Anti-TROP-2 antibody-exatecan analogue conjugate and its medical use

Abstract

The present invention provides an anti-TROP-2 antibody-exatecan analogue conjugate and its pharmaceutical use. Specifically, the present invention provides an anti-TROP-2 antibody-exatecan analogue conjugate represented by the general formula (Pc-LYD), where Pc is an anti-TROP-2 antibody or an antigen-binding fragment thereof. JPEG2023510905000066.jpg7182

Description

[Technical Field]

[0001] This application claims priority from a Chinese patent application filed on January 22, 2020 (application number CN 202010073438.8).

[0002] The present disclosure relates to anti-TROP-2 antibodies, anti-TROP-2 antibody-exatecan analogue conjugates, methods for their preparation, pharmaceutical compositions containing them, and their use in the preparation of medicaments for treating diseases or conditions mediated by TROP-2, particularly in the preparation of anti-cancer drugs. [Background technology]

[0003] Nothing herein necessarily constitutes prior art, but rather merely provides background information relevant to the present disclosure.

[0004] TROP-2 (human trophoblast cell surface glycoprotein antigen 2, also known as tumor-associated calcium signal transducer 2 (TACSTD2), epithelial glycoprotein 1 (EGP-1), gastrointestinal tumor-associated antigen (GA733-1), surface marker 1 (M1S1)) is a cell surface glycoprotein encoded and expressed by the Tacstd2 gene on chromosome 1p32. TROP-2 belongs to the GA733 protein family and shares relatively high structural and sequence similarity with epithelial cell adhesion molecule (EpCAM, Trop1, or TACSTD1), with a homology of 49%.

[0005] The primary structure of the TROP-2 protein is a polypeptide of approximately 36 kD consisting of approximately 323 amino acids, which is post-translationally modified by N-terminal glycosylation to form a type I plasma membrane glycoprotein, i.e., the TROP-2 protein, which is distinct from EpCAM. The TROP-2 protein spans the plasma membrane, with the N-terminus comprising an extracellular domain (Trop2EC), which is connected to a short intracellular tail (Trop2IC), a hydrophobic polypeptide consisting of 26 amino acid residues, by a single unidirectional transmembrane helix (TM), thereby anchoring it to the plasma membrane.

[0006] TROP-2 has now been found to play an important role in embryonic development and tumor cell proliferation and metastasis. TROP-2 was first discovered in trophoblast cells and is used as a surface marker. Trophoblasts are derived from extraembryonic trophoblasts, and TROP-2 contributes to embryo implantation and placental tissue formation, and plays an important role in maintaining the proliferation properties of embryonic stem cells and organ development. TROP-2 is also an important tumor progression factor. It is highly expressed in various tumors, including pancreatic cancer, breast cancer, colon cancer, gastric cancer, oral squamous cell carcinoma, and ovarian cancer, and can promote tumor cell proliferation, invasion, and metastasis. High expression is closely associated with shortened survival and poor prognosis in tumor subjects, making research into antitumor agents targeting TROP-2 of great significance.

[0007] Antibody drug conjugates (ADCs) connect monoclonal antibodies or antibody fragments to biologically active cytotoxins via linker compounds, taking advantage of the binding specificity of antibodies to surface antigens on normal and tumor cells and the high efficiency of cytotoxic substances, while avoiding the drawbacks of antibodies, such as the relatively small therapeutic effect and the excessive toxicity and side effects of toxic substances. This means that, compared with traditional chemotherapy drugs, antibody drug conjugates can kill tumor cells more precisely and reduce the impact on normal cells. Summary of the Invention

[0008] The present disclosure relates to anti-TROP-2 antibodies, ADCs thereof, and uses thereof. The disclosure provides ADC drugs conjugated to an anti-TROP-2 antibody or antigen-binding fragment and a cytotoxic exatecan analogue.

[0009] The present disclosure provides a ligand-drug conjugate represented by the general formula (Pc-LYD) or a pharmaceutically acceptable salt thereof: [ka] Among them, Y is -O-(CR aR b ) m -CR 1 R 2 -C(O)-, -O-CR 1 R 2 -(CR a R b ) m -, -O-CR 1 R 2 -, -NH-(CR a R b ) m -CR 1 R 2 -C(O)- and -S-(CR a R b ) m -CR 1 R 2 -C(O)-, R a and R b are the same or different and are each independently selected from a hydrogen atom, a deuterium atom, a halogen, an alkyl group, a halogenated alkyl group, a deuterated alkyl group, an alkoxy group, a hydroxy group, an amino group, a cyano group, a nitro group, a hydroxyalkyl group, a cycloalkyl group, and a heterocyclyl group, or R a and R b together with the carbon atoms to which they are attached form a cycloalkyl or heterocyclyl group, R 1 is selected from halogen, halogenated alkyl groups, deuterated alkyl groups, cycloalkyl groups, cycloalkylalkyl groups, alkoxyalkyl groups, heterocyclyl groups, aryl groups, and heteroaryl groups; R 2 is selected from a hydrogen atom, a halogen, a halogenated alkyl group, a deuterated alkyl group, a cycloalkyl group, a cycloalkylalkyl group, an alkoxyalkyl group, a heterocyclyl group, an aryl group, and a heteroaryl group, or R 1 and R 2 together with the carbon atoms to which they are attached form a cycloalkyl or heterocyclyl group, Or, R a and R 2 together with the carbon atoms to which they are attached form a cycloalkyl or heterocyclyl group, m is an integer from 0 to 4, and in non-limiting examples, m is selected from 0, 1, 2, 3, and 4; n is 1 to 10, and n is a decimal or an integer; L is a linker unit, Pc is an anti-TROP-2 antibody or an antigen-binding fragment thereof.

[0010] In some embodiments, the ligand-drug conjugate represented by the general formula (Pc-LYD) or a pharmaceutically acceptable salt thereof, wherein the anti-TROP-2 antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 having the same sequences as those of the heavy chain variable region represented by SEQ ID NO: 3, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3 having the same sequences as those of the light chain variable region represented by SEQ ID NO: 4.

[0011] In some embodiments, the ligand-drug conjugate represented by the general formula (Pc-LYD) described in any one of the above, or a pharmaceutically acceptable salt thereof, wherein the anti-TROP-2 antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 represented by SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7, respectively, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3 represented by SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10, respectively.

[0012] In some embodiments, in the ligand-drug conjugate represented by the general formula (Pc-LYD) or a pharmaceutically acceptable salt thereof described in any one of the above, the anti-TROP-2 antibody is a murine antibody, a chimeric antibody, a humanized antibody, or a human antibody.

[0013] In some embodiments, the ligand-drug conjugate represented by the general formula (Pc-LYD) or a pharmaceutically acceptable salt thereof described in any one of the above aspects, wherein the anti-TROP-2 antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein the amino acid sequence of the heavy chain variable region is represented by SEQ ID NO: 3 or has at least 90% to 100% identity thereto, including, but not limited to, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% identity thereto, and the amino acid sequence of the light chain variable region is represented by SEQ ID NO: 4 or has at least 90% to 100% identity thereto, including, but not limited to, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% identity thereto.

[0014] In some embodiments, the ligand-drug conjugate represented by the general formula (Pc-LYD) or a pharmaceutically acceptable salt thereof described in any one of the above, wherein the anti-TROP-2 antibody or antigen-binding fragment thereof comprises a heavy chain variable region whose sequence is represented by SEQ ID NO:3 and a light chain variable region whose sequence is represented by SEQ ID NO:4.

[0015] In some embodiments, the ligand-drug conjugate represented by the general formula (Pc-LYD) or a pharmaceutically acceptable salt thereof described in any one of the above aspects, wherein the anti-TROP-2 antibody or antigen-binding fragment thereof comprises a heavy chain constant region and a light chain constant region, preferably the heavy chain constant region is selected from the constant regions of human IgG1, IgG2, IgG3, and IgG4, and the light chain constant region is selected from the constant regions of human antibody κ and λ chains, more preferably the antibody comprises a heavy chain constant region whose sequence is set forth in SEQ ID NO: 11 and a light chain constant region whose sequence is set forth in SEQ ID NO: 12.

[0016] In some embodiments, the ligand-drug conjugate represented by the general formula (Pc-LYD) or a pharmaceutically acceptable salt thereof described in any one of the above, wherein the anti-TROP-2 antibody comprises a heavy chain represented by SEQ ID NO: 13 and a light chain represented by SEQ ID NO: 14.

[0017] In some embodiments, the anti-TROP-2 antibody of the ligand-drug conjugate represented by the general formula (Pc-LYD) or a pharmaceutically acceptable salt thereof comprises a heavy chain whose sequence is represented by SEQ ID NO: 15 and a light chain whose sequence is represented by SEQ ID NO: 16, or a heavy chain whose sequence is represented by SEQ ID NO: 17 and a light chain whose sequence is represented by SEQ ID NO: 18.

[0018] In some embodiments, in the ligand-drug conjugate represented by general formula (Pc-LYD) or a pharmaceutically acceptable salt thereof described in any one of the above items, n is 2 to 10, preferably 4 to 8, and n is a decimal or an integer. In some embodiments, n is 0 to 10, preferably 1 to 10, and more preferably an average of 1 to 8, or 2 to 8, or 2 to 7, or 2 to 4, or 3 to 8, or 3 to 7, or 3 to 6, or 4 to 7, or 4 to 6, or 4 to 5. In some embodiments, n is an average of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0019] In some embodiments, the ligand-drug conjugate represented by the general formula (Pc-LYD) or a pharmaceutically acceptable salt thereof according to any one of the preceding claims, Among them, Y is -O-(CR a R b ) m -CR 1 R 2 -C(O)-, R a and R b are the same or different and are each independently selected from a hydrogen atom, a deuterium atom, a halogen atom, and an alkyl group; R 1 is a halogenated alkyl group or C 3-6 is a cycloalkyl group, R 2 is a hydrogen atom, a halogenated alkyl group, and C 3-6 cycloalkyl groups, Or, R 1 and R 2 C along with the carbon atoms attached to them 3-6 forming a cycloalkyl group, m is 0 or 1.

[0020] In some embodiments, in the ligand-drug conjugate represented by the general formula (Pc-LYD) or a pharmaceutically acceptable salt thereof according to any one of the preceding claims, Y is [ka] Selected from Among them, the O-end of Y is connected to the linker unit L.

[0021] In some embodiments, in the ligand-drug conjugate represented by the general formula (Pc-LYD) or a pharmaceutically acceptable salt thereof according to any one of the preceding claims, the linker unit -L- is -L 1 -L 2 -L 3 -L 4 - and L 1 -(succinimide-3-yl-N)-WC(O)-, -CH2-C(O)-NR 3 -WC(O)- and -C(O)-WC(O)-, wherein W is C 1-8 Alkyl group, C 1-8 alkyl-cycloalkyl groups and straight-chain heteroalkyl groups of 1 to 8 atoms, wherein the heteroalkyl groups contain 1 to 3 heteroatoms selected from N, O and S, and the C 1-8 the alkyl group, the cycloalkyl group, and the straight-chain heteroalkyl group are each independently optionally further substituted with one or more substituents selected from halogen, hydroxyl, cyano, amino, alkyl, chloroalkyl, deuterated alkyl, alkoxy, and cycloalkyl groups; L 2 is -NR4 (CH2CH2O)pCH2CH2C(O)-, -NR 4 (CH2CH2O)pCH2C(O)-, -S(CH2)pC(O)- or a chemical bond, wherein p is an integer of 1 to 20; L 3 is a peptide residue consisting of 2 to 7 amino acid residues, wherein the amino acid residues are selected from amino acid residues formed by amino acids selected from phenylalanine, glycine, valine, lysine, citrulline, serine, glutamic acid, and aspartic acid, and are optionally further substituted with one or more substituents selected from halogen, a hydroxy group, a cyano group, an amino group, an alkyl group, a chloroalkyl group, a deuterated alkyl group, an alkoxy group, and a cycloalkyl group; L 4 is -NR 5 (CR 6 R 7 ) t -, -C(O)NR 5 -, -C(O)NR 5 (CH2) t - and a chemical bond, wherein t is an integer from 1 to 6; R 3 , R 4 and R 5 are the same or different and are each independently selected from a hydrogen atom, an alkyl group, a halogenated alkyl group, a deuterated alkyl group, and a hydroxyalkyl group; R 6 and R 7 are the same or different and are each independently selected from a hydrogen atom, a halogen, an alkyl group, a halogenated alkyl group, a deuterated alkyl group, and a hydroxyalkyl group.

[0022] In some embodiments, in the ligand-drug conjugate represented by the general formula (Pc-LYD) or a pharmaceutically acceptable salt thereof according to any one of the preceding claims, the linker unit -L- is -L 1 -L 2 -L 3 -L 4 - and L 1-(succinimide-3-yl-N)-WC(O)-, -CH2-C(O)-NR 3 -WC(O)- and -C(O)-WC(O)-, wherein W is C 1-8 Alkyl group, C 1-8 alkyl-cycloalkyl groups and straight-chain heteroalkyl groups of 1 to 8 chain atoms, said heteroalkyl groups containing 1 to 3 heteroatoms selected from N, O and S, said C 1-8 the alkyl group, the cycloalkyl group, and the straight-chain heteroalkyl group are each independently optionally further substituted with one or more substituents selected from halogen, hydroxyl, cyano, amino, alkyl, chloroalkyl, deuterated alkyl, alkoxy, and cycloalkyl groups; L 2 is -NR 4 (CH2CH2O)pCH2CH2C(O)-, -NR 4 (CH2CH2O)pCH2C(O)-, -S(CH2)pC(O)- or a chemical bond, wherein p is an integer of 1 to 20; L 3 is a peptide residue consisting of 2 to 7 amino acid residues, wherein the amino acid residues are selected from amino acid residues formed by amino acids selected from phenylalanine (F), glycine (G), valine (V), lysine (K), citrulline, serine (S), glutamic acid (Q), and aspartic acid (D), and are optionally further substituted with one or more substituents selected from halogen, hydroxy group, cyano group, amino group, alkyl group, chloroalkyl group, deuterated alkyl group, alkoxy group, and cycloalkyl group; L 4 is -NR 5 (CR 6 R 7 ) t -, -C(O)NR 5 -, -C(O)NR 5 (CH2) t - and a chemical bond, where t is an integer from 1 to 6, and in non-limiting examples, 1, 2, 3, 4, 5, and 6; R 3 , R 4 and R5 are the same or different and are each independently selected from a hydrogen atom, an alkyl group, a halogenated alkyl group, a deuterated alkyl group, and a hydroxyalkyl group; R 6 and R 7 are the same or different and are each independently selected from a hydrogen atom, a halogen, an alkyl group, a halogenated alkyl group, a deuterated alkyl group, and a hydroxyalkyl group.

