ANTI-C-MET CONJUGATES - PHARMACEUTICALS INCLUDING CAMPTOTHECIN DERIVATIVES AND PHARMACEUTICALS CONTAINING THIS CONJUGATE

Abstract

The invention relates to an antibody-drug conjugate consisting of a compound with formula 1 or its pharmaceutical salt, stereoisomer, tautomer, prodrug, hydrate, solvate, or isotopically labeled analogue, bound to an anti-c-Met antibody via a α-linked group; and a pharmaceutical composition comprising such conjugate. The antibody-drug conjugate according to the invention is capable of selectively expressing the effect of compound with formula 1 on cancer cells expressing cMet.

Description

Anti-C-MET antibody-drug conjugates comprising camptothecin derivatives

[0001] The present invention relates to an anti-c-MET antibody-drug conjugate having a structure in which a compound according to chemical formula 1 is linked to an antibody via a linker.

[0002] In addition, the present invention relates to a method for treating or preventing cancer using the antibody-drug conjugate, a pharmaceutical composition for treating or preventing cancer comprising the antibody-drug conjugate as an active ingredient, a method for treating or preventing cancer comprising a step of administering the same, and a use thereof.

[0003] Cancer is a serious disease, and according to statistics from the World Health Organization (WHO), the incidence of cancer is increasing worldwide every year, creating a pressing need in the pharmaceutical industry to develop novel anticancer drugs. Camptothecin (CPT), an alkaloid isolated from plants in the Davidiaceae family, has been shown to exhibit cytotoxic anticancer properties by inhibiting topoisomerase. Camptothecin-based drugs such as irinotecan (CPT-11), topotecan, 10-hydroxy camptothecin, belotecan (CKD-602), and exatecan have been developed both domestically and internationally. Recently, a substance called DXd has also been developed, indicating that the development of novel camptothecin derivatives is ongoing.

[0004] However, cytotoxic anticancer agents lack cell selectivity and attack not only cancer cells but also normal cells, causing unexpected side effects. Therefore, they are sometimes manufactured as antibody-drug conjugates (ADCs) by conjugating antibodies that bind to specific target antigens on the surface of cancer cells with drugs that have cytotoxic effects.

[0005] Meanwhile, c-MET protein, a member of the MET family of proteins, is a type of tyrosine kinase receptor expressed on the surface of various epithelial cells. Hepatocyte growth factor receptor (HGF) is a ligand for the c-MET receptor, and when HGF binds to c-MET, a series of intracellular signals that mediate embryonic development and wound healing in normal cells are initiated. However, in various types of major cancer cells, such as breast, stomach, lung, and colon cancer, amplification, mutation, and protein overexpression related to the c-MET gene occur, promoting tumor development and progression. Therefore, c-MET and its associated signaling pathways are attracting attention as clinically important therapeutic targets in anticancer treatment.

[0006] Accordingly, the development of antibody-drug conjugates that contain camptothecin derivative compounds as drugs and have high cytotoxicity and act specifically on cancer cells expressing c-MET is becoming a new challenge.

[0007] Accordingly, the object of the present invention is to provide an anti-c-MET antibody-drug conjugate comprising a camptothecin derivative having excellent cytotoxicity activity and excellent targeting specificity for cells expressing c-MET, and a pharmaceutical use thereof.

[0008] To solve the above problem, the present invention provides an antibody-drug conjugate having a structure in which a compound according to chemical formula 1 or a pharmaceutically acceptable salt, stereoisomer, tautomer, prodrug, hydrate, solvate or isotopically labeled analog thereof is linked to an anti-c-MET antibody via a linker.

[0009] Another aspect of the present invention provides a method for treating or preventing cancer, comprising the step of contacting the antibody-drug conjugate with a cancer cell.

[0010] Another aspect of the present invention provides a pharmaceutical composition for treating or preventing cancer, comprising the antibody-drug conjugate as an active ingredient, a method for treating cancer, and a use thereof, comprising a step of administering the same.

[0011] The antibody-drug conjugate according to the present invention can exhibit the effect of the compound drug of chemical formula 1 in a target-specific manner on cells expressing c-MET.

[0012] The compound of formula 1 is an active camptothecin derivative designed to bind to DDX5 protein and E3 ligase, wherein the compound can bind to E3 ligase as a molecular glue; and / or can kill target cells expressing DDX5 protein through a molecular glue mechanism (MoA); and / or has a mechanism of action (MoA) to degrade oncoprotein DDX5 together with the ability to inhibit type 1 topoisomerase.

[0013] The antibody-drug conjugate of the present invention readily penetrates cell membranes due to the hydrophobicity of the compound of formula 1. Upon entering cancer cells, the linker is specifically cleaved by a lysosomal protease highly expressed in tumors, releasing the drug of formula 1 and killing the cancer cells. The drug is then released and can induce a bystander effect, killing surrounding cancer cells. This allows it to exhibit an excellent killing effect not only on cancer cells that highly express c-MET but also on cancer cells with low expression.

[0014] Figure 1 illustrates the size exclusion chromatography (SEC) results of the antibody-drug conjugate (ADC) of Example 3.

[0015] Figure 2 shows the results of flow cytometry analysis for a commercially available monoclonal antibody (FITC-mAb, 11-8858-42) and anti-c-MET antibody & FITC-anti human IgG Fc (410720) antibodies in the process of selecting a cell line expressing c-MET protein.

[0016] Figure 3 shows the results of an in-vitro cell viability comparison evaluation when the T47D breast cancer cell line was treated with a cytotoxic compound or ADC.

[0017] Figure 4 shows the results of an in-vitro cell viability comparison evaluation when the SNU-5 gastric cancer cell line was treated with a cytotoxic compound or ADC.

[0018] Figure 5 shows the results of an in-vitro cell viability comparison evaluation when the NCI-H1573 lung cancer cell line was treated with a cytotoxic compound or ADC.

[0019] Figure 6 shows the results of an in-vitro cell viability comparison evaluation when the HT29 colon cancer cell line was treated with a cytotoxic compound or ADC.

[0020] Figure 7 shows the results of an in-vitro cell viability comparison evaluation when the NCI-H1975 lung cancer cell line was treated with a cytotoxic compound or ADC.

[0021] Figure 8 is a graph showing the binding affinity of the M01 antibody to c-MET proteins from human, mouse, rat, dog, and monkey origin.

[0022] Figure 9 is a graph showing the binding affinity of the ADC (M01-PBX-7016) and M01 antibody of Example 3 of the present invention to c-MET proteins derived from humans and monkeys.

[0023] Figure 10 is a graph showing that the M01 antibody binds to a cell line expressing c-MET protein in a dose-dependent manner.

[0024] Figure 11 is a graph showing the change in body weight of mice during the in-vivo experiment on the Hs746T gastric cancer cell line.

[0025] Figures 12 and 13 are tumor growth curves during the in-vivo experimental period for the Hs746T gastric cancer cell line.

[0026] Figure 14 is a graph showing the change in body weight of mice during the in-vivo experimental period for the LU-01-0439 lung cancer cell line.

[0027] Figure 15 is a tumor growth curve during the in-vivo experimental period for the LU-01-0439 lung cancer cell line.

[0028] Figure 16 is a graph showing the change in body weight of mice during the in-vivo experimental period for the NCI-H441 lung cancer cell line.

[0029] Figure 17 is a tumor growth curve during the in-vivo experimental period for the NCI-H441 lung cancer cell line.

[0030] The present invention will be described in more detail below.

[0031] Embodiments of the present invention may be modified in various forms, and the scope of the present invention is not limited to the embodiments described below. Furthermore, these embodiments of the present invention are provided to more completely explain the present invention to those of ordinary skill in the art. Furthermore, throughout the specification, the term "comprising" a certain element does not exclude other elements, but rather implies the inclusion of other elements, unless specifically stated otherwise.

[0032] According to one aspect of the present invention, an antibody-drug conjugate (ADC) is provided, wherein a compound according to the following chemical formula 1 or a pharmaceutically acceptable salt, stereoisomer, tautomer, prodrug, hydrate, solvate or isotopically labeled analog thereof has a structure in which the compound is linked to an anti-c-MET antibody via a linker:

[0033] [Chemical Formula 1]

[0034]

[0035] Here,

[0036] X1 and X3 are each independently carbon, oxygen, nitrogen, or sulfur, and X1 and X3 may be the same or different,

[0037] X2 is carbon, oxygen, nitrogen, sulfur, a single bond or a double bond,

[0038] n is 0, 1, or 2,

[0039] Y1, Y2 and Y3 may each independently be hydrogen or a functional group containing oxygen, nitrogen, phosphorus or sulfur.

[0040] Here, when X2 is a single bond or a double bond, n may be 0. More specifically, it may be a single bond or a double bond that binds X1 and X3.

[0041] Here, non-limiting examples of functional groups containing oxygen, nitrogen, phosphorus or sulfur include -CHO, -COOH, -NH2, -SH, -CONH2, -PO3H, -PO4H2, -OPO4H, -PO2(OR 1 )(OR 2 )(R 1 , R 2 =C s H t N u O w S x P y X z, n H 2n+1 , 0≤n≤16), -NR3 + X - (R= C n H m , 0≤n≤16, 0≤m≤34, X = OH, Cl or Br), NR4 + X - (R= C n H m , 0≤n≤16, 0≤m≤34, X = OH, Cl or Br), -COSH, -COOCO-, -CORCO- (R = C n H m , 0≤n≤3, 0≤m≤2l+1), -COOR, -CN, -N3, -N2, -NROH(R = C s H t N u O w S x P y X z , X = -F, -Cl, -Br or -I, 0≤s≤20, 0≤t≤2(s+u)+1, 0≤u≤2s, 0≤w≤2s, 0≤x≤2s, 0≤y≤2s, 0≤z≤2s), -NR 1 NR 2 R 3 (R 1 , R 2 , R 3 = C s H t N u O w S x P y X z , X = -F, -Cl, -Br or -I, 0≤s≤20, 0≤t≤2(s+u)+1, 0≤u≤2s, 0≤w≤2s, 0≤x≤2s, 0≤y≤2s, 0≤z≤2s), -CONHNR 1 R 2 (R 1 , R2 = C s H t N u O w S x P y X z , X = -F, -Cl, -Br or -I, 0≤s≤20, 0≤t≤2(s+u)+1, 0≤u≤2s, 0≤w≤2s, 0≤x≤2s, 0≤y≤2s, 0≤z≤2s), -NR 1 R 2 R 3 X'(R 1 , R 2 , R 3 = C s H t N u O w S x P y X z , X = -F, -Cl, -Br or -I, X'= F-,Cl-,Br-, or I-, 0≤s≤20, 0≤t≤2(s+u)+1, 0≤u≤2s, 0≤w≤2s, 0≤x≤2s, 0≤y≤2s, 0≤z≤2s), -OH, -O-, >C=O, -SS-, -SO-, -NO2, -COX(X = F, Cl, Br or I), -COOCO-, -CONH-, -CN, -SCOCH3, -SCN, -NCS, -NCO, -OCN, -CN, -F, -Cl, -I, -Br, epoxy group, -hydrazone, -ONO2, -PO(OH)2, -C=NNH2, -HC=CH-, -C=C-, -C ≡C- and hydrocarbons with two or more carbon atoms. It may include a functional group selected from the group consisting of:

[0042] According to one aspect of the present invention, an antibody-drug conjugate (ADC) is provided, wherein a compound according to the following chemical formula 1 or a pharmaceutically acceptable salt, stereoisomer, tautomer, prodrug, hydrate, solvate or isotopically labeled analog thereof has a structure in which the compound is linked to an anti-c-MET antibody via a linker:

[0043] [Chemical Formula 1]

[0044]

[0045] Here,

[0046] X1 and X3 are each independently carbon, oxygen, nitrogen, or sulfur, and X1 and X3 may be the same or different,

[0047] X2 is carbon, oxygen, nitrogen, sulfur, a single bond or a double bond,

[0048] X1, (X2)n and X3 can form a 6-membered or 7-membered ring together, where n is 1 or 2,

[0049] Y1, Y2 and Y3 may each independently be hydrogen or a functional group containing oxygen, nitrogen, phosphorus or sulfur.

[0050] Here, non-limiting examples of functional groups containing oxygen, nitrogen, phosphorus or sulfur include -CHO, -COOH, -NH2, -SH, -CONH2, -PO3H, -PO4H2, -OPO4H, -PO2(OR 1 )(OR 2 )(R 1 , R 2 =C s H t N u O w S x P y X z , n H 2n+1 , 0≤n≤16), -NR3 + X - (R= C n H m , 0≤n≤16, 0≤m≤34, X = OH, Cl or Br), NR4 + X - (R= Cn H m , 0≤n≤16, 0≤m≤34, X = OH, Cl or Br), -COSH, -COOCO-, -CORCO- (R = C n H m , 0≤n≤3, 0≤m≤2l+1), -COOR, -CN, -N3, -N2, -NROH(R = C s H t N u O w S x P y X z , X = -F, -Cl, -Br or -I, 0≤s≤20, 0≤t≤2(s+u)+1, 0≤u≤2s, 0≤w≤2s, 0≤x≤2s, 0≤y≤2s, 0≤z≤2s), -NR 1 NR 2 R 3 (R 1 , R 2 , R 3 = C s H t N u O w S x P y X z , X = -F, -Cl, -Br or -I, 0≤s≤20, 0≤t≤2(s+u)+1, 0≤u≤2s, 0≤w≤2s, 0≤x≤2s, 0≤y≤2s, 0≤z≤2s), -CONHNR 1 R 2 (R 1 , R 2 = C s H t N u O w S x P y X z , X = -F, -Cl, -Br or -I, 0≤s≤20, 0≤t≤2(s+u)+1, 0≤u≤2s, 0≤w≤2s, 0≤x≤2s, 0≤y≤2s, 0≤z≤2s), -NR 1 R 2 R 3 X'(R 1 , R 2 , R 3 = Cs H t N u O w S x P y X z , X = -F, -Cl, -Br or -I, X'= F-,Cl-,Br-, or I-, 0≤s≤20, 0≤t≤2(s+u)+1, 0≤u≤2s, 0≤w≤2s, 0≤x≤2s, 0≤y≤2s, 0≤z≤2s), -OH, -O-, >C=O, -SS-, -SO-, -NO2, -COX(X = F, Cl, Br or I), -COOCO-, -CONH-, -CN, -SCOCH3, -SCN, -NCS, -NCO, -OCN, -CN, -F, -Cl, -I, -Br, epoxy group, -hydrazone, -ONO2, -PO(OH)2, -C=NNH2, -HC=CH-, -C=C-, -C ≡C- and hydrocarbons with two or more carbon atoms. It may include a functional group selected from the group consisting of:

[0051] According to one aspect of the present invention, in the chemical formula 1,

[0052] X1, X2 and X3 can each independently be carbon,

[0053] n can be 1,

[0054] Y1 and Y2 can each independently be hydrogen,

[0055] Y3 may be hydrogen or a functional group containing oxygen, nitrogen, phosphorus or sulfur.

