ANTIBODY CONJUGATIONS AGAINST TISSUE FACTORS - PHARMACOLOGICAL SUBSTANCES INCLUDING CAMPTOTHECIN DERIVATIVES AND PHARMACEUTICALS CONTAINING THESE CONJUGATIONS

Abstract

The invention relates to an antibody-drug conjugate of a structure in which a compound of formula 1 or a pharmaceutical salt, stereoisomer, tautomer, prodrug, hydrate, solvate or isotopic analogue is conjugated to an antibody against tissue factor via a binding group; and a pharmaceutical composition comprising such conjugate. The antibody-drug conjugate of the invention is capable of selectively expressing the effect of compound of formula 1 against cancer cells expressing tissue factor.

Description

Anti-TISSUE FACTOR antibody-drug conjugates containing camptothecin derivatives

[0001] The present invention relates to an anti-Tissue Factor 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, tissue factor (TF), also known as thromboplastin, coagulation factor III, or CD142, is a protein present in subendothelial tissues, platelets, and leukocytes that is required for the initiation of thrombin formation from prothrombin. As a cell surface receptor for the serine protease factor VIIa (FVIIa), tissue factor (TF) enables cells to initiate the blood coagulation cascade. Meanwhile, as tissue factor (TF) expression has been observed in various types of cancer, including pancreatic, cervical, and skin cancers, it is gaining attention as a clinically important therapeutic target for 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 tissue factor (TF) is becoming a new challenge.

[0007] Accordingly, the object of the present invention is to provide an anti-TF antibody-drug conjugate comprising a camptothecin derivative having excellent cytotoxicity activity and excellent targeting specificity for cells expressing tissue factor (TF), 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-TF 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 or preventing cancer comprising a step of administering the same, and a use thereof.

[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 tissue factor (TF).

[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 entry into 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 for an excellent killing effect not only on cancer cells that highly express tissue factor (TF), 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 commercially available monoclonal antibodies, PE-mAb (12-1429-42) and APC-mAb (365205), in the process of selecting cell lines expressing tissue factor (TF).

[0016] Figure 3 shows the process of selecting cell lines expressing tissue factor (TF) using Tisotumab & AF647-F(ab)2 anti-human IgG-F(ab)2 (109-606-006) and Tivdak. TM (Lot:196169) & AF647-F(ab)2 anti-human IgG-F(ab)2 (109-606-006) are the results of flow cytometry analysis.

[0017] Figure 4 shows the results of in-vitro cell viability evaluation when ADC was treated in T47D (breast cancer cell line).

[0018] Figure 5 shows the results of in-vitro cell viability evaluation when ADC was treated in MCF-7 (breast cancer cell line).

[0019] Figure 6 shows the results of in-vitro cell viability evaluation when ADC was treated in KYSE-520 (esophageal cancer cell line).

[0020] Figure 7 shows the results of in-vitro cell viability evaluation when ADC was treated in Caski (cervical cancer cell line).

[0021] Figure 8 shows the results of in-vitro cell viability evaluation when ADC was treated in HPAF-II (pancreatic cancer cell line).

[0022] Figure 9 shows the results of in-vitro cell viability evaluation when ADC was treated in A431 (skin cancer cell line).

[0023] Figure 10 shows the group-wise body weight changes of female BALB / c nude mice transplanted with KYSE520.

[0024] Figure 11 shows the tumor growth curves by group of female BALB / c nude mice implanted with KYSE520.

[0025] Figure 12 shows the group-wise body weight changes of female CB17 SCID mice transplanted with HPAFII.

[0026] Figure 13 shows the tumor growth curves by group in female CB17 SCID mice transplanted with HPAFII.

[0027]

[0028] The present invention will be described in more detail below.

[0029] 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" or "including" a component does not exclude other components, but rather implies the inclusion of other components, unless specifically stated otherwise.

[0030] 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-TF antibody via a linker:

[0031] [Chemical Formula 1]

[0032]

[0033] Here,

[0034] X1 and X3 are each independently carbon, oxygen, nitrogen, or sulfur, and X1 and X3 may be the same or different,

[0035] X2 is carbon, oxygen, nitrogen, sulfur, a single bond or a double bond,

[0036] n is 0, 1, or 2,

[0037] Y1, Y2 and Y3 may each independently be hydrogen or a functional group containing oxygen, nitrogen, phosphorus or sulfur.

[0038] 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.

[0039] 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 또는 -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 또는 -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 또는 -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 having two or more carbon atoms It may contain a functional group selected from the group.

[0040] 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-TF antibody via a linker:

[0041] [Chemical Formula 1]

[0042]

[0043] Here,

[0044] X1 and X3 are each independently carbon, oxygen, nitrogen, or sulfur, and X1 and X3 may be the same or different,

[0045] X2 is carbon, oxygen, nitrogen, sulfur, a single bond or a double bond,

[0046] X1, (X2)n and X3 can form a 6-membered or 7-membered ring together, where n is 1 or 2,

[0047] Y1, Y2 and Y3 may each independently be hydrogen or a functional group containing oxygen, nitrogen, phosphorus or sulfur.

[0048] 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 (R1 , R 2 , R 3 = C s H t N u O w S x P y X z , X = -F, -Cl, -Br 또는 -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 또는 -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 having two or more carbon atoms It may contain a functional group selected from the group.

[0049] According to one aspect of the present invention, in the chemical formula 1,

[0050] X1, X2 and X3 can each independently be carbon,

[0051] n can be 1,

[0052] Y1 and Y2 can each independently be hydrogen,

[0053] Y3 may be hydrogen or a functional group containing oxygen, nitrogen, phosphorus or sulfur.

[0054] Here, non-limiting examples of functional groups containing oxygen, nitrogen, phosphorus or sulfur may be the same as the examples above.

[0055] According to one aspect of the present invention, in the chemical formula 1,

[0056] X1 and X3 are each independently carbon, oxygen, nitrogen, or sulfur, and X1 and X3 may be the same or different,

[0057] X2 is carbon, oxygen, nitrogen, sulfur, a single bond or a double bond,

[0058] n is 0, 1, or 2,

[0059] Y1, Y2 and Y3 may each independently be hydrogen or a functional group containing oxygen, nitrogen, phosphorus or sulfur.

[0060] The functional group containing the above nitrogen may be -NZ1Z2,

[0061] 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,

[0062] 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.