[0023] In some embodiments, in the ligand-drug conjugate represented by the general formula (Pc-LYD) or a pharmaceutically acceptable salt thereof according to any one of the preceding claims, the linker unit -L- is -L 1 -L 2 -L 3 -L 4 - and L 1 teeth [ka] and s 1 is an integer from 2 to 8, and in non-limiting examples, is 2, 3, 4, 5, 6, 7, and 8; L 2 is a chemical bond, L 3 is a tetrapeptide residue, preferably L 3 is the tetrapeptide residue of GGFG, L 4 is -NR 5 (CR 6 R 7 ) t - and R 5 , R 6 or R 7 are the same or different and each independently represent a hydrogen atom or an alkyl group; t is 1 or 2; Among them, the above L 1 The end is connected to the Pc and L 4 The end is connected to the Y.

[0024] In some embodiments, in the ligand-drug conjugate represented by the general formula (Pc-LYD) or a pharmaceutically acceptable salt thereof according to any one of the preceding claims, -L- is [ka] is.

[0025] In some embodiments, in the ligand-drug conjugate represented by the general formula (Pc-LYD) or a pharmaceutically acceptable salt thereof according to any one of the preceding embodiments, -LY- is optionally [ka] Choose from.

[0026] In some embodiments, the ligand-drug conjugate represented by the general formula (Pc-LYD) or a pharmaceutically acceptable salt thereof according to any one of the preceding claims, a -YD) or a pharmaceutically acceptable salt thereof, [ka] Among them, W, L 2 , L 3 , R 5 , R 6 , R 7 is as defined above for the linker unit -L-; Pc, n, R 1 , R 2 , m is as defined in the general formula (Pc-LYD).

[0027] In some embodiments, the ligand-drug conjugate represented by the general formula (Pc-LYD) or a pharmaceutically acceptable salt thereof according to any one of the preceding claims, b -YD) or a pharmaceutically acceptable salt thereof, [ka] Among them, s 1 is an integer from 2 to 8, Pc, R 1 , R 2 , R 5 , R 6 , R 7 , m and n are represented by the general formula (Pc-L a -YD).

[0028] In some embodiments, the ligand-drug conjugate represented by the general formula (Pc-LYD) or a pharmaceutically acceptable salt thereof according to any one of the preceding claims, wherein the ligand-drug conjugate is [ka] Selected from Among them, Pc and n are as defined in the general formula (Pc-LYD).

[0029] In some embodiments, the ligand-drug conjugate represented by the general formula (Pc-LYD) or a pharmaceutically acceptable salt thereof according to any one of the preceding claims, wherein the ligand-drug conjugate is [ka] Selected from Among them, n is 4 to 8, and n is a decimal or an integer; Pc is an anti-TROP-2 antibody, which comprises a heavy chain represented by SEQ ID NO:13 and a light chain represented by SEQ ID NO:14.

[0030] In some embodiments, the ligand-drug conjugate represented by the general formula (Pc-LYD) or a pharmaceutically acceptable salt thereof is [ka] and Among them, n is 1 to 8, n is a decimal number or an integer, preferably an integer or decimal number of 2 to 4 or 4 to 8, more preferably an integer or decimal number of 4 to 6, Pc is an anti-TROP-2 antibody, which comprises a heavy chain represented by SEQ ID NO: 13 and a light chain represented by SEQ ID NO: 14; Alternatively, it comprises a heavy chain represented by SEQ ID NO: 15 and a light chain represented by SEQ ID NO: 16, Alternatively, it comprises a heavy chain represented by SEQ ID NO:17 and a light chain represented by SEQ ID NO:18.

[0031] In some embodiments, the ligand-drug conjugate represented by the general formula (Pc-LYD) or a pharmaceutically acceptable salt thereof is [ka] Selected from Among them, n is an integer or decimal number of 1 to 8, preferably an integer or decimal number of 2 to 4 or 4 to 8, and more preferably an integer or decimal number of 4 to 6; PD3 is an anti-TROP-2 antibody, which comprises a heavy chain represented by SEQ ID NO: 13 and a light chain represented by SEQ ID NO: 14; hRS7 is an anti-TROP-2 antibody, or comprises a heavy chain represented by SEQ ID NO: 15 and a light chain represented by SEQ ID NO: 16; TINA is an anti-TROP-2 antibody, or alternatively, comprises a heavy chain represented by SEQ ID NO:17 and a light chain represented by SEQ ID NO:18.

[0032] The present disclosure further provides an anti-TROP-2 antibody or antigen-binding fragment thereof, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 having the same sequences as those of the heavy chain variable region represented by SEQ ID NO: 3, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3 having the same sequences as those of the light chain variable region represented by SEQ ID NO: 4.

[0033] In some embodiments, the anti-TROP-2 antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 represented by SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:7, respectively, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3 represented by SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:10, respectively.

[0034] In some embodiments, the anti-TROP-2 antibody or antigen-binding fragment thereof of any one of the preceding claims is a murine antibody, a chimeric antibody, a humanized antibody, or a human antibody.

[0035] In some embodiments, the anti-TROP-2 antibody or antigen-binding fragment thereof of any one of the preceding claims comprises a heavy chain variable region and a light chain variable region, wherein the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO: 3 or has at least 90% to 100% identity thereto, including, but not limited to, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% identity thereto, and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 4 or has at least 90% to 100% identity thereto, including, but not limited to, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% identity thereto.

[0036] In some embodiments, the anti-TROP-2 antibody or antigen-binding fragment thereof of any one of the preceding claims comprises an antibody heavy chain constant region and a light chain constant region, preferably the heavy chain constant region is selected from the constant regions of human IgG1, IgG2, IgG3, and IgG4 and common variants thereof, and the light chain constant region is selected from the chain constant regions of human antibody κ and λ and common variants thereof, more preferably the antibody comprises a heavy chain constant region represented by SEQ ID NO: 11 and a light chain constant region represented by SEQ ID NO: 12.

[0037] In some embodiments, the anti-TROP-2 antibody or antigen-binding fragment thereof of any one of the preceding claims comprises a heavy chain represented by SEQ ID NO: 13 and a light chain represented by SEQ ID NO: 14.

[0038] In another aspect, the present disclosure provides a nucleic acid molecule encoding the anti-TROP-2 antibody. In another aspect, the present disclosure provides a nucleic acid molecule encoding the anti-TROP-2 antibody or antigen-binding fragment thereof.

[0039] In another aspect, the disclosure provides a host cell comprising the nucleic acid molecule.

[0040] The present disclosure comprises the following steps: [ka] After reducing Pc, the general formula (L a -YD), and the compound represented by the general formula (Pc-L a -YD), Among them, Pc is an anti-TROP-2 antibody or an antigen-binding fragment thereof; n, m, W, L 2 , L 3 , R 1 , R 2 , R 5 , R 6 and R 7 is the general formula (Pc-L a-YD), a The present invention further provides a method for preparing a ligand-drug conjugate represented by formula (I) or a pharmaceutically acceptable salt thereof.

[0041] The present disclosure comprises the following steps: [ka] The method includes a step of reducing Pc, and then coupling with a compound represented by formula (L'-D) to obtain a compound represented by general formula (Pc-L'-D), wherein: Pc is the anti-TROP-2 antibody or an antigen-binding fragment thereof, There is further provided a method for preparing a ligand-drug conjugate represented by the general formula (Pc-L'-D), wherein n is as defined in the general formula (Pc-LYD).

[0042] In another aspect, the present disclosure provides a pharmaceutical composition comprising the ligand-drug conjugate or pharmaceutically acceptable salt thereof described in any one of the preceding claims, or the anti-TROP-2 antibody or antigen-binding fragment thereof described in any one of the preceding claims, and one or more pharmaceutically acceptable excipients, diluents, or vectors. In some embodiments, the unit dose of the pharmaceutical composition comprises 0.1 to 3000 mg or 1 to 1000 mg of the anti-TROP-2 antibody or antibody-drug conjugate.

[0043] In another aspect, the present disclosure provides use of the ligand-drug conjugate or pharmaceutically acceptable salt thereof described in any one of the preceding claims, or the anti-TROP-2 antibody or antigen-binding fragment thereof described in any one of the preceding claims, or a pharmaceutical composition comprising the same, as a drug.

[0044] In another aspect, the present disclosure provides use of the ligand-drug conjugate or a pharmaceutically acceptable salt thereof described in any one of the preceding claims, or the anti-TROP-2 antibody or antigen-binding fragment thereof or a pharmaceutical composition comprising the same, in the preparation of a medicament for treating a disease or condition or tumor mediated by TROP-2, wherein the disease or condition mediated by TROP-2 is a cancer with high, moderate or low TROP-2 expression.

[0045] In another aspect, the present disclosure provides use of the ligand-drug conjugate or pharmaceutically acceptable salt thereof according to any one of the preceding claims, or the anti-TROP-2 antibody or antigen-binding fragment thereof according to any one of the preceding claims, or a pharmaceutical composition comprising the same, in the preparation of a medicament for treating or preventing a tumor, wherein the tumor and cancer are preferably head and neck squamous cell carcinoma, head and neck cancer, brain cancer, glioma, glioblastoma multiforme, neuroblastoma, central nervous system cancer, neuroendocrine tumor, pharyngeal cancer, pharyngeal squamous cell carcinoma, oral squamous cell carcinoma, nasopharyngeal cancer, esophageal cancer, thyroid cancer, malignant pleural mesothelioma, lung cancer, breast cancer, liver cancer, hepatobiliary cancer, pancreatic cancer, stomach cancer, gastrointestinal cancer, intestinal cancer, colon cancer, colorectal cancer, kidney cancer, clear cell renal cell carcinoma, ovarian cancer, endometrial cancer, cervical cancer, bladder cancer, prostate cancer, testicular cancer, or skin cancer. The lymphomas are selected from the group consisting of cancer, melanoma, leukemia, lymphoma, bone cancer, chondrosarcoma, myeloma, multiple myeloma, myelodysplastic syndrome, Krukenberg's tumor, myeloproliferative neoplasm, squamous cell carcinoma, Ewing's sarcoma, urothelial carcinoma, and Merkel cell carcinoma, and more preferably, the lymphomas are selected from the group consisting of Hodgkin's lymphoma, non-Hodgkin's lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, and primary mediastinal large B-cell lymphoma. , mantle cell lymphoma, small lymphocytic lymphoma, T-cell / histiocyte-rich large B-cell lymphoma, and lymphoplasmacytic lymphoma; the lung cancer is selected from non-small cell lung cancer and small cell lung cancer; and the leukemia is selected from chronic myeloid leukemia, acute myeloid leukemia, lymphocytic leukemia, lymphoblastic leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, and myeloid cell leukemia.

[0046] In another aspect, the present disclosure further relates to a method for treating and / or preventing a tumor, the method comprising administering to a subject in need thereof a therapeutically effective amount of the ligand-drug conjugate or pharmaceutically acceptable salt thereof described in any one of the preceding claims, or the anti-TROP-2 antibody or antigen-binding fragment thereof or pharmaceutically acceptable salt thereof described in any one of the preceding claims, or a pharmaceutical composition comprising them, wherein the tumor is preferably a cancer associated with high, intermediate or low TROP-2 expression.

[0047] In another aspect, the present disclosure further relates to a method for treating or preventing tumors or cancers, the method comprising administering to a subject in need thereof a therapeutically effective amount of the ligand-drug conjugate or pharmaceutically acceptable salt thereof described in any one of the preceding claims, or the anti-TROP-2 antibody or antigen-binding fragment thereof described in any one of the preceding claims, or a pharmaceutical composition comprising the same, wherein the tumors and cancers are preferably head and neck squamous cell carcinoma, head and neck cancer, brain cancer, glioma, glioblastoma multiforme, neuroblastoma, central nervous system cancer, neuroendocrine tumor, pharyngeal cancer, pharyngeal squamous cell carcinoma, oral squamous cell carcinoma, nasopharyngeal cancer, esophageal cancer, thyroid cancer, malignant pleural mesothelioma, lung cancer, breast cancer, liver cancer, hepatobiliary cancer, pancreatic cancer, gastric cancer, gastrointestinal cancer, intestinal cancer, colon cancer, colorectal cancer, renal cancer, clear cell renal cell carcinoma, ovarian cancer, endometrial cancer, cervical cancer, bladder cancer, Prostate cancer, testicular cancer, skin cancer, melanoma, leukemia, lymphoma, bone cancer, chondrosarcoma, myeloma, multiple myeloma, myelodysplastic syndrome, Krueckenberg's tumor, myeloproliferative neoplasm, squamous cell carcinoma, Ewing's sarcoma, urothelial carcinoma, and Merkel cell carcinoma, and more preferably, the lymphoma is Hodgkin's lymphoma, non-Hodgkin's lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, primary mediastinal large cell lymphoma, The lung cancer is selected from B-cell lymphoma, mantle cell lymphoma, small lymphocytic lymphoma, T-cell / histiocyte-rich large B-cell lymphoma, and lymphoplasmacytic lymphoma; the lung cancer is selected from non-small cell lung cancer and small cell lung cancer; and the leukemia is selected from chronic myeloid leukemia, acute myeloid leukemia, lymphocytic leukemia, lymphoblastic leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, and myeloid cell leukemia.

[0048] In another aspect, the present disclosure further provides the anti-TROP-2 antibody or antibody-drug conjugate thereof as a drug, preferably as a drug for treating cancer or tumor, more preferably as a drug for treating cancer mediated by TROP-2.

[0049] The active compound (e.g., a ligand-drug conjugate described herein or a pharmaceutically acceptable salt thereof) can be prepared in a form suitable for administration by any suitable route, and preferably the active compound is in a unit dose form or in a form that allows a subject to self-administer a unit dose. The unit dose form of the active compound or composition described herein may be a tablet, capsule, cachet, bottled drug solution, drug powder, granules, lozenge, suppository, reconstituted powder, or liquid formulation.