[0056] Here, non-limiting examples of functional groups containing oxygen, nitrogen, phosphorus or sulfur may be the same as the examples described above.

[0057] According to one aspect of the present invention, in the chemical formula 1,

[0058] X1 and X3 are each independently carbon, oxygen, nitrogen, or sulfur, and X1 and X3 may be the same or different,

[0059] X2 is carbon, oxygen, nitrogen, sulfur, a single bond or a double bond,

[0060] n is 0, 1, or 2,

[0061] Y1, Y2 and Y3 may each independently be hydrogen or a functional group containing oxygen, nitrogen, phosphorus or sulfur.

[0062] The functional group containing the above nitrogen may be -NZ1Z2,

[0063] Z1 and Z2 are each independently hydrogen, halogen, hydroxyl, C 1-3 Alkyl, C(=O)H, C(=O)(C 1-3 alkyl), C(=O)(C 2-3 alkenyl), C(=O)NH2, C(=O)NH(C 1-3 alkyl), (CH2) 2-4 OH, (CH2) 2-4 O(C 1-3 alkyl), (CH2) 2-4 NH2, (CH2) 2-4 NH(C 1-3 alkyl), (CH2) 2-4 N(C 1-3 alkyl)2 or C(=O)CR 1 R 2 R 3 It may include a functional group selected from the group consisting of,

[0064] At this time R 1 , R 2 and R 3 are each independently hydrogen, hydroxyl, C 1-3 Alkyl, C 3-7 Cycloalkyl or C 1-3 It may be haloalkyl.

[0065] According to one aspect of the present invention, in the chemical formula 1,

[0066] X1, X2 and X3 can each independently be carbon,

[0067] n can be 1,

[0068] Y1 and Y2 can each independently be hydrogen,

[0069] Y3 may be hydrogen or a functional group containing oxygen, nitrogen, phosphorus or sulfur.

[0070] Here, non-limiting examples of functional groups containing oxygen, nitrogen, phosphorus or sulfur may be the same as the examples described above.

[0071] According to one aspect of the present invention, in the chemical formula 1,

[0072] X1 and X3 are each independently carbon, oxygen, nitrogen, or sulfur, and X1 and X3 may be the same or different,

[0073] X2 is carbon, oxygen, nitrogen, sulfur, a single bond or a double bond,

[0074] X1, (X2)n and X3 can form a 6-membered or 7-membered ring together, where n is 1 or 2,

[0075] Y1, Y2 and Y3 may each independently be hydrogen or a functional group containing oxygen, nitrogen, phosphorus or sulfur.

[0076] The functional group containing the above nitrogen may be -NZ1Z2,

[0077] Z1 and Z2 are each independently hydrogen, halogen, hydroxyl, C 1-3 Alkyl, C(=O)H, C(=O)(C 1-3 alkyl), C(=O)(C 2-3 alkenyl), C(=O)NH2, C(=O)NH(C 1-3 alkyl), (CH2) 2-4 OH, (CH2) 2-4 O(C 1-3 alkyl), (CH2) 2-4 NH2, (CH2) 2-4 NH(C 1-3 alkyl), (CH2) 2-4 N(C 1-3 alkyl)2 or C(=O)CR 1 R 2R 3 It may include a functional group selected from the group consisting of,

[0078] At this time R 1 , R 2 and R 3 are each independently hydrogen, hydroxyl, C 1-3 Alkyl, C 3-7 Cycloalkyl or C 1-3 It may be haloalkyl.

[0079] According to one aspect of the present invention, in the chemical formula 1,

[0080] X1, X2 and X3 can each independently be carbon,

[0081] n can be 1,

[0082] Y1 and Y2 can each independently be hydrogen,

[0083] Y3 can be -NZ1Z2,

[0084] Z1 and Z2 are each independently hydrogen, halogen, hydroxyl, C 1-3 Alkyl, C(=O)H, C(=O)(C 1-3 alkyl), C(=O)(C 2-3 alkenyl), C(=O)NH2, C(=O)NH(C 1-3 alkyl), (CH2) 2-4 OH, (CH2) 2-4 O(C 1-3 alkyl), (CH2) 2-4 NH2, (CH2) 2-4 NH(C 1-3 alkyl), (CH2) 2-4 N(C 1-3 alkyl)2 or C(=O)CR 1 R 2 R 3 It may include a functional group selected from the group consisting of,

[0085] At this time R 1 , R 2 and R 3 are each independently hydrogen, hydroxyl, C 1-3 Alkyl, C3-7 Cycloalkyl or C 1-3 It may be haloalkyl.

[0086] According to one aspect of the present invention, in the chemical formula 1,

[0087] X1, X2 and X3 can each independently be carbon,

[0088] n can be 1,

[0089] Y1 and Y2 can each independently be hydrogen,

[0090] Y3 can be -NZ1Z2,

[0091] Z1 and Z2 are each independently hydrogen, C(=O)H, C(=O)(C 1-3 alkyl), C(=O)(C 2-3 alkenyl), C(=O)NH2, C(=O)NH(C 1-3 alkyl) or C(=O)CR 1 R 2 R 3 It can be,

[0092] At this time R 1 , R 2 and R 3 are each independently hydrogen, hydroxyl, C 1-3 Alkyl, C 3-7 Cycloalkyl or C 1-3 It may be haloalkyl.

[0093] According to one aspect of the present invention, in the chemical formula 1,

[0094] X1, X2 and X3 can each independently be carbon,

[0095] n can be 1,

[0096] Y1 and Y2 can each independently be hydrogen,

[0097] Y3 can be -NZ1Z2,

[0098] Z1 is hydrogen, and Z2 is C(=O)(C 2-3 alkenyl) or C(=O)CR 1 R 2R 3 It may be,

[0099] At this time R 1 , R 2 and R 3 are each independently hydrogen, hydroxyl, C 1-3 Alkyl, C 3-5 Cycloalkyl or C 1-3 It may be haloalkyl.

[0100] According to one aspect of the present invention, in the chemical formula 1,

[0101] X1, X2 and X3 can each independently be carbon,

[0102] n can be 1,

[0103] Y1 and Y2 can each independently be hydrogen,

[0104] Y3 can be -NZ1Z2,

[0105] Z1 is hydrogen, and Z2 is C(=O)CR 1 R 2 R 3 It may be,

[0106] At this time R 1 , R 2 and R 3 are each independently hydrogen, hydroxyl or C 1-3 It could be an alkyl.

[0107] According to one aspect of the present invention, the compound of formula 1 may be an antibody-drug conjugate, which is a compound represented by any one of the following formulae 2 to 11, or a pharmaceutically acceptable salt, stereoisomer, tautomer, prodrug, hydrate, solvate or isotopically labeled analog thereof.

[0108] [Chemical Formula 2]

[0109]

[0110] [Chemical Formula 3]

[0111]

[0112] [Chemical Formula 4]

[0113]

[0114] [Chemical Formula 5]

[0115]

[0116] [Chemical Formula 6]

[0117]

[0118] [Chemical Formula 7]

[0119]

[0120] [Chemical Formula 8]

[0121]

[0122] [Chemical Formula 9]

[0123]

[0124] [Chemical Formula 10]

[0125]

[0126] [Chemical Formula 11]

[0127]

[0128] According to one aspect of the present invention, the compound of formula 1 may be an active camptothecin derivative designed to bind to DDX5 protein and E3 ligase.

[0129] According to one aspect of the present invention, the compound of the above chemical formula 1 may bind to E3 ligase as a molecular glue.

[0130] According to one aspect of the present invention, the compound of chemical formula 1 can kill target cells expressing DDX5 protein through a molecular glue mechanism (MoA).

[0131] The target cells may be cancer cells or senescent cells. Senescent cells also include cells that no longer perform the organ's specific functions.

[0132] According to one aspect of the present invention, the compound of the above chemical formula 1 may have a mechanism of action (MoA) that degrades oncoprotein DDX5 together with the ability to inhibit type 1 topoisomerase.

[0133] The term "drug" or "payload" used in the present invention may mean any one compound selected from the group consisting of the above chemical formulas 1 to 11. The drug or payload of the present invention may be combined with a linker and an anti-c-MET antibody to produce an antibody-drug conjugate.

[0134] The term "antibody-drug conjugate (ADC)" used in the present invention allows a monoclonal antibody or an antigen-binding fragment thereof to be linked to a biologically active cytotoxin via a chemically stable linker, thereby maximizing the specificity of antibody binding to cancer cell surface antigens and the high efficiency of cytotoxins, while avoiding the low efficacy of antibody therapeutics or the toxicity of cytotoxins. This allows the ADC to precisely bind to cancer cells and reduce the effects on normal cells, compared to conventional anticancer therapeutics. The antibody-drug conjugate of the present invention specifically binds to cells expressing the c-MET protein, thereby enabling specific drug targeting.

[0135] The above antibody-drug conjugate is characterized in that it can easily penetrate cell membranes due to the hydrophobicity of the compound of chemical formula 1, and when it enters cancer cells, the linker is specifically cleaved by a lysosomal protease highly expressed in tumors, releasing the drug of chemical formula 1 and killing the cancer cells.

[0136] The above antibody-drug conjugate is characterized by having an excellent killing effect even on cancer cells with low c-MET expression, by causing a bystander effect in which the drug is released after cell death and kills surrounding cancer cells.

[0137] The term "antibody" as used in the present invention may mean an antibody or an antigen-binding fragment thereof, and may mean a macromolecular compound capable of recognizing and binding to an antigen or receptor associated with a target cell population to which it binds. The antibody may be, but is not limited to, a murine antibody, a humanized antibody, a chimeric antibody, a rabbit antibody, a phage display antibody, a yeast display antibody, a CDR-grafted antibody, or a recombinant human antibody. In particular, the term antibody as used in the present invention means an "anti-c-MET antibody," which may mean an antibody or an antigen-binding fragment thereof that specifically binds to c-MET.

[0138] The antibody of the present invention may preferably be a monoclonal antibody, and the antigen-binding fragment may be one or more fragments of an antibody having a specific binding ability to an antigen.

[0139] The antibody of the present invention can form a bond with a linker through a heteroatom on the antibody.

[0140] The antibody of the present invention is an anti-c-MET antibody, and non-limiting examples of the anti-c-MET antibody include Telisotuzumab, Emibetuzumab (LY2875358), Onartuzumab, ARGX-111, HLX55, M01, etc. Here, the M01 antibody may be an antibody having CDRs of SEQ ID NOs: 1 to 6. In one aspect of the present invention, the M01 may be an antibody comprising i) a heavy chain having an amino acid sequence of SEQ ID NO: 9 and ii) a light chain having an amino acid sequence of SEQ ID NO: 10.

[0141] The antibody of the present invention may be an antibody or antigen-binding fragment thereof having i) a heavy chain comprising CDR-H1, CDR-H2 and CDR-H3 having amino acid sequences of SEQ ID NOs: 1, 2 and 3, respectively, and ii) a light chain comprising CDR-L1, CDR-L2 and CDR-L3 having amino acid sequences of SEQ ID NOs: 4, 5 and 6, respectively.

[0142] The antibody of the present invention may be an antibody or an antigen-binding fragment thereof comprising i) a heavy chain variable region having an amino acid sequence of SEQ ID NO: 7 and ii) a light chain variable region having an amino acid sequence of SEQ ID NO: 8.

[0143] The antibody of the present invention may be an antibody or an antigen-binding fragment thereof comprising i) a heavy chain having an amino acid sequence of SEQ ID NO: 9 and ii) a light chain having an amino acid sequence of SEQ ID NO: 10.

[0144] The Drug to Antibody Ratio (DAR), which refers to the average number of drugs linked per antibody of the present invention, may be in the range of about 1 to about 20, and specifically may be in the range of about 1 to about 8, about 2 to about 8, about 3 to about 8, about 4 to about 8, about 5 to about 8, about 6 to about 8, about 7 to about 8, or about 7.5 to about 8. Additionally, specifically, it may be in the range of about 3 to 5, about 3.5 to about 4.5, or about 3 to about 4.

[0145] In one aspect of the present invention, the DAR of the antibody-drug conjugate of the present invention may be in the range of 7 to 8 or 3 to 4. In a preferred aspect of the present invention, the DAR of the antibody-drug conjugate of the present invention may be in the range of 7 to 8. In a preferred aspect of the present invention, the DAR of the antibody-drug conjugate of the present invention may be in the range of 3 to 4.

[0146] The term "linker" as used in the present invention refers to a chemical structural fragment or bond that is linked to an antibody at one end and to a drug at the other end, or is linked to another linker and then to a drug.

[0147] More specifically, the linker may be cleavable or non-cleavable, and the cleavable linker not only functions to connect the antibody and the drug together, but can also be selectively cleaved by a specific pH, enzyme (protease, phosphatase, glycosidase, sulfatase), and glutathione (GSH) level of the cancer surrounding environment to release the drug.

[0148] More specifically, the linker is C 1-6It may include at least one selected from the group consisting of alkylene, carbonyl, maleimide, succinimide -O-, sugar, sugar acid, β-glucuronide, β-galactoside, phosphodiester, PEG, PSAR (polysarcosine), disulfide, PAB (p-aminobenzyloxycarbonyl), triazole, hydrazone, phosphate, diester, thioether and peptide, but is not limited thereto.

[0149] More specifically, the peptide residue may be a peptide residue composed of one or more amino acids selected from the group consisting of alanine (A), asparagine (N), glutamine (Q), ornithine, proline (P), threonine (T), phenylalanine (F), glycine (G), valine (V), lysine (K), citrulline, serine (S), glutamic acid (E), and aspartic acid (D), and more specifically, may be a peptide residue composed of one or more amino acids selected from the group consisting of valine, citrulline, phenylalanine, and glycine, and more specifically, may be a dipeptide, tripeptide, or tetrapeptide residue, and more specifically, may be a dipeptide residue of valine-citrulline, valine-alanine, and phenylalanine-glycine, or a tetrapeptide residue of GGFG (glycine-glycine-phenylalanine-glycine). Here, some of the amino acid residues of the peptide residues can be optionally substituted.

[0150] The linker of the present invention may be represented by the following chemical formula 12:

[0151] [Chemical Formula 12]

[0152]

[0153] Here,

[0154] R X and R Y are each independently hydrogen or C 1-6 It is alkyl,

[0155] p and q are each independently integers from 1 to 6,

[0156] * is the part that binds to the payload.

[0157] According to one aspect of the present invention, in the chemical formula 12,

[0158] R X and R Y are each independently hydrogen or C 1-3 It can be alkyl,

[0159] p can be an integer from 4 to 6,

[0160] q can be an integer from 1 to 3.