[0063] According to one aspect of the present invention, in the chemical formula 1,

[0064] X1, X2 and X3 can each independently be carbon,

[0065] n can be 1,

[0066] Y1 and Y2 can each independently be hydrogen,

[0067] Y3 may be hydrogen or a functional group containing oxygen, nitrogen, phosphorus or sulfur.

[0068] Here, non-limiting examples of functional groups containing oxygen, nitrogen, phosphorus or sulfur may be the same as the examples above.

[0069] According to one aspect of the present invention, in the chemical formula 1,

[0070] X1 and X3 are each independently carbon, oxygen, nitrogen, or sulfur, and X1 and X3 may be the same or different,

[0071] X2 is carbon, oxygen, nitrogen, sulfur, a single bond or a double bond,

[0072] X1, (X2)n and X3 can form a 6-membered or 7-membered ring together, where n is 1 or 2,

[0073] Y1, Y2 and Y3 may each independently be hydrogen or a functional group containing oxygen, nitrogen, phosphorus or sulfur.

[0074] The functional group containing the above nitrogen may be -NZ1Z2,

[0075] 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,

[0076] At this time R 1 , R 2 and R 3are each independently hydrogen, hydroxyl, C 1-3 Alkyl, C 3-7 Cycloalkyl or C 1-3 It may be haloalkyl.

[0077] According to one aspect of the present invention, in the chemical formula 1,

[0078] X1, X2 and X3 can each independently be carbon,

[0079] n can be 1,

[0080] Y1 and Y2 can each independently be hydrogen,

[0081] Y3 can be -NZ1Z2,

[0082] 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,

[0083] 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.

[0084] According to one aspect of the present invention, in the chemical formula 1,

[0085] X1, X2 and X3 can each independently be carbon,

[0086] n can be 1,

[0087] Y1 and Y2 can each independently be hydrogen,

[0088] Y3 can be -NZ1Z2,

[0089] 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,

[0090] 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.

[0091] According to one aspect of the present invention, in the chemical formula 1,

[0092] X1, X2 and X3 can each independently be carbon,

[0093] n can be 1,

[0094] Y1 and Y2 can each independently be hydrogen,

[0095] Y3 can be -NZ1Z2,

[0096] Z1 is hydrogen, and Z2 is C(=O)(C 2-3 alkenyl) or C(=O)CR 1 R 2 R 3 It may be,

[0097] At this time R 1 , R 2 and R 3 are each independently hydrogen, hydroxyl, C1-3 Alkyl, C 3-5 Cycloalkyl or C 1-3 It may be haloalkyl.

[0098] According to one aspect of the present invention, in the chemical formula 1,

[0099] X1, X2 and X3 can each independently be carbon,

[0100] n can be 1,

[0101] Y1 and Y2 can each independently be hydrogen,

[0102] Y3 can be -NZ1Z2,

[0103] Z1 is hydrogen, and Z2 is C(=O)CR 1 R 2 R 3 It may be,

[0104] At this time R 1 , R 2 and R 3 are each independently hydrogen, hydroxyl or C 1-3 It could be an alkyl.

[0105] According to one aspect of the present invention, the compound of the above 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.

[0106] [Chemical Formula 2]

[0107]

[0108] [Chemical Formula 3]

[0109]

[0110] [Chemical Formula 4]

[0111]

[0112] [Chemical Formula 5]

[0113]

[0114] [Chemical Formula 6]

[0115]

[0116] [Chemical Formula 7]

[0117]

[0118] [Chemical Formula 8]

[0119]

[0120] [Chemical Formula 9]

[0121]

[0122] [Chemical Formula 10]

[0123]

[0124] [Chemical Formula 11]

[0125] .

[0126] According to one aspect of the present invention, the compound of formula 1 may be an active camptothecin derivative designed to bind to the DDX5 protein and E3 ligase. According to one aspect of the present invention, the compound of formula 1 may bind to the E3 ligase as a molecular glue.

[0127] 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).

[0128] The target cells may be cancer cells or senescent cells. Senescent cells also include cells that no longer perform the organ's specific functions.

[0129] 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.

[0130] 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-TF antibody to produce an antibody-drug conjugate.

[0131] 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 ADCs to bind precisely to cancer cells and reduce their effects on normal cells, compared to conventional anticancer therapeutics. The antibody-drug conjugate of the present invention specifically binds to cells expressing tissue factor (TF), thereby enabling specific drug targeting.

[0132] 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.

[0133] The above antibody-drug conjugate is characterized by having an excellent killing effect even on cancer cells with low tissue factor (TF) expression, by causing a bystander effect in which the drug is released after cell death and kills surrounding cancer cells.

[0134] The terms "tissue factor", "TF", "Tissue Factor", "CD142", "tissue factor antigen", "TF antigen" and "CD142 antigen" as used herein are used interchangeably herein and, unless otherwise specified, include any variants, isoforms and species homologs of human tissue factor expressed naturally by a cell or expressed on a cell transfected with the tissue factor gene.

[0135] 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-TF antibody," which may mean an antibody or an antigen-binding fragment thereof that specifically binds to tissue factor (TF).

[0136] 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.

[0137] The antibody of the present invention can form a bond with a linker through a heteroatom on the antibody.

[0138] The antibody of the present invention is an anti-TF antibody, and non-limiting examples of the anti-TF antibody include Tisotumab, or an antibody selected from the antibodies used in ADCs such as Exelixis' XB-002, Shanghai Miracogen's MRG-004A, AmMax's AMB101, and Sutro biopharma's STRO-004.

[0139] The antibody of the present invention may be Tisotumab.

[0140] 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.

[0141] 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.

[0142] 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.

[0143] 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.

[0144] 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.

[0145] 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.

[0146] 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.

[0147] More specifically, the linker is C 1-6 It 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.

[0148] 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.

[0149] The “linker” of the present invention may be represented by the following chemical formula 12:

[0150] [Chemical Formula 12]

[0151]

[0152] Here,

[0153] R X and R Y are each independently hydrogen or C 1-6 It is alkyl,

[0154] p and q are each independently integers from 1 to 6,

[0155] * is the part that binds to the payload.

[0156] According to one aspect of the present invention, in the chemical formula 12,

[0157] R X and R Y are each independently hydrogen or C 1-3 It can be alkyl,

[0158] p can be an integer from 4 to 6,

[0159] q can be an integer from 1 to 3.

[0160] According to one aspect of the present invention, in the chemical formula 12,

[0161] R X and R Y can each independently be hydrogen,

[0162] p can be 5,

[0163] q can be 1.

[0164] According to one aspect of the present invention, the compound of chemical formula 12 may be a compound of chemical formula 13.

[0165] [Chemical Formula 13]

[0166]

[0167] Here, * is the part that binds to the payload.

[0168] 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.

[0169] 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.

[0170] [Chemical Formula 14]

[0171]

[0172] Here, * represents a site that binds to the payload, and ** represents a site that binds to the antibody.

[0173] 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.

[0174] 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.

[0175] 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.

[0176] 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.

[0177] 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.

[0178] 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.

[0179] 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.

[0180] 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).

[0181] 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.

[0182] 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.

[0183] 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.

[0184] 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.

[0185] 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.

[0186] 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.

[0187] 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.

[0188] 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.

[0189] 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.

[0190] 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, fallopian tube cancer, ovarian epithelial cancer, ovarian germ cell cancer, male breast cancer, brain cancer, pituitary adenoma, multiple myeloma, gallbladder cancer, biliary tract cancer, colon cancer, head and neck 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, One selected from the group consisting of glioma, renal pelvic cancer, kidney cancer, 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 cord cancer, acoustic neuroma, pancreatic cancer, salivary gland cancer, Kaposi's sarcoma, Paget's disease, tonsillar 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 This may include, but is not limited to, primary cancer as well as metastatic cancer.

[0191] According to one aspect of the present invention, the cancer may be at least one selected from the group consisting of cervical cancer, endometrial cancer, ovarian cancer, peritoneal cancer, fallopian tube cancer, breast cancer, head and neck cancer, esophageal cancer, bladder cancer, and pancreatic cancer.

[0192] In another aspect, the present invention relates to a method for treating or preventing cancer, comprising administering the antibody-drug conjugate to a subject.

[0193] In the present invention, the term “subject” is used synonymously with “individual,” and the subject may be a mammal, for example, a human, a rat, a cow, a horse, a pig, a dog, a sheep, a goat, or a cat.

[0194] Any description that overlaps with the pharmaceutical composition of the present invention described above is omitted.

[0195] In another aspect, the present invention relates to the use of the antibody-drug conjugate for the treatment or prevention of cancer.

[0196] Any description that overlaps with the pharmaceutical composition of the present invention described above is omitted.

[0197] In another aspect, the present invention relates to the use of the antibody-drug conjugate for preparing a pharmaceutical composition for treating or preventing cancer.

[0198] Any description that overlaps with the pharmaceutical composition of the present invention described above is omitted.

[0199] All matters mentioned in the compositions, treatment methods and uses of the present invention apply equally unless they are contradictory.

[0200] [Example]

[0201] 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.

[0202]

[0203] The method for producing a camptothecin derivative according to the present invention is as follows.

[0204] Manufacturing Example: Synthesis of a PBX Payload

[0205] Manufacturing Example 1: Synthesis of active camptothecin derivatives of chemical formula 2 or chemical formula 3 (PBX-7011 and PBX-7012)

[0206]

[0207] 1-1. Synthesis of compound 2

[0208]

[0209] 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.

[0210] 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.

[0211] SC_ACID: m / z 294.2 [M+H] +

[0212]

[0213] 1-2. Synthesis of compound 3

[0214]

[0215] 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).

[0216] SC_ACID: m / z 264.0 [M+H] +

[0217]

[0218] 1-3. Synthesis of compound 4

[0219]

[0220] To a solution of 7-iodobenzo[d][1,3]dioxol-5-amine (6.39 g, 24.29 mmol) in dichloromethane (49 mL) were added acetic anhydride (2.75 mL, 29.2 mmol) and triethylamine (4.06 mL, 29.2 mmol). 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 in ice-cold EtOAc (5-10 ml). The two batches were combined and further dried to obtain an off-white solid.

[0221] Total yield: 5.0 g, 66%

[0222] SC_ACID: m / z 306.0 [M+H] +

[0223] 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).

[0224]

[0225] 1-4. Synthesis of compound 5

[0226]

[0227] 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.

[0228] SC_ACID: m / z 264.4 [M+H] +

[0229]

[0230] 1-5. Synthesis of compound 6

[0231]

[0232] 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 bubbled with hydrogen and additional Pd / C (2.97 g, 1.397 mmol). The reaction mixture was bubbled with hydrogen for 10 min and stirred overnight at 45 °C under hydrogen. 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%.

[0233] SC_ACID: 266.2 [M+H] +

[0234] 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).

[0235]

[0236] 1-6. Synthesis of compound 7

[0237]

[0238] 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%

[0239] SC_ACID: m / z 248.2 [M+H] +

[0240] 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).

[0241]

[0242] 1-7. Synthesis of compound 8

[0243]

[0244] 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 obtain 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 obtain a gray solid (32 mg, 52%) with a purity of 80-90%. A higher purity sample can be obtained by purification using preparative MPLC.

[0245] SC_ACID: m / z 305.4 [M+H] +

[0246] 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).

[0247]

[0248] 1-8. Synthesis of compound 9

[0249]

[0250] 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.

[0251] SC_ACID: m / z 263.0 [M+H] +

[0252] 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).

[0253]

[0254] 1-9. Synthesis of compound 10

[0255]

[0256] (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.

[0257] 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.

[0258] 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.

[0259] LCMS analysis showed two diastereoisomers.

[0260] SC_ACID: m / z 490.2 [M+H] +

[0261] 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).

[0262]

[0263] 1-10. Synthesis of PBX-7011 and PBX-7012

[0264]

[0265] 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.

[0266] 1st eluting isomer: PBX-7011, 23 mg, 34% yield

[0267] U_AN_ACID: m / z 448.4 [M+H] +

[0268] 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).

[0269]

[0270] 2nd eluting isomer: PBX-7012, 28 mg, 41% yield

[0271] U_AN_ACID: m / z 448.2 [M+H] +

[0272] 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).

[0273]

[0274] Manufacturing Example 2: Synthesis of active camptothecin derivatives of chemical formula 4 or 5 (PBX-7014 and PBX-7015)

[0275] 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).

[0276]

[0277]

[0278] 2-1: Synthesis of PBX-7014

[0279]

[0280] A stock solution of activated glycolic acid was prepared according to the following procedure:

[0281] 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.

[0282] 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%

[0283] U_AN_ACID: m / z 506.2 [M+H] +

[0284] 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).

[0285]

[0286] 2-2: Synthesis of PBX-7015

[0287]

[0288] A stock solution of activated acid was prepared according to the following procedure:

[0289] 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.

[0290] 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%

[0291] U_AN_ACID: 506.2 [M+H] +

[0292] 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).

[0293]

[0294] Manufacturing Example 3: Synthesis of an active camptothecin derivative of chemical formula 6 (PBX-7016) or an active camptothecin derivative of chemical formula 7 (PBX-7017)

[0295] This example describes the synthesis of compound PBX-7016 starting from the Exatecan-hybrid compound (PBX-7011).

[0296]

[0297] A stock solution of activated D-lactic acid was prepared according to the following procedure.

[0298] 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.

[0299] 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.

[0300] Yield: 22 mg, 52%

[0301] U_AN_ACID: m / z 520.2 [M+H] +

[0302] 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).

[0303] 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.

[0304]

[0305] Manufacturing Example 4: Synthesis of an active camptothecin derivative of chemical formula 8 (PBX-7018)

[0306]

[0307] (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.

[0308] 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%)

[0309] The above PBX-7011 can be obtained by the method described in PCT / KR2023 / 009854, but is not limited thereto.

[0310]

[0311] U_AN_ACID: m / z 574.2 [M+H] +

[0312] 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).

[0313]

[0314] Manufacturing Example 5: Synthesis of an active camptothecin derivative of chemical formula 9 (PBX-7020)

[0315]

[0316] (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.

[0317] 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%)

[0318]

[0319] U_AN_ACID: m / z 534.2 [M+H]+

[0320] 1 H 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).

[0321]

[0322] Manufacturing Example 6: Synthesis of an active camptothecin derivative of chemical formula 10 (PBX-7022)

[0323]

[0324] 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%)

[0325]

[0326] U_AN_ACID: m / z 502.2 [M+H] +

[0327]

[0328] 1 H 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).

[0329]

[0330] Manufacturing Example 7: Synthesis of an active camptothecin derivative of chemical formula 11 (PBX-7024)

[0331]

[0332]

[0333] PBX-7024 was prepared in high yield by coupling (2S)-2-cyclopropyl-2-hydroxyacetic acid to the PBX-7011 compound.

[0334] (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.

[0335] Yield: 32 mg, 65%

[0336] U_AN_ACID: m / z 520.2 [M+H] +

[0337] 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).

[0338]

[0339] Manufacturing Example 8: Synthesis of linker-payload (mc-GGFG-PBX-7016) compound

[0340]

[0341] 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.

[0342] Yield: 510 mg, 63%

[0343] U_AN_ACID: m / z 1060.02 [M+H] +

[0344] 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).

[0345]

[0346] Example: Anti-TF ADC comprising a compound of formula 1 (PBX payload)

[0347] Manufacturing of antibody-drug conjugates (ADCs)

[0348] Anti-TF ADCs containing the PBX payload manufactured in the above manufacturing example were manufactured (Table 1).

[0349] No. Payload Linker Antibody Example 1 PBX-7011mc-GGFG Anti-TF antibody (Tisotumab, anti-TF mAb) Example 2 PBX-7014 Example 3 PBX-7016 Example 4 PBX-7018 Example 5 PBX-7020 Example 6 PBX-7022 Example 7 PBX-7024

[0350] 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 the structure of Chemical Formula 13. The linker was reacted with the payload to first generate a linker-payload conjugate. For example, the linker-payload included in Example 3 was synthesized by the method described in Preparation Example 8.

[0351] Furthermore, ADC was synthesized by conjugation with Tisotumab (Medchemexpress) as an anti-TF antibody. The ADC was prepared by the following method. The prepared Tisotumab 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 thiol sites necessary for the reaction from the disulfide of the antibody.

[0352] 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 reduced Tisotumab at 4°C for 1 hour. As a result, Tisotumab-PBX-7016 (anti-TF-PBX-7016) ADC, in which the linker-payload compound was conjugated to an anti-TF antibody (anti-TF mAb), was prepared.

[0353]

[0354] Evaluation of antibody-drug conjugates (ADCs)

[0355] The purity of the ADC monomer of Example 3 was determined using size exclusion chromatography (SEC) as shown in Figure 1 (2x PBS, 1 mg / ml, 280 nm).

[0356] The DAR was evaluated by mass (Da)-area analysis via LC-MS and is shown in Table 2. The SEC value was >98%.

[0357] Example 3 Mass (Da) Area Ratio DAR Light Chain D0 2 3 3 7 5.5 5 10 9 7.7 7.1 0.9 7.9 D1 2 4 4 3 5.4 6 9 9 5.8 9 2.9 D2--0.0 Heavy Chain D0 5 0 27 7.1-0.0 3.0 D1 5 1 3 3 7 1-0.0 D2 5 2 3 9 7.1-0.0 D3 5 3 4 5 7.1-0.0 6.1 1.0 0.0

[0358] Comparative Example: Manufacturing of Isotype ADCs and Anti-TF ADCs with Different Payloads

[0359] ADCs conjugated with anti-influenza A antibody (Navivumab, P23) and anti-SARS-CoV-2 antibody (Regdanvimab, P59) using PBX-7016 as a payload were manufactured using the method described in the above examples. P23 ​​and P59 were used as isotype controls for this experiment. Meanwhile, Tivdak TM was purchased and used from Jupiter Research Service LLC.

[0360] Finally, an ADC with MMAE (Monomethyl auristatin E) as a payload and an anti-influenza A antibody (Navivumab, P23) conjugated thereto was prepared by the method described in the above examples. The anti-influenza A antibody (P23) 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.

[0361] MMAE was purchased from MedChemExpress in the form of payload and linker-payload (MC-vc-PAB-MMAE). ADCs with PBX-7016 as a payload were used in the form of linker-payload (mc-GGFG-PBX-7016) using mc-GGFG as a linker.

[0362] The SEC and DAR values ​​for each ADC above are as described in Table 3 below.

[0363] ADCSECDAR1P23-PBX-7016>98%82P59-PBX-7016>98%83Tivdak (Tisotumab-MMAE)>98%44P23-MMAE>98%4

[0364] Experimental Example 1: Cell viability assay of PBX payload anti-TF ADC

[0365] Experimental Example 1-1. Selection of Tissue Factor (TF)-Expressing Cell Lines

[0366] Cell lines expressing tissue factor (TF) were selected using flow cytometry.

[0367] The cell lines used were T47D (breast cancer cell line), MCF-7 (breast cancer cell line), HPAF-II (pancreatic cancer cell line), KYSE-520 (esophageal cancer cell line), Caski (cervical cancer cell line), and A431 (skin cancer cell line). Antibody binding and flow cytometry analysis were performed using commercially available monoclonal antibodies, PE-mAb (12-1429-42) and APC-mAb (365205) (see Fig. 2), and Tisotumab & AF647-F(ab)2 anti-human IgG-F(ab)2 (109-606-006) and Tivdak. TM Antibody binding and flow cytometry analysis were performed using (Lot:196169) & AF647-F(ab)2 anti-human IgG-F(ab)2 (109-606-006) (see Fig. 3).

[0368] High levels of tissue factor (TF) expression were observed in HPAF-II (pancreatic cancer cell line), KYSE-520 (esophageal cancer cell line), Caski (cervical cancer cell line), and A431 (skin cancer cell line), and the mean fluorescence intensity (MFI) and staining index in each cell line are as shown in Table 4 below.

[0369] Cell line tissue (carcinoma) MFI staining index expression no staining conjugated mAb T47D breast (Breast) 45922 Negative or (+) MCF-7 breast (Breast) 5351510 Negative or (+) KYSE-520 Esophagus (Esophagus) 6456, 067876+++ Caski cervix (Cervix) 4929, 318598+++ HPAF-Ⅱ Pancreas (Pancreas) 7633, 610442+++ A431 Skin (Skin) 7821, 016269+++

[0370]

[0371] Experimental Example 1-2. Cell viability assay

[0372] 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.

[0373] Specifically, 1,000 to 3,000 cell lines listed in Table 3 were seeded per well in a 96-well plate and incubated (37°C, 5% CO2). After 24 hours, 100 μl of payload compounds or ADC drugs at 9 concentrations (serial dilutions of 1 / 4 each 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.

[0374] As a result, it was confirmed that the ADC of Example 3 (Tisotumab-PBX-7016) had excellent cell inhibition ability. The specific IC for each ADC 50 The values ​​(unit: nM) are as shown in Table 5 below (see Figures 4 to 9).

[0375] Cell linesPBX-7016MMAEP23-PBX-7016(Isotype ADC)P59-PBX-7016(Isotype ADC)Tisotumab-PBX-7016(Example 3)TivdakT47D0.460.1655.257.384137MCF-76.020.82256294357442KYSE-5202.060.12115104< 0.015< 0.015Caski0.750.03469.568.5< 0.015< 0.015HPAF-II2.870.0452511650.018< 0.015A4311.940.03271.5126.50.065< 0.015

[0376] Specifically, the ADC of Example 3 showed superior IC compared to the PBX-7016 compound itself or the isotype ADC (P23-PBX-7016, P59-PBX-7016) in HPAF-II (pancreatic cancer cell line), KYSE-520 (esophageal cancer cell line), Caski (cervical cancer cell line) and A431 (skin cancer cell line), which are high-expressing cell lines in which tissue factor (TF) is expressed in large amounts. 50 It was observed that PBX-7016 had a value. Therefore, 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 tissue factor (TF). In addition, the ADC of Example 3 was Tivdak TM IC comparable to 50 Since it was observed that the value was present, it was confirmed that the inhibitory effect on the target cell line was excellent.

[0377] Accordingly, it can be seen that the Tisotumab-PBX-7016 ADC of the present invention, which has a camptothecin derivative compound of chemical formula 1 as a payload and is specific for tissue factor (TF), exhibits an excellent inhibitory effect on cells expressing tissue factor (TF), and can be used for cancer treatment or prevention.

[0378]

[0379] Experimental Example 2: In vivo antitumor efficacy evaluation (1)

[0380] Experimental Example 2-1. Confirmation of anticancer effects in the KYSE520 human esophageal cancer xenograft model.

[0381] Experimental design

[0382] To evaluate the antitumor efficacy using the KYSE520 cell line human esophageal cancer transplant model in BALB / c nude mice, the experiment was designed as follows (Table 6). Eighty-seven female mice, aged 6 to 8 weeks and weighing 18 to 20 g, and 33 spare mice were prepared.

[0383] Group Number of mice (N) Therapeutic dose (mg / kg) Dosage volume (mL / kg) Dosage route Schedule G110 Isotype mAb (P23) 105i.v.QW x 3 weeks G27 Tisotumab (Anti-TF mAb) 105i.v.QW x 3 weeks G310 Isotype-PBX-7016 105i.v.QW x 3 weeks G410 Tivdak 15i.v.QW x 3 weeks G510 Tivdak 35i.v.QW x 3 weeks G610 Tivdak 105i.v.QW x 3 weeks G710 Tisotumab-PBX-7016 15i.v.QW x 3 weeks G810 Tisotumab-PBX-7016 35i.v.QW x 3 weeksG910Tisotumab-PBX-7016105i.v.QW x 3 weeks

[0384] Specifically, in the above Table 6, Isotype mAb (P23) is an anti-influenza A mAb called Navivumab, and Isotype-PBX-7016 is an ADC (P23-PBX-7016) that uses P23 as an isotypine control. Meanwhile, the Tisotumab-PBX-7016 ADC has a structure in which Tisotumab (anti-TF mAb) and PBX-7016 are linked by a mc-GGFG linker, and Tivdak™ is an ADC in which MMAE is bound to Tisotumab.

[0385] cell culture

[0386] KYSE520 tumor cells were maintained in vitro in RPMI 1640 medium supplemented with 10% fetal bovine serum and 1% antibiotic-antimycotic at 37°C under 5% CO2 in air. Tumor cells were routinely passaged twice a week by trypsin-EDTA treatment, and cells growing in the exponential growth phase were harvested and counted for tumor inoculation.

[0387]

[0388] Tumor inoculation and test substance administration

[0389] KYSE520 tumor cells (5 × 10 ) were injected into the right flank of each mouse with 0.2 mL of PBS and Matrigel (1:1 mixture). 6 Cells / mouse) were subcutaneously inoculated. Animals were randomly assigned, and 10 days after tumor inoculation, the average tumor volume was 175 mm 3 Treatment was initiated when tumor volume reached . The treatment start date was set as Day 0, and animals were assigned to groups using Excel-based randomization software that performs hierarchical randomization according to tumor volume. Each group consisted of 10 or 7 tumor-bearing mice. Mice were administered the test substances according to the regimens shown in Table 6 above.

[0390]

[0391] Tumor measurements and statistical analysis

[0392] The primary objective was to determine whether the treatment could delay tumor growth or cure mice. Tumor volume was measured in two dimensions twice a week using calipers, and the volume (mm 3 ) is expressed using the following formula:

[0393] V = 0.5 axb 2

[0394] (a: long diameter of the tumor, b: short diameter of the tumor)

[0395] Tumor size was used to calculate T / C (tumor volume relative to control volume) and TGI (tumor growth inhibition) values. The T / C value (%) indicates the antitumor effect, where T and C are the mean tumor volumes of the treatment and control groups on a given day, respectively. The T / C value (%) was calculated by dividing the tumor volume of the treatment group by the tumor volume of the vehicle group on the same day. The TGI of each group was calculated using the following formula:

[0396] TGI(%) = [1-(T i -T0) / (V i -V0)] ×100%

[0397] (T i : Mean tumor volume of the treatment group on a specific day, T0: Mean tumor volume of the treatment group on the first day of treatment, V i : T i (V0: mean tumor volume of the control group on the first day of treatment)

[0398] The mean and mean error (including SEM) for tumor volume in each group were determined at each time point. Statistical analysis was performed on data obtained 35 days after the start of treatment to determine differences in tumor volume and tumor weight between groups. The Kruskal-Wallis test was performed, followed by Dunn's post hoc analysis, to compare tumor volume and tumor weight. All data were analyzed using SPSS 29.0 (p < 0.05 was considered statistically significant).

[0399]

[0400] Animal body weights were regularly monitored as an indirect measure of toxicity. Body weight change (%) and tumor growth (Tumor volume, mm) were measured by group. 3) curves are shown in Figures 10 and 11. The average tumor volume over time is shown in Table 7, and the TGI was calculated based on the tumor volume measurement on the 35th day after the start of treatment and is shown in Table 8. T / C is calculated based on the tumor weight measurement on the 35th day after the start of treatment. weight The results were calculated and shown in Table 9.

[0401] Days b Tumor volume (mm 3 )) a G1G2G3G4G5G6G7G8G90175 ± 8175 ± 10175 ± 8175 ± 8175 ± 8175 ± 8175 ± 8175 ± 8175 ± 84274 ± 16270 ± 25250 ± 11279 ± 20241 ± 23243 ± 22252 ± 17224 ± 14231 ± 127362 ± 31329 ± 31319 ± 13360 ± 29279 ± 24259 ± 24353 ± 26279 ± 22273 ± 1911502 ± 42448 ± 47410 ± 22489 ± 37281 ± 31234 ± 23454 ± 38291 ± 20248 ± 1414680 ± 53589 ± 62505 ± 30672 ± 51270 ± 31196 ± 16534 ± 48296 ± 31213 ± 1218883 ± 82731 ± 93519 ± 41771 ± 64174 ± 17122 ± 11575 ± 59203 ± 16143 ± 6211,083 ± 98878 ± 111521 ± 42865 ± 75154 ± 17 (n = 9) c101 ± 7606 ± 65185 ± 16130 ± 7251,425 ± 1091,116 ± 142589 ± 541,168 ± 101142 ± 15 (n = 9)88 ± 7718 ± 83174 ± 16123 ± 8281,675 ± 1351,347 ± 158675 ± 861,497 ± 121129 ± 16(n = 9)69 ± 5917 ± 125161 ± 16111 ± 8321,895 ± 1491,597 ± 148810 ± 1161,634 ± 126115 ± 16(n = 9)58 ± 51,086 ± 158145 ± 1598 ± 7352,151 ± 1761,847 ± 175947 ± 1611,893 ± 16699 ± 14 (n = 9)45 ± 61,298 ± 192125 ± 1261 ± 4

[0402] a. Mean ± SEM

[0403] b. Number of days since starting treatment

[0404] c. On day 18, one mouse was sacrificed if its body weight decreased by more than 20%. The average tumor volume was calculated from the surviving mice.

[0405] Group Number of mice (N) Tumor volume (mm) 3 )) a 35T / C on day b (%)TGI c (%)pvalue d G1102,151 ± 176---G271,847 ± 17585.8615.40>0.999G310947 ± 16144.0560.92>0.999G4101,893 ± 16688.0313.03>0.999G59 e 99 ± 144.58103.89<0.001G61045 ± 62.08106.62<0.001G7101,298 ± 19260.3643.16>0.999G810125 ± 125.82102.540.007G91061 ± 42.83105.80<0.001

[0406] a. Mean ± SEM

[0407] b. T / C (%) = T / C × 100% (T and C are the average volumes on day 35 after treatment in the treatment and control groups, respectively)

[0408] c. TGI (%) = [1-(T 35 -T0) / (V 35 -V0)] ×100%

[0409] d. p values ​​are calculated based on tumor volume

[0410] e. One mouse was sacrificed on day 18 due to a weight loss of more than 20%.

[0411] Group number of mice (N) Tumor weights (g) a 35T / C on day weight b (%)pvalue c G1102.157 ± 0.205--G271.767 ± 0.22481.94>0.999G3100.987 ± 0.15945.74>0.999G4101.869 ± 0.13886.67>0.999G59 d 0.089 ± 0.0134.120.002G6100.042 ± 0.0041.93<0.001G7101.383 ± 0.20564.12>0.999G8100.106 ± 0.0134.900.005G9100.048 ± 0.0052.22<0.001

[0412] a. Mean ± SEM

[0413] b. T / C (%) = T / C × 100% (T and C are the average tumor weights on day 35 after treatment in the treatment and control groups, respectively)

[0414] c. p values ​​are calculated based on tumor weight

[0415] d. On day 18, one mouse was sacrificed due to a weight loss of more than 20%.

[0416]

[0417] All test substances of the tested dosing regimens were confirmed to be safe in female BALB / c nude mice implanted with KYSE520. On the 18th day after the start of treatment, one mouse in G5 showed a weight loss of more than 20% and was necropsied, but no abnormalities were found.

[0418] As shown in Tables 8 to 10, the average tumor volume of G1 was 2151 mm on day 35 after the start of administration. 3 Compared with G1, the mean tumor volume on day 35 after the start of treatment was 99 mm in G5 (T / C = 4.58%, TGI = 103.89%, p<0.001), G6 (T / C = 2.08%, TGI = 106.62%, p<0.001), G8 (T / C = 5.82%, TGI = 102.54%, p = 0.007), and G9 (T / C = 2.83%, TGI = 105.80%, p<0.001), respectively. 3 , 45 mm 3 , 125 mm 3 and 61 mm 3 Statistically significant antitumor activity was observed. The tumor weight results were consistent with the tumor volume results. Therefore, it can be seen that the antibody-drug conjugate (ADC) of the present invention exhibits excellent tumor growth inhibitory activity.

[0419]

[0420] Experimental Example 3: In vivo antitumor efficacy evaluation (2)

[0421] Experimental Example 3-1. Confirmation of anticancer effects in the HPAFII human pancreatic cancer xenograft model.

[0422] Experimental design

[0423] To evaluate the antitumor efficacy using the HPAFII human pancreatic cancer transplantation model of the CB17 SCID mouse cell line, the experiment was designed as follows (Table 10). One hundred and twenty female mice, aged 6 to 8 weeks and weighing 17 to 22 g, and 60 spare mice were prepared.

[0424] Group Number of mice (n) Therapeutic dose (mg / kg) Dosage volume (mL / kg) Dosage route Schedule G110 Isotype mAb (P23) 35i.v.QW x 3G210 Tisotumab (Anti-TF mAb) 35i.v.QW x 3G310 Isotype-MMAE 35i.v.QW x 3G410 Tivdak 0.35i.v.QW x 3G510 Tivdak 15i.v.QW x 3G610 Tivdak 15i.v.QW x 1G710 Tivdak 35i.v.QW x 1G810 Isotype-PBX-7016 35i.v.QW x 3G910 Tisotumab-PBX-7016 0.35ivQW x 3G1010Tisotumab-PBX-701615i.v.QW x 3G1110Tisotumab-PBX-701615i.v.QW x 1G1210Tisotumab-PBX-701635i.v.QW

[0425] Specifically, in the above Table 10, Isotype mAb (P23) is an anti-influenza A mAb called Navivumab, and Isotype-PBX-7016 is an ADC (P23-PBX-7016) that uses P23 as an isotypine control. Meanwhile, the Tisotumab-PBX-7016 ADC has a structure in which Tisotumab (anti-TF mAb) and PBX-7016 are linked by a mc-GGFG linker, and Tivdak™ is an ADC in which MMAE is bound to Tisotumab.

[0426] cell culture

[0427] HPAFII tumor cells were maintained in vitro in EMEM medium supplemented with 10% fetal bovine serum and 1% antibiotic-antimycotic at 37°C under 5% CO2 in air. Tumor cells were routinely passaged twice a week by trypsin-EDTA treatment, and cells growing in the exponential growth phase were harvested and counted for tumor inoculation.

[0428]

[0429] Tumor inoculation and test substance administration

[0430] HPAFII tumor cells (3 × 10 ) were injected into the right flank of each mouse with 0.2 mL of PBS and Matrigel (1:1 mixture). 6 Cells / mouse) were subcutaneously inoculated. Animals were randomly assigned, and the average tumor volume was 235 mm on day 9 after tumor inoculation. 3 Treatment was initiated when tumor volume was reached. The treatment start date was set as Day 0, and animals were assigned to groups using Excel-based randomization software that performs hierarchical randomization according to tumor volume. Each group consisted of 10 tumor-bearing mice. The test substances were administered to the mice according to the regimen shown in Table 10 above.

[0431] Tumor measurements and statistical analysis

[0432] Tumor measurements and statistical analysis were performed using data obtained 28 days after the start of treatment.

[0433]

[0434] As an indirect measure of toxicity, animal body weight was regularly monitored, and body weight change (Body weight change %) and tumor growth (Tumor volume, mm) were recorded by group. 3 ) curves are shown in Figures 12 and 13. The average tumor volume over time is shown in Tables 11 and 12, and the TGI is calculated based on the tumor volume measurement on the 28th day after the start of treatment and is shown in Table 13. T / C is calculated based on the tumor weight measurement on the 28th day after the start of treatment. weight The results were calculated and shown in Table 14.

[0435] Days b Tumor volume (mm 3 )) aG1G2G3G4G5G60235 ± 17235 ± 17235 ± 17235 ± 17235 ± 17235 ± 174359 ± 50290 ± 24357 ± 38313 ± 34190 ± 12217 ± 167443 ± 64362 ± 38424 ± 46361 ± 49124 ± 11141 ± 1611646 ± 78527 ± 53603 ± 79476 ± 6485 ± 987 ± 914903 ± 90690 ± 57799 ± 98576 ± 6970 ± 8103 ± 14181,209 ± 105996 ± 961,055 ± 125,737 ± 9154 ± 6161 ± 31211,470 ± 1251,262 ± 1381,306 ± 147934 ± 11340 ± 4213 ± 34251,955 ± 1451,581 ± 1591,626 ± 1821,219 ± 14133 ± 4344 ± 49282,354 ± 1652,004 ± 1722,142 ± 1641,607 ± 14426 ± 3486 ± 73

[0436] a. Mean ± SEM

[0437] b. Number of days since starting treatment

[0438] Days b Tumor volume (mm 3 )) aG7G8G9G10G11G120235 ± 17235 ± 17235 ± 17235 ± 17235 ± 17235 ± 174216 ± 19352 ± 41362 ± 38342 ± 30355 ± 32326 ± 317128 ± 10371 ± 41389 ± 39285 ± 31295 ± 25221 ± 261169 ± 8481 ± 46498 ± 52287 ± 43295 ± 26173 ± 201456 ± 4682 ± 59657 ± 73254 ± 43354 ± 44133 ± 121839 ± 3786 ± 56906 ± 124303 ± 69464 ± 73140 ± 132130 ± 21,071 ± 771,098 ± 129337 ± 83564 ± 90147 ± 152526 ± 21,397 ± 1191,474 ± ​​143460 ± 129819 ± 146168 ± 262819 ± 11,772 ± 1571,636 ± 165499 ± 1461,007 ± 195180 ± 35