[0050] The dosage of the active compound or composition used in the therapeutic methods of the present disclosure generally varies depending on the severity of the disease, the weight of the subject, and the potency of the active compound. As a general guide, a suitable unit dose may be from 0.1 mg to 1000 mg.

[0051] The pharmaceutical compositions of the present disclosure may contain one or more additives in addition to the active compound, and the additives may be selected from components such as fillers, diluents, binders, wetting agents, disintegrants, or excipients. Depending on the method of administration, the composition may contain 0.1 to 99% by weight of the active compound.

[0052] The TROP-2 antibodies and antibody-drug conjugates provided by the present disclosure have good affinity for cell surface antigens, good cell endocytosis efficiency, and strong tumor-inhibiting efficiency, and are suitable for broader drug application and clinical pharmaceutical use. [Brief explanation of the drawings]

[0053] [Figure 1] 1 shows the results of a binding experiment between anti-TROP-2 antibodies and cells expressing TROP-2. [Figure 2]1 shows the bystander killing activity of ADCs against BxPC3 cells and MiaPaCa2 mixed cells. [Figure 3] 1 shows the inhibitory activity of different ADCs against FaDu-implanted tumors in mice. [Figure 4] 1 shows the inhibitory activity of different doses of ADC against SKOV3-implanted tumors in mice. [Figure 5] 1 shows the inhibitory activity of different doses of ADC against Colo205 xenograft tumors in mice. DETAILED DESCRIPTION OF THE INVENTION

[0054] 1. Terminology Unless otherwise limited, all technical and scientific terms used herein are consistent with those commonly understood by those skilled in the art. Although any methods and materials similar or equivalent to those described herein can be used to practice or test the present disclosure, the preferred methods and materials are described herein. In describing and claiming the present disclosure, the following terms will be used in accordance with the definitions set forth below.

[0055] When trade names are used in this disclosure, it is intended to include the formulation of the trade name product, the drug and active drug portion of the trade name product.

[0056] Unless stated to the contrary, terms used in the specification and claims have the following meanings.

[0057] The term "drug" refers to a cytotoxic drug, which can have a chemical molecule that is relatively powerful in disrupting the normal growth of tumor cells. Cytotoxic drugs can, in principle, kill cells at sufficiently high concentrations, but due to their lack of specificity, they can also induce apoptosis of normal cells while killing tumor cells, resulting in severe side effects. The term also refers to toxins, such as small molecule toxins or enzymatically active toxins derived from bacteria, fungi, plants, or animals; radioisotopes (e.g., At 211 , I 131 , I 125 , Y 90 ,Re 186,Re 188 , Sm 153 , Bi 212 , P 32 and radioactive isotopes of Lu), chemotherapy drugs, antibiotics and nucleolytic enzymes.

[0058] The term "linker unit," "linker," "connecting unit," or "connecting fragment" refers to a chemical structural fragment or bond that is connected at one end to a ligand and at the other end to a drug, and may be connected to other linkers, which in turn may be connected to further ligands or drugs.

[0059] The linker may comprise one or more linker elements. Exemplary linker elements include 6-maleimidocaproyl ("MC"), maleimidopropionyl ("MP"), valine-citrulline ("val-cit" or "vc"), alanine-phenylalanine ("ala-phe"), p-aminobenzyloxycarbonyl ("PAB"), N-succinimidyl 4-(2-pyridylthio)pentanoate ("SPP"), N-succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate ("SMCC", also referred to herein as "MCC"), and N-succinimidyl (4-iodo-acetyl)aminobenzoate ("SIAB"). The linker may be selected from the following elements: a stretcher, a spacer, and an amino acid unit, or a combination thereof. The linker can be synthesized by methods known in the art, for example, as described in US2005-0238649A1. The linker may be a "cleavable linker" that facilitates drug release in cells. For example, an acid-labile linker (e.g., hydrazone), a protease-sensitive (e.g., peptidase-sensitive) linker, a photolabile linker, a dimethyl linker, or a disulfide-containing linker (Chari et al., Cancer Research 52:127-131 (1992); U.S. Patent No. 5,208,020) may be used.

[0060] The linker elements are: MC=6-maleimidocaproyl, which has the following structure: [ka] Val-Cit or "vc" = valine-citrulline (an exemplary dipeptide in a protease-cleavable linker); Citrulline = 2-amino-5-ureidopentanoic acid, PAB = p-aminobenzyloxycarbonyl (an example of a "self-immolative" linker element); Me-Val-Cit = N-methyl-valine-citrulline (in which the linker peptide bond is modified so that it is not cleaved by cathepsin B); MC(PEG)6-OH = maleimidocaproyl-polyethylene glycol (can be attached to antibody cysteines); SPP = N-succinimidyl 4-(2-pyridylthio)pentanoate, SPDP = N-succinimidyl 3-(2-pyridyldithio)propionate, SMCC = succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate, and Including, but not limited to, IT = iminothiolane.

[0061] The term "ligand-drug conjugate" refers to a ligand connected to a biologically active drug via a linking unit, preferably an "antibody-drug conjugate." In the present disclosure, an "antibody drug conjugate" (ADC) refers to a monoclonal antibody or antibody fragment connected to a biologically active toxic drug via a linking unit. The antibody may be conjugated to the drug directly or via a linker. n is the average number of drug modules per antibody, which may be an integer or a decimal. The range is, for example, about 0 to about 20 drug modules per antibody. In some embodiments, it is 1 to about 10 drug modules per antibody. In some embodiments, it is 1 to about 8 drug modules per antibody, for example, 2, 3, 4, 5, 6, 7, or 8 drug modules. In the antibody-drug conjugate mixture composition of the present disclosure, the average drug load of each antibody is about 1 to about 10, including, but not limited to, about 3 to about 7, about 3 to about 6, about 3 to about 5, about 1 to about 9, about 7, or about 4.

[0062] The three-letter and one-letter codes for amino acids used in this disclosure are as described in J. Biol. Chem, 243, p. 3558 (1968).

[0063] The term "antibody" refers to an immunoglobulin, which has a tetrapeptide chain structure consisting of two heavy chains and two light chains connected by interchain disulfide bonds. Immunoglobulins are divided into five types based on the amino acid composition and sequence order of the immunoglobulin heavy chain constant region, or may be referred to as immunoglobulin isotypes: IgM, IgD, IgG, IgA, and IgE, with the corresponding heavy chains being μ, δ, γ, α, and ε chains, respectively. Ig of the same type can be further divided into different subclasses based on differences in the amino acid composition of the hinge region and the number and position of heavy chain disulfide bonds; for example, IgG may be divided into IgG1, IgG2, IgG3, and IgG4. Light chains are divided into κ or λ chains based on the constant region. Each of the five types of Ig may have either κ or λ chains.

[0064] In full-length antibody heavy and light chains, approximately 110 amino acids near the N-terminus are highly variable and form the variable region (Fv region), while the remaining amino acid sequence near the C-terminus is relatively stable and forms the constant region. The variable region contains three hypervariable regions (HVRs) and four framework regions (FRs) whose sequences are relatively conserved. The three hypervariable regions determine the specificity of the antibody and are also called complementarity-determining regions (CDRs). Each light chain variable region (LCVR) and heavy chain variable region (HCVR) consists of three CDR regions and four FR regions, arranged in the following order from the amino terminus to the carboxy terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The three CDR regions of the light chain are LCDR1, LCDR2, and LCDR3, and the three CDR regions of the heavy chain are HCDR1, HCDR2, and HCDR3.

[0065] The terms "fully humanized antibody," "fully human antibody," "human antibody," or "fully human antibody," also referred to as "fully human monoclonal antibody," refer to antibodies whose variable and constant regions are both derived from humans, eliminating immunogenicity, toxicity, and side effects. The main technologies related to the preparation of fully human antibodies include human hybridoma technology, EBV-transformed B lymphocyte technology, phage display technology, transgenic mouse antibody preparation technology, and single B cell antibody preparation technology.

[0066] The term "antigen-binding fragment" refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. Fragments of a full-length antibody can perform the antigen-binding function of an antibody. The binding fragments included in the term "antigen-binding fragment" are selected from antigen-binding fragments of Fab, Fab', F(ab')2, single-chain antibodies (scFv), dimerized V regions (diabodies), disulfide-stabilized V regions (dsFv), and peptides containing CDRs, and examples include: (i) a Fab fragment, which is a monovalent fragment consisting of the VL, VH, CL, and CH1 domains; (ii) an F(ab')2 fragment, which is a bivalent fragment comprising two Fab fragments connected by a disulfide bridge in the hinge region; (iii) an Fd fragment consisting of the VH and CH1 domains; (iv) an Fv fragment consisting of the VH and VL domains of a single antibody arm; (v) a single domain or dAb fragment consisting of the VH domain (Ward et al., (1989) Nature 341:544-546); and (vi) an isolated complementarity-determining region (CDR) or (vii) a combination of two or more isolated CDRs optionally connected by a synthetic linker. Furthermore, the two domains of an Fv fragment, VL and VH, are encoded by separate genes, but can be recombinantly linked with a synthetic linker to generate a single protein chain in which the VL and VH regions pair to form a monovalent molecule (called a single-chain Fv (scFv); see, for example, Bird et al. (1988) Science 242:423-426 and Huston et al. (1988) Proc. Natl. Acad. Sci USA 85:5879-5883). Such single-chain antibodies are also intended to be encompassed by the term "antigen-binding fragment" of an antibody. Such antibody fragments can be obtained by conventional techniques known to those skilled in the art, and, like intact antibodies, fragments are selected for their functionality. Antigen-binding portions can be produced by recombinant DNA technology or by enzymatic or chemical cleavage of intact immunoglobulins. The antibodies may be of different isotypes, such as, for example, IgG (eg, IgG1, IgG2, IgG3 or IgG4 subtypes), IgA1, IgA2, IgD, IgE or IgM antibodies.

[0067] Typically, Fab is an antibody fragment with a molecular weight of approximately 50,000 and antigen-binding activity obtained by treating an IgG antibody molecule with the protease papain (e.g., cleaving the heavy chain at amino acid residue 224), in which the N-terminal portion of the heavy chain and the light chain are linked together by a disulfide bond.

[0068] Typically, F(ab')2 is an antibody fragment containing two Fab regions connected at the hinge, with a molecular weight of approximately 100,000, and having antigen-binding activity, obtained by digesting the lower portion of the disulfide bond in the IgG hinge region with the enzyme pepsin.

[0069] Generally, Fab' is an antibody fragment having a molecular weight of about 50,000 and having antigen-binding activity, which is obtained by cleaving the disulfide bond in the hinge region of the above-mentioned F(ab')2.

[0070] Alternatively, Fab' can be produced by inserting DNA encoding the Fab' fragment into a prokaryotic or eukaryotic expression vector and introducing the vector into a prokaryote or eukaryote to express the Fab'.

[0071] The terms "single-chain antibody," "single-chain Fv," or "scFv" refer to a molecule comprising an antibody heavy chain variable domain (or VH) and an antibody light chain variable domain (or VL) connected by a linker. Such scFv molecules may have the general structure NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH. Suitable prior art linkers consist of repeated GGGGS amino acid sequences or variants thereof, for example, 1-4 repeat variants (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90:6444-6448). Other linkers for use in the present disclosure are described in Alfthan et al. (1995), Protein Eng. 8:725-731, Choi et al. (2001), Eur. J. Immunol. 31:94-106, Hu et al. (1996), Cancer Res. 56:3055-3061, Kipriyanov et al. (1999), J. Mol. Biol. 293:41-56, and Roovers et al. (2001), Cancer Immunol.

[0072] The term "CDR" refers to one of the six hypervariable regions in the variable domain of an antibody that primarily mediates antigen binding. Typically, each heavy chain variable region has three CDRs (HCDR1, HCDR2, and HCDR3), and each light chain variable region has three CDRs (LCDR1, LCDR2, and LCDR3). The "Kabat" numbering convention (see Kabat et al. (1991), "Sequences of Proteins of Immunological Interest," 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD), the "Chothia" numbering convention (see Al-Lazikani et al. (1997) JMB 273:927-948), and the ImMunoGenTics (IMGT) numbering convention (see Lefranc MP, Immunologist, 7, 132-136 (1999); Lefranc, MP et al., Dev. Comp. Immunol., 27, The amino acid sequence boundaries of the CDRs can be determined by any one of a variety of known methods, including those described in the Kabat Rules (1995) 55-77 (2003). For example, in a typical format, the CDR amino acid residues in the heavy chain variable region (VH) are numbered 31-35 (HCDR1), 50-65 (HCDR2), and 95-102 (HCDR3), and the CDR amino acid residues in the light chain variable region (VL) are numbered 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3), according to the Kabat Rules. According to the Chothia Rules, the CDR amino acid residues in the VH are numbered 31-35 (HCDR1), 50-65 (HCDR2), and 95-102 (HCDR3). The CDRs are 26-32 (HCDR1), 52-56 (HCDR2), and 95-102 (HCDR3), and the amino acid residue numbers in the VL are 26-32 (LCDR1), 50-52 (LCDR2), and 91-96 (LCDR3). According to the combined Kabat and Chothia CDR definition, the CDRs are composed of amino acid residues 26-35 (HCDR1), 50-65 (HCDR2), and 95-102 (HCDR3) in the human VH and amino acid residues 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3) in the human VL.According to the IMGT rules, the CDR amino acid residues in VH are numbered approximately 26-35 (CDR1), 51-57 (CDR2), and 93-102 (CDR3), and the CDR amino acid residues in VL are numbered approximately 27-32 (CDR1), 50-52 (CDR2), and 89-97 (CDR3). According to the IMGT rules, the CDR regions of an antibody can be determined by the program IMGT / DomainGapAlign.

[0073] The term "framework region" refers to the part of a variable domain VL or VH that serves as a scaffold for the antigen binding loops (CDRs) of that variable domain. In effect, it is a variable domain without the CDRs.

[0074] The term "epitope" or "antigenic determinant" refers to a site on an antigen that is bound by an immunoglobulin or antibody. An epitope usually includes at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 consecutive or non-consecutive amino acids in a unique spatial conformation. See, e.g., Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, G.E. Morris, Ed. (1996).

[0075] The terms "specific binding," "selective binding," "selectively binds," and "specifically binds" refer to the binding of an antibody or fragment thereof to a predetermined epitope on an antigen. Typically, an antibody or fragment thereof binds to an epitope on an antigen within a range of about 10 -7 Less than m, e.g., about 10 -8 M, 10 -9 M or 10 -10 It binds with an affinity (KD) less than or equal to M.

[0076] The term "KD" refers to the dissociation equilibrium constant of an antibody-antigen interaction. Typically, antibodies or antigen-binding fragments of the present disclosure have a dissociation equilibrium constant of about 10 -7 Less than m, e.g., about 10 -8 M or 10 -9It binds to TROP-2 or an epitope thereof with a dissociation equilibrium constant (KD) of less than M. For example, in the present disclosure, the affinity between an antibody and a cell surface antigen is measured as a KD value by the FACS method.

[0077] The term "nucleic acid molecule" refers to a DNA molecule or an RNA molecule. A nucleic acid molecule may be single-stranded or double-stranded, with double-stranded DNA being preferred. A nucleic acid is "operatively linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, a promoter or enhancer is operatively linked to a coding sequence if it affects the transcription of the coding sequence.

[0078] "Identity" of amino acid sequences is the percentage of amino acid residues in a first sequence that are similar to those in a second sequence, with gaps introduced as necessary during the alignment process to achieve the greatest percentage of sequence identity, and with any conservative substitutions not considered part of the sequence identity. To measure the percentage of amino acid sequence identity, alignment can be achieved by several methods within the skill of the art, such as publicly available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2, or Megalign (DNASTAR) software. Those skilled in the art can determine the parameters to be applied to measure alignment, including any algorithms necessary to achieve maximum alignment over the entire length of the sequences being compared.

[0079] The term "expression vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it is linked. In one embodiment, the vector is a "plasmid," which refers to a circular double-stranded DNA loop into which other DNA segments can be linked. In another embodiment, the vector is a viral vector into which other DNA segments can be linked to the viral genome. The vectors disclosed herein can either autonomously replicate in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors), or can integrate into the genome of a host cell after introduction and thereby replicate along with the host genome (e.g., non-episomal mammalian vectors).

[0080] Methods for producing and purifying antibodies and antigen-binding fragments well known in the art are described, for example, in Chapters 5-8 and 15 of the Cold Spring Harbor Manual of Antibody Laboratory Techniques. The antibodies or antigen-binding fragments described in the present invention have one or more human FR regions added to non-human CDR regions by genetic engineering. Human FR germline sequences can be obtained from the ImMunoGeneTics (IMGT) website by aligning the IMGT human antibody variable region germline gene database with MOE software, or from Immunoglobulin Journal, Lefranc, G., The Immunoglobulin Facts Book, Academic Press, 2001, ISBN 012441351.

[0081] The term "host cell" refers to a cell into which an expression vector has already been introduced. Host cells may include microbial (e.g., bacterial), plant, or animal cells. Bacteria amenable to transformation include members of the Enterobacteriaceae family, such as strains of Escherichia coli and Salmonella, Bacillaceae, such as Bacillus subtilis, Pneumococcus, Streptococcus, and Haemophilus influenzae. Suitable microorganisms include Saccharomyces cerevisiae and Pichia pastoris. Suitable animal host cell lines include CHO (Chinese hamster ovary cell line) and NS0 cells.

[0082] The engineered antibodies or antigen-binding fragments of the present disclosure can be prepared and purified by conventional methods. For example, cDNA sequences encoding the heavy and light chains can be cloned and recombined into expression vectors. The recombinant immunoglobulin expression vector can be stably transfected into host cells. In one preferred conventional technique, a mammalian expression system results in glycosylation of the antibody, particularly at the N-terminal end of the Fc region. Positive clones are expanded in a bioreactor culture to produce the antibody. The culture medium secreting the antibody can be purified by conventional techniques, for example, through an A or G Sepharose FF column. Nonspecifically bound components are washed away. The bound antibody is then eluted using a pH gradient, and the antibody fragments are detected and collected by SDS-PAGE. The antibody can be filtered and concentrated by conventional methods. Soluble admixtures and multimers may be removed by conventional methods, such as molecular sieving or ion exchange. The resulting product should be immediately frozen, for example, at -70°C, or lyophilized.

[0083] The term "peptide" refers to a molecule consisting of two or more amino acid molecules joined together by peptide bonds, and is a structural and functional fragment of a protein.

[0084] The term "alkyl group" refers to a saturated aliphatic hydrocarbon group, a straight or branched chain group containing 1 to 20 carbon atoms, with alkyl groups containing 1 to 12 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12) carbon atoms being preferred, alkyl groups containing 1 to 10 carbon atoms being more preferred, and alkyl groups containing 1 to 6 carbon atoms (e.g., 1, 2, 3, 4, 5, or 6 carbon atoms) being most preferred. Non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 2,2-dimethylbutyl, 1,3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2,3-dimethylbutyl, n-heptyl, 2-methylhexyl, 3-methylhexyl, 4-methylhexyl, 5-methylhexyl groups, 2,3-dimethylpentyl, 2,4-dimethylpentyl, 2,2-dimethylpentyl, 3,3-dimethylpentyl, 2-ethylpentyl, 3-ethylpentyl, n-octyl, 2,3-dimethylhexyl, 2,4-dimethylhexyl, 2,5-dimethylhexyl, 2,2-dimethylhexyl, 3,3-dimethylhexyl, 4,4-dimethylhexyl, 2-ethylhexyl, 3-ethylhexyl, 4-ethylhexyl, 2-methyl-2-ethylpentyl, 2-methyl-3-ethylpentyl, n-nonyl, 2-methyl-2-ethylhexyl, 2-methyl-3-ethylhexyl, 2,2-diethylpentyl, n-decyl, 3,3-diethylhexyl, 2,2-diethylhexyl, and various branched chain isomers thereof.More preferred are lower alkyl groups containing 1 to 6 carbon atoms, non-limiting examples of which include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 2,2-dimethylbutyl, 1,3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2,3-dimethylbutyl, and the like. The alkyl group may be substituted or unsubstituted. When substituted, the substituents may be substituted at any available attachment site, and the substituents are preferably independently one or more groups selected from alkyl groups, alkenyl groups, alkynyl groups, alkoxy groups, alkylthio groups, alkylamino groups, halogens, mercapto groups, hydroxy groups, nitro groups, cyano groups, cycloalkyl groups, heterocycloalkyl groups, aryl groups, heteroaryl groups, cycloalkoxy groups, heterocycloalkoxy groups, cycloalkylthio groups, heterocycloalkylthio groups, and oxo groups.

[0085] The term "heteroalkyl group" refers to an alkyl group containing one or more heteroatoms selected from N, O, or S, wherein alkyl groups are as defined above.

[0086] The term "alkylene group" refers to a saturated, straight-chain or branched-chain aliphatic hydrocarbon group having a residue derived by removing two hydrogen atoms from the same carbon atom or from two different carbon atoms of an alkane parent, and is a straight-chain or branched-chain group containing 1 to 20 carbon atoms, preferably 1 to 12 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12) carbon atoms, and more preferably 1 to 6 carbon atoms (e.g., 1, 2, 3, 4, 5, or 6 carbon atoms). Non-limiting examples of alkylene groups include, but are not limited to, methylene (-CH-), 1,1-ethylidene (-CH(CH)-), 1,2-ethylidene (-CHCH)-, 1,1-propylidene (-CH(CHCH)-), 1,2-propylidene (-CHCH(CH)-), 1,3-propylidene (-CHCHCHCH-), 1,4-butylidene (-CHCHCHCHCH-), and 1,5-butylidene (-CHCHCHCHCHCH-). The alkylene group may be substituted or unsubstituted, and when substituted, the substituents may be substituted at any available attachment site, and the substituents are preferably independently one or more groups selected from alkyl groups, alkenyl groups, alkynyl groups, alkoxy groups, alkylthio groups, alkylamino groups, halogens, mercapto groups, hydroxy groups, nitro groups, cyano groups, cycloalkyl groups, heterocyclyl groups, aryl groups, heteroaryl groups, cycloalkoxy groups, heterocycloalkoxy groups, cycloalkylthio groups, heterocycloalkylthio groups, and oxo groups.

[0087] The term "alkoxy group" refers to -O-(alkyl group) and -O-(unsubstituted cycloalkyl group), where alkyl group and cycloalkyl group are defined above. Non-limiting examples of alkoxy groups include methoxy group, ethoxy group, propoxy group, butoxy group, cyclopropoxy group, cyclobutoxy group, cyclopentyloxy group, and cyclohexyloxy group. An alkoxy group may be optionally substituted or unsubstituted. When substituted, the substituents are preferably independently one or more groups selected from alkyl group, alkenyl group, alkynyl group, alkoxy group, alkylthio group, alkylamino group, halogen, mercapto group, hydroxy group, nitro group, cyano group, cycloalkyl group, heterocycloalkyl group, aryl group, heteroaryl group, cycloalkoxy group, heterocycloalkoxy group, cycloalkylthio group, and heterocycloalkylthio group.

[0088] The term "halogenated alkyl group" refers to a group in which one or more hydrogens on the alkyl group have been replaced with halogens, wherein the alkyl group is as defined above.

[0089] The term "deuterated alkyl group" refers to an alkyl group in which a hydrogen atom on the alkyl group has been replaced with one or more deuterium atoms, wherein the alkyl group is as defined above.

[0090] The term "hydroxyalkyl group" refers to an alkyl group in which a hydrogen is replaced with one or more hydroxy groups, wherein the alkyl group is as defined above.

[0091] The term "hydroxy group" refers to an --OH group.

[0092] The term "halogen" refers to fluorine, chlorine, bromine or iodine.

[0093] The term "amino group" refers to -NH2.

[0094] The term "nitro group" refers to -NO2.

[0095] The term "cyano" refers to -CN.

[0096] The present disclosure further includes various deuterated forms of the compound of formula (Pc-LYD). Each available hydrogen atom connected to a carbon atom can be independently replaced with a deuterium atom. Those skilled in the art can synthesize deuterated forms of the compound of formula (Pc-LYD) by referring to relevant literature. When preparing the deuterated form of the compound of formula (Pc-LYD), commercially available deuterated starting materials may be used, or the compound may be synthesized by conventional techniques using deuterated reagents, including, but not limited to, deuterated borane, tritiated borane in tetrahydrofuran, deuterated lithium aluminum, deuterated iodoethane, deuterated iodomethane, etc.

[0097] "Optional" or "optionally" means that the subsequently described event or circumstance may, but need not, occur, and the phrase includes instances where the event or circumstance occurs and instances where it does not occur. For example, "a heterocyclyl group optionally substituted with an alkyl group" means that an alkyl group may, but need not, be present, and the phrase includes instances where the heterocyclyl group is substituted with an alkyl group and instances where the heterocyclyl group is not substituted with an alkyl group.

[0098] "Substituted" means that one or more hydrogen atoms in a group, preferably at most 5, more preferably 1, 2 or 3 hydrogen atoms, are independently replaced with a substituent. Substituents are located only in their chemically possible positions, and a person skilled in the art can determine (by experiment or theory) possible or impossible substitutions without undue effort. For example, an amino group or hydroxy group having free hydrogen may be unstable if it is bonded to a carbon atom having an unsaturated (e.g., olefinic) bond.

[0099] The term "pharmaceutical composition" refers to a mixture of one or more compounds described herein or physiologically / pharmaceutically acceptable salts or prodrugs thereof with other chemical components, and other components such as physiologically / pharmaceutically acceptable vectors and excipients, to facilitate administration to a living body and contribute to the absorption of the active ingredients to further exert their biological activity.

[0100] The term "pharmaceutically acceptable salt" or "pharmaceutically available salt" refers to a salt of an antibody-drug conjugate according to the present disclosure, which salt is safe and effective when administered to a subject and possesses the desired biological activity. The ligand-drug conjugate according to the present disclosure contains at least one amino group and can therefore form a salt with an acid. Non-limiting examples of pharmaceutically available salts include hydrochloride, hydrobromide, hydroiodide, sulfate, hydrogensulfate, citrate, acetate, succinate, ascorbate, oxalate, nitrate, sorbate, hydrogen phosphate, dihydrogen phosphate, salicylate, hydrogen citrate, tartrate, maleate, fumarate, formate, benzoate, methanesulfonate, ethanesulfonate, benzenesulfonate, and p-toluenesulfonate.

[0101] The term "drug loading" or "average drug load" refers to the average number of cytotoxic drugs loaded onto each ligand in a ligand-drug conjugate, and may be expressed as the ratio of the drug amount to the antibody amount. The drug loading ranges from 0 to 12, preferably 1 to 10, cytotoxic drugs attached to each ligand (Pc). In an embodiment of the present disclosure, the drug loading is represented by n, also referred to as the drug-antibody ratio (DAR) value, and is illustratively the average of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. The average drug amount on each ADC molecule after the coupling reaction can be characterized by conventional methods such as UV / visible spectroscopy, mass spectrometry, ELISA, and HPLC.

[0102] In one embodiment of the present disclosure, the cytotoxic agent is conjugated to a sulfhydryl group of the antibody by a linking unit.

[0103] The following non-limiting methods: (1) controlling the molar ratio of the coupling reagent to the monoclonal antibody; (2) controlling the reaction time and temperature; (3) selecting different reaction reagents; The loading of the ligand-drug conjugate can be controlled by methods including, but not limited to,

[0104] The preparation of common pharmaceutical compositions is set out in the Chinese Pharmacopoeia.

[0105] The term "vector," as used in the present disclosure, refers to a system that can change the drug's entry into the human body and its distribution within the body, control the drug's release rate, and transport the drug to target organs. The drug vector's release and targeting system can reduce drug degradation and loss, lower side effects, and improve bioavailability. For example, polymer surfactants used as vectors can self-assemble to form various types of aggregates due to their unique amphiphilic structure, including micelles, microemulsions, gels, liquid crystals, and vesicles. These aggregates have the ability to encapsulate drug molecules and good membrane permeability, making them effective drug vectors.

[0106] The term "excipient" refers to an additive other than the main ingredient in a pharmaceutical formulation, which may also be called an auxiliary material. For example, binders, fillers, disintegrants, and lubricants in tablets, the matrix portion in semi-solid preparations such as ointments and creams, and preservatives, antioxidants, flavoring agents, fragrances, cosolvents, emulsifiers, solubilizers, osmotic pressure adjusters, and coloring agents in liquid preparations may all be called excipients.

[0107] The term "diluent," also known as a filler, is primarily used to increase the weight and volume of tablets. Adding a diluent not only ensures a consistent volume, but also reduces the dosage deviation of the active ingredient and improves the compressibility of the drug. When a tablet contains an oily ingredient, an absorbent must be added to absorb the oily substance to maintain a "dry" state and facilitate tablet formation. Examples include starch, lactose, inorganic calcium salts, and microcrystalline cellulose.

[0108] The pharmaceutical compositions may be in the form of a sterile injectable aqueous solution. Acceptable solvents and vehicles used include water, Ringer's solution, and isotonic sodium chloride solution. The sterile injectable preparation may also be a sterile injectable oil-in-water microemulsion in which the active ingredient is dissolved in the oil phase. For example, the active ingredient may be dissolved in a mixture of soybean oil and lecithin. The oil solution is then added to a mixture of water and glycerin and processed to form a microemulsion. The injectable solution or microemulsion can be infused into the subject's bloodstream via local bolus injection. Alternatively, solutions and microemulsions are preferably administered in a manner that maintains a constant, cyclical concentration of the compounds of the present disclosure. To maintain such a constant concentration, a continuous intravenous administration device can be used. An example of such a device is the Deltec CADD-PLUS™ 5400 intravenous infusion pump.

[0109] The pharmaceutical compositions may be in the form of sterile injectable aqueous or oily suspensions for intramuscular and subcutaneous administration. Such suspensions can be prepared using suitable dispersing or wetting agents and suspending agents as described above according to known techniques. Sterile injectable preparations may also be sterile injectable solutions or suspensions prepared in non-toxic, gastrointestinal-acceptable diluents or solvents, such as solutions prepared in 1,3-butanediol. Sterile fixed oils are also conveniently used as solvents or suspending media. For this purpose, any suitable fixed oil, including synthetic monoglycerides or diglycerides, can be used. Fatty acids, such as oleic acids, can also be used to prepare injectables.

[0110] 2. Synthesis method To achieve the synthesis objectives, the following synthesis technology scheme is adopted:

[0111] General formula (Pc-L a -YD), which is a process for preparing a compound of the formula [ka] After reducing Pc, the general formula (L a -YD) and the general formula (Pc-L a -YD), wherein TCEP is preferred as the reducing agent, and disulfide bonds on reduced antibodies are particularly preferred; Among them, Pc, W, L 2 , L 3 , R 1 , R 2 , R 5 , R 6 , R 7 , m and n are the same as those in the general formula (Pc-L a -YD).

[0112] The above specification provides details of one or more embodiments of the present disclosure. Although the present disclosure can be practiced or tested using any methods and materials similar or equivalent to those described herein, the preferred methods and materials are described below. Other features, objects, and advantages of the present disclosure will become apparent from the specification and claims. In the specification and claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Unless otherwise defined, all technical and scientific terms used herein have the ordinary meaning understood by those skilled in the art. All patents and publications cited in the specification are incorporated by reference. The following examples are presented to more fully illustrate preferred embodiments of the present disclosure. These examples should not be construed in any way as limiting the scope of the present disclosure, which is limited only by the claims. [Example]

[0113] In the examples or test examples of the present invention, experimental methods for which specific conditions are not specified generally follow common conditions or conditions recommended by the manufacturers of raw materials or products. See Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, and Contemporary Methods in Molecular Biology, Ausubel et al., Greene Publishing Company, Wiley Interscience, NY. Reagents for which specific sources are not specified are commonly available commercially.

[0114] 1. Preparation of antibodies Example 1-1: Construction of a cell line with high TROP-2 expression The pCDH-hTROP-2 lentiviral expression vector plasmid and Lipofectamine 3000 transfection reagent for pVSV-G and pCMV-dR8.91 lentiviral packaging vectors were transfected into 293T virus packaging cells. The virus-containing culture supernatant was collected, filtered, and centrifuged at ultra-high speed. The concentrated virus was then used to infect Chinese hamster ovary cells CHO-K1. After selection with puromycin for 2-3 weeks, FACS single-cell sorting was performed.

[0115] The amount of TROP-2 expression on the surface of lentivirus-infected CHO-K1 cells was detected by FACS, and CHO-K1 / hTROP-2 monoclonal cell lines with high TROP-2 expression were selected.

[0116] The TROP-2 amino acid sequence (Genbank: NP_002344.2) is as follows: [ka] SEQ ID NO: 1, The Trop2-His amino acid sequence is as follows: [ka] Sequence number 2.

[0117] Example 1-2: Preparation of anti-human TROP-2 monoclonal antibody The anti-human TROP-2 monoclonal antibody in this application was prepared according to the method disclosed in Patent WO03074566. Using the antibody variable region gene of hRS7 as a template, point mutations were designed in the CDRs using computer software. The molecular clone was inserted into the protein expression vector Phr-IgG (with signal peptide and constant region gene (CH1-Fc / CL) fragment) and expressed in HEK293 and Expi-CHO-S cells. The antibody was purified using conventional methods. Activity was tested using CHO-K1 cells overexpressing huTROP-2 protein and huTROP-2 protein (His27-Thr274, registration number # NP_002344.2). Antibodies with relatively good target binding activity were selected, among which the PD3 variable region sequence is as follows: PD3 heavy chain variable region: [ka] SEQ ID NO: 3, PD3 light chain variable region: [ka] SEQ ID NO: 4, Note: The underlined regions are the CDR regions as determined by the Kabat numbering convention.

[0118] [Table 1] The antibody heavy chain constant region may be selected from the constant regions of human IgG1, IgG2, IgG4, and variants thereof, and the light chain constant region may be selected from the light chain constant region of human κ or λ chains or variants thereof. Illustratively, the antibody heavy chain constant region is selected from human IgG1, the sequence of which is set forth in SEQ ID NO: 11, and the light chain constant region is selected from the constant region of human κ chain, the sequence of which is set forth in SEQ ID NO: 12.

[0119] Human IgG1 heavy chain constant region: [ka] SEQ ID NO: 11, Human kappa light chain constant region: [ka] SEQ ID NO: 12,

[0120] Illustratively, the light chain / heavy chain constant regions are combined with the variable regions of the aforementioned PD3 antibody to form a complete antibody, the light chain / heavy chain sequences of which are as follows: PD3 heavy chain: [ka] SEQ ID NO: 13, PD3 light chain: [ka] SEQ ID NO: 14,

[0121] The control molecule hRS7 used in the present disclosure was constructed with reference to patent WO03074566, and the TINA antibody was constructed with reference to patent WO2015098099A1, and their sequences are as follows, respectively: hRS7 heavy chain: [ka] SEQ ID NO: 15, hRS7 light chain: [ka] SEQ ID NO: 16, TINA Heavy Chain: [ka] SEQ ID NO: 17, TINA light chain: [ka] Sequence number 18.

[0122] II. Preparation of Compounds Experimental methods in the examples of this disclosure for which specific conditions are not specified generally follow common conditions or those recommended by the manufacturers of raw materials or products. Reagents for which a specific source is not specified are commonly available commercially.

[0123] The structures of the compounds were determined by nuclear magnetic resonance (NMR) or mass spectrometry (MS). NMR measurements were performed using a Bruker AVANCE-400 nuclear magnetic spectrometer. The solvents used were deuterated dimethyl sulfoxide (DMSO-d), deuterated chloroform (CDCl), and deuterated methanol (CD3OD). The internal standard was tetramethylsilane (TMS). The chemical shifts were 10 -6 Expressed in ppm.

[0124] MS measurements were performed using a FINNIGAN LCQAd(ESI) mass spectrometer (manufacturer: Thermo, model number: Finnigan LCQ advantage MAX).

[0125] UPLC measurements were performed using a Waters Acquity UPLC SQD liquid chromatograph mass spectrometer.

[0126] HPLC measurements were performed using an Agilent 1200DAD high performance liquid chromatograph (Sunfire C18 150 x 4.6 mm column) and a Waters 2695-2996 high performance liquid chromatograph (Gimini C18 150 x 4.6 mm column).

[0127] UV-HPLC measurements were performed using a Thermo nanodrop 2000 ultraviolet spectrophotometer.

[0128] Growth inhibition rate and IC 50 The values ​​were measured using a PHERA starFS microplate reader (BMG, Germany).

[0129] Yantai Huanghai HSGF254 or Qingdao GF254 silica gel plates are used as silica gel plates for thin layer chromatography (TLC). The silica gel plate specifications for thin layer chromatography (TLC) are 0.15 mm to 0.2 mm, while the silica gel plate specifications for separating and purifying products by thin layer chromatography are 0.4 mm to 0.5 mm.

[0130] Column chromatography generally used Yantai Huanghai 200-300 mesh silica gel as the vector.

[0131] Known starting materials according to the present disclosure may be synthesized by adopting or following methods known in the art, or may be purchased commercially from companies such as ABCR GmbH & Co. KG, Acros Organics, Aldrich Chemical Company, Shaoyuan Chemical Technology (Accela ChemBio Inc.), and Darui Chemical.

[0132] In the examples, all reactions were carried out in an argon or nitrogen atmosphere unless otherwise specified.

[0133] An argon or nitrogen atmosphere refers to an argon or nitrogen balloon with a volume of about 1 L connected to the reaction flask.

[0134] A hydrogen atmosphere refers to a hydrogen balloon with a volume of approximately 1 L attached to the reaction flask.

[0135] The pressurized hydrogenation reaction was carried out using a Parr 3916EKX hydrogenation apparatus and a Seiran QL-500 hydrogen generator or an HC2-SS hydrogenation apparatus.

[0136] The hydrogenation reaction usually involves three cycles of evacuation and refilling with hydrogen.

[0137] The microwave reaction was carried out using a CEM Discover-S 908860 microwave reactor.

[0138] In the examples, unless otherwise specified, the solutions used in the reactions refer to aqueous solutions.

[0139] Unless otherwise specified in the examples, the reaction temperature is room temperature.

[0140] Room temperature is the optimum reaction temperature, with the temperature range being 20°C to 30°C. Preparation of PBS buffer solution with pH 6.5 in the example: 8.5 g of KH2PO4, 8.56 g of K2HPO4.3H2O, 5.85 g of NaCl, and 1.5 g of EDTA were taken and placed in a flask, and the volume was adjusted to 2 L. The solution was completely dissolved by ultrasonication and then shaken to obtain the solution.

[0141] The eluent system for column chromatography and the developing solvent system for thin-layer chromatography used to purify the compound include A: dichloromethane and isopropyl alcohol system, B: dichloromethane and methanol system, and C: petroleum ether and ethyl acetate system, and the volume ratio of the solvents may be adjusted depending on the polarity of the compound, or may be adjusted by adding a small amount of triethylamine and an acidic or basic reagent, etc.

[0142] Some of the compounds of the present disclosure have been characterized by Q-TOF LC / MS, which utilized an Agilent 6530 accurate mass quadrupole-time of flight mass spectrometer and an Agilent 1290-Infinity ultra-high performance liquid chromatograph (Agilent Poroshell 300SB-C8 5 μm, 2.1 × 75 mm column).

[0143] For the YD drug moiety of the antibody-drug conjugates of the present disclosure, reference is made to PCT / CN2019 / 107873, and the synthesis and testing of related compounds are cited in this patent, the synthesis of which, in non-limiting examples, is cited as follows:

[0144] Example 2-1 N-((1S,9S)-9-Ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)-1-hydroxycyclopropane-1-carboxamide 1 [ka] Exatecan mesylate 1b (2.0 mg, 3.76 μmol, prepared by the method disclosed in patent application EP0737686A1) was added to 1 mL of N,N-dimethylformamide and cooled to 0-5°C in an ice-water bath. One drop of triethylamine was added and the reaction mixture was stirred until the solution became clear. 1-Hydroxycyclopropylformate 1a (1.4 mg, 3.7 μmol, prepared by the known method disclosed in Tetrahedron Letters, 25(12), 1269-72, 1984) and 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (3.8 mg, 13.7 μmol) were added sequentially to the reaction mixture. After addition, the mixture was stirred at 0-5°C for 2 hours. The reaction was quenched by adding 5 mL of water to the reaction mixture, and the reaction mixture was extracted with ethyl acetate (8 mL × 3). The organic phases were combined and washed with saturated sodium chloride solution (5 mL × 2). The organic phase was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure. The resulting residue was purified by thin-layer chromatography using developing solvent system B to give the title product 1 (1.6 mg, yield: 82.1%).

[0145] MS m / z (ESI): 520.2 [M+1]. 1H NMR (400 MHz, CDCl3): δ 7.90-7.84 (m, 1H), 7.80-7.68(m, 1H), 5.80-5.70 (m, 1H), 5.62-5.54(m, 2H), 5.44-5.32 (m, 2H), 5.28-5.10(m, 2H), 3.40-3.15 (m, 3H), 2.44 (s, 3H), 2.23(t, 1H), 2.06-1.75 (m, 2H), 1.68-1.56 (m, 1H), 1.22-1.18 (m, 2H), 1.04-0.98 (m, 2H), 0.89 (t, 3H).

[0146] Example 2-2 (S)-2-Cyclopropyl-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)-2-hydroxyacetamide 2-A (R)-2-Cyclopropyl-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)-2-hydroxyacetamide 2-B [ka] To 1b (4 mg, 7.53 μmol), 2 mL of ethanol and 0.4 mL of N,N-dimethylformamide were added, the atmosphere was purged with argon three times, and the mixture was cooled to 0-5°C in an ice-water bath. 0.3 mL of N-methylmorpholine was added dropwise and stirred until the reaction mixture became clear. 2-Cyclopropyl-2-hydroxyacetic acid 2a (2.3 mg, 19.8 μmol, prepared according to the method disclosed in patent application WO2013106717), 1-hydroxybenzotriazole (3 mg, 22.4 μmol), and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (4.3 mg, 22.4 μmol) were added sequentially to the reaction mixture. After addition, the mixture was stirred at 0-5°C for 1 hour. The ice-water bath was removed, and the mixture was heated to 30°C and stirred for 2 hours. The reaction mixture was concentrated under reduced pressure, and the resulting crude product, compound 2, was purified by high-performance liquid chromatography (separation conditions: column: XBridge Prep C18 OBD 5 μm 19 × 250 mm, mobile phase: A - water (10 mmol NHOAc), B - acetonitrile, gradient elution, flow rate: 18 mL / min). The corresponding components were collected and concentrated under reduced pressure to give the title products (2-A: 1.5 mg, 2-B: 1.5 mg).

[0147] MS m / z (ESI): 534.0 [M+1]. Single-configuration compound 2-B (relatively short retention time) UPLC analysis: Retention time: 1.06 min, Purity: 88% (Column: ACQUITY UPLC BEHC18 1.7 um 2.1 x 50 mm, Mobile phase: A - water (5 mmol NH4OAc), B - acetonitrile). 1H NMR (400 MHz, DMSO-d6): δ 8.37 (d, 1H), 7.76 (d, 1H), 7.30 (s, 1H), 6.51 (s, 1H), 5.58-5.56 (m, 1H), 5.48 (d, 1H), 5.41 (s, 2H), 5.32-5.29 (m, 2H), 3.60 (t, 1H), 3.19-3.13 (m, 1H), 2.38 (s, 3H), 2.20-2.14 (m, 1H), 1.98 (q, 2H), 1.87-1.83 (m, 1H), 1.50-1.40 (m, 1H), 1.34-1.28 (m, 1H), 0.86 (t, 3H), 0.50-0.39 (m, 4H).

[0148] Compound 2-A (with a single formulation) (comparatively long retention time) UPLC analysis: retention time: 1.10 minutes, purity: 86% (color: ACQUITY UPLC BEHC18 1.7 μm 2.1×50 mm, mobile phase: A-water (5 mmol NH4OAc), B-acetone). 1 H NMR (400 MHz, DMSO-d6): δ 8.35 (d, 1H), 7.78 (d, 1H), 7.31 (s, 1H), 6.52 (s, 1H), 5.58-5.53 (m, 1H), 5.42 (s, 2H), 5.37 (d, 1H), 5.32 (t, 1H), 3.62 (t, 1H), 3.20-3.15 (m, 2H), 2.40 (s, 3H), 2.25-2.16 (m, 1H), 1.98 (q, 2H), 1.87-1.82 (m, 1H), 1.50-1.40 (m, 1H), 1.21-1.14 (m, 1H), 0.87 (t, 3H), 0.47-0.35 (m, 4H).

[0149] Example 2-3 (S)-N-((1S,9S)-9-Ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)-3,3,3-trifluoro-2-hydroxypropanamide 3-A (R)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)-3,3,3-trifluoro-2-hydroxypropanamide 3-B [ka] 1b (5.0 mg, 9.41 μmol) was added to 2 mL of ethanol and 0.4 mL of N,N-dimethylformamide, cooled to 0-5°C in an ice-water bath, and 0.3 mL of N-methylmorpholine was added dropwise and stirred until the reaction mixture became clear. 3,3,3-trifluoro-2-hydroxypropionic acid 3a (4.1 mg, 28.4 μmol, supplier: Alfa), 1-hydroxybenzotriazole (3.8 mg, 28.1 μmol), and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (5.4 mg, 28.2 μmol) were added sequentially to the reaction mixture. After addition, the mixture was stirred at 0-5°C for 10 minutes. The ice-water bath was removed, and the mixture was heated to 30°C and stirred for 8 hours. The reaction mixture was concentrated under reduced pressure, and the resulting crude product, compound 3, was purified by high-performance liquid chromatography (separation conditions: column: XBridge Prep C18 OBD 5 μm 19 × 250 mm, mobile phase: A-water (10 mmol NH4OAc), B-acetonitrile, gradient elution, flow rate: 18 mL / min). The corresponding components were collected and concentrated under reduced pressure to give the title product (1.5 mg, 1.5 mg).

[0150] MS m / z (ESI): 561.9 [M+1]. Single-configuration compounds (relatively short retention times) UPLC analysis: retention time: 1.11 minutes, purity: 88% (color: ACQUITY UPLC BEHC18 1.7 μm 2.1 × 50 mm, mobile phase: A-water (5 mmol NH4OAc), B-acetone). 1 H NMR (400 MHz, DMSO-d6): δ 8.94 (d, 1H), 7.80 (d, 1H), 7.32 (s, 1H), 7.20 (d, 1H), 6.53 (s, 1H), 5.61-5.55 (m, 1H), 5.45-5.23 (m, 3H), 5.15-5.06 (m, 1H), 4.66-4.57 (m, 1H), 3.18-3.12 (m, 1H), 2.40 (s, 3H), 2.26-2.20 (m, 1H), 2.16-2.08 (m, 1H), 2.02-1.94 (m, 1H), 1.89-1.82 (m, 1H), 1.50-1.40 (m, 1H), 0.87 (t, 3H).

[0151] Compounds with a single formulation (with relatively long retention time) UPLC analysis: retention time: 1.19 minutes, purity: 90% (color: ACQUITY UPLC BEHC18 1.7 μm 2.1 × 50 mm, mobile phase: A-water (5 mmol NH4OAc), B-acetone). 1 H NMR (400 MHz, DMSO-d6): δ 8.97 (d, 1H), 7.80 (d, 1H), 7.31 (s, 1H), 7.16 (d, 1H), 6.53 (s, 1H), 5.63-5.55 (m, 1H), 5.45-5.20 (m, 3H), 5.16-5.07 (m, 1H), 4.66-4.57 (m, 1H), 3.18-3.12 (m, 1H), 2.40 (s, 3H), 2.22-2.14 (m, 1H), 2.04-1.95 (m, 2H), 1.89-1.82 (m, 1H), 1.50-1.40 (m, 1H), 0.87 (t, 3H).

[0152] Example 2-4 N-((1S,9S)-9-Ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)-1-hydroxycyclopentane-1-carboxamide 4 [ka] 1 mL of N,N-dimethylformamide was added to 1b (3.0 mg, 5.64 μmol), cooled to 0-5°C in an ice-water bath, and one drop of triethylamine was added and stirred until the reaction mixture became clear. 1-Hydroxycyclopentaneformic acid 4a (2.2 mg, 16.9 μmol, prepared according to the method disclosed in patent application WO2013106717) and 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (4.7 mg, 16.9 μmol) were added to the reaction mixture in that order. After addition, the mixture was stirred at 0-5°C for 1 hour. The reaction was quenched by adding 5 mL of water to the reaction mixture, and the reaction mixture was extracted with ethyl acetate (10 mL × 3). The organic phases were combined and washed with saturated sodium chloride solution (5 mL × 2). The organic phase was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure. The resulting residue was purified by thin-layer chromatography using developing solvent system B to give the title product 4 (2.5 mg, yield: 80.9%).

[0153] MS m / z (ESI): 548.0 [M+1]. 1 H NMR (400 MHz, CDCl3): δ 7.73-7.62 (m, 2H), 5.75-5.62 (m, 1H), 5.46-5.32 (m, 2H), 5.26-5.10 (m, 1H), 3.30-3.10 (m, 1H), 2.43 (s, 3H), 2.28-2.20 (m, 2H), 2.08-1.84 (m, 8H), 1.69-1.58 (m, 2H), 1.04-1.00 (m, 2H), 0.89 (t, 3H).

[0154] Examples 2-5 N-((1S,9S)-9-Ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)-1-(hydroxymethyl)cyclopropane-1-carboxamide 5 [ka] 1 mL of N,N-dimethylformamide was added to 1b (2.0 mg, 3.76 μmol), cooled to 0-5°C in an ice-water bath, and one drop of triethylamine was added and stirred until the reaction mixture became clear. 1-(hydroxymethyl)-cyclopropanecarboxylic acid 5a (0.87 mg, 7.5 μmol, prepared according to the method disclosed in patent application WO201396771) and 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (2 mg, 7.24 μmol) were added sequentially to the reaction mixture. After addition, the mixture was stirred at 0-5°C for 2 hours. The reaction was quenched by adding 5 mL of water to the reaction mixture, and the reaction mixture was extracted with ethyl acetate (8 mL × 3). The organic phases were combined and washed with saturated sodium chloride solution (5 mL × 2). The organic phase was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure. The resulting residue was purified by thin-layer chromatography using developing solvent system B to give the title product 5 (1.0 mg, yield: 50%).

[0155] MS m / z (ESI): 533.9 [M+1]. 1H NMR (400 MHz, CDCl3): δ 8.07 (s, 1H), 7.23-7.18 (m, 2H), 6.71-6.64 (m, 1H), 6.55-6.51 (m, 1H), 5.36-5.27 (m, 2H), 4.67-4.61 (m, 2H), 3.53-3.48 (m, 1H), 3.30-3.22 (m, 2H), 3.18-3.13 (m, 1H), 2.71-2.61 (m, 2H), 2.35-2.28 (m, 1H), 2.04-1.91 (m, 4H), 1.53-1.40 (m, 3H), 0.91-0.75 (m, 4H).

[0156] Examples 2-6 N-((1S,9S)-9-Ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)-1-(hydroxymethyl)cyclobutane-1-carboxamide 6 [ka] 1b (3.0 mg, 5.64 μmol) was added to 1 mL of N,N-dimethylformamide and cooled to 0-5°C in an ice-water bath. One drop of triethylamine was added and the reaction mixture was stirred until the solution became clear. 1-(hydroxymethyl)cyclobutane-1-formic acid 6a (2.2 mg, 16.9 μmol, prepared according to the method disclosed in "Journal of the American Chemical Society, 2014, vol. 136, #22, pp. 8138-8142") and 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (4.7 mg, 16.9 μmol) were added sequentially to the reaction mixture. After addition, the mixture was stirred at 0-5°C for 1 hour. The reaction was quenched by adding 5 mL of water to the reaction mixture, and the reaction mixture was extracted with ethyl acetate (10 mL × 3). The organic phases were combined and washed with saturated sodium chloride solution (5 mL × 2). The organic phase was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure. The resulting residue was purified by thin-layer chromatography using developing solvent system B to give the title product 6 (2.1 mg, yield: 67.9%).

[0157] MS m / z (ESI): 548.0 [M+1]. 1 H NMR (400 MHz, DMSO-d6): δ 7.85-7.62 (m, 1H), 6.88 (br,1H), 5.87-5.48 (m,2H), 5.47-5.33 (m,1H), 5.31-5.06 (m,1H), 4.25-3.91 (m, 2H), 3.25 (br, 1H), 2.60-2.32 (m, 3H), 2.23 (t, 1H), 2.15-1.95 (m, 3H), 1.70-1.56 (m, 2H), 1.41-1.17 (m, 9H), 1.03 (s, 1H), 0.95-0.80 (m, 2H).

[0158] Examples 2-7 N-((1S,9S)-9-Ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)-1-hydroxycyclobutane-1-carboxamide 7 [ka] 1b (3.0 mg, 5.64 μmol) was added to 2 mL of ethanol and 0.4 mL of N,N-dimethylformamide, cooled to 0-5°C in an ice-water bath, and 0.3 mL of N-methylmorpholine was added dropwise. The reaction mixture was stirred until clear. 1-Hydroxycyclobutaneformic acid 7a (2.0 mg, 17.22 μmol, supplied by Yakuseki), 1-hydroxybenzotriazole (2.3 mg, 17.0 μmol), and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (3.2 mg, 16.7 μmol) were added sequentially to the reaction mixture. After addition, the mixture was stirred at 0-5°C for 10 minutes. The ice-water bath was removed, and the mixture was stirred at room temperature for 2 hours. The reaction mixture was concentrated under reduced pressure, and the resulting residue was purified by thin-layer chromatography using developing solvent system B to obtain the title product 7 (2.5 mg, yield: 83.1%).

[0159] MS m / z (ESI): 534.0 [M+1]. 1 H NMR (400 MHz, DMSO-d6): δ 8.28 (d, 1H), 7.75 (d, 1H), 7.29 (s, 1H), 6.51 (s, 1H), 6.12 (s, 1H), 5.59-5.51 (m, 1H), 5.41 (s, 2H), 5.20-5.01 (m, 2H), 3.27-3.17 (m, 1H), 3.15-3.05 (m, 1H), 2.71-2.63 (m, 1H), 2.37 (s, 3H), 2.12-2.05 (m, 1H), 2.03-1.94 (m, 2H), 1.92-1.78 (m, 4H), 1.50-1.42 (m, 1H), 0.90-0.83 (m, 4H).

[0160] Examples 2-8 1-(((S)-7-Benzyl-20-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-3,6,9,12,15-pentaoxo-2,5,8,11,14-pentaazaicosyl)oxy)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)cyclopropane-1-carboxamide 8 [ka]

[0161] Step 1 Benzyl 1-((2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetamido)methoxy)cyclopropane-1-carboxylate 8c Benzyl 1-hydroxycyclopropane-1-carboxylate 8a (104 mg, 0.54 mmol, prepared by the method disclosed in patent application US2005 / 20645) and (2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetamido)methyl acetate 8b (100 mg, 0.27 mmol, prepared by the method disclosed in patent application CN105829346A) were added to a reaction flask, 5 mL of tetrahydrofuran was added, the mixture was purged with argon three times, and the temperature was lowered to 0-5°C in an ice-water bath. Potassium tert-butoxide (61 mg, 0.54 mmol) was added, the ice-water bath was removed, the mixture was warmed to room temperature, and the mixture was stirred for 10 min. 20 mL of ice water was added, and the mixture was extracted with ethyl acetate (5 mL × 2) and chloroform (5 mL × 5). The combined organic phase was concentrated. The resulting residue was dissolved in 3 mL of 1,4-dioxane, 0.6 mL of water was added, and sodium bicarbonate (27 mg, 0.32 mmol) and 9-fluorenylmethyl chloroformate (70 mg, 0.27 mmol) were added. The mixture was stirred at room temperature for 1 hour. 20 mL of water was added, and the mixture was extracted with ethyl acetate (8 mL × 3). The organic phase was washed with saturated sodium chloride solution (20 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography using developing solvent system B to give the title product 8c (100 mg, yield: 73.6%). MS m / z (ESI): 501.0 [M+1].

[0162] Step 2 1-((2-((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)acetamido)methoxy)cyclopropane-1-carboxylic acid 8d 8c (50 mg, 0.10 mmol) was dissolved in 3 mL of a mixed solvent of tetrahydrofuran and ethyl acetate (V:V = 2:1), and palladium on carbon (25 mg, 10% content) was added. The mixture was purged with hydrogen three times and stirred at room temperature for 1 hour. The reaction mixture was filtered through diatomaceous earth, the filter cake was rinsed with tetrahydrofuran, and the filtrate was concentrated to give the title product 8d (41 mg, 100% yield). MS m / z (ESI): 411.0 [M+1].

[0163] Step 3 (9H-Fluoren-9-yl)methyl (2-(((1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)carbamoyl)cyclopropoxy)methyl)amino)-2-oxoethyl)carbamate 8e 1b (7 mg, 0.013 mmol) was added to a reaction flask, 1 mL of N,N-dimethylformamide was added, the atmosphere was purged with argon three times, and the mixture was cooled to 0-5 °C in an ice-water bath. One drop of triethylamine was added, and a solution of 8d (7 mg, 0.017 mmol) in 0.5 mL of N,N-dimethylformamide was added. 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (7 mg, 0.026 mmol) was added, and the mixture was stirred in an ice bath for 35 min. 10 mL of water was added, and the mixture was extracted with ethyl acetate (5 mL × 3). The organic phase was washed with saturated sodium chloride solution (10 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The resulting residue was purified by thin-layer chromatography using developing solvent system B to give the title product 8e (8.5 mg, yield: 78.0%). MS m / z (ESI): 828.0 [M+1].

[0164] Step 4 1-((2-Aminoacetamido)methoxy)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)cyclopropane-1-carboxamide 8f 8e (4 mg, 4.84 μmol) was dissolved in 0.2 mL of dichloromethane, 0.1 mL of diethylamine was added, and the mixture was stirred at room temperature for 2 hours. The reaction mixture was concentrated under reduced pressure, 2 mL of toluene was added, and the mixture was concentrated under reduced pressure. This procedure was repeated twice, 3 mL of n-hexane was added, and the upper layer of n-hexane was poured off. This procedure was repeated three times, and the mixture was concentrated under reduced pressure to give the crude product 8f (2.9 mg). This product was used directly in the next step without further purification. MS m / z (ESI): 606.0 [M+1].

[0165] Step 5 1-(((S)-7-Benzyl-20-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-3,6,9,12,15-pentaoxo-2,5,8,11,14-pentaazaicosyl)oxy)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)cyclopropane-1-carboxamide 8 Crude product 8f (2.9 mg, 4.84 μmol) was dissolved in 0.5 mL of N,N-dimethylformamide, purged with argon three times, and cooled to 0-5°C in an ice-water bath. A 0.3 mL N,N-dimethylformamide solution of (S)-2-(-2-(-2-(6-(-2,5-dioxo-1H-pyrrol-1-yl)hexaneamino)acetamino)acetamino)-3-phenylpropionic acid 8g (2.7 mg, 5.80 μmol, prepared by the method disclosed in patent application "EP2907824") was added, followed by the addition of 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (2.7 mg, 9.67 μmol). The mixture was stirred in an ice bath for 30 minutes, the ice bath was removed, the mixture was warmed to room temperature, and the mixture was stirred for 15 minutes. The reaction mixture was purified by high-performance liquid chromatography (separation conditions: column: XBridge Prep C18 OBD 5 μm 19 × 250 mm, mobile phase: A-water (10 mmol NH4OAc), B-acetonitrile, gradient elution, flow rate: 18 mL / min), and the corresponding components were collected and concentrated under reduced pressure to give the title product 8 (2 mg, yield 39.0%).

[0166] MS m / z (ESI): 1060.0 [M+1]. 1H NMR (400 MHz, DMSO-d6): δ 9.01 (d, 1H), 8.77 (t, 1H), 8.21 (t, 1H), 8.08-7.92 (m, 2H), 7.73 (d, 1H), 7.28 (s, 1H), 7.24-7.07 (m, 4H), 6.98 (s, 1H), 6.50 (s, 1H), 5.61 (q, 1H), 5.40 (s, 2H), 5.32 (t, 1H), 5.12 (q, 2H), 4.62 (t, 1H), 4.52 (t, 1H), 4.40-4.32 (m, 1H), 3.73-3.47 (m, 8H), 3.16-3.04 (m, 2H), 2.89 (dd, 1H), 2.69-2.55 (m, 2H), 2.37-2.23 (m, 4H), 2.12-1.93 (m, 4H), 1.90-1.74 (m, 2H), 1.52-1.38 (m, 4H), 1.33-1.11 (m, 5H), 0.91-0.81 (m, 4H).

[0167] Examples 2-9 N-((2R,10S)-10-Benzyl-2-cyclopropyl-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-1,6,9,12,15-pentaoxo-3-oxa-5,8,11,14-tetraazahexadecan-16-yl)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamide 9-A N-((2S,10S)-10-Benzyl-2-cyclopropyl-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-1,6,9,12,15-pentaoxo-3-oxa-5,8,11,14-tetraazahexadecan-16-yl)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamide 9-B [ka]

[0168] Step 1 2-Cyclopropyl-2-hydroxybenzyl acetate 9a 2a (1.3 g, 11.2 mmol, prepared according to the method disclosed in patent application WO2013 / 106717) was dissolved in 50 mL of acetonitrile, and potassium carbonate (6.18 g, 44.8 mmol), benzyl bromide (1.33 mL, 11.2 mmol), and tetrabutylammonium iodide (413 mg, 1.1 mmol) were added sequentially. The reaction mixture was stirred at room temperature for 48 hours, filtered through diatomaceous earth, and the filter cake was rinsed with ethyl acetate (10 mL). The combined filtrate was concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography using eluent system C to give the title product 9a (2 g, 86.9% yield).

[0169] Step 2 10-Cyclopropyl-1-(9H-fluoren-9-yl)-3,6-dioxo-2,9-dioxa-4,7-diazaundecan-11-oate benzyl ester 9b 9a (120.9 mg, 0.586 mmol) and 8b (180 mg, 0.489 mmol) were added to a reaction flask, 4 mL of tetrahydrofuran was added, and the mixture was purged with argon three times. The mixture was cooled to 0-5 °C in an ice-water bath, potassium tert-butoxide (109 mg, 0.98 mmol) was added, the ice-water bath was removed, the mixture was warmed to room temperature, and stirred for 40 min. 10 mL of ice-water was added, and the mixture was extracted with ethyl acetate (20 mL × 2) and chloroform (10 mL × 5). The combined organic phase was concentrated. The resulting residue was dissolved in 4 mL of dioxane, 2 mL of water was added, sodium bicarbonate (49.2 mg, 0.586 mmol), and 9-fluorenylmethyl chloroformate (126 mg, 0.49 mmol) were added, and the mixture was stirred at room temperature for 2 h. After adding 20 mL of water, the mixture was extracted with ethyl acetate (10 mL × 3). The organic phase was washed with saturated sodium chloride solution (20 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography using developing solvent system C to give the title product 9b (48 mg, yield: 19%). MS m / z (ESI): 515.0 [M+1].

[0170] Step 3 10-Cyclopropyl-1-(9H-fluoren-9-yl)-3,6-dioxo-2,9-dioxa-4,7-diazaundecan-11-oic acid 9c 9b (20 mg, 0.038 mmol) was dissolved in 4.5 mL of a mixed solvent of tetrahydrofuran and ethyl acetate (V:V = 2:1), and palladium on carbon (12 mg, 10% content, dry) was added. The mixture was purged with hydrogen three times and stirred at room temperature for 1 hour. The reaction mixture was filtered through diatomaceous earth, the filter cake was rinsed with ethyl acetate, and the filtrate was concentrated to give the crude title product 9c (13 mg), which was used directly in the next step without further purification. MS m / z (ESI): 424.9 [M+1].

[0171] Step 4 (9H-Fluoren-9-yl)methyl(2-((1-cyclopropyl-2-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-2-oxoethoxy)methyl)amino)-2-oxoethyl)carbamate 9d 1b (10 mg, 18.8 μmol) was added to a reaction flask, 1 mL of N,N-dimethylformamide was added, and the mixture was purged with argon three times. The mixture was cooled to 0-5 °C in an ice-water bath, and one drop of triethylamine was added. Crude product 9c (13 mg, 30.6 μmol) and 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (16.9 mg, 61.2 μmol) were added, and the mixture was stirred in an ice bath for 40 min. 10 mL of water was added, and the mixture was extracted with ethyl acetate (10 mL × 3). The organic phases were combined. The organic phase was washed with saturated sodium chloride solution (10 mL × 2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The resulting residue was purified by thin-layer chromatography using developing solvent system B to obtain the title product 9d (19 mg, yield: 73.6%). MS m / z (ESI): 842.1 [M+1].

[0172] Step 5 2-((2-Aminoacetamido)methoxy)-2-cyclopropyl-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)acetamide 9e 9d (19 mg, 22.6 μmol) was dissolved in 2 mL of dichloromethane, 1 mL of diethylamine was added, and the mixture was stirred at room temperature for 2 hours. The reaction mixture was concentrated under reduced pressure, and 1 mL of toluene was added and concentrated under reduced pressure. This procedure was repeated twice. The residue was pulped with 3 mL of n-hexane, and after standing, the supernatant was poured off and the solid was retained. The solid residue was concentrated under reduced pressure and dried using an oil pump to obtain the crude title product 9e (17 mg), which was used directly in the next step without further purification. MS m / z (ESI): 638.0 [M+18].

[0173] Step 6 N-((2R,10S)-10-Benzyl-2-cyclopropyl-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-1,6,9,12,15-pentaoxo-3-oxa-5,8,11,14-tetraazahexadecan-16-yl)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamide 9-A N-((2S,10S)-10-Benzyl-2-cyclopropyl-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-1,6,9,12,15-pentaoxo-3-oxa-5,8,11,14-tetraazahexadecan-16-yl)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamide 9-B The crude product 9e (13.9 mg, 22.4 μmol) was dissolved in 0.6 mL of N,N-dimethylformamide, purged with argon three times, and cooled to 0-5°C in an ice-water bath. 8 g (21.2 mg, 44.8 μmol) of 0.3 mL of N,N-dimethylformamide was added, and 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (18.5 mg, 67.3 μmol) was added. The mixture was stirred in an ice bath for 10 minutes, the ice bath was removed, the mixture was warmed to room temperature, and the mixture was stirred for 1 hour to produce compound 9. The reaction mixture was purified by high-performance liquid chromatography (separation conditions: column: XBridge Prep C18 OBD 5 μm 19 × 250 mm, mobile phase: A-water (10 mmol NH4OAc), B-acetonitrile, gradient elution, flow rate: 18 mL / min), and the corresponding components were collected and concentrated under reduced pressure to give the title products (9-A: 2.4 mg, 9-B: 1.7 mg). MS m / z (ESI): 1074.4 [M+1].

[0174] Single-configuration compound 9-A (relatively short retention time): UPLC analysis: Retention time: 1.14 min, Purity: 85% (Column: ACQUITY UPLC BEHC18 1.7 um 2.1 x 50 mm, Mobile phase: A - water (5 mmol NH4OAc), B - acetonitrile). 1H NMR (400 MHz, DMSO-d6): δ 8.60 (t, 1H), 8.51-8.49 (d, 1H), 8.32-8.24 (m, 1H), 8.13-8.02 (m, 2H), 8.02-7.96 (m, 1H), 7.82-7.75 (m, 1H), 7.31 (s, 1H), 7.26-7.15 (m, 4H), 6.99 (s, 1H), 6.55-6.48 (m, 1H), 5.65-5.54 (m, 1H), 5.41 (s, 2H), 5.35-5.15 (m, 3H), 4.74-4.62 (m, 1H), 4.54-4.40 (m, 2H), 3.76-3.64 (m,4H), 3.62-3.48 (m, 2H), 3.20-3.07 (m, 2H), 3.04-2.94 (m, 1H), 2.80-2.62 (m, 1H), 2.45-2.30 (m, 3H), 2.25-2.15 (m, 2H), 2.15-2.04 (m, 2H), 1.93-1.78 (m, 2H), 1.52-1.39 (m, 3H), 1.34-1.12 (m, 5H), 0.87 (t, 3H), 0.64-0.38 (m, 4H).

[0175] Compound 9-B in a single configuration (comparatively long retention time): UPLC analysis: retention time: 1.16 minutes, purity: 89% (Kurarum: ACQUITY UPLC BEHC18 1.7 um 2.1×50 mm, mobile phase: A-water (5 mmol NH4OAc), B-アセトニトリル). 1H NMR (400 MHz, DMSO-d6): δ 8.68-8.60 (m, 1H), 8.58-8.50 (m, 1H), 8.32-8.24 (m, 1H), 8.13-8.02 (m, 2H), 8.02-7.94 (m, 1H), 7.82-7.75 (m, 1H), 7.31 (s, 1H), 7.26-7.13 (m, 3H), 6.99 (s, 1H), 6.55-6.48 (m, 1H), 5.60-5.50 (m, 1H), 5.41 (s, 2H), 5.35-5.15 (m, 2H), 4.78-4.68 (m, 1H), 4.60-4.40 (m, 2H), 3.76-3.58 (m, 4H), 3.58-3.48 (m, 1H), 3.20-3.10 (m, 2H), 3.08-2.97 (m, 2H), 2.80-2.72 (m, 2H), 2.45-2.30 (m, 3H), 2.25-2.13 (m, 2H), 2.13-2.04 (m, 2H), 2.03-1.94 (m, 2H), 1.91-1.78 (m, 2H), 1.52-1.39 (m, 3H), 1.34-1.12 (m, 4H), 0.91-0.79 (m, 3H), 0.53-0.34 (m, 4H).

[0176] 3. Preparation of ADC Example 3-1 ADC-1 [ka] At 37°C, the prepared tris(2-carboxyethyl)phosphine (TCEP) aqueous solution (10 mM, 67.5 μL, 675 nmol) was added to a PBS buffer solution of antibody PD3 (0.05 M PBS buffer solution, pH 6.5, 10.0 mg / mL, 4.0 mL, 270 nmol), placed in a water bath oscillator, and allowed to oscillate at 37°C for 3 hours before quenching. The reaction solution was then cooled to 25°C in a water bath. Compound 9-A (2.9 mg, 2700 nmol) was dissolved in 180 μL of DMSO and added to the reaction mixture. The mixture was placed in a water bath oscillator and allowed to oscillate at 25 °C for 3 hours to terminate the reaction. The reaction mixture was desalted and purified using a Sephadex G25 gel column (eluent: 0.05 M PBS buffer solution, pH 6.5, containing 0.001 M EDTA) to obtain the exemplary product ADC-1 (1.93 mg / mL, 18.4 mL) represented by the formula PD3-9-A in PBS buffer, which was then stored at 4 °C. The average value calculated by UV-Vis: n=3.77.

[0177] Example 3-2 ADC-2 At 37°C, the prepared tris(2-carboxyethyl)phosphine (TCEP) aqueous solution (10 mM, 143.1 μL, 1431 nmol) was added to a PBS buffer solution of antibody PD3 (0.05 M PBS buffer solution, pH 6.5, 10.0 mg / mL, 4.0 mL, 270 nmol), placed in a water bath oscillator, and allowed to oscillate at 37°C for 3 hours before quenching. The reaction solution was then cooled to 25°C in a water bath.

[0178] Compound 9-A (4.35 mg, 4050 nmol) was dissolved in 270 μL of DMSO and added to the reaction mixture. The mixture was placed in a water bath oscillator and oscillated at 25 °C for 3 hours to terminate the reaction. The reaction mixture was desalted and purified on a Sephadex G25 gel column (eluent: 0.05 M PBS buffer solution, pH 6.5, containing 0.001 M EDTA) to obtain the exemplary product ADC-2 (1.69 mg / mL, 17.8 mL) represented by the formula PD3-9-A in PBS buffer, which was then stored at 4 °C.

[0179] The average value calculated by UV-Vis: n=6.59.

[0180] Example 3-3 ADC-3 [ka] At 37°C, an aqueous solution of antibody PD3 in PBS buffer (0.05 M PBS buffer, pH 6.5, 10.0 mg / mL, 0.3 mL, 20.3 nmol) was added to the prepared aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 5.1 μL, 51 nmol), placed in a water bath oscillator, and allowed to oscillate at 37°C for 3 hours before quenching. The reaction solution was then cooled to 25°C in a water bath. Compound 12h (0.194 mg, 203 nmol; see WO2017063509 for compound preparation) was dissolved in 20 μL of acetonitrile and added to the reaction mixture. The mixture was placed in a water bath oscillator and oscillated at 25°C for 3 hours to terminate the reaction. The reaction mixture was desalted and purified on a Sephadex G25 gel column (eluent: 0.05 M PBS aqueous buffer solution, pH 6.5, containing 0.001 M EDTA) to obtain the title product ADC-3 in PBS buffer (4.3 mg / mL, 0.6 mL), which was then stored at 4°C. The average value was calculated by UV-Vis: n=4.14.

[0181] Example 3-4 ADC-4 [ka] For the synthesis process, see Example 19 of WO2015098099. At 37°C, the prepared tris(2-carboxyethyl)phosphine (TCEP) solution (10 mM, 33.8 μL, 338 nmol) was added to a PBS buffer solution of antibody TINA (0.05 M PBS buffer solution, pH 6.5, 10.0 mg / mL, 2.0 mL, 135.4 nmol), placed in a water bath oscillator, and allowed to oscillate at 37°C for 3 hours before quenching. The reaction solution was then cooled to 25°C in a water bath. Compound 58 (1.4 mg, 1354 nmol) was dissolved in 70 μL of DMSO and added to the reaction mixture. The mixture was placed in a water bath oscillator and oscillated at 25 °C for 3 hours to terminate the reaction. The reaction mixture was desalted and purified on a Sephadex G25 gel column (eluent: 0.05 M PBS aqueous buffer solution, pH 6.5, containing 0.001 M EDTA) to obtain the exemplary title product ADC-4 (1.13 mg / mL, 15.1 mL) represented by the formula TINA-58 in PBS buffer, which was then stored at 4 °C. The average value was calculated by UV-Vis: n=3.99.

[0182] Example 3-5 ADC-5 [ka] At 37°C, an aqueous solution of antibody PD3 in PBS buffer (0.05 M PBS buffer, pH 6.5, 10.0 mg / mL, 1.5 mL, 101.4 nmol) was added to the prepared aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 25.3 μL, 253 nmol), placed in a water bath oscillator, and allowed to oscillate at 37°C for 3 hours before quenching. The reaction solution was then cooled to 25°C in a water bath. Compound 58 (1.05 mg, 1014 nmol, see Example 58 on page 163 of the patent "CN104755494A") was dissolved in 60 μL of DMSO and added to the reaction solution. The reaction was stopped by placing it in a water bath oscillator and oscillating it at 25 °C for 3 hours. The reaction solution was desalted and purified on a Sephadex G25 gel column (eluent: 0.05 M PBS buffer solution at pH 6.5) to obtain the exemplary title product ADC-5 represented by formula PD3-58 in PBS buffer (0.82 mg / mL, 13.5 mL), which was stored at 4 °C. The average value calculated by UV-Vis: n=3.88.

[0183] Example 3-6 ADC-6 [ka] At 37°C, an aqueous solution of antibody hRS7 in PBS buffer (0.05 M PBS buffer, pH 6.5, 10.0 mg / mL, 1.4 mL, 94.60 nmol) was added to the prepared aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 50.1 μL, 501 nmol). The mixture was placed in a water bath oscillator and allowed to oscillate at 37°C for 3 hours, after which the reaction was stopped. The reaction solution was then cooled to 25°C in a water bath. Compound SN38 (synthesized with reference to Example 1 on page 59 of Patent CN105407891A, 2.1 mg, 1419 nmol) was dissolved in 50 μL of DMSO and added to the reaction solution. The reaction was stopped by placing it in a water bath oscillator and oscillating it at 25 °C for 3 hours. The reaction solution was desalted and purified on a Sephadex G25 gel column (eluent: 0.05 M PBS buffer solution, pH 6.5) to obtain the exemplary title product ADC-6 (1.03 mg / mL, 11.5 mL) represented by the formula hRS7-SN38 in PBS buffer, which was then stored at 4 °C. The mean value was calculated using CE-SDS: n=7.56.

[0184] Example 3-7 ADC-7 [ka] At 37°C, an aqueous solution of antibody hRS7 in PBS buffer (0.05 M PBS buffer, pH 6.5, 10.0 mg / mL, 2.18 mL, 147.3 nmol) was added to the prepared aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 36.8 μL, 368 nmol). The mixture was placed in a water bath oscillator and allowed to oscillate at 37°C for 3 hours, after which the reaction was stopped. The reaction solution was then cooled to 25°C in a water bath. Compound 9-A (1.58 mg, 1471 nmol) was dissolved in 100 μL of DMSO and added to the reaction mixture. The mixture was placed in a water bath oscillator and allowed to oscillate at 25° C. for 3 hours to terminate the reaction. The reaction mixture was desalted and purified on a Sephadex G25 gel column (eluent: 0.05 M PBS aqueous buffer solution, pH 6.5) to obtain the exemplary title product ADC-7 (1.10 mg / mL, 16.4 mL) represented by the formula hRS7-9-A in PBS buffer, which was then stored at 4° C. The average value calculated by UV-Vis: n=3.72.

[0185] Example 3-8 ADC-8 [ka] At 37°C, an aqueous solution of antibody hRS7 in PBS buffer (0.05 M PBS buffer, pH 6.5, 10.0 mg / mL, 2.18 mL, 147.3 nmol) was added to the prepared aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 36.8 μL, 368 nmol). The mixture was placed in a water bath oscillator and allowed to oscillate at 37°C for 3 hours, after which the reaction was stopped. The reaction solution was then cooled to 25°C in a water bath. Compound 58 (1.52 mg, 1473 nmol, see Example 58 on page 163 of the patent "CN104755494A") was dissolved in 100 μL of DMSO and added to the reaction solution. The reaction was stopped by placing it in a water bath oscillator and oscillating it at 25 °C for 3 hours. The reaction solution was desalted and purified on a Sephadex G25 gel column (eluent: 0.05 M PBS buffer solution at pH 6.5) to obtain the exemplary title product ADC-8 (1.02 mg / mL, 16.8 mL) represented by the formula hRS7-58 in PBS buffer, which was stored at 4 °C. The average value calculated by UV-Vis: n=3.93.

[0186] Example 3-9 ADC-9 [ka] At 37°C, the prepared tris(2-carboxyethyl)phosphine (TCEP) solution (10 mM, 16.0 μL, 160 nmol) was added to a PBS buffer solution of antibody TINA (0.05 M PBS buffer solution, pH 6.5, 10.0 mg / mL, 0.95 mL, 64.2 nmol), placed in a water bath oscillator, and allowed to oscillate at 37°C for 3 hours to terminate the reaction. The reaction solution was then cooled to 25°C in a water bath. Compound 9-A (0.69 mg, 642 nmol) was dissolved in 30 μL of DMSO and added to the reaction mixture. The mixture was placed in a water bath oscillator and allowed to oscillate at 25 ° C for 3 hours to terminate the reaction. The reaction mixture was desalted and purified on a Sephadex G25 gel column (elution phase: 0.05 M PBS buffer solution at pH 6.5) to obtain the exemplary title product ADC-9 (0.99 mg / mL, 7.0 mL) represented by the formula TINA-9-A in PBS buffer, which was then stored at 4 ° C. The average value was calculated by UV-Vis: n=3.99.

[0187] Analysis of drug loading in ADC stock solutions ADCs are antibody-linked drugs whose treatment mechanism is to deliver toxin molecules to cells via antibody targeting, resulting in cell death. The drug loading plays a crucial role in efficacy.

[0188] 1. UV-Vis calculation method The drug loading of the ADC stock solution was measured by ultraviolet light.

[0189] Experimental Method The cuvettes containing the sodium succinate buffer solution were placed in the reference absorption cell and the sample measurement absorption cell, respectively, and after subtracting the solvent blank, the cuvette containing the sample solution was placed in the sample measurement absorption cell, and the absorbance at 280 nm and 370 nm was measured.

[0190] Result calculation The loading amount of the ADC stock solution was measured by ultraviolet spectrophotometry (instrument used: Thermo nanodrop 2000 ultraviolet spectrophotometer). The principle is that the total absorbance value of the ADC stock solution at a certain wavelength is equal to the cumulative absorbance value of the cytotoxic drug and monoclonal antibody at that wavelength, i.e., (1)A 280 nm =ε mab-280 bC mab +ε Drug-280 bC Drug ε Drug-280 : The drug has an average molar extinction coefficient of 5100 at 280 nm, C Drug : drug concentration, ε mab-280 : The average molar extinction coefficient at 280 nm of the monoclonal antibody stock solution is 214600, C mab : Concentration of monoclonal antibody stock solution, b: The optical path length is 1 cm.

[0191] Similarly, the total absorbance equation for the sample at 370 nm can be obtained, i.e., (2)A 370 nm =ε mab-370 bC mab +ε Drug-370 bC Drug ε Drug-370 : The drug has an average molar extinction coefficient of 19000 at 370 nm, C Drug : drug concentration, ε mab-370 : The extinction coefficient of the monoclonal antibody stock solution at 370 nm is 0, C mab : Concentration of monoclonal antibody stock solution, b: The optical path length is 1 cm.

[0192] Using equations (1) and (2), the drug loading can be calculated using the extinction coefficients and concentration data for the monoclonal antibody and drug at the two detection wavelengths. Drug loading dose = C Drug / Cmab .

[0193] 2. Calculation method based on CE-SDS Reagents and equipment SDS-MW Analysis Kit: Manufactured by Beckman, product number 390953. This reagent kit includes SDS-MW separation gel buffer, SDS-MW sample buffer, acidic wash solution (0.1 mol / L hydrochloric acid solution), basic wash solution (0.1 mol sodium hydroxide solution), and internal standard (10 kDa). The SDS reagent kit (product number BSYK018) manufactured by Beijing Bosiya Biochemical Technology Research Institute may also be used. This reagent kit includes CE-SDS gel buffer and CE-SDS sample buffer. Alkylation solution (0.25 mol iodoacetamide solution): Weigh out approximately 0.046 g of iodoacetamide and add 1 mL of ultrapure water to dissolve. Mix uniformly and store at 2-8 °C, protected from light, for 7 days. Capillary electrophoresis apparatus: SCIEX PA800plus. Capillary: An uncoated fused silica capillary (inner diameter 50 μm) was cut to a total length of 30.2 cm, with an effective separation length of 20 cm for the high-resolution method.

[0194] Preparation of sample solution The test sample was diluted to 1 mg / mL with SDS sample buffer. 95 μL of the test sample solution (1 mg / mL) was taken, and 5 μL of 0.8 mol / L iodoacetamide solution was added and swirled to mix uniformly. 95 μL of the blank control solution was taken, and 5 μL of 0.8 mol / L iodoacetamide solution was added and swirled to mix uniformly. 75 μL of each sample tube was then removed and placed in a sample vial for immediate analysis.

[0195] Measurement method 1) Capillary pretreatment: Wash with 0.1 mol / L sodium hydroxide solution at 60 psi for 3 minutes, then with 0.1 mol / L hydrochloric acid solution at 60 psi for 2 minutes, and finally with pure water at 70 psi for 1 minute. This should be done before each run. 2) Pre-filling the capillary: Wash with SDS separation gel buffer at 50 psi for 15 minutes. This should be done before each run. Sample injection: 10 kV reversed-phase polarity electric injection, sample was reduced and injected for 20 seconds. Separation: Operated at 15 kV for 40 minutes, reversed phase polarity. Sample chamber temperature: 18-25°C. Capillary temperature: 18-25°C.

[0196] Results analysis Since all sulfhydryl groups liberated from the released disulfide bonds in the antibody were conjugated to the corresponding drug, the data were analyzed using Beckman software, and the ratio of the calibrated peak areas of the heavy chain, non-glycosylated heavy chain, and light chain to the total calibrated peak area was calculated. The weighted average value of the ADC was finally calculated using the formula: DAR = [4 × heavy chain (H) peak area + 2 × hapten (HL) peak area + 4 × double chain (HH) peak area + 2 × heavy-heavy-light chain (HHL) peak area] / [heavy chain (H) peak area / 2 + hapten (HL) peak area / 2 + double chain (HH) peak area + heavy-heavy-light chain (HHL) peak area + complete antibody peak area].

[0197] The activity of the antibody of the present disclosure will be verified by biochemical test methods below.

[0198] Test Example 1: Antibody protein level binding experiment hTROP-2 protein was diluted to 1 μg / mL in pH 7.4 PBS (Genbyou Bioscience, B320) buffer and added to a 96-well microplate at a volume of 100 μL per well and incubated overