 1: 4)

[0280] Trastuzumab antibody (4 mg / ml) and 20-fold molar equivalents of TCEP were reacted in reduction buffer (150 mM NaCl, 50 mM Histidine pH 6.0) at 25°C for 2 hours. Residual TCEP was removed using a PD-10 column, and 2 mg / ml antibody and 1.80-fold molar equivalents of compound of formula 1 (hereinafter referred to as compound 1) were reacted in reaction buffer (25 mM Histidine pH 6.0) containing 10% DMSO at 25°C for 1 hour. Subsequently, compound 7 (12-fold molar equivalents) was added and reacted at 25°C for 1 hour. Thereafter, the residual linker-payload was removed using a PD-10 column. When DAR was analyzed using LC-MS, the DAR of compound 1 was confirmed to be 1.62 and that of compound 7 was 6.24. The ratio of the two linker-payloads is confirmed to be 1:3.85 in order.

[0281]

[0282] 1-3. Preparation of ADC 3 (Total DAR 8, Drug DAR Ratio 1:5)

[0283] Trastuzumab antibody (4 mg / ml) and 20-fold molar equivalents of TCEP were reacted in reduction buffer (150 mM NaCl, 50 mM Histidine pH 6.0) at 25°C for 2 hours. Residual TCEP was removed using a PD-10 column, and 2 mg / ml antibody and 1.53-fold molar equivalents of compound 1 were reacted in reaction buffer (25 mM Histidine pH 6.0) containing 10% DMSO at 25°C for 1 hour. Subsequently, 12-fold molar equivalents of compound 7 were added and reacted at 25°C for 1 hour. Residual linker-payload was removed using a PD-10 column. When DAR was analyzed using LC-MS, the DAR of compound 1 was confirmed to be 1.24 and that of compound 7 was confirmed to be 6.57. The ratio of the two linker-payloads was confirmed to be 1:5.30, respectively.

[0284]

[0285] 1-4. Manufacturing of ADC 4 (Total DAR 8, Drug DAR Ratio 1:6)

[0286] Trastuzumab antibody (4 mg / ml) and 20-fold molar equivalents of TCEP were reacted in reduction buffer (150 mM NaCl, 50 mM Histidine pH 6.0) at 25°C for 2 hours. Residual TCEP was removed using a PD-10 column, and 2 mg / ml antibody and 1.34-fold molar equivalents of compound 1 were reacted in reaction buffer (25 mM Histidine pH 6.0) containing 10% DMSO at 25°C for 1 hour. Subsequently, 12-fold molar equivalents of compound 7 were added and reacted at 25°C for 1 hour. Residual linker-payload was removed using a PD-10 column. When DAR was analyzed using LC-MS, the DAR of compound 1 was confirmed to be 1.08 and that of compound 7 was 6.73. The ratio of the two linker-payloads was confirmed to be 1:6.23, respectively.

[0287]

[0288] 1-5. Preparation of ADC 5 (Total DAR 8, Drug DAR Ratio 1:7)

[0289] Trastuzumab antibody (4 mg / ml) and 20-fold molar equivalents of TCEP were reacted in reduction buffer (150 mM NaCl, 50 mM Histidine pH 6.0) at 25°C for 2 hours. Residual TCEP was removed using a PD-10 column, and 2 mg / ml antibody and 1.20-fold molar equivalents of compound 1 were reacted in reaction buffer (25 mM Histidine pH 6.0) containing 10% DMSO at 25°C for 1 hour. Subsequently, 12-fold molar equivalents of compound 7 were added and reacted at 25°C for 1 hour. Residual linker-payload was removed using a PD-10 column. When DAR was analyzed using LC-MS, the DAR of compound 1 was confirmed to be 0.98 and that of compound 7 was 6.81. The ratio of the two linker-payloads was confirmed to be 1:6.95, respectively.

[0290]

[0291] 1-6. Preparation of ADC 6 (Total DAR 8, Drug DAR Ratio 1:8)

[0292] Trastuzumab antibody (4 mg / ml) and 20-fold molar equivalents of TCEP were reacted in reduction buffer (150 mM NaCl, 50 mM Histidine pH 6.0) at 25°C for 2 hours. Residual TCEP was removed using a PD-10 column, and 2 mg / ml antibody and 1.09-fold molar equivalents of compound 1 were reacted in reaction buffer (25 mM Histidine pH 6.0) containing 10% DMSO at 25°C for 1 hour. Subsequently, 12-fold molar equivalents of compound 7 were added and reacted at 25°C for 1 hour. Residual linker-payload was removed using a PD-10 column. When DAR was analyzed using LC-MS, the DAR of compound 1 was confirmed to be 0.86 and that of compound 7 was 6.80. The ratio of the two linker-payloads was confirmed to be 1:7.91, respectively.

[0293]

[0294] [Example 2]

[0295] The DAR of the first drug (MMAE) is the manufacturing of a single ADC

[0296] In this Example 2, when the DAR of the first drug (MMAE) is 1, the effects according to changes in the first drug and the overall DAR are examined. In this Example 2, Trastuzumab was used as the antibody.

[0297]

[0298] Preparation of carrier-drug conjugates

[0299] 2-1. Manufacturing of ADC 7 (Total DAR 9, Drug DAR Ratio 1:8)

[0300] Trastuzumab used an antibody comprising mutations of T362W (kabat number) for heavy chain 1 (knob heavy chain) and T362S, L364A, Y403V, ​​and K443C (kabat number) for heavy chain 2 (hole heavy chain). The antibody in this example comprises a gene sequence encoding the amino acid sequences of SEQ ID NOs: 1 to 3.

[0301] 4 mg / ml antibody and 20-fold molar equivalents of TCEP were reacted in reduction buffer (150 mM NaCl, 50 mM Histidine pH 6.0) at 25°C for 2 hours. Residual TCEP was removed using a PD-10 column, and 2 mg / ml antibody and 20-fold molar equivalents of dhAA (Dehydroascorbic acid) were reacted at 25°C for 1 hour. Residual dhAA was removed using a PD-10 column, and 2 mg / ml antibody and 2-fold molar equivalents of compound 7 were reacted in reaction buffer (25 mM Histidine pH 6.0) containing 10% DMSO at 25°C for 1 hour. Residual linker-payload was removed using a PD-10 column, and 4 mg / ml antibody and 20-fold molar equivalents of TCEP were reacted in reduction buffer at 25°C for 2 hours. Afterwards, excess TCEP was removed using a PD-10 column, and 2 mg / ml antibody and 12-fold molar equivalents of compound 7 were reacted in a reaction buffer containing 10% DMSO at 25°C for 1 hour. The residual linker-payload was removed using a PD-10 column.

[0302] As a result of DAR analysis using LC-MS, the DAR of compound 1 in the manufactured ADC was confirmed to be 0.95, and the DAR of compound 7 was confirmed to be 7.88. The ratio of the two linker-payloads was confirmed to be 1:8.29, respectively.

[0303]

[0304] 2-2. Preparation of ADC 8 (Total DAR 7, Drug DAR Ratio 1:6)

[0305] Trastuzumab used an antibody containing mutations C225A, T362W (kabat number) for heavy chain 1 and T362S, L364A, Y403V (kabat number) for heavy chain 2. The antibody in this example contains a gene sequence encoding the amino acid sequences of SEQ ID NOs: 4 to 6.

[0306] 4 mg / ml antibody and 20-fold molar equivalents of TCEP were reacted in reduction buffer (150 mM NaCl, 50 mM Histidine pH 6.0) at 25°C for 2 hours. Residual TCEP was removed using a PD-10 column, and 2 mg / ml antibody and 20-fold molar equivalents of dhAA (Dehydroascorbic acid) were reacted at 25°C for 1 hour. Residual dhAA was removed using a PD-10 column, and 2 mg / ml antibody and 2-fold molar equivalents of compound 1 were reacted in reaction buffer (25 mM Histidine pH 6.0) containing 10% DMSO at 25°C for 1 hour. Residual linker-payload was removed using a PD-10 column, and 4 mg / ml antibody and 20-fold molar equivalents of TCEP were reacted in reduction buffer at 25°C for 2 hours. Afterwards, excess TCEP was removed using a PD-10 column, and 2 mg / ml antibody and 12-fold molar equivalents of compound 7 were reacted in a reaction buffer containing 10% DMSO at 25°C for 1 hour. The residual linker-payload was removed using a PD-10 column.

[0307] When DAR was analyzed using LC-MS, the DAR of compound 1 was confirmed to be 0.97, and that of compound 7 was confirmed to be 5.78. The ratio of the two linker-payloads was confirmed to be 1:5.96, respectively.

[0308]

[0309] 2-3. Preparation of ADC 9 (Total DAR 6, Drug DAR Ratio 1:5)

[0310] In Trastuzumab, heavy chains 1 and 2 used antibodies containing a mutation of C225A (kabat number). The antibody in this example includes a gene sequence encoding the amino acid sequences of SEQ ID NOs: 7 and 8.

[0311] Trastuzumab antibody (4 mg / ml) and 20-fold molar equivalents of TCEP were reacted in reduction buffer (150 mM NaCl, 50 mM Histidine pH 6.0) at 25°C for 2 hours. Residual TCEP was removed using a PD-10 column, and 2 mg / ml antibody and 1.20-fold molar equivalents of compound 1 were reacted in reaction buffer (25 mM Histidine pH 6.0) containing 10% DMSO at 25°C for 1 hour. Subsequently, 12-fold molar equivalents of compound 7 were added and reacted at 25°C for 1 hour. Residual linker-payload was removed using a PD-10 column.