[0161] According to one aspect of the present invention, in the chemical formula 12,

[0162] R X and R Y can each independently be hydrogen,

[0163] p can be 5,

[0164] q can be 1.

[0165] According to one aspect of the present invention, the compound of chemical formula 12 may be a compound of chemical formula 13.

[0166] [Chemical Formula 13]

[0167]

[0168] Here, * is the part that binds to the payload.

[0169] In one aspect of the present invention, the compound of formula 12 can have a maleimide ring linked to an amino acid residue of an antibody. In a preferred aspect of the present invention, the compound of formula 12 can have any one of the carbons of the -CH2=CH2- structure of the maleimide ring linked to a cysteine ​​among the amino acid residues of the antibody.

[0170] In one aspect of the present invention, when the compound of the above chemical formula 13 is combined with an antibody, it can be linked in the form of chemical formula 14.

[0171] [Chemical Formula 14]

[0172]

[0173] Here, * represents a site that binds to the payload, and ** represents a site that binds to the antibody.

[0174] In one aspect of the present invention, ** shown in the compound of formula 14 is a site that binds to an antibody. In a preferred aspect of the present invention, the compound of formula 14 can bind to cysteine ​​among the amino acid residues of the antibody at the ** site.

[0175] The term “mc-GGFG” used in the present invention means a linker having a structure of a compound of chemical formula 13 or a compound of chemical formula 14.

[0176] The compound of formula 1 according to the present invention may exist in the form of a pharmaceutically acceptable salt, stereoisomer, tautomer, prodrug, hydrate, solvate or isotopically labeled analog. Accordingly, the category of the compound of the present invention includes the compound of formula 1 and pharmaceutically acceptable salts, stereoisomers, tautomers, prodrugs, hydrates, solvates or isotopically labeled analogs thereof, and can be similarly applied to drug-linker compounds and antibody-drug conjugates based thereon.

[0177] The term "pharmaceutically acceptable salt" as used herein means any organic or inorganic acid addition salt of the compound, which has an effective action at a concentration that is relatively non-toxic and harmless to the patient, and wherein the side effects attributable to the salt do not diminish the beneficial effects of the parent compound.

[0178] In particular, the pharmaceutically acceptable salt may be an acid addition salt formed by a free acid. Here, the acid addition salt can be obtained from inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, nitrous acid, phosphorous acid, etc.; non-toxic organic acids such as aliphatic mono- and dicarboxylates, phenyl-substituted alkanoates, hydroxyalkanoates and alkanedioates, aromatic acids, aliphatic and aromatic sulfonic acids, etc.; organic acids such as trifluoroacetic acid, acetate, benzoic acid, citric acid, lactic acid, maleic acid, gluconic acid, methanesulfonic acid, 4-toluenesulfonic acid, tartaric acid, fumaric acid, etc.

[0179] Such pharmaceutically acceptable salts may include sulfates, sulfites, nitrates, phosphates, pyrophosphates, chlorides, bromides, iodides, fluorides, acetates, trifluoroacetates, propionates, decanoates, caprylates, acrylates, formates, isobutyrates, caprates, heptanoates, propiolates, oxalates, malonates, succinates, suberates, sebacates, fumarates, maleate, benzoate, phthalates, benzenesulfonates, toluenesulfonates, phenylacetates, phenylpropionates, phenylbutyrates, citrates, lactates, glycolates, malate, tartrate, mandelates, and the like.

[0180] The above acid addition salt can be prepared by a conventional method, for example, by dissolving the compound in an organic solvent such as methanol, ethanol, acetone, methylene chloride, acetonitrile, etc., adding an organic acid or inorganic acid, filtering and drying the resulting precipitate, or by distilling the solvent and an excess acid under reduced pressure, drying, and crystallizing in the presence of an organic solvent.

[0181] In addition, the pharmaceutically acceptable salt may be a salt or metal salt obtained using a base. As an example of a metal salt, an alkali metal or alkaline earth metal salt can be obtained by dissolving a compound in an excess alkali metal hydroxide or alkaline earth metal hydroxide solution, filtering out the undissolved compound salt, and evaporating and drying the filtrate. As an alkali metal salt, sodium, potassium, or calcium salts may be pharmaceutically suitable. In addition, a corresponding salt can be obtained by reacting an alkali metal or alkaline earth metal salt with a suitable silver salt (e.g., silver nitrate).

[0182] In addition, the compound of the present invention may include hydrates and solvates. The hydrate refers to a compound or a salt thereof that further contains a stoichiometric or non-stoichiometric amount of water bound by non-covalent intermolecular forces. The hydrate includes, for example, a monohydrate, a dihydrate, a trihydrate, a tetrahydrate, etc. The solvate refers to a solvent-containing compound formed by the association of one or more solvent molecules with the compound of the present invention. The solvate includes, for example, a monosolvate, a disolvate, a trisolvate, and a disolvate. The hydrate and solvate can be prepared using a known method, and are preferably non-toxic and water-soluble. In particular, the hydrate and solvate may be water, an alcoholic solvent (particularly, methanol, ethanol, etc.), trifluoroacetic acid, formic acid, and ammonia, but are not limited thereto.

[0183] Additionally, the compounds of the present invention may be prepared in the form of prodrugs, which, although having little or no pharmacological activity themselves, can be converted into compounds with the desired activity, for example, by hydrolysis, when administered internally. Such prodrugs may be derivatives of compounds containing a biologically reactive functional group, wherein the biologically reactive functional group can be cleaved from the compound or otherwise reacted under biological conditions (in vivo or in vitro) to yield the compound. Typically, the prodrug is inactive, or at least has a lower activity than the compound, which allows the compound to exhibit its activity after cleavage from the biologically reactive functional group. The biologically reactive functional group can be hydrolyzed or oxidized under biological conditions to yield the compound. For example, the prodrug may contain a biologically hydrolyzable group. Examples of biologically hydrolyzable groups include, but are not limited to, biologically hydrolyzable phosphates, biologically hydrolyzable esters, biologically hydrolyzable amides, biologically hydrolyzable carbonic esters, biologically hydrolyzable carbamates, and biologically hydrolyzable ureides.

[0184] In addition, the compounds of the present invention may have the form of stereoisomers. The stereoisomers refer to compounds that have the same chemical formula or molecular formula but are sterically different, and include enantiomers, diastereomers, geometric isomers (or cis / trans isomers), rotamers, and atropisomers, and each of these isomers and mixtures thereof are also included in the scope of the present invention. For example, the compounds of the present invention may have a chiral carbon center, and thus individual enantiomers or mixtures thereof (e.g., racemic mixtures) may also fall within the scope of the present invention. In addition, the compound of the present invention may have the form of a diastereomer including a conformational isomer such as a rotamer and a geometric isomer (or cis / trans isomer), and all such stereoisomers and mixtures thereof may all fall within the scope of the present invention. In this respect, since the formula (I) of the present invention does not specify a stereochemical structure, it may include the stereoisomers of the formula (I). Specifically, in the chemical structural formula, a solid bond ( ) is a wedge-shaped solid line combination representing the absolute arrangement of the stereocenter. ) and wedge-shaped dotted line combination ( ) may be included. It should also be construed that stereoisomers of the exemplary compounds described herein are also included in the scope of the present invention.

[0185] Additionally, the compounds of the present invention may exist in the form of tautomers. Tautomers can have a structure in which a proton is shifted from one atom of a molecule to another atom of the same molecule, and can be interconverted by the movement of hydrogen atoms accompanying the conversion of single bonds and adjacent double bonds. In a solution in which tautomerization is possible, a chemical equilibrium of tautomers will exist. The exact ratio of tautomers depends on several factors, including temperature, solvent, and pH.

[0186] In addition, the compounds of the present invention further include isotopically labeled analogs. Specifically, the compounds of the present invention further include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, and chlorine (respectively). 2 H, 3 H, 13 C, 11 C, 14 C, 15 N, 18 O, 17 O, 31 P, 32 P, 35 S, 18 F and 36 Cl). All compounds of the present invention containing the above isotopes and / or other isotopes of other atoms fall within the scope of the present invention.

[0187] According to one aspect of the present invention, a method for treating or preventing cancer is provided, comprising the step of contacting the antibody-drug conjugate with a cancer cell.

[0188] The antibody-drug conjugate according to the present invention can inhibit the activity of cancer cells. Accordingly, the present invention provides a pharmaceutical composition or anticancer agent for treating or preventing cancer, comprising the antibody-drug conjugate as an active ingredient.

[0189] According to one aspect of the present invention, the present invention provides a method for treating or preventing cancer comprising administering an antibody-drug conjugate to a subject.

[0190] According to one aspect of the present invention, the present invention provides a use of an antibody-drug conjugate for the treatment or prevention of cancer.

[0191] According to one aspect of the present invention, the present invention provides the use of an antibody-drug conjugate for preparing a pharmaceutical composition for treating or preventing cancer.

[0192] Here, “treatment” means any action that improves or beneficially changes the symptoms of the disease by administering the compound, and “prevention” means any action that inhibits or delays the occurrence, spread, and recurrence of the disease by administering the compound.

[0193] Here, “administration” means providing a given substance to a subject in need thereof by any appropriate method, and the route of administration of the antibody-drug conjugate of the present invention can be administered through any general route, such as oral or parenteral, as long as it can reach the target tissue.

[0194] Here, “cancer” includes any cancer treatable by inhibition of topoisomerase I and / or inhibition of any one or more cancer-associated survival genes selected from the group consisting of DDX5, survivin, Mcl-1, XIAP and cIAP2, and may be a solid cancer or a hematological cancer. For example, the cancer is 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, 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, It may be at least one selected from the group consisting of 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 tumor, gastrointestinal stromal cancer, Wilms' cancer, breast cancer, 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 tumor, 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, but is not limited thereto. Additionally, the cancer includes not only primary cancer but also metastatic cancer.

[0195] According to one aspect of the present invention, the cancer may be at least one selected from the group consisting of liver cancer, colon cancer, lung cancer, non-small cell lung cancer, stomach cancer, kidney cancer, and pancreatic cancer.

[0196] [Example]

[0197] Hereinafter, the composition of the present invention is specifically described through examples. However, the following examples are intended only to aid understanding of the present invention and are not intended to limit the scope of the present invention. The reagents, solvents, and starting materials described in the examples below are readily available commercially, and their sources of acquisition are not specifically described.

[0198] The method for producing a camptothecin derivative according to the present invention is as follows.

[0199] Manufacturing Example: Synthesis of a PBX Payload

[0200] Manufacturing Example 1: Synthesis of active camptothecin derivatives of chemical formula 2 or chemical formula 3 (PBX-7011 and PBX-7012)

[0201]

[0202] 1-1. Synthesis of compound 2

[0203]

[0204] Silver triflate (57.7 g, 224 mmol) and iodine (57.0 g, 224 mmol) were added to a solution of 5-nitrobenzo[d][1,3]dioxole (25 g, 150 mmol) in a dichloromethane (748 ml) flask. The solution was stirred in a dark room at room temperature under an N2 atmosphere.

[0205] AgI was removed by filtration and the solid was washed with dichloromethane (100 mL). The solvent was removed under reduced pressure and the residue was partitioned between EtOAc (250 mL) and 5% (v / v) NH4OH / H2O solution (200 mL). The organic layer was separated and washed with 1 M Na2SO3 (5 x 200 mL) and brine (200 mL), dried over Na2SO4, treated with activated charcoal, and filtered through Celite. The solvent was evaporated under reduced pressure to give the crude as a brown solid. This was triturated with ice-cold EtOH (400 mL), filtered, and the solid was washed with ice-cold EtOH (100 mL) to give the product as a pale brown-gray solid (14.3 g). The solvent was removed from the filtrate and the crude was triturated a second time with ice-cold EtOH (300 mL). The solid was collected by filtration and washed with EtOH (50 mL) to give an additional amount of product (10.2 g) as a dark brown-gray solid. Both batches were used as is without further purification.

[0206] SC_ACID: m / z 294.2 [M+H] +

[0207] 1-2. Synthesis of compound 3

[0208]

[0209] Iron powder (6.67 g, 119 mmol) and ammonium chloride (6.39 g, 119 mmol) were added to a suspension of 4-iodo-6-nitrobenzo[d][1,3]dioxole (8.75 g, 29.9 mmol) in a mixture of water (80 mL), methanol (40.0 mL), and tetrahydrofuran (40.0 mL). The suspension was heated to 75 °C and stirred for 16.5 h. The reaction mixture was cooled to room temperature and concentrated under reduced pressure. The resulting black solid was suspended in ethyl acetate (100 mL), and the solution was decanted. The residue was washed with EtOAc (3 x 50 mL). The mixture was transferred to a separatory funnel, water was added, and the aqueous layer was removed. The organic layer was washed with saturated aqueous NaHCO3 solution (150 mL) and brine (150 mL). The organic layer was dried over Na2SO4, and the solvent was removed under reduced pressure to give a brown solid (4.75 g, 60% yield). Additional product was recovered by thoroughly washing the iron residue with ethyl acetate. The organic fraction was then washed with saturated aqueous NaHCO3 solution (150 mL), brine (150 mL), and dried over Na2SO4. The solvent was removed under reduced pressure to give a brown solid (1.74 g, 22% yield).

[0210] SC_ACID: m / z 264.0 [M+H] +

[0211] 1-3. Synthesis of compound 4

[0212]

[0213] 7-Iodobenzo[d][1,3]dioxol-5-amine (6.39 g) in dichloromethane (49 ml)

[0214] To a solution of 24.29 mmol) acetic anhydride (2.75 mL, 29.2 mmol) and triethylamine (4.06 mL, 29.2 mmol) were added. The reaction mixture was stirred at room temperature overnight. After several hours, additional DCM (10 mL) was added. The suspension was filtered through a sintered funnel and washed with ice-cold DCM (10 mL). The product was further dried under reduced pressure to give a light gray solid (4.46 g). The filtrate was concentrated under reduced pressure, and the residue was dissolved in EtOAc, washed with water and brine, dried over Na2SO4, and concentrated under reduced pressure to give a brown solid (~3 g). The brown solid was purified by flash column chromatography (80 g Si, 0-100% ethyl acetate in heptane). All batches of the product were triturated with ice-cold EtOAc (5-10 ml). The two batches were combined and further dried to obtain a grayish-white solid.

[0215] Total yield: 5.0 g, 66%

[0216] SC_ACID: m / z 306.0 [M+H] +

[0217] 1H NMR (400 MHz, DMSO-d6) δ 9.88 (s, 1H), 7.39 (d, J = 1.9 Hz, 1H), 7.17 (d, J = 1.9 Hz, 1H), 6.04 (s, 2H), 1.99 (s, 3H).

[0218] 1-4. Synthesis of compound 5

[0219]

[0220] A slurry of N-(7-iodobenzo[d][1,3]dioxol-5-yl)acetamide (5.06 g, 16.59 mmol), but-3-enoic acid (1.69 mL, 19.90 mmol), and potassium carbonate (2.98 g, 21.56 mmol) in acetonitrile (40 mL) was cooled to 0–5°C in a three-neck flask equipped with a condenser. Water (13.33 mL) was slowly added to evolve gas. When gas evolution ceased, the mixture was degassed with Ar for 30 min. Tri-o-tolylphosphine (0.505 g, 1.659 mmol) and palladium acetate (0.186 g, 0.829 mmol) were added, and the mixture was again degassed for 30 min and then heated to reflux under Ar. The reaction mixture was cooled to room temperature and filtered through Celite. The filter cake was washed with H2O and EtOAc. The organic solvent was removed from the filtrate under vacuum, and the aqueous phase was acidified with a solution of concentrated HCl until the pH reached 1-2. The aqueous layer was extracted with EtOAc, and the combined organic phases were washed with brine, dried over Na2SO4, filtered, and concentrated under reduced pressure to give the crude product. The crude material was triturated with ice-cold EtOAc (30 mL), and the solid was collected by filtration to give the product as a brown solid. The mother liquor was concentrated under reduced pressure and purified by flash chromatography (80 g Si, 0-100% EtOAc in heptane). The product fractions were concentrated under reduced pressure to give a light brown foam. Total yield: 3.46 g, 75%. A mixture of E / Z isomers was obtained.

[0221] SC_ACID: m / z 264.4 [M+H] +

[0222] 1-5. Synthesis of compound 6

[0223]

[0224] A suspension of 4-(6-acetamidobenzo[d][1,3]dioxol-4-yl)but-3-enoic acid (3.47 g, 13.18 mmol) in tetrahydrofuran (50 mL) / water (50 mL) was degassed with N2 for 15 min. Pd / C (2.97 g, 1.397 mmol) was added and the suspension was degassed with H2 for 5 min before heating to 40 °C under H2 (balloon). After 24 h, the reaction mixture was added nitrogen and additional Pd / C (2.97 g, 1.397 mmol). The reaction mixture was bubbled with hydrogen for 10 min and stirred at 45 °C under hydrogen overnight. The reaction was filtered through Celite. The filter cake was washed with H2O (50 mL) and EtOAc (50 mL). The EtOAc (50 mL) and organic solvent were removed from the filtrate under vacuum. The aqueous solution was acidified with concentrated HCl until the pH = 1, and a dark brown / green precipitate was collected by filtration through a sintered funnel. The aqueous filtrate was extracted. The combined organic phases were washed with brine, dried over Na2SO4, filtered, and the solvent was removed under vacuum to give a brown oil. Since the product recovery was low, the filter cake was washed with EtOAc (50 mL), water (200 mL), and MeOH (400 mL). The water / EtOAc flush was added to the flask containing the solid from the MeOH flush. The aqueous layer was acidified to a concentration. HCl and an off-white precipitate formed until the pH = 1, which was collected by filtration through a sintered funnel. After extracting the aqueous filtrate with EtOAc (3x200 mL), LCMS indicated that all product had been removed from the aqueous phase. The combined organic phases were washed with brine, dried over Na2SO4, filtered, and the solvent was removed in vacuo to give a light brown solid. All product batches were combined and purified by flash column chromatography (40 g Si, 0-10% MeOH in DCM). The product fractions were concentrated to give a light brown solid. Yield: 2.52 g, 72%.

[0225] SC_ACID: 266.2 [M+H] +

[0226] 1H NMR (400 MHz, DMSO-d6) δ 12.07 (s, 1H), 9.78 (s, 1H), 7.14 (d, J = 2.0 Hz, 1H), 6.80 (d, J = 2.2 Hz, 1H), 5.95 (s, 2H), 2.50 - 2.46 (m, 2H), 2.23 (t, J = 7.4 Hz, 2H), 1.98 (s, 3H), 1.84 - 1.70 (m, 2H).

[0227] 1-6. Synthesis of compound 7

[0228]

[0229] A suspension of 4-(6-acetamidobenzo[d][1,3]dioxol-4-yl)butanoic acid (150 mg, 0.565 mmol) in TFA (433 μL, 5.65 mmol) was cooled on ice. TFAA (157 μL, 1.131 mmol) was added. The mixture was stirred at 0-5°C for 1 h, over which time it turned into a dark solution. The reaction mixture was added dropwise to ice-cold saturated aqueous NaHCO3 solution (10 mL), and the aqueous solution was extracted with ethyl acetate (3 x 25 mL). The combined organic layers were dried over sat. NaHCO3 and brine, filtered, and the solvent was removed in vacuo to give a salmon pink solid. Yield: 140 mg, 100%

[0230] SC_ACID: m / z 248.2 [M+H] +

[0231] 1H NMR (400 MHz, DMSO-d6) δ 12.34 (s, 1H), 8.11 (s, 1H), 6.13 (s,2H), 2.80 (t, J = 6.2 Hz, 2H), 2.66 - 2.58 (m, 2H), 2.12 (s, 3H), 2.01 - 1.91(m, 2H).

[0232] 1-7. Synthesis of compound 8

[0233]

[0234] A suspension of N-(6-oxo-6,7,8,9-tetrahydronaphtho[1,2-d][1,3]dioxol-5-yl)acetamide (50 mg, 0.202 mmol) in tetrahydrofuran (1.2 mL) was cooled to 0°C and potassium tert-butoxide (27.2 mg, 0.243 mmol) and isoamyl nitrite (35.0 μL, 0.263 mmol) were added. The dark green mixture was stirred on ice (< 5°C) for 1.5 h. Acetic acid (170 μL, 2.94 mmol), acetic anhydride (170 μL, 1.810 mmol) and zinc dust (66.1 mg, 1.011 mmol) were added to the reaction mixture. The suspension was stirred at 0°C for 2 h. The reaction mixture was filtered over celite and flushed with DCM. The filtrate was concentrated under reduced pressure to give a black oily substance. The crude product was purified by flash column chromatography (4 g Si, 0-4% MeOH in DCM). The product fractions were concentrated to give a gray solid (32 mg, 52%) with 80-90% purity. Higher purity samples can be obtained by preparative MPLC.

[0235] SC_ACID: m / z 305.4 [M+H] +

[0236] 1H NMR (400 MHz, DMSO-d6) δ 12.10 (s, 1H), 8.21 (d, J = 8.0Hz, 1H), 8.12 (s, 1H), 6.15 (d, J = 9.3 Hz, 2H), 4.66 - 4.56 (m, 1H), 2.93(dd, J = 8.9, 4.0 Hz, 2H), 2.14 (s, 3H), 2.13 - 1.93 (m, 2H), 1.91 (s, 3H).

[0237] 1-8. Synthesis of compound 9

[0238]

[0239] N,N'-(6-Oxo-6,7,8,9-tetrahydronaphtho[1,2-d][1,3]dioxole-5,7-diyl)diacetamide (244 mg, 0.802 mmol) was suspended in 2 M hydrochloric acid (4.69 mL, 9.38 mmol) in ethanol / water (5 / 1). The mixture was heated to 55 °C for 4 h. The black mixture was cooled to 0-5 °C. Triethylamine (1.4 mL, 10.04 mmol) was added dropwise with stirring. The mixture was then diluted with EtOH and evaporated to dryness. The residue was partitioned between water and DCM. The layers were separated, and the aqueous layer was extracted once with DCM. The combined organic layers were dried over Na2SO4 and concentrated to give the product as a brown solid (178 mg, 73%) in 86% purity.

[0240] SC_ACID: m / z 263.0 [M+H] +

[0241] 1H NMR (400 MHz, DMSO) δ 8.04 (d, J = 8.0 Hz, 1H), 6.20 (s, 1H), 5.94 (d, J = 6.0 Hz, 2H), 4.48 - 4.41 (m, 1H), 3.08 (s, 2H), 2.88 - 2.69(m, 2H), 2.15 - 2.03 (m, 1H), 1.88 (s, 3H), 1.86 - 1.75 (m, 1H).

[0242] 1-9. Synthesis of compound 10

[0243]

[0244] (4S)-4-Ethyl-7,8-dihydro-4-hydroxy-1H-pyrano[3,4-f]indolizine-3,6,10(4H)-trione (146 mg, 0.555 mmol) and N-(5-amino-6-oxo-6,7,8,9-tetrahydronaphtho[1,2-d][1,3]dioxol-7-yl)acetamide (112 mg, 0.427 mmol) were added to dry toluene (4.5 ml). PPTS (21 mg, 0.085 mmol) was added, and the reaction mixture was stirred at 115°C for 40 h.

[0245] The reaction mixture was cooled to room temperature. The suspension was diluted with 2 mL of DCM and filtered. A black residue (210 mg) was obtained.

[0246] The crude product was purified by column chromatography (12 g Si, 0-7% methanol in DCM) to give the product (45 mg, 21%) as a brown solid.

[0247] LCMS analysis showed two diastereoisomers.

[0248] SC_ACID: m / z 490.2 [M+H] +

[0249] 1H NMR (400 MHz, DMSO-d6) δ 8.47 (t, J = 9.3 Hz, 1H), 7.42 (s, 1H), 7.24 (s, 1H), 6.49 (s, 1H), 6.29 (d, J = 5.2 Hz, 2H), 5.57 - 5.49 (m, 1H), 5.41 (s, 2H), 5.23 - 5.07 (m, 2H), 3.09 - 3.00 (m, 2H), 2.11 - 2.01 (m, 2H), 1.91 (s, 3H), 1.89 - 1.79 (m, 2H), 0.87 (t, J = 7.1 Hz, 3H).

[0250] 1-10. Synthesis of PBX-7011 and PBX-7012

[0251]

[0252] N-((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)acetamide (73.5 mg, 0.150 mmol) was dissolved in 1.0 mL 6N HCl and stirred at 90 °C for 8 h and then at room temperature overnight. The reaction mixture was concentrated under reduced pressure and purified by acidic preparative MPLC twice (Luna2-30). The fractions containing the separated diastereomers were acidified with 5 drops of 3N HCl and lyophilized to give the product as a yellow solid.

[0253] 1st eluting isomer: PBX-7011, 23 mg, 34% yield

[0254] U_AN_ACID: m / z 448.4 [M+H] +

[0255] 1H NMR (400 MHz, DMSO-d6) δ 8.54 (s, 3H), 7.50 (s, 1H), 7.27 (s, 1H), 6.52 (s, 1H), 6.34 (d, J = 13.3 Hz, 2H), 5.77 (d, J = 19.3 Hz, 1H), 5.44(s, 2H), 5.37 (d, J = 19.3 Hz, 1H), 5.05 (s, 1H), 3.19 - 3.02 (m, 2H), 2.46(s, 1H), 2.20 - 2.03 (m, 1H), 1.94 - 1.81 (m, 2H), 0.88 (t, J = 7.3 Hz, 3H).

[0256] 2nd eluting isomer: PBX-7012, 28 mg, 41% yield

[0257] U_AN_ACID: m / z 448.2 [M+H] +

[0258] 1H NMR (400 MHz, DMSO-d6) δ 8.57 (d, J = 4.7 Hz, 3H), 7.51 (s, 1H), 7.27 (s, 1H), 6.52 (s, 1H), 6.34 (d, J = 12.2 Hz, 2H), 5.76 (d, J = 19.4 Hz, 1H), 5.44 (s, 2H), 5.37 (d, J = 19.4 Hz, 1H), 5.08 (s, 1H), 3.16 - 2.98(m, 2H), 2.46 (s, 1H), 2.18 - 2.06 (m, 1H), 1.94 - 1.80 (m, 2H), 0.87 (t, J = 7.3 Hz, 3H).

[0259] Manufacturing Example 2: Synthesis of active camptothecin derivatives of chemical formula 4 or 5 (PBX-7014 and PBX-7015)

[0260] This example describes the synthesis of compounds PBX-7014 and PBX-7015 starting from two separate diastereomers of the Exatecan-hybrid compound (PBX-7011 and PBX-7012).

[0261]

[0262] 2-1: Synthesis of PBX-7014

[0263]

[0264] A stock solution of activated glycolic acid was prepared according to the following procedure:

[0265] Glycolic acid (17 mg, 0.224 mmol) was dissolved in 1 mL of N,N-dimethylformamide. HOSu (25.7 mg, 0.223 mmol) and EDC (42.8 mg, 0.223 mmol) were added. The reaction mixture was stirred at room temperature for 1 h.

[0266] Next, 0.4 mL of activated acid solution was added to a suspension of (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 (40 mg, 0.089 mmol) and triethylamine (0.025 mL, 0.179 mmol) in N,N-dimethylformamide (2.5 mL). The mixture was stirred at room temperature for 3 h. 0.05 mL of freshly prepared activated acid solution was added. The reaction mixture was then stirred at room temperature for an additional 2 h. The reaction mixture was evaporated to dryness. The crude product was purified by column chromatography (0-8% methanol in chloroform). This gave a yellow solid containing the PBX-7014 product and residual succinimide. The product was further purified by acidic preparative MPLC (Luna 5-40) to give a light yellow solid after lyophilization of the product fraction. Yield: 20 mg, 50%

[0267] U_AN_ACID: m / z 506.2 [M+H] +

[0268] 1H NMR (400 MHz, DMSO-d6) δ 8.40 (d, J = 8.9 Hz, 1H), 7.40 (s, 1H), 7.23 (s, 1H), 6.49 (s, 1H), 6.28 (d, J = 4.6 Hz, 2H), 5.61 - 5.45 (m, 2H), 5.45 - 5.35 (m, 2H), 5.19 - 5.06 (m, 2H), 3.95 (s, 2H), 3.13 - 2.96 (m, 2H), 2.21 - 2.02 (m, 2H), 1.94 - 1.77 (m, 2H), 0.87 (t, J = 7.3 Hz, 3H).

[0269] 2-2: Synthesis of PBX-7015

[0270]

[0271] A stock solution of activated acid was prepared according to the following procedure:

[0272] Glycolic acid (17.00 mg, 0.223 mmol) was dissolved in 1 mL of N,N-dimethylformamide. HOSu (25.7 mg, 0.223 mmol) and EDC (42.8 mg, 0.223 mmol) were added. The reaction mixture was stirred at room temperature for 1 h.

[0273] Next, 0.25 mL of activated acid solution was added to a suspension of (1R,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 (25 mg, 0.056 mmol) and triethylamine (0.016 mL, 0.112 mmol) in N,N-dimethylformamide (2.5 mL). The mixture was stirred at room temperature overnight. 0.03 mL of freshly prepared activated acid solution was added. The reaction mixture was then stirred at room temperature for 2 h. The reaction mixture was combined with the previous smaller batch and evaporated to dryness. The crude product was purified by column chromatography. This gave a yellow solid containing the PBX-7015 product and residual succinimide. The product was purified by acidic preparative MPLC (Luna 5-40). The product fraction was lyophilized to give a light yellow solid. Yield: 15 mg, 38%

[0274] U_AN_ACID: 506.2 [M+H] +

[0275] 1H NMR (400 MHz, DMSO) δ 8.44 (d, J = 9.0 Hz, 1H), 7.41 (s, 1H), 7.24 (s, 1H), 6.48 (s, 1H), 6.29 (d, J = 2.3 Hz, 2H), 5.61 - 5.44 (m, 2H), 5.44 - 5.36 (m, 2H), 5.20 - 5.07 (m, 2H), 3.96 (s, 2H), 3.10 - 2.96 (m, 2H), 2.20 - 2.06 (m, 2H), 1.95 - 1.79 (m, J = 7.3 Hz, 2H), 0.87 (t, J = 7.3 Hz, 3H).

[0276] Manufacturing Example 3: Synthesis of active camptothecin derivatives of chemical formulas 6 and 7 (PBX-7016 and PBX-7017)

[0277] This example describes the synthesis of compound PBX-7016 starting from the Exatecan-hybrid compound (PBX-7011).

[0278]

[0279] A stock solution of activated D-lactic acid was prepared according to the following procedure.

[0280] D-lactic acid (22 mg, 0.244 mmol) was dissolved in 1 mL of N,N-dimethylformamide. HOSu (27 mg, 0.235 mmol) and EDC (38.6 mg, 0.201 mmol) were added. The reaction mixture was stirred at room temperature for 2 h.

[0281] Next, 0.3 mL of activated acid solution was added to a solution of (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 (36 mg, 0.080 mmol) and triethylamine (0.022 ml, 0.161 mmol) in N,N-dimethylformamide (2.5 mL). The mixture was stirred at room temperature for 6 h. 0.05 mL of the prepared activated acid solution was added. The reaction mixture was then stirred at room temperature overnight. The reaction mixture was purified directly by acid preparative MPLC (Luna10-50), and the product fraction was lyophilized to give a light yellow solid.

[0282] Yield: 22 mg, 52%

[0283] U_AN_ACID: m / z 520.2 [M+H] +

[0284] 1H NMR (400 MHz, DMSO) δ 8.43 (d, J = 9.1 Hz, 1H), 7.41 (s, 1H), 7.23 (s, 1H), 6.50 (s, 1H), 6.29 (d, J = 2.1 Hz, 2H), 5.62 - 5.58 (m, 1H), 5.58 - 5.51 (m, 1H), 5.41 (s, 2H), 5.21 - 5.01 (m, 2H), 4.17 - 4.07 (m, 1H), 3.14 - 2.95 (m, 2H), 2.19 - 2.04 (m, 2H), 1.92 - 1.78 (m, 2H), 1.39 (d, J = 6.8 Hz, 3H), 0.87 (t, J = 7.3 Hz, 3H).

[0285] As described above, PBX-7016 of chemical formula 6 can be synthesized from PBX-7011 of chemical formula 2, and thus PBX-7017 of chemical formula 7 can be synthesized from PBX-7012 of chemical formula 3 using the same method.

[0286] Manufacturing Example 4: Synthesis of an active camptothecin derivative of chemical formula 8 (PBX-7018)

[0287]

[0288] (S)-3,3,3-Trifluoro-2-hydroxypropanoic acid (11.59 mg, 0.080 mmol) was dissolved in N,N-dimethylformamide (2 ml) and HATU (Hexafluorophosphate Azabenzotriazole Tetramethyl Uronium) (30.6 mg, 0.080 mmol) was added.

[0289] The mixture was stirred for 3 min, then (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 (30 mg, 0.067 mmol) and DIPEA (N,N-Diisopropylethylamine) (0.047 ml, 0.268 mmol) designated PBX-7011 were added, and the reaction mixture was stirred at room temperature for 1.5 h. The reaction mixture was purified directly by acidic preparative MPLC (Luna10-50), and the product fractions were lyophilized to obtain the product as an off-white solid. (Yield: 19 mg, 49%)

[0290] The above PBX-7011 can be obtained by the method described in PCT / KR2023 / 009854, but is not limited thereto.

[0291] U_AN_ACID: m / z 574.2 [M+H] +

[0292] 1H NMR (400 MHz, DMSO-d6) δ 8.95 (d,J= 8.6 Hz, 1H), 7.42 (s, 1H), 7.24 (s, 1H), 7.18 (d,J= 6.2 Hz, 1H), 6.48 (s, 1H), 6.29 (d,J= 7.1 Hz, 2H), 5.61 - 5.52 (m, 1H), 5.46 - 5.35 (m, 2H), 5.21 (d,J= 19.2 Hz, 1H), 5.06 (d,J= 19.2 Hz, 1H), 4.66 - 4.55 (m, 1H), 3.08 - 2.97 (m, 2H), 2.18 - 2.03 (m, 2H), 1.93 - 1.77 (m, 2H), 0.87 (t,J= 7.3 Hz, 3H).

[0293] Manufacturing Example 5: Synthesis of an active camptothecin derivative of chemical formula 9 (PBX-7020)

[0294]

[0295] (R)-2-Hydroxybutyric acid (20.9 mg, 0.201 mmol) was dissolved in 1 mL DMF. HOSu (23.1 mg, 0.201 mmol) and EDC (38.6 mg, 0.201 mmol) were added, and the reaction mixture was stirred at room temperature for 2 h.

[0296] Then, 0.3 mL of the activated acid solution was added to a suspension of PBX-7011 (30 mg, 0.067 mmol) and triethylamine (0.019 ml, 0.134 mmol) in N,N-dimethylformamide (2 ml). The mixture was stirred at room temperature for 23 h. The reaction mixture was directly purified by acid preparative MPLC (Luna10-50) which gave an off-white solid after lyophilization of the product fractions. (Yield: 25 mg, 69%)

[0297] U_AN_ACID: m / z 534.2 [M+H] +

[0298] 1H NMR (400 MHz, DMSO) δ 8.39 (d, J = 8.9 Hz, 1H), 7.41 (s, 1H), 7.23 (s, 1H), 6.48 (s, 1H), 6.29 (d, J = 3.6 Hz, 2H), 5.57 - 5.50 (m, 1H), 5.48 - 5.44 (m, 1H), 5.41 (s, 2H), 5.13 (s, 2H), 3.92 - 3.86 (m, 1H), 3.06 - 3.00 (m, 2H), 2.15 - 2.05 (m, 2H), 1.92 - 1.74 (m, 3H), 1.71 - 1.62 (m, 1H), 0.93 (t, J = 7.4 Hz, 3H), 0.87 (t, J = 7.3 Hz, 3H).

[0299] Manufacturing Example 6: Synthesis of an active camptothecin derivative of chemical formula 10 (PBX-7022)

[0300]

[0301] A suspension of PBX-7011 (30 mg, 0.067 mmol) and DIPEA (0.047 mL, 0.268 mmol) in dichloromethane (1 mL) was cooled to 0°C. Acryloyl chloride solution (5.44 μl, 0.067 mmol) in 0.1 mL of DCM was added dropwise. The reaction mixture was stirred at 0°C, and after 30 min, 0.15 equivalents of acryloid chloride as a stock solution in DCM was added. After stirring for a total of 2 h, the reaction mixture was concentrated under reduced pressure. The residue was dissolved in DMSO and purified by acidic preparative MPLC (Reprosil 10-50 method). The product fractions were collected and lyophilized to give a pale yellow, fluffy solid. (Yield: 22 mg, 65%)

[0302] U_AN_ACID: m / z 502.2 [M+H] +

[0303] 1H NMR (400 MHz, DMSO-d6) δ 8.71 (d,J= 8.8 Hz, 1H), 7.43 (s, 1H), 7.23 (s, 1H), 6.49 (s, 1H), 6.30 (d,J= 4.2 Hz, 2H), 6.28 - 6.21 (m, 2H), 5.69 (dd,J= 8.5, 3.7 Hz, 1H), 5.65 - 5.58 (m, 1H), 5.40 (s, 2H), 5.25 - 5.12 (m, 1H), 5.12 - 4.99 (m, 1H), 3.10 - 3.02 (m, 2H), 2.16 - 2.06 (m, 2H), 1.92 - 1.78 (m, 2H), 0.86 (t,J= 7.3 Hz, 3H).

[0304] Manufacturing Example 7: Synthesis of an active camptothecin derivative of chemical formula 11 (PBX-7024)

[0305]

[0306] PBX-7024 was prepared in high yield by coupling (2S)-2-cyclopropyl-2-hydroxyacetic acid to the PBX-7011 compound.

[0307] (2S)-2-Cyclopropyl-2-hydroxyacetic acid (26 mg, 0.244 mmol) was dissolved in 1 mL of N,N-dimethylformamide. HOSu (26 mg, 0.226 mmol) and EDC (42 mg, 0.219 mmol) were added. The reaction mixture was stirred at room temperature for 2 h. Next, 0.6 mL of activated acid solution was added to a solution of (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 (40 mg, 0.089 mmol) and DIPEA (0.047 ml, 0.268 mmol) in N,N-dimethylformamide (2.5 ml). The mixture was stirred at room temperature overnight. The reaction mixture was purified directly by acidic preparative MPLC (Luna10-50) to give a bright white solid after lyophilization of the product fraction.

[0308] Yield: 32 mg, 65%

[0309] U_AN_ACID: m / z 520.2 [M+H] +

[0310] 1H NMR (400 MHz, DMSO-d6) δ 8.33 (d, J = 8.7 Hz, 1H), 7.39 (s, 1H), 7.23 (s, 1H), 6.48 (s, 1H), 6.28 (d, J = 4.9 Hz, 2H), 5.50 (q, J = 6.7) Hz, 1H), 5.40 (s, 3H), 5.23 - 5.06 (m, 2H), 3.62 (d, J = 6.3 Hz, 1H), 3.03 (q, J = 6.2 Hz, 2H), 2.21 - 2.02 (m, 2H), 1.92 - 1.79 (m, 2H), 1.18 - 1.08 (m, 1H), 0.87 (t, J = 7.3 Hz, 3H), 0.47 - 0.30 (m, 4H).

[0311] Manufacturing Example 8: Synthesis of linker-payload (mc-GGFG-PBX-7016) compound

[0312]

[0313] PBX-7011 (410 mg, 754 umol) and (2R,10S)-10-benzyl-23-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-2-methyl-6,9,12,15,18-penta-oxo-3-oxa-5,8,11,14,17-pentaazatricosanoic acid)(466 mg, 739 umol) having the mc-GGFG structure It was dissolved in 10 mL of N,N-dimethylformamide. DIPEA (439 mg, 3.39 mmol) and HATU (430 mg, 1.13 mmol) were dissolved thereafter and stirred at room temperature for 30 minutes. The mixture was then purified three times by acidic preparative MPLC (Luna10-50) and lyophilized to obtain mc-GGFG-PBX-7016 as a light yellow solid.

[0314] Yield: 510 mg, 63%

[0315] U_AN_ACID: m / z 1060.02 [M+H] +

[0316] 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).

[0317] Example: Preparation of an anti-c-MET antibody-drug conjugate (ADC) comprising a compound of formula 1 (PBX payload)

[0318] Anti-c-MET ADCs containing the PBX payload manufactured in the above manufacturing example were manufactured (Table 1).

[0319] No. Payload Linker Antibody Example 1 PBX-7011mc-GGFG Anti-c-MET Antibody (M01) Example 2 PBX-7014 Example 3 PBX-7016 Example 4 PBX-7018 Example 5 PBX-7020 Example 6 PBX-7022 Example 7 PBX-7024

[0320] A linker-payload containing a PBX payload and introducing an enzymatically cleavable linker system, GGFG, was synthesized. Specifically, the molecular structure of the linker-payload had an mc-GGFG-PBX payload, and the linker used had a structure represented by Chemical Formula 13.

[0321] The linker was first reacted with a payload to produce a linker-payload conjugate. For example, the linker-payload included in Example 3 was synthesized using the method described in Preparation Example 8.

[0322] Furthermore, ADC was synthesized by conjugation with an anti-c-MET antibody (M01) having CDRs of SEQ ID NOs: 1 to 6. The ADC was prepared by the following method. The prepared anti-c-MET antibody was buffer-exchanged with Reaction buffer (150 mM NaCl, 50 mM Histidine pH 6.0) using a NAP-5 column (Cytiva) or PD-10 desalting column (Cytiva), and then 30 equivalents of 50 mM TCEP (Tris(2-carboxyethyl)phosphine hydrochloride) (Merck) were treated with the antibody at 25°C for 2 hours to create a thiol site necessary for the reaction from the disulfide of the antibody.

[0323] Afterwards, excess TCEP was removed using a NAP-5 column or PD-10 desalting column and exchanged with reduction buffer (25 mM Histidine, pH 6.0). The conjugation reaction was performed by reacting 12 equivalents of the 5 mM linker-payload compound prepared above with the reduced anti-c-MET antibody at 4°C for 1 hour. As a result, an ADC in which the linker-payload compound was conjugated to the anti-c-Met antibody was prepared.

[0324] Evaluation of antibody-drug conjugates (ADCs)

[0325] The monomer purity of the anti-c-MET ADC of Example 3 was confirmed using size exclusion chromatography (SEC) and is shown in Fig. 1.

[0326] DAR was evaluated by mass (Da) and area analysis via LC-MS and is shown in Table 2.

[0327] Example 3 Mass (Da) Area Drug Standard Calculation Ratio DAR Light Chain D023814.0---1.08.0 D124874.9326425.9326425.9100.0 D2---- Heavy Chain D050538.2---3.0 D151598.1---D252658.0---D353718.9154691.0154691.0100.0

[0328] Comparative Example: Preparation of Isotype ADC and Anti-c-MET ADC with Different Payloads

[0329] An ADC containing known camptothecin and MMAE (Monomethyl auristatin E) as payloads and conjugated with an anti-c-MET antibody (M01) was prepared by the method described in the above examples. In addition, an ADC containing PBX-7016 as a payload and conjugated with an anti-SARS-CoV-2 antibody (P63) and an anti-influenza A antibody (Navivumab, P23) was prepared by the method described in the above examples. Finally, an ADC containing MMAE (Monomethyl auristatin E) as a payload and conjugated with an anti-influenza A antibody (Navivumab, P23) was prepared by the method described in the above examples. The anti-SARS-CoV-2 antibody (P63) is an antibody comprising a heavy chain having an amino acid sequence of SEQ ID NO: 11 and a light chain having an amino acid sequence of SEQ ID NO: 12. The above anti-influenza A antibody (Navivumab, P23) is an antibody comprising a heavy chain having an amino acid sequence of SEQ ID NO: 13 and a light chain having an amino acid sequence of SEQ ID NO: 14.

[0330] Camptothecin was synthesized and used as a compound of formula I (the structure including the linker is formula V) disclosed in US 2022 / 0378935, and MMAE was purchased from MedChemExpress in the form of payload and linker-payload (mc-vc-PAB-MMAE). An ADC with PBX-7016 as a payload was used in the form of linker-payload (mc-GGFG-PBX-7016) using mc-GGFG as a linker.

[0331] The SEC and DAR values ​​for each ADC are as shown in Table 3 below.

[0332] ADCSECDAR1M01-camptothecin>97%6.32M01-MMAE>97%3.53P63-PBX-7016>97%84P23-PBX-7016>97%85P23-MMAE>97%3.1

[0333] Experimental Example 1: Cell viability assay of PBX payload anti-c-MET ADC

[0334] Experimental Example 1-1. Selection of c-MET-expressing cell lines

[0335] Cell lines expressing c-MET protein were selected using flow cytometry.

[0336] The cell lines used were T47D (ATCC), SNU-5 (KCLB), H1573 (KCLB), HT29 (KCLB), and H1975 (Elab science), and antibody binding and flow cytometry analysis were performed using commercially available monoclonal antibodies (FITC-mAb, 11-8858-42) and anti-c-MET antibodies (M01) & FITC-anti human IgG Fc (410720) (Fig. 2).

[0337] The highest level of c-MET expression was observed in the gastric cancer cell line SNU-5, and the mean fluorescence intensity (MFI) and staining index in each cell line are as shown in Table 4 below.

[0338] Cell line tissue (carcinoma) MFI staining index expression no staining conjugated mAb T47D breast (ductal) 9111-SNU-5 stomach (gastric) 425,995143+++ NCI-H1573 lung (adenocarcinoma) 1954929+(+) HT29 colon (adenocarcinoma) 1416212+ NCI-H1975 lung (adenocarcinoma) 1213912+

[0339] Experimental Example 1-2. Cell viability assay

[0340] An in-vitro cell viability assay was performed on the cell line selected in Experimental Example 1-1 using the ADC, PBX-7016 payload compound, and MMAE payload compound manufactured in Example 3 and Comparative Example above.

[0341] Specifically, 3,000 cells per well of the cell lines described in Table 4 were seeded in a 96-well plate and incubated (37°C, 5% CO2). After 24 hours, 100 μl of payload compounds or ADCs at 9 concentrations (serial dilutions of 1 / 5 from 1,000 nM) were treated to the cells. At this time, an untreated control group (drug concentration 0) was also prepared. After incubation for 6 days (37°C, 5% CO2), 100 μl of CellTiter-Glo reagent (CellTiter-Glo® Luminescent Cell Viability Assay kit (Promega, G7571)) was added to each well and pipetted. After incubation for 10 minutes at RT, fluorescence was measured. When the fluorescence value at 0 drug concentration is considered 100%, the concentration that shows 50% of the fluorescence value is called IC 50 It was measured by value.

[0342] As a result, it was confirmed that the anti-c-MET ADC of Example 3 had excellent cell inhibition ability. The specific IC for each ADC 50 The values ​​(unit: nM) are as shown in Table 5 below (Figs. 3 to 7).

[0343] Cell line MMAEPBX-7016 M01-PBX-7016 (Example 3) M01-camptothecin M01-MMAEP63-PBX-7016 (Isotype ADC) T47D0.5 10.45 32.6 58.2 16 9 14.6 SNU-50.425 210.110.90.0 454.5 NCI-H15 730.422.5 56.147.56 532.5 HT2 90.143.88.754.50.96 1.4 NCI-H19 750.39.8 182.12 83.73 1.797.6

[0344] Specifically, in the SNU-5 cell line, where c-MET is expressed in large amounts, the anti-c-MET ADC of Example 3 had an IC that was approximately 50 times better than the PBX-7016 compound itself and approximately 500 times better than the isotype ADC. 50 It was observed that the values ​​had improved IC compared to the isotype ADC (P63-PBX-7016) in c-MET low-expressing cell lines NCI-H1573 and HT29. 50 By showing the values, it was confirmed that when PBX-7016 was developed as an ADC, it could exhibit a particularly excellent inhibitory effect on target cell lines expressing a large amount of c-MET. In addition, the anti-c-MET ADC of Example 3 showed a superior IC compared to the competing drug M01-camptothecin ADC, which conjugated another camptothecin-based payload to an anti-c-MET antibody, on all tested cell lines. 50 It was observed that the inhibitory effect on the target cell line was superior, as it had a higher value.

[0345] Accordingly, the ADC of the present invention, which has a camptothecin derivative compound of chemical formula 1 as a payload and is specific for c-MET protein, exhibits an excellent inhibitory effect on cells expressing c-MET, and can be used for cancer treatment or prevention.

[0346] Experimental Example 2: c-MET binding assay of PBX payload anti-c-MET ADC

[0347] When manufacturing an ADC by combining a linker and payload with an anti-c-MET antibody, we aimed to determine the effect on binding affinity. To this end, an assay for c-MET was performed using the anti-c-MET ADC of Example 3 in Table 1 and the anti-c-MET antibody (M01) constituting it.

[0348] ELISA assay

[0349] First, a binding assay using the M01 antibody alone was performed. Specifically, the binding affinity of the M01 antibody to c-MET proteins from human, mouse, rat, dog, and monkey (cynomolgus) was examined using an ELISA assay. First, the M01 antibody was diluted and cross-reactivity tested against c-MET proteins from various species using an ELISA assay. As a result, it was confirmed that the M01 antibody specifically binds to recombinant human c-MET and monkey c-MET, but does not bind to c-MET from mouse, rat, or dog (Fig. 8).

[0350] Based on the above results, ELISA assays were performed on recombinant human c-Met (human c-Met) and monkey c-MET (cynomolgus c-Met) of the anti-c-MET ADC and M01 antibody of Example 3. As a result, similar EC values ​​were found for the anti-c-MET ADC and M01 antibody. 50 The values ​​were confirmed, and the specific values ​​are as shown in Table 6 below (Fig. 9).

[0351] EC 50 (nM)M01-PBX-7016 (Example 3)M01 Antibody c-MET Antigen Origin Monkey 0.110.14 Human 0.120.14

[0352] As a result of the above in-vitro experiment, it was confirmed that both the anti-c-MET ADC and M01 antibody of Example 3 bound to recombinant human and monkey c-MET proteins in a concentration-dependent manner.

[0353] Specifically, EC for monkey-derived c-MET protein 50 The values ​​were confirmed to be 0.11 nM and 0.14 nM for the anti-c-MET ADC of the present invention, and 0.12 nM and 0.14 nM for the human c-MET protein. Thus, it was confirmed that the binding characteristics did not change even when the M01 antibody was bound to the linker and payload in the anti-c-MET ADC of the present invention.

[0354] BLI assay

[0355] Afterwards, biolayer interferometry (BLI) analysis was performed to determine the binding affinity of the anti-c-MET ADC and M01 antibody of the present invention to c-MET protein.

[0356] The binding affinity constants (KD) of the anti-c-MET ADC and M01 antibody of Example 3 for human and monkey-derived c-MET proteins are shown in Table 7 below.

[0357] These results also showed that the binding affinity of the anti-c-MET ADC and the M01 antibody of the present invention to human-derived c-MET protein was similar, confirming that the binding affinity of the ADC was maintained even after conjugation. Similarly, the anti-c-MET ADC and the M01 antibody of the present invention to monkey-derived c-MET protein also had similar binding affinity to human-derived c-MET protein.

[0358] c-MET protein-derived loading sample KD (nM) K on (1 / Ms)K dis (1 / s)Full R 2Human M01-PBX-7016 (Example 3) 0.7748.13E+046.29E-050.9421 M01 antibody 0.6056.74E+044.07E-050.9518 Monkey M01-PBX-7016 (Example 3) 0.9171.00E+059.17E-050.9608 M01 antibody 0.7078.05E+045.69E-050.9587

[0359] CELISA and immunofluorescence staining

[0360] Finally, we sought to confirm the binding of the anti-c-MET ADC and M01 antibody of the present invention to c-MET protein expressed on the surface of tumor cells. This was analyzed based on cell-based ELISA (CELISA) and the expression levels of c-MET protein on the surface of cell lines of each cancer type.

[0361] Quantification of c-MET protein on the cell surface was assessed by indirect immunofluorescence staining of cell surface antigens. The cell lines used in the present invention have various c-MET protein expression levels. This is quantified by c-MET antigen binding capacity (ABC), which represents the number of cell surface c-MET protein molecules, and is expressed as TPM (transcripts per kilobase million).

[0362] As shown in Table 8 below, TPM values ​​and cell surface c-MET protein expression levels showed a significant correlation.

[0363] Cell line cMet expression (binding sites / cell) TPM value Lung cancer EBC-1~ 216,000 2633.62 NCI-H1373~ 69,000 N / AHCC-827~ 53,000 310.45 NCI-H1975~ 24,000 230.67 NCI-H1573~ 14,000 439.89 Gastric cancer SNU-5~ 343,000 1860.61 Hs746T~ 188,000 3540.54 SNU-620~ 124,000 2298.88 MKN-45~ 94,000 2523.17 SNU-1 (cMet expression negative) Control) 084.45 Colorectal cancer HT-29~ 24,000 227.74 NCI-H747~ 22,000 257.43 HCT-116~ 10,000 239.87 SW48~ 1003.55

[0364] In summary, the M01 antibody binds to cell lines expressing the c-MET protein in a dose-dependent manner (Fig. 10), and the binding of the anti-c-MET ADC of the present invention to each cell line exhibited a similar pattern. In other words, it was confirmed that linking the M01 antibody to a payload via a linker did not significantly affect the binding affinity of the antibody.

[0365] Experimental Example 3: Confirmation of the anticancer effect of a PBX payload anti-c-MET ADC.

[0366] Experimental Example 3-1. Confirmation of anticancer effects in the Hs746T human gastric cancer xenograft model.

[0367] The anticancer effect of the ADC of the present invention was confirmed in the Hs746T human gastric cancer xenograft model of BALB / c nude mice (Zhejiang Vital River Laboratory Animal Technology Co., LTD).

[0368] cell culture

[0369] Hs746T tumor cells (ATCC, catalog no. HTB-135) were cultured as a monolayer in DMEM medium supplemented with 10% FBS and 1% antibiotic-antimycotic at 37°C in an atmosphere of 5% CO2. Hs746T tumor cells were routinely passaged three times a week by treatment with trypsin-EDTA. Exponentially growing cells were harvested and counted for tumor inoculation.

[0370] Tumor inoculation and animal grouping

[0371] Mice were randomly assigned to groups of six, and tumor cells were inoculated into the mice according to the experimental conditions shown in Table 9 below. Specifically, Hs746T tumor cells (2Х10) were injected into the right flank of each mouse together with 0.1 ml of PBS containing Matrigel. 6 Cells / mouse) were subcutaneously inoculated. After inoculation, the average tumor volume was 197 mm 3 Treatment was started on the 15th day after reaching the target, and the treatment start date was set as Day 0. This experiment was conducted for 27 days.

[0372] Group Number of mice Treatment dose (mg / kg) Dosage (mL / kg) Dosage route Schedule 16P23 (Isotype mAb) 35i.v. Single dose 26M01 (Anti-cMet mAb) 35i.v. Single dose 36P23-PBX-7016 0.35iv Single dose 46P23-PBX-7016 15i.v. Single dose 56P23-PBX-7016 35i.v. Single dose 66M01-PBX-7016 (Example 3) 0.35iv Single dose 76M01-PBX-7016 (Example 3) 15i.v. Single dose 86M01-PBX-7016 (Example 3) 35i.v. Single dose96M01-MMAE0.35i.v.Single dose106M01-MMAE15i.v.Single dose116M01-MMAE35i.v.Single dose

[0373] Tumor measurement and endpoint confirmation

[0374] The experimental endpoint was set as the cessation of tumor growth or cure of the mice. Tumor size and volume were measured twice a week in two and three dimensions using calipers, and these values ​​were used to calculate tumor volume over control volume (T / C) and tumor growth inhibition (TGI) values.

[0375] In the above T / C value (%), T and C represent the average volume of the experimental group and the control group on a specific day, respectively, and the T / C value is an indicator of the antitumor effect.

[0376] The above TGI value is a numerical value calculated using the following formula, where T i is the mean tumor volume of the treatment group on a given date, T0 is the mean tumor volume of the treatment group on the first day of treatment, V i is T i V0 is the mean tumor volume of the vehicle control group on the same day, and V0 is the mean tumor volume of the vehicle group on the first day of treatment.

[0377] TGI (%) = [1-(T i -T0) / (V i -V0)] Х100

[0378] Statistics and Results Analysis

[0379] The mean and standard error of the mean (SEM) of tumor volume over time for each group are shown in Tables 10 and 11 below.

[0380] At 13 days after the start of the experiment, the tumor volume of individual mice in the isotype mAb control group reached 3000 mm, the criterion for sacrifice. 3, and therefore, statistical analysis was performed based on the results of tumor volumes measured on day 13. Because the data did not follow a normal distribution, the Kruskal-Wallis test was performed to compare tumor volumes between groups, followed by Dunn's post hoc analysis. All data were analyzed using SPSS 29.0. p < 0.05 was considered statistically significant.

[0381] Tumor volume over time (groups 1 to 5) Days Tumor volume (mm) 3 )P23(Isotype mAb)3 mg / kgM013 mg / kgP23-PBX-70160.3 mg / kgP23-PBX-70161 mg / kgP23-PBX-70163 mg / kg0197 ± 22197 ± 24197 ± 24197 ± 24197 ± 233323 ± 73291 ± 54309 ± 37298 ± 36281 ± 556523 ± 126378 ± 80512 ± 87476 ± 80318 ± 7210982 ± 268562 ± 146873 ± 170807 ± 171308 ± 80131,575 ± 419910 ± 2331,205 ± 2531,189 ± 243405 ± 125172,037 ± 382(n=5)1,323 ± 2871,714 ± 3741,706 ± 325669 ± 192202,105 ± 72(n=4)1,562 ± 3422149 ± 4752,089 ± 416826 ± 223243,016 ± 278(n=4)2,141 ± 5201,968 ± 619(n=4)1,918 ± 502(n=4)1,140 ± 306273336(n=1)1,512 ± 394(n=3)2,175 ± 874(n=3)1,873 ± 359(n=3)1,646 ± 416

[0382] Tumor volume over time (groups 6 to 11) Days Tumor volume (mm) 3)M01-PBX-7016 (Example 3) 0.3 mg / kgM01-PBX-7016 (Example 3) 1 mg / kgM01-PBX-7016 (Example 3) 3 mg / kgM01-MMAE0.3 mg / kgM01-MMAE1 mg / kgM01-MMAE3 mg / kg0197 ± 22197 ± 25197 ± 23197 ± 22197 ± 24197 ± 253281 ± 30300 ± 64286 ± 50310 ± 65307 ± 25253 ± 526357 ± 40334 ± 89234 ± 31459 ± 68387 ± 27221 ± 7510455 ± 74279 ± 110149 ± 27713 ± 129534 ± 35193 ± 9713677 ± 125271 ± 137115 ± 231,034 ± 132737 ± 48221 ± 128171,092 ± 227310 ± 23365 ± 131,705 ± 1461,015 ± 59356 ± 199201,335 ± 284362 ± 31731 ± 52,169 ± 2831,188 ± 107434 ± 234241,851 ± 365506 ± 45523 ± 42542 ± 256(n=5)1,477 ± 124630 ± 318271,648 ± 122(n=4)810 ± 71815 ± 23,230 ± 346(n=4)2,120 ± 192826 ± 385

[0383] Mortality, morbidity, and weight gain / loss

[0384] Mice's body weight was regularly monitored as an indirect measure of toxicity. No significant weight loss was observed during the experimental period. Body weight changes in each treatment group are shown in Figure 11.

[0385] Tumor volume

[0386] The average tumor volume over time in the experimental mice is shown in Tables 10 and 11 above.

[0387] Tumor growth inhibition assay and tumor growth curve

[0388] Tumor growth inhibition assays in experimental mice were performed 13 days after the start of the experiment and calculated based on tumor volume measurements. The results are shown in Table 12. Tumor growth curves are shown in Figures 12 and 13.

[0389] Number of treated tumors (Tumor volume) (mm) 3 )13 daysT / C(%)TGI(%)p valueP23(Isotype mAb), 3 mg / kg61,575 ± 419---M01, 3 mg / kg6910 ± 23357.7548.28>0.999P23-PBX-7016, 0.3 mg / kg61,205± 25376.4926.87>0.999P23-PBX-7016, 1 mg / kg61,189 ± 24375.5227.98>0.999P23-PBX-7016, 3 mg / kg6405 ± 12525.7184.880.209M01-PBX-7016 (Example 3), 0.3 mg / kg6677 ± 12542.9765.17>0.999M01-PBX-7016 (Example 3), 1 mg / kg6271 ± 13717.2294.590.052M01-PBX-7016 (Example 3), 3 mg / kg6115 ± 237.28105.950.003M01-MMAE, 0.3 mg / kg61,034 ± 13265.6739.22>0.999M01-MMAE, 1 mg / kg6737 ± 4846.7860.83>0.999M01-MMAE, 3 mg / kg6221 ± 12814.0498.230.009

[0390] result

[0391] To confirm the anticancer effect of the ADC of the present invention in the Hs746T human gastric cancer subcutaneous xenograft mouse model, mouse mortality, body weight increase / decrease rate, tumor volume, tumor growth inhibition rate, tumor weight, and tumor growth curve were analyzed.

[0392] The average tumor volume in the group administered P23 (Isotype mAb) was 1,575 mm on the 13th day after the start of administration.3 Compared with the isotype mAb group, P23-PBX-7016 showed 3 mg / kg (T / C = 25.71%, TGI = 84.88%, p=0.209), M01-PBX-7016 showed 1 mg / kg (T / C = 17.22%, TGI = 94.59%, p=0.052), M01-PBX-7016 showed 3 mg / kg (T / C = 7.28%, TGI = 105.95%, p=0.003), and M01-MMAE showed 3 mg / kg (T / C = 14.04%, TGI = 98.23%, p=0.009), and when treated, the average tumor volume was 405 mm on the 13th day after the start of treatment. 3 , 271 mm 3 , 115 mm 3 , 221 mm 3 The anti-tumor effect was shown. Therefore, it can be seen that the antibody-drug conjugate (ADC) of the present invention exhibits excellent tumor growth inhibition activity.

[0393] Meanwhile, no significant weight loss was observed during the experiment.

[0394] Experimental Example 3-2. Confirmation of anticancer effects in a LU-01-0439 human lung cancer xenograft model.

[0395] The anticancer effect of the ADC of the present invention was confirmed in the LU-01-0439 human lung cancer xenograft model of BALB / c nude mice (Zhejiang Vital River Laboratory Animal Technology Co., LTD).

[0396] Creation of PDX models

[0397] A lung cancer PDX model of LU-01-0439 was generated by transplanting surgically resected clinical samples into immunodeficient mice, establishing it as passage 0 (P0). Subsequently, the cells were passaged through serial transplantation, and passages after P0 were defined as passage 1 (P1). Tumors recovered from frozen P2 tumor tissue were designated FP3, and FP4 tumor tissues were finally prepared for experiments.

[0398] The H-score assessed by immunohistochemical staining with SP44 (Ventana), an anti-c-Met antibody of LU-01-0439, was evaluated as 179 points according to the criteria in Table 13 below.

[0399] 0 No staining 1 + Mild intensity (clearly visible at 200x magnification) 2 + Medium intensity (clearly visible at 100x magnification) 3 + Strong intensity (clearly visible at 40x magnification)

[0400] Tumor transplantation and animal grouping

[0401] Mice were randomly assigned to groups of eight, and tumors were implanted into the mice according to the experimental conditions shown in Table 14 below. Specifically, LU-01-0439 FP4 tumor fragments (20–30 mm) were implanted into the right flank of each mouse. 3 ) were subcutaneously transplanted. After tumor transplantation, the average tumor volume was 175 mm 3 Treatment was started on the 24th day after reaching the target, and the treatment start date was set as Day 0. This experiment was conducted for 35 days.

[0402] Group Number of mice Treatment Dose (mg / kg) Dosage (mL / kg) Dosage route Schedule 18P23 (Isotype mAb) 105i.v.QW x 2 weeks 28M01 (Anti-cMet mAb) 105i.v.QW x 2 weeks 38P23-PBX-7016 105i.v.QW x 2 weeks 48M01-MMAE 15i.v.QW x 2 weeks 58M01-MMAE 35i.v.QW x 2 weeks 68M01-MMAE 105i.v.QW x 2 weeks 78M01-PBX-7016 (Example 3) 15i.v.QW x 2 weeks 88M01-PBX-7016 (Example 3) 35i.v.QW x 2 weeks98M01-PBX-7016(Example 3)105i.v.QW x 2 weeks

[0403] Tumor measurement and endpoint confirmation

[0404] Tumor size was measured using the same method as in Experimental Example 3-1.

[0405] Statistics and Results Analysis

[0406] The mean and standard error of the mean (SEM) of tumor volume over time for each group are shown in Tables 15 and 16 below.

[0407] Statistical analysis was performed on data obtained 35 days after the start of the experiment to determine differences in tumor volume and tumor weight between groups. Because the data were not normally distributed, the Kruskal-Wallis test was performed, followed by Dunn's post hoc analysis, to compare tumor volumes between groups. All data were analyzed using SPSS 29.0. A p < 0.05 value was considered statistically significant.

[0408] Tumor volume over timeTumor volume (groups 1 to 6)DaysTumor volume (mm3)P23(Isotype mAb)10 mg / kgM0110 mg / kgP23-PBX-701610 mg / kgM01-MMAE1 mg / kgM01-MMAE3 mg / kgM01-MMAE10 mg / kg0175 ± 15175 ± 16175 ± 15175 ± 15175 ± 15175 ± 153219 ± 21228 ± 25209 ± 22217 ± 25201 ± 22208 ± 247281 ± 30299 ± 34238 ± 22273 ± 36240 ± 35237 ± 3010343 ± 42407 ± 45247 ± 19399 ± 58297 ± 41284 ± 3514455 ± 58538 ± 55217 ± 18572 ± 92401 ± 64403 ± 5817568 ± 74738 ± 82217 ± 28710 ± 120487 ± 82543 ± 8721736 ± 113894 ± 106205 ± 42830 ± 139556 ± 92635 ± 12624874 ± 1351,168 ± 134263 ± 61973 ± 148690 ± 95805 ± 160281,098 ± 1881,425 ± 167393 ± 901,258 ± 202852 ± 106966 ± 159311,344 ± 2281,690 ± 214536 ± 1171,523 ± 2281,027 ± 113(n=7)c1,169 ± 175351,460 ± 2401,655 ± 245630 ± 1371,650 ± 2391,141 ± 138(n=7)1,378 ± 227

[0409] Tumor volume over time (groups 7 to 9) Days Tumor volume (mm) 3)M01-PBX-7016 (Example 3) 1 mg / kgM01-PBX-7016 (Example 3) 3 mg / kgM01-PBX-7016 (Example 3) 10 mg / kg0 175 ± 15 175 ± 15 175 ± 16 3 222 ± 22 234 ± 28 231 ± 357 231 ± 26 223 ± 28 212 ± 28 10 218 ± 28 174 ± 25 171 ± 23 14 202 ± 24 114 ± 21 112 ± 21 17 192 ± 2490 ± 2084 ± 14 21 152 ± 2742 ± 842 ± 11 24 170 ± 3634 ± 633 ± 928256 ± 6026 ± 523 ± 631362 ± 9518 ± 415 ± 535394 ± 897 ± 47 ± 3

[0410] Mortality, morbidity, and weight gain / loss

[0411] Twenty-eight days after the start of the experiment, one mouse in Group 5 (M01-MMAE, 3 mg / kg) died due to severe weight loss. Autopsy revealed pale liver and kidneys, and numerous subcutaneous hemorrhages. However, similar findings were not observed in other mice, indicating a low incidence and a lack of dose-dependent responses. Therefore, it remains unclear whether administration of the experimental substance contributed to the deaths.

[0412] Mice's body weight was regularly monitored as an indirect measure of toxicity. No significant weight loss was observed during the experimental period, except for the aforementioned mice. Body weight changes in each treatment group are shown in Figure 14.

[0413] Tumor volume

[0414] The average tumor volume over time in the experimental mice is shown in Tables 15 and 16 above.

[0415] Tumor growth inhibition assay

[0416] Tumor growth inhibition assays in experimental mice were performed 35 days after the start of the experiment and calculated based on tumor volume measurements. The results are shown in Table 17.

[0417] Number of treated tumors (Tumor volume) (mm) 3 )35 daysT / C(%)TGI(%)p valueP23(Isotype mAb), 10 mg / kg81,460 ± 240---M01, 10 mg / kg81,655 ± 245113.37-15.18>0.999P23-PBX-7016, 10 mg / kg8630 ± 13743.1164.62>0.999M01-MMAE, 1 mg / kg81,650 ± 239113.00-14.77>0.999M01-MMAE, 3 mg / kg71,141 ± 13878.1124.86>0.999M01-MMAE, 10 mg / kg81,378 ± 22794.366.40>0.999M01-PBX-7016 (Example 3), 1 mg / kg8394 ± 8927.0182.920.307M01-PBX-7016 (Example 3), 3 mg / kg87 ± 40.45113.080.001M01-PBX-7016 (Example 3), 10 mg / kg87 ± 30.45113.090.003

[0418] Tumor weight

[0419] Tumor weight analysis of experimental mice was performed 35 days after the start of the experiment. The results are shown in Table 18.

[0420] Number of treated subjectsTumor weight(g)T / Cp value35 days(%)P23(Isotype mAb), 10 mg / kg81.307 ± 0.237--M01, 10 mg / kg81.307 ± 0.204100.00>0.999P23-PBX-7016, 10 mg / kg80.561 ± 0.11742.93>0.999M01-MMAE, 1 mg / kg81.628 ± 0.270124.52>0.999M01-MMAE, 3 mg / kg71.171 ± 0.16089.55>0.999M01-MMAE, 10 mg / kg81.375 ± 0.247105.13>0.999M01-PBX-7016 (Example 3), 1 mg / kg80.417 ± 0.09531.890.859M01-PBX-7016 (Example 3), 3 mg / kg80.007 ± 0.0030.540.002M01-PBX-7016 (Example 3), 10 mg / kg80.009 ± 0.0020.720.006

[0421] Tumor growth curve

[0422] The tumor growth curves in each group are shown in Figure 15.

[0423] result

[0424] To confirm the anticancer effect of the ADC of the present invention in the LU-01-0439 human lung cancer subcutaneous xenograft mouse model, mouse mortality rate, body weight increase / decrease rate, tumor volume, tumor growth inhibition rate, tumor weight, and tumor growth curve were analyzed.

[0425] Except for one mouse in Group 5 (M01-MMAE, 3 mg / kg), which showed significant weight loss on day 28 from the start of the experiment, no significant weight loss was observed in the other mice. Even in the mice in which weight loss was observed, no significant correlation was observed between the administered substance and the weight loss.

[0426] The average tumor volume of the group administered P23 (Isotype mAb) was 1,460 mm on day 35 after the start of administration. 3Compared with the isotype mAb group, the M01-PBX-7016 (Example 3) 3 mg / kg and 10 mg / kg (T / C = 0.45% and 0.45%, TGI = 113.08% and 113.09%, p = 0.001 and 0.003) groups had a mean tumor volume of 7 mm on day 35 after the start of administration, respectively. 3 and 7mm 3 , and statistically significant antitumor activity was confirmed. Therefore, it can be seen that the antibody-drug conjugate (ADC) of the present invention exhibits excellent tumor growth inhibition activity.

[0427] Experimental Example 3-3. Confirmation of anticancer effects in the NCI-H441 human lung cancer xenograft model.

[0428] The anticancer effect of the ADC of the present invention was confirmed in the NCI-H441 human lung cancer xenograft model of BALB / c nude mice (Zhejiang Vital River Laboratory Animal Technology Co., LTD).

[0429] cell culture

[0430] NCI-H441 tumor cells (ATCC, catalog no. HTB-174) were maintained in vitro in RPMI 1640 medium supplemented with 10% fetal bovine serum and 1% antibiotic-antimycotic at 37°C in an atmosphere of 5% CO2. NCI-H441 tumor cells were routinely passaged twice a week by treatment with trypsin-EDTA. Exponentially growing cells were harvested and counted for tumor inoculation.

[0431] Tumor inoculation and animal grouping

[0432] Mice were randomly assigned to groups of 10 each, and tumor cells were inoculated into the mice according to the experimental conditions shown in Table 19 below. Specifically, NCI-H441 tumor cells (5х10) were injected into the right flank of each mouse together with 0.2 ml of PBS containing Matrigel (1:1). 6 Cells / mouse) were inoculated subcutaneously. After inoculation, the average tumor volume was 180 mm 3 Treatment was started on the 14th day after reaching the target, and the treatment start date was set as Day 0. This experiment was conducted for 35 days.

[0433] Group Number of mice Treatment Dose (mg / kg) Dosage (mL / kg) Route Schedule 1 10P23 (Isotype mAb) 35i.v.QW x 2 weeks 2 10M01 (Anti-cMet mAb) 35i.v.QW x 2 weeks 3 10P23-MMAE 35i.v.QW x 2 weeks 4 10P23-PBX-7016 35i.v.QW x 2 weeks 5 10M01-MMAE 0.35i.v.QW x 2 weeks 6 10M01-MMAE 15i.v.QW x 2 weeks 7 10M01-MMAE 35i.v.QW x 2 weeks 8 10M01-PBX-7016 (Example 3) 0.35ivQW x 2 weeks910M01-PBX-7016(Example 3)15i.v.QW x 2 weeks1010M01-PBX-7016(Example 3)35i.v.QW x 2 weeks

[0434] Tumor measurement and endpoint confirmation

[0435] Tumor size was measured using the same method as in Experimental Example 3-1.

[0436] Statistics and Results Analysis

[0437] The mean and standard error of the mean (SEM) of tumor volume over time for each group are shown in Tables 20 and 21 below.

[0438] Statistical analysis was performed on data obtained 35 days after the start of the experiment to determine differences in tumor volume and tumor weight between groups. Because the data were not normally distributed, the Kruskal-Wallis test was performed, followed by Dunn's post hoc analysis, to compare tumor volumes between groups. All data were analyzed using SPSS 29.0. A p < 0.05 value was considered statistically significant.

[0439] Tumor volume over time (groups 1 to 5) Days Tumor volume (mm) 3 )P23(Isotype mAb)3 mg / kgM013 mg / kgP23-MMAE3 mg / kgP23-PBX-70163 mg / kgM01-MMAE0.3 mg / kg0180 ± 13180 ± 13180 ± 13180 ± 13180 ± 124204 ± 17199 ± 13208 ± 19198 ± 16212 ± 177246 ± 22234 ± 16244 ± 26171 ± 14229 ± 1811293 ± 26254 ± 20283 ± 36140 ± 14264 ± 2214335 ± 31273 ± 25287 ± 39131 ± 14285 ± 2218506 ± 49348 ± 36358 ± 66134 ± 15373 ± 4021639 ± 59467 ± 47396 ± 71138 ± 17434 ± 5325826 ± 74734 ± 86563 ± 93155 ± 20584 ± 7728983 ± 84882 ± 108708 ± 119190 ± 33751 ± 96321,286 ± 1151,151 ± 156977 ± 193235 ± 42973 ± 130351,593 ± 1401,431 ± 1571,222 ± 218303 ± 541,301 ± 172

[0440] Tumor volume over time (groups 6 to 10) Days Tumor volume (mm) 3)M01-MMAEM01-PBX-7016 (Example 3)1 mg / kg3 mg / kg0.3 mg / kg1 mg / kg3 mg / kg0180 ± 13180 ± 13180 ± 13180 ± 13180 ± 134189 ± 15184 ± 18184 ± 16191 ± 15192 ± 207164 ± 17176 ± 26140 ± 13126 ± 14128 ± 1611146 ± 16147 ± 25121 ± 12101 ± 13104 ± 1414136 ± 16121 ± 21106 ± 1188 ± 985 ± 1318118 ± 15100 ± 1978 ± 768 ± 768 ± 1021100 ± 1379 ± 1568 ± 654 ± 553 ± 825121 ± 1966 ± 1264 ± 648 ± 449 ± 728162 ± 2657 ± 1156 ± 736 ± 241 ± 732223 ± 4046 ± 953 ± 1030 ± 237 ± 735305 ± 5942 ± 855 ± 1518 ± 128 ± 12

[0441] Mortality, morbidity, and weight gain / loss

[0442] Mice's body weight was regularly monitored as an indirect measure of drug toxicity. No significant weight loss was observed during the experimental period, and body weight changes in each treatment group are shown in Figure 16. No disease or death occurred.

[0443] Tumor volume

[0444] The average tumor volume over time in the experimental mice is shown in Tables 20 and 21 above.

[0445] Tumor growth inhibition assay

[0446] Tumor growth inhibition assays in experimental mice were performed 35 days after the start of the experiment and calculated based on tumor volume measurements. The results are shown in Table 22.

[0447] Number of treated tumors (Tumor volume) (mm) 3 )on day 35T / C(%)TGI(%)p valueP23(Isotype mAb), 3 mg / kg101,593 ± 140---M01, 3 mg / kg101,431 ± 15789.8511.44>0.999P23-MMAE, 3 mg / kg101,222 ± 21876.7026.27>0.999P23-PBX-7016, 3 mg / kg10303 ± 5419.0091.340.203M01-MMAE, 0.3 mg / kg101,301 ± 17281.6720.67>0.999M01-MMAE, 1 mg / kg10305 ± 5919.1491.180.286M01-MMAE, 3 mg / kg1042 ± 82.61109.83<0.001M01-PBX-7016 (Example 3), 0.3 mg / kg1055 ± 153.48108.840.002M01-PBX-7016 (Example 3), 1 mg / kg1018 ± 11.15111.47<0.001M01-PBX-7016 (Example 3), 3 mg / kg1028 ± 121.76110.78<0.001

[0448] Tumor weight

[0449] Tumor weight analysis of experimental mice was performed 35 days after the start of the experiment. The results are shown in Table 23.

[0450] Treatment Number of Tumors Tumor weight (g) on ​​day 35T / C weight (%) p value P23 (Isotype mAb), 3 mg / kg 101.670 ± 0.158--M01, 3 mg / kg 101.510 ± 0.145 90.47 >0.999 P23-MMAE, 3 mg / kg 101.342 ± 0.247 80.39 >0.999 P23-PBX-7016, 3 mg / kg 100.318 ± 0.05 919.05 0.177 M01-MMAE, 0.3 mg / kg 101.368 ± 0.186 81.95 >0.999 M01-MMAE, 1 mg / kg 100.343 ± 0.06720.570.395M01-MMAE, 3 mg / kg100.048 ± 0.0122.86<0.001M01-PBX-7016(Example 3),0.3 mg / kg100.058 ± 0.0143.480.002M01-PBX-7016(Example 3), 1 mg / kg100.019 ± 0.0011.14<0.001M01-PBX-7016(Example 3), 3 mg / kg100.033 ± 0.0131.99<0.001

[0451] Tumor growth curve

[0452] The tumor growth curves in each group are shown in Figure 17.

[0453] result

[0454] To confirm the anticancer effect of the ADC of the present invention in the NCI-H441 human lung cancer subcutaneous xenograft mouse model, mouse mortality, body weight increase / decrease rate, tumor volume, tumor growth inhibition rate, tumor weight, and tumor growth curve were analyzed.

[0455] The average tumor volume of the group administered P23 (Isotype mAb) was 1,593 mm on day 35 after the start of administration. 3Compared with the isotype mAb group, the mean tumor volume on the 35th day after the start of administration was 42 mm in the 3 mg / kg M01-MMAE (T / C = 2.61%, TGI = 109.83%, p<0.001), 0.3 mg / kg M01-PBX-7016 (T / C = 3.48%, TGI = 108.84%, p=0.002), 1 mg / kg M01-PBX-7016 (T / C = 1.15%, TGI = 111.47%, p<0.001), and 3 mg / kg M01-PBX-7016 (T / C = 1.76%, TGI = 110.78%, p<0.001) groups, respectively. 3 , 55 mm 3 , 18 mm 3 , 28 mm 3 , and statistically significant antitumor activity was confirmed. Therefore, it can be seen that the antibody-drug conjugate (ADC) of the present invention exhibits excellent tumor growth inhibition activity.

[0456] Sequence list

[0457] The sequence list of antibodies used in the present invention is as described in Table 24 below.

[0458] 서열번호서열 명칭서열1M01(anti-c-MET antibody) CDR-H1GYIFTAYT2M01(anti-c-MET antibody) CDR-H2IKPNNGLA3M01(anti-c-MET antibody) CDR-H3ARSEITTEFDY4M01(anti-c-MET antibody) CDR-L1ESVDSYANSF5M01(anti-c-MET antibody) CDR-L2RAS6M01(anti-c-MET antibody) CDR-L3QQSKEDPLT7M01(anti-c-MET antibody) heavy chain variable regionQVQLVQSGAEVKKPGASVKVSCKASGYIFTAYTMHWVRQAPGQGLEWMGWIKPNNGLANYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCARSEITTEFDYWGQGTLVTVSS8M01(anti-c-MET antibody) light chain variable regionDIVMTQSPDSLAVSLGERATINCKSSESVDSYANSFLHWYQQKPGQPPKLLIYRASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQSKEDPLTFGGGTKVEIK9M01(anti-c-MET antibody) heavychainQVQLVQSGAEVKKPGASVKVSCKASGYIFTAYTMHWVRQAPGQGLEWMGWIKPNNGLANYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCARSEITTEFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK10M01(anti-c-MET antibody) light chainDIVMTQSPDSLAVSLGERATINCKSSESVDSYANSFLHWYQQKPGQPPKLLIYRASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQSKEDPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC11P63(SARS-CoV-2 mAb) heavychainEVQLVQSGGGLIQPGGSLRLSCAASEFIVSSNYMTWVRQAPGKGLEWVSIIYPGGSTYYADSVKGRFTISRDNSKNTLYLRMNSLRAEDTAVYYCARDLPLTGTTLDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK12P63(SARS-CoV-2 mAb) light chainELVLTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPLTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC13Navivumab(anti-influenza A mAb) heavychainMDWTWSILFLVAAATGAHSQVQLVQSGAEVKKPGASVKVSCKTSGYSFSTYGVSWVRQAPGQGPEWVGWISAYTGITDYAQKFQGRVTLTTDATTATAFLDLRSLRPDDTATYFCARDKVQGRVEVGSGGRHDYWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEGLHNHYTQKSLSLSPGK14Navivumab(anti-influenza A mAb) light chainMETPAQLLFLLLLWLPDTTGEVVLTQSPGTLALPPGERATLSCRASHRVGSTYIAWYQQKSGQAPRRLIYGASNRATDIPDRFSGSGSGTDFTLTIRRLEPEDSAVYYCQQFSVSPWTFGQGTRVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

Claims

1. An antibody-drug conjugate (ADC) having a structure in which a compound according to the following chemical formula 1 or a pharmaceutically acceptable salt, stereoisomer, tautomer, prodrug, hydrate, solvate or isotope-labeled analog thereof is linked to an anti-c-MET antibody via a linker: [Chemical Formula 1] Here, X1 and X3 are each independently carbon, oxygen, nitrogen, or sulfur, X1 and X3 may be the same or different, X2 is carbon, oxygen, nitrogen, sulfur, a single bond, or a double bond, n is 0, 1, or 2, Y1, Y2 and Y3 are each independently hydrogen or a functional group containing oxygen, nitrogen, phosphorus or sulfur.

2. In paragraph 1, X 1, X2 and X3 are each independently carbon, n is 1, Y1 and Y2 are each independently hydrogen, Y3 is -NZ1Z2, Z1 and Z2 are each independently hydrogen, halogen, hydroxyl, C 1-3 Alkyl, C(=O)H, C(=O)(C 1-3 alkyl), C(=O)(C 2-3 alkenyl), C(=O)NH2, C(=O)NH(C 1-3 alkyl), (CH2) 2-4 OH, (CH2) 2-4 O(C 1-3 alkyl), (CH2) 2-4 NH2, (CH2) 2-4 NH(C 1-3 alkyl), (CH2) 2-4 N(C 1-3 alkyl)2 or C(=O)CR 1 R 2 R 3 Contains a functional group selected from the group consisting of, At this time R 1 , R 2 and R 3 are each independently hydrogen, hydroxyl, C 1-3 Alkyl, C 3-7 Cycloalkyl or C 1-3 Haloalkylene, antibody-drug conjugate.

3. In paragraph 1, X 1, X2 and X3 are each independently carbon, n is 1, Y1 and Y2 are each independently hydrogen, Y3 is -NZ1Z2, Z1 is hydrogen, and Z2 is C(=O)(C 2-3 alkenyl) or C(=O)CR 1 R 2 R 3 It can be, At this time R 1 , R 2 and R 3 are each independently hydrogen, hydroxyl, C 1-3 Alkyl, C 3-7 Cycloalkyl or C 1-3 Haloalkylene, antibody-drug conjugate.

4. In paragraph 1, The compound of the above chemical formula 1 is an antibody-drug conjugate represented by any one of the following chemical formulas 2 to 11: [Chemical Formula 2] [Chemical Formula 3] [Chemical Formula 4] [Chemical Formula 5] [Chemical Formula 6] [Chemical Formula 7] [Chemical Formula 8] [Chemical Formula 9] [Chemical Formula 10] [Chemical Formula 11] .

5. In paragraph 1, The above linker is an antibody-drug conjugate represented by the following chemical formula 12: [Chemical Formula 12] Here, R X and R Y are each independently hydrogen or C 1-6 It is alkyl, p and q are each independently integers from 1 to 6, * is the part that binds to the payload.

6. In paragraph 1, An antibody-drug conjugate, wherein the anti-c-MET antibody is any one selected from the group consisting of Telisotuzumab, Emibetuzumab (LY2875358), Onartuzumab, ARGX-111, HLX55, and M01.

7. In paragraph 1, An antibody-drug conjugate, wherein the anti-c-MET antibody is an antibody or antigen-binding fragment thereof having i) a heavy chain comprising CDR-H1, CDR-H2 and CDR-H3 having amino acid sequences of SEQ ID NOs: 1, 2 and 3, respectively, and ii) a light chain comprising CDR-L1, CDR-L2 and CDR-L3 having amino acid sequences of SEQ ID NOs: 4, 5 and 6, respectively.

8. In paragraph 7, An antibody-drug conjugate, wherein the anti-c-MET antibody is an antibody or an antigen-binding fragment thereof comprising i) a heavy chain variable region having an amino acid sequence of SEQ ID NO: 7 and ii) a light chain variable region having an amino acid sequence of SEQ ID NO:

8.

9. In paragraph 7, An antibody-drug conjugate, wherein the anti-c-MET antibody is an antibody or an antigen-binding fragment thereof comprising i) a heavy chain having an amino acid sequence of SEQ ID NO: 9 and ii) a light chain having an amino acid sequence of SEQ ID NO:

10.

10. A pharmaceutical composition for treating or preventing cancer, comprising an antibody-drug conjugate according to any one of claims 1 to 9 as an active ingredient.

11. In paragraph 10, The above cancers are 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, 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, heart cancer, A pharmaceutical composition selected from the group consisting of 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 tumor, gastrointestinal stromal cancer, Wilms' cancer, breast cancer, 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 tumor, 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.

12. In paragraph 10, A pharmaceutical composition, wherein the cancer is selected from the group consisting of liver cancer, colon cancer, lung cancer, non-small cell lung cancer, stomach cancer, kidney cancer, and pancreatic cancer.

13. A method for treating or preventing cancer, comprising administering to a subject an antibody-drug conjugate according to any one of claims 1 to 9.

14. Use of an antibody-drug conjugate according to any one of claims 1 to 9 for the treatment or prevention of cancer.

15. Use of an antibody-drug conjugate according to any one of claims 1 to 9 for preparing a pharmaceutical composition for treating or preventing cancer.

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