[0439] a. Mean ± SEM

[0440] b. Number of days since starting treatment

[0441] Group Number of mice (N) Tumor volume (mm) 3 )) a 28T / C on day b (%)TGI c (%)pvalue d G1102,354 ± 165--<0.001G2102,004 ± 17285.1416.51G3102,142 ± 16490.9910.01G4101,607 ± 14468.2935.24G51026 ± 31.09109.91G610486 ± 7320.6788.15G71019 ± 10.80110.22G8101,772 ± 15775.3127.44G9101,636 ± 16569.5333.86G1010499 ± 14621.1887.58G11101,007 ± 19542.7863.58G1210180 ± 357.64102.63

[0442] a. Mean ± SEM

[0443] b. T / C (%) = T / C × 100% (T and C are the average volumes on day 28 after treatment in the treatment and control groups, respectively)

[0444] c. TGI (%) = [1-(T 28 -T0) / (V 28 -V0)] ×100%

[0445] d. p values ​​are calculated based on tumor volume

[0446] Group number of mice (N) Tumor weights (g) a 28T / C on day weight b (%)pvalue c G1102.137 ± 0.144-<0.001G2101.856 ± 0.14886.85G3101.933 ± 0.17790.45G4101.461 ± 0.14668.36G5100.022 ± 0.0031.03G6100.454 ± 0.08821.23G7100.012 ± 0.0010.54G8101.810 ± 0.13784.70G9101.691 ± 0.11879.15G10100.449 ± 0.13821.01G11101.070 ± 0.23850.05G12100.144 ± 0.0366.72

[0447] a. Mean ± SEM

[0448] b. T / C (%) = T / C × 100% (T and C are the average tumor weights on day 28 after treatment in the treatment and control groups, respectively)

[0449] c. p values ​​are calculated based on tumor weight

[0450]

[0451] All test substances in the tested dosing regimens were found to be safe in female CB17 SCID mice implanted with HPAFII, and no significant body weight loss was observed during the experiment.

[0452] As shown in Tables 11 to 14, the average tumor volume of G1 was 2354 mm on day 28 after the start of administration. 3 Compared with G1, G5 (T / C = 1.09%, TGI = 109.91%), G6 (T / C = 20.67%, TGI = 88.15%), G7 (T / C = 0.80%, TGI = 110.22%), G10 (T / C = 21.18%, TGI = 87.58%), and G12 (T / C = 7.64%, TGI = 102.63%) had a mean tumor volume of 26 mm, respectively, on day 28 after the start of treatment. 3 , 486 mm 3 , 19 mm 3 , 499 mm 3 and 180 mm 3 Statistically significant antitumor activity was observed. The tumor weight results were consistent with the tumor volume results. Therefore, it can be seen that the antibody-drug conjugate (ADC) of the present invention exhibits excellent tumor growth inhibitory activity.

[0453]

[0454] Sequence list

[0455] The sequence list of antibodies used in the present invention is as described in Table 15 below.

[0456] 서열번호서열명칭서열1Anti-TF mAb CDR-H1GFTFSNYA2Anti-TF mAb CDR-H2ISGSGDYT3Anti-TF mAb CDR-H3ARSPWGYYLDS4Anti-TF mAb CDR-L1QGISSR5Anti-TF mAb CDR-L2AAS6Anti-TF mAb CDR-L3QQYNSYPYT7Anti-TF mAb heavy chain variable regionEVQLLESGGGLVQPGGSLRLSCAASGFTFSNYAMSWVRQAPGKGLEWVSSISGSGDYTYYTDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARSPWGYYLDSWGQGTLVTVSS8Anti-TF mAb light chain variable regionDIQMTQSPPSLSASAGDRVTITCRASQGISSRLAWYQQKPEKAPKSLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSYPYTFGQGTKLEIK9Anti-TF mAb heavy chainEVQLLESGGGLVQPGGSLRLSCAASGFTFSNYAMSWVRQAPGKGLEWVSSISGSGDYTYYTDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARSPWGYYLDSWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK10Anti-TF mAb lightchainDIQMTQSPPSLSASAGDRVTITCRASQGISSRLAWYQQKPEKAPKSLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSYPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC11Isotype mAb (P23) heavy chainMDWTWSILFLVAAATGAHSQVQLVQSGAEVKKPGASVKVSCKTSGYSFSTYGVSWVRQAPGQGPEWVGWISAYTGITDYAQKFQGRVTLTTDATTATAFLDLRSLRPDDTATYFCARDKVQGRVEVGSGGRHDYWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEGLHNHYTQKSLSLSPGK*12Isotype mAb (P23) 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-Tissue Factor antibody via a linker: [Chemical Formula 1] Here, X1 and X3 are each independently carbon, oxygen, nitrogen, or sulfur, and 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-Tissue Factor antibody is selected from the group consisting of antibodies of Tisotumab, XB-002, MRG-004A, AMB101 and STRO-004.

7. In paragraph 1, The above anti-Tissue Factor antibody is an antibody-drug conjugate called Tisotumab.

8. In paragraph 1, An antibody-drug conjugate, wherein the anti-Tissue Factor 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.

9. In paragraph 1, An antibody-drug conjugate wherein the anti-Tissue Factor 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.

10. In paragraph 1, An antibody-drug conjugate, wherein the anti-Tissue Factor 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.

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

12. In paragraph 11, 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, fallopian tube cancer, ovarian epithelial cancer, ovarian germ cell cancer, male breast cancer, brain cancer, pituitary adenoma, multiple myeloma, gallbladder cancer, biliary tract cancer, colon cancer, head and neck 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, A pharmaceutical composition selected from the group consisting of renal pelvic cancer, kidney cancer, 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, tonsillar 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.

13. In paragraph 11, A pharmaceutical composition, wherein the cancer is selected from the group consisting of cervical cancer, endometrial cancer, ovarian cancer, peritoneal cancer, fallopian tube cancer, breast cancer, head and neck cancer, esophageal cancer, bladder cancer, and pancreatic cancer.

14. A method for treating or preventing cancer, comprising administering to a subject an antibody-drug conjugate according to any one of claims 1 to 10.

15. Use of an antibody-drug conjugate according to any one of claims 1 to 10 for the treatment or prevention of cancer.

16. Use of an antibody-drug conjugate according to any one of claims 1 to 10 for preparing a pharmaceutical composition for treating or preventing cancer.

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