[0312] When DAR was analyzed using LC-MS, the DAR of compound 1 was confirmed to be 0.94, and that of compound 7 was confirmed to be 4.54. The ratio of the two linker-payloads was confirmed to be 1:4.83, respectively.

[0313]

[0314] 2-4. Preparation of ADC 10 (Total DAR 5, Drug DAR Ratio 1: 4)

[0315] Trastuzumab used an antibody containing mutations of heavy chain 1, C223A, C225A, and T362W (kabat number), and heavy chain 2, C225A, T362S, L364A, and Y403V (kabat number). The antibody in this example contains a gene sequence encoding the amino acid sequence of SEQ ID NOs: 9 to 11.

[0316] 4 mg / ml antibody and 20-fold molar equivalents of TCEP were reacted in reduction buffer (150 mM NaCl, 50 mM Histidine pH 6.0) at 25°C for 2 hours. Residual TCEP was removed using a PD-10 column, and 2 mg / ml antibody and 20-fold molar equivalents of dhAA (Dehydroascorbic acid) were reacted at 25°C for 1 hour. Residual dhAA was removed using a PD-10 column, and 2 mg / ml antibody and 2-fold molar equivalents of compound 1 were reacted in reaction buffer (25 mM Histidine pH 6.0) containing 10% DMSO at 25°C for 1 hour. Residual linker-payload was removed using a PD-10 column, and 4 mg / ml antibody and 20-fold molar equivalents of TCEP were reacted in reduction buffer at 25°C for 2 hours. Afterwards, excess TCEP was removed using a PD-10 column, and 2 mg / ml antibody and 12-fold molar equivalents of compound 7 were reacted in a reaction buffer containing 10% DMSO at 25°C for 1 hour. The residual linker-payload was removed using a PD-10 column.

[0317] When DAR was analyzed using LC-MS, the DAR of compound 1 was confirmed to be 0.92, and that of compound 7 was confirmed to be 3.82. The ratio of the two linker-payloads was confirmed to be 1:4.15, respectively.

[0318]

[0319] [Example 3]

[0320] Manufacturing of additional ADCs

[0321] 3-1. Preparation of ADC 11 (Total DAR 8, Drug DAR Ratio 2:6 (1:3))

[0322] Trastuzumab antibody (4 mg / ml) and 20-fold molar equivalents of TCEP were reacted in reduction buffer (150 mM NaCl, 50 mM Histidine pH 6.0) at 25°C for 2 hours. Residual TCEP was removed using a PD-10 column, and 2 mg / ml antibody and 2.20-fold molar equivalents of compound of formula 1 (hereinafter referred to as compound 1) were reacted in reaction buffer (25 mM Histidine pH 6.0) containing 10% DMSO at 25°C for 1 hour. Subsequently, compound 7 (12-fold molar equivalents) was added and reacted at 25°C for 1 hour. Thereafter, the residual linker-payload was removed using a PD-10 column. When DAR was analyzed using LC-MS, the DAR of compound 1 was confirmed to be 2.02 and that of compound 7 was 5.81. The ratio of the two linker-payloads is confirmed to be 1:2.88 in order.

[0323]

[0324] 3-2. Preparation of ADC 12 (Total DAR 4, Drug DAR Ratio 0: 4)

[0325] Trastuzumab antibody (4 mg / ml) and 9-fold molar equivalents of TCEP were reacted in reduction buffer (150 mM NaCl, 50 mM Histidine pH 6.0) at 25°C for 2 hours. Residual TCEP was removed using a PD-10 column, and 2 mg / ml antibody and 6-fold molar equivalents of compound of formula 7 (hereinafter referred to as compound 7) were added in reaction buffer (25 mM Histidine pH 6.0) containing 10% DMSO, and the reaction was performed at 25°C for 1 hour. Residual linker-payload was removed using a PD-10 column. When DAR was analyzed using LC-MS, the DAR of compound 7 was confirmed to be 3.97.

[0326]

[0327] [Example 4]

[0328] Cell viability assay

[0329] 4-1. Total DAR is fixed at 8, and each drug has a different DAR.

[0330] For cell lines of various cancer types, 3000 cell lines (KPL4, MDA-MB-468) were seeded per well in a 96-well plate and incubated (37°C, 5% CO2). After 24 hours, ADC samples (100 μl) at 9 concentrations (serial dilution of 1 / 5 each from 1000 nM) were treated to the cells. At this time, single payload ADC ADC1 (DAR8) of Example 1-1 and dual payload ADC Tra-ADC series (ADC2 to ADC6 of Example 1) samples were treated, and a control group (ADC concentration 0) that was not treated with ADC samples was also prepared. After incubation for 6 days (37℃, 5% CO2), 100 µl of CellTiter-Glo reagent (CellTiter-Glo® Luminescent Cell Viability Assay kit (Promega, G7571)) was added to each well, pipetted, and luminescence was measured after incubation at room temperature (RT) for 10 minutes. When the luminescence value when the ADC concentration is 0 is considered 100%, the concentration that shows 50% of the luminescence value is the IC50 value.

[0331] The results of this experiment are shown in Table 1, Figures 2 and 3 below. Referring to Figure 2, it can be seen that when the total DAR is the same, ADC2 to ADC6 according to the present invention have a higher cell killing effect on HER2+ (positive) cancer cell lines than ADCs containing a single drug. On the other hand, in HER2- (negative) cancer cell lines, the results of ADC1 and ADC2 to ADC6 were similar, confirming that there was no toxicity due to the use of heterologous drugs on non-target cells (see Figure 3).

[0332] ADCsKPL-4IC50 (nM)MDA-MB-468IC50 (nM)1:3 (ADC 11)0.2730.071:4 (ADC 2)0.2943.791:5 (ADC 3)0.30102.51:6 (ADC 4)0.17150.01:7 (ADC 5)0.30121.71:8 (ADC 6)0.31137.80:8 (ADC 1)4.8698.48

[0333] This can be confirmed more clearly by referring to Fig. 4 and Table 2. Fig. 4 and Table 2 show the results comparing the toxicity of each ADC in HER2-positive cell lines (KPL4) and negative cell lines (MDA-MB-468) based on the above results.

[0334] Compound 1 (MMAE)): DAR (Compound 7 (TopI): Compound 1 / Compound 7 ratio 1 setKPL4 (HER2 +)IC50 (nM) 2 setKPL4 (HER2 +)IC50 (nM) 3 setKPL4 (HER2 +)IC50 (nM) 1 setMDA-MB-468 (HER2 -)IC50 (nM) 2 setMDA-MB-468 (HER2 -)IC50 (nM) 3 setMDA-MB-468 (HER2 -)IC50 (nM) (-) IC50 average / (+) IC50 average ratio 1:4 ADC 20.250.290.240.443.7939.1233.5125.171:5 ADC 30.20.30.190.31102.5130.2193.5532.751:6 ADC 40.170.170.190.52150.0139.3210.7568.181:7 ADC 50.140.30.20.54121.7110.74221.91436.881:8 ADC 60.130.310.130.63137.8125.3210.7442.800:8 ADC 104.863.175.2498.48100.78110.1423.32

[0335] Referring to Table 2 above, for ADC3, ADC4, ADC5, and ADC6, it can be confirmed that the average IC50 value (HER2(-) IC50 average / HER2(+) IC50 average ratio) is significantly higher in the HER2(-) cell line than in the HER2(+) cell line. That is, according to this example, when the total DAR is about 8 and the DAR of the third drug (microtubule inhibitor) is about 1, it can be seen that the DAR of the first drug (topoisomerase I inhibitor) should be 5 to 8 (more preferably 6.5 to 7.5) to have high selectivity for target cells.

[0336]

[0337] 4-2. Conditions where the total DAR and the DAR of each drug are different

[0338] For cell lines of various cancer types, 3000 cell lines (KPL4, MDA-MB-468) were seeded per well in a 96-well plate and incubated (37°C, 5% CO2). After 24 hours, ADC samples (100 μl) at 9 concentrations (serial dilution of 1 / 5 each from 1000 nM) were treated to the cells. At this time, single payload ADC ADC1 (DAR8) of Example 1-1 and dual payload ADC Tra-ADC series (ADC7 to ADC10 of Example 2, ADC5 of Example 1) samples were treated, and a control group (ADC concentration 0) that was not treated with ADC samples was also prepared. After incubation for 6 days (37℃, 5% CO2), 100 µl of CellTiter-Glo reagent (CellTiter-Glo® Luminescent Cell Viability Assay kit (Promega, G7571)) was added to each well, pipetted, and luminescence was measured after incubation at room temperature (RT) for 10 minutes. When the luminescence value when the ADC concentration is 0 is considered 100%, the concentration that shows 50% of the luminescence value is the IC50 value.

[0339] The results of this experiment are shown in Table 3, Figures 5 and 6 below. Referring to Figure 5, it can be seen that when the total DAR is the same, the cell killing effect on HER2+ (positive) cancer cell lines is higher in ADC5 and ADC7 to ADC10 containing heterologous drugs according to the present invention than in ADC1 containing a single drug. Among them, the effect was particularly noticeable in ADC5 and ADC7, which had a total DAR of 8 to 9. On the other hand, in HER2- (negative) cancer cell lines, the results of ADC1, ADC5, and ADC7 to ADC10 were similar, confirming that there was no toxicity due to the use of heterologous drugs for non-target cells (see Figure 6).

[0340] ADCsKPL-4IC50 (nM)MDA-MB-468IC50 (nM)0:8(DAR 8)ADC 14.998.261:8 (DAR9)ADC 70.1894.871:7 (DAR8)ADC 50.27115.51:6 (DAR 7)ADC 80.42132.41:5 (DAR 6)ADC 90.28103.11:4 (DAR 5)ADC 100.4559.05

[0341] Table 4 below shows the results comparing the toxicity of each ADC to HER2-positive cell lines (KPL4) and negative cell lines (MDA-MB-468) based on the above experimental results.

[0342] ADC1ADC7ADC5ADC8ADC9ADC10KPL4(HER2 +)IC50 (nM)4.90.180.270.420.280.45MDA-MB-468(HER2 -)IC50 (nM)98.2694.87115.5132.4103.159.05(-) IC50 / (+) IC50 ratio20.03526.27435.22309.06371.07130.97

[0343] Referring to Table 4 above, it can be confirmed that the ADC comprising heterologous drugs manufactured according to the present invention has superior selectivity for the target (HER2-positive cancer cell line) compared to ADC1 comprising a single drug. Among the ADCs comprising heterologous drugs, the selectivity is high in ADC7, ADC5, ADC8, and ADC9, and thus the overall DAR of the ADC comprising heterologous drugs is preferably about 6 to 9, and most preferably 8 to 9.

[0344] [Example 5]

[0345] Mouse model experimental analysis (in vivo data)

[0346] 5-1. Experimental method

[0347] Isotype IgG control ADCs G2, G3, and G4 were manufactured using mouse IgG antibodies and in the same manner as in Examples 1-1, 3-2, and 1-5, respectively, except that only the required drugs were conjugated. Single payload ADCs G5 and G6 were also manufactured using the same manner as in Examples 1-1 and 3-2, respectively, except that only the drugs were conjugated.

[0348] To confirm the effectiveness of ADCs containing heterologous drugs, the inventors administered isotype IgG control ADCs (G2, G3, G4), single payload ADCs (G5, G6), and dual payload ADCs (G7: ADC 5) to the NCI-N87 transplant mouse model, and then evaluated the in vivo efficacy.

[0349] Isotype IgG ADC was administered to confirm off-target toxicity, and the efficacy on tumor proliferation after administration of each substance was investigated. All animal experiments were conducted after approval by the Animal Experiment Ethics Committee of the CRO company based on the Animal Protection Act. NOD SCID mice (6-7 weeks old) were brought in and allowed to acclimatize for 6 days in the animal room where the test was conducted, and healthy animals were selected after checking their health and suitability for the test. NCI-N87 cells (1 x 10 8 Dilute in DPBS to a concentration of 10 cells / mL and mix 1:1 with Matrigel (Corning, Cat No. 356237, Lot No. 13823004) and dispense 100 μL (5 X 10) per individual. 6 cells) were subcutaneously transplanted into the flank. Tumor size was measured from the third day after transplantation, and the tumor size was approximately 123-220 mm. 3Upon reaching the target, mice were randomly assigned to vehicle or each of the six ADC treatment groups (n = 6). After group separation, vehicle or each of the six ADCs was administered intravenously once, totaling 3 mg / kg.

[0350] For all animals, tumor size was measured using an electronic caliper, and tumor volume was calculated using the following formula.

[0351] Tumor volume (mm 3 ) = [length (mm) x width (mm) 2 ] x 0.5

[0352] All experimental results obtained in the experiment were expressed as mean ± standard deviation (SD) and mean ± standard error (SEM) and were tested using SPSS (version 20, IBM SPSS Statistics, USA). Levene's test was performed to compare the homogeneity of variance for all data, and one-way analysis of variance (ANOVA) was performed to find significant differences between groups. When significance was observed between groups in ANOVA, the LSD test was performed if the variance was homogeneous to identify the test group with a significant difference from the control group, and Dunnett's T3 test was performed as a post-hoc test if the variance was heterogeneous.

[0353]

[0354] 5-2. Tumor size and weight analysis results

[0355] According to the above Example 5-2, the changes in tumor size and body weight according to the administration of G1 to G7 are as shown in Figs. 8 and 9. Referring to Fig. 8, it can be confirmed that the tumor size was significantly reduced in G6 and G7, and the body weight also did not show any particular change, confirming that it can be used safely.

[0356]

[0357] 5-3. Confirmation of tumor growth inhibition effect

[0358] Based on the experimental results obtained in Example 5-1, the effects of drug administration on tumor growth in each group were analyzed. Referring to Figure 10, in this experiment, G5, G6, and G7 all exhibited significant effects on tumor growth inhibition.

[0359]

[0360] Group Ratio [Tra-ADC] / [Isotype-ADC] G5 (Tra-compound 7, 3mg / kg) 5.9 G6 (Tra-compound 1, 3mg / kg) 3.45 G7 (ADC 5, 3mg / kg) 8.5

[0361]

[0362] In addition, when each group was compared with each isotype control, as shown in Table 5 above, G7 (ADC 5, 3 mg / kg) showed a ratio of 8.5, and thus, in the case of ADC containing a single drug in G6, the minimum effective dose is low, so the efficacy is high but the safety is greatly reduced, whereas when using ADC containing heterologous drugs, the safety is maintained at the level of the safer drug of the two drugs, while the efficacy can be improved compared to the existing single drug ADC, and the specificity for the target can be confirmed to be increased accordingly.

[0363]

[0364] [Example 6]

[0365] Confirmation of yield differences according to the knob-in-hole structure and use of different MMAE bonding positions in the knob-in-hole structure.

[0366] 6-1. Preparation of antibody-dual drug conjugate (AD2C) using knob-in-hole antibody

[0367] In this example, an antibody-dual drug conjugate (AD2C) was prepared using a Trastuzumab variant containing a Knob-in-hole (KIH) sequence (hereinafter, “Trastuzumab-KIH”).

[0368] First, the Trastuzumab-KIH antibody was constructed by introducing the T362W mutation (based on the Kabat number) into the knob heavy chain and the T362S, L364A, and Y403V mutations into the hole heavy chain. Furthermore, the C225A mutation was incorporated into both heavy chains of the Trastuzumab-KIH antibody, and the S239C mutation was additionally introduced into the hole heavy chain, which were utilized in the conjugation reaction.

[0369] The specific manufacturing process is as follows (see Fig. 13):

[0370] i) Trastuzumab-KIH antibody (4 mg / ml) was mixed with 20-fold molar equivalents of TCEP in reducing buffer (150 mM NaCl, 50 mM Histidine, pH 6.0) and reacted at 25°C for 2 hours. Afterwards, residual TCEP was removed using a PD-10 column.

[0371] ii) The reduced antibody (2 mg / ml) was reacted with 20-fold molar equivalents of dehydroascorbic acid (dhAA) in a reaction buffer (25°C, 1 hour), and then the residual dhAA was removed using a PD-10 column.

[0372] iii) The purified antibody (2 mg / ml) was reacted with 6-fold molar equivalents of linker-drug 1 in a reaction buffer containing 10% DMSO (25 mM Histidine, pH 6.0) at 25°C for 1 hour. After the reaction, the residual linker-drug 1 was removed using a PD-10 column.

[0373] iv) Next, the antibody (4 mg / ml) was reacted with 20-fold molar equivalents of TCEP in a reducing buffer at 25°C for 2 hours, and then the residual TCEP was removed using a PD-10 column.

[0374] v) Finally, the reduced antibody (2 mg / ml) was reacted with 12-fold molar equivalents of linker-drug 2 in a reaction buffer containing 10% DMSO at 25°C for 1 hour. After completion of the reaction, the residual linker-drug 2 was removed using a PD-10 column, thereby obtaining the desired antibody-dual drug conjugate (AD2C).

[0375]

[0376] The DAR and production yield of the linker-drug 1, linker-drug 2 used in this example and the manufactured antibody-dual drug conjugate are as shown in Tables 6 and 7 below.

[0377] Linker-Drug 1Linker-Drug 2DAR (DAR Ratio)Production Yield (%)mc-vc-MMAEFormula 70.98:5.77 (1:5.89)65.7Deruxtecan0.95:5.87 (1:6.18)61.8GGFG-Exatecan0.92:5.64 (1:6.13)63.3Formula 61.10:5.89 (1:5.35)66.9Formula 110.97:5.87 (1:6.05)59.6Formula 8-11.12:5.64 (1:5.04)67.4

[0378] Linker-Drug 1Linker-Drug 2DAR (DAR Ratio)Production Yield (%)mc-vc-MMAEChemical Formula 70.98:5.77 (1:5.89)65.7mc-va-MMAE0.95:5.87 (1:6.18)61.8mc-glucuronide-MMAE0.92:5.64 (1:6.13)63.3mc-glucuronide-MMAF1.10:5.89 (1:5.35)66.9

[0379] [Chemical Formula 6]

[0380]

[0381] [Chemical Formula 7]

[0382]

[0383] [Chemical Formula 11]

[0384]

[0385] [Chemical Formula 8-1]

[0386]

[0387] According to this example, after manufacturing an antibody-dual drug conjugate (AD2C), SEC analysis results comparing it with the existing antibody, Trastuzumab (Figures 14a and 14b) showed that the peak profiles of the two samples were almost similar, and the changes in purity were also not significant, with both samples exhibiting a high purity of over 95%. This suggests that the conjugation of the two types of drugs did not significantly alter the physical properties of the antibodies.

[0388] Meanwhile, in the HIC analysis shown in Figures 14c and 14d, the main peak of AD2C shifted by approximately 4 minutes compared to the peak retention time (RT) of Trastuzumab, which is believed to be due to the hydrophobicity of the drug. The peak was mainly detected as a result of the formation of one mc-vc-MMAE as linker-drug 1 and six compounds of chemical formula 7 as linker-drug 2. Finally, the manufacturing yield of AD2C was confirmed to be approximately 51.62%.

[0389]

[0390] 6-2. Preparation of antibody-dual drug conjugate (AD2C) without using knob-in-hole antibody

[0391] In this example, an antibody-dual drug conjugate (AD2C) was prepared using a generic Trastuzumab antibody (without mutations) (see Figure 15).

[0392] First, the antibody (4 mg / ml) was reacted with 20-fold molar equivalents of TCEP in a reducing buffer (150 mM NaCl, 50 mM Histidine, pH 6.0) at 25°C for 2 hours, and then the residual TCEP was removed using a PD-10 column. The purified antibody (2 mg / ml) was reacted with 2-fold molar equivalents of linker-drug 1 in a reaction buffer containing 10% DMSO (25 mM Histidine, pH 6.0) at 25°C for 1 hour, and then with 12-fold molar equivalents of linker-drug 2 under the same conditions for 1 hour. The reaction product was purified through HIC-FPLC analysis, and during this process, only the ADC with a DAR ratio of 1:7 was selectively recovered, ultimately obtaining AD2C.

[0393]

[0394] The DAR and production yield of the linker-drug 1, linker-drug 2 used in this example and the manufactured antibody-dual drug conjugate are as shown in Table 8 below.

[0395] Linker-Drug 1Linker-Drug 2DAR (DAR Ratio)Production Yield (%)mc-vc-MMAEChemical Formula 70.98:6.56 (1:5.89)6.98

[0396] In this example, since a general antibody was used, the DAR values ​​of linker-drug 1 (mc-vc-MMAE) and linker-drug 2 (chemical formula 7) are given as an average. Therefore, after completion of manufacturing, the desired DAR combination of 1:7 was selectively purified through HIC-FPLC. The HIC profile for purification showed mixed DAR ratios of 0:8, 1:7, 2:6, etc. (Figure 16), and only the shaded 1:7 peak was isolated and obtained.

[0397] Analysis of purified AD2C revealed that the SEC peak profiles were nearly identical to those of the original antibody, Trastuzumab (Figures 17a and 17b), and both showed high purity of over 95%, with no significant variation in purity. This suggests that the conjugation of the two drugs did not significantly affect the physical properties of the antibody.

[0398] In addition, in HIC analysis, the main peak RT (retention time) of AD2C shifted by approximately 4 minutes compared to Trastuzumab, which is believed to be due to the hydrophobicity of the drug (Figs. 17c and 17d). The peaks were mainly detected in the form of one linker-drug 1 (mc-vc-MMAE) and seven linker-drug 2 (chemical formula 7), confirming that the purification was successful. However, the final manufacturing yield was relatively low at approximately 6.98% due to losses occurring during the HIC purification process.

[0399]

[0400] In summary, when manufacturing AD2C using a general antibody, linker-drug 1 and linker-drug 2 linkages are random, resulting in a variety of DAR combinations (e.g., 0:8, 1:7, 2:6, etc.). Furthermore, obtaining the desired DAR combination requires an additional HIC-FPLC purification process. This resulted in a relatively low final yield of only approximately 6.98%.

[0401] On the other hand, when an antibody with a knob-in-hole (KIH) mutation was used, the drug was selectively conjugated to a specific position, allowing the target DAR combination (DAR 1 of the first drug conjugate) to be directly obtained with high purity, and the final yield was also significantly improved. Therefore, it was confirmed that using the KIH antibody of the present invention in the production of an antibody-dual drug conjugate (AD2C) in which the binding ratio of the first drug conjugate is DAR 1 is an effective approach that can secure high yield and reproducibility.

[0402]

[0403] The present invention has been described above, focusing on preferred embodiments thereof. Those skilled in the art will appreciate that the present invention can be implemented in modified forms without departing from its essential characteristics. Therefore, the disclosed embodiments should be considered illustrative rather than limiting. The scope of the present invention is set forth in the claims, not the foregoing description, and all differences within the scope equivalent thereto should be construed as being encompassed by the present invention.

[0404]

[0405] Sequence number 1

[0406] Antibody 1: Heavy chain 1

[0407] EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQ GTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDK THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0408]

[0409] Sequence number 2

[0410] Antibody 1: Heavy chain 2

[0411] EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGC

[0412]

[0413] 서열번호 3

[0414] Antibody 1: Light chain

[0415] DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0416]

[0417] 서열번호 4

[0418] Antibody 2: Heavy chain 1

[0419] EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHCCPPAPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0420]

[0421] säkävälödäh 5

[0422] Antibody 2: Heavy chain 2

[0423] EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0424]

[0425] 서열번호 6

[0426] Antibody 2: Light chain

[0427] DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0428]

[0429] 서열번호 7

[0430] Antibody 3: Heavy chain 1 & 2

[0431] EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPAPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0432]

[0433] 서열번호 8

[0434] Antibody 3: Light chain

[0435] DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0436]

[0437] 서열번호 9

[0438] Antibody 4: Heavy chain 1

[0439] EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTSSASTKGPSVFPLAPSSKSTGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTAPPAPAPELLGGPSVFLFPPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0440]

[0441] säkävälödäh 10

[0442] Antibody 4: Heavy chain 2

[0443] EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHCCPPAPAPELLGGPSVFLFPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0444]

[0445] säkävälödäh 11

[0446] Antibody 4: Light chain

[0447] DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

Claims

1. In an antibody-drug conjugate in which one or more drugs are combined, The antibody has a knob-in-hole structure, and the first drug is bound at a ratio of about DAR 1.

2. In paragraph 1, The above first drug is an antibody comprising a microtubule polymerization inhibitory compound.

3. In paragraph 2, The first drug is a drug selected from the group consisting of MMAE (monomethyl auristatin E), MMAF (monomethyl auristatin F), MMAU (monomethyl auristatin U), auristatin E, DM1 (emtansine), DM4, eribulin, SC209, chemical formula 1-1 and pharmaceutically acceptable salts, hydrates, isomers and prodrugs thereof, an antibody: [Chemical Formula 1-1] .

4. In paragraph 1, A second drug is additionally bound to the above antibody, The second drug is an antibody that contains a compound that inhibits topoisomerase I activity.

5. In paragraph 4, The second drug is bound to the antibody at a ratio of about DAR 5 to 8.

6. In paragraph 4, The second drug is an antibody selected from the group consisting of DXd, Exatecan, FL118, SN-38, Camptothecin, Chemical Formula 4, Chemical Formula 4-1, Chemical Formula 4-2, and pharmaceutically acceptable salts, hydrates, isomers, and prodrugs thereof. [Chemical Formula 4] , [Chemical Formula 4-1] , [Chemical Formula 4-2] .

7. In an antibody-drug conjugate in which one or more drugs are combined, An antibody-drug conjugate wherein the antibody has a knob-in-hole structure and the first drug is bound at a ratio of about DAR 1.

8. In paragraph 7, An antibody-drug conjugate wherein the first drug is a drug comprising a microtubule polymerization inhibitory compound.

9. In paragraph 7, The first drug is a drug selected from the group consisting of MMAE (Monomethyl auristatin E), MMAF (Monomethyl auristatin F), MMAU (Monomethyl auristatin U), auristatin E, DM1 (Emtansine), DM4, Eribulin, SC209, Chemical Formula 1-1 and pharmaceutically acceptable salts, hydrates, isomers and prodrugs thereof, an antibody-drug conjugate: [Chemical Formula 1-1] .

10. In paragraph 7, A second drug is additionally bound to the above antibody, An antibody-drug conjugate wherein the second drug is a drug comprising a topoisomerase I activity inhibitory compound.

11. In paragraph 10, The second drug is an antibody-drug conjugate selected from the group consisting of DXd, Exatecan, FL118, SN-38, camptothecin, chemical formula 4, chemical formula 4-1, chemical formula 4-2 and pharmaceutically acceptable salts, hydrates, isomers and prodrugs thereof. [Chemical Formula 4] , [Chemical Formula 4-1] , [Chemical Formula 4-2] .

12. In paragraph 10, An antibody-drug conjugate wherein the second drug is bound to the antibody at a ratio of about DAR 5 to 8.

13. In paragraph 7, An antibody-drug conjugate wherein the drug is linked to an antibody via a linker.

14. Antibody; comprising a first drug and a second drug bound to the antibody, The above first drugs are combined in a ratio of about DAR 1, The above antibody is an antibody-dual drug conjugate having a knob-in-hole structure.

15. In paragraph 14, An antibody-dual drug conjugate wherein the first drug is a drug comprising a microtubule polymerization inhibitory compound.

16. In paragraph 14, An antibody-dual drug conjugate wherein the second drug is a drug comprising a topoisomerase I activity inhibitory compound.

17. In paragraph 14, The first drug is a drug selected from the group consisting of MMAE (Monomethyl auristatin E), MMAF (Monomethyl auristatin F), MMAU (Monomethyl auristatin U), auristatin E, DM1 (Emtansine), DM4, Eribulin, SC209, Chemical Formula 1-1 and pharmaceutically acceptable salts, hydrates, isomers and prodrugs thereof, an antibody-dual drug conjugate: [Chemical Formula 1-1] .

18. In paragraph 14, The second drug is an antibody-dual drug conjugate selected from the group consisting of DXd, Exatecan, FL118, SN-38, Camptothecin, Chemical Formula 4, Chemical Formula 4-1, Chemical Formula 4-2 and pharmaceutically acceptable salts, hydrates, isomers and prodrugs thereof: [Chemical Formula 4] , [Chemical Formula 4-1] , [Chemical Formula 4-2] .

19. In paragraph 14, An antibody-dual drug conjugate, wherein the first drug and the second drug are each linked to an antibody via a linker.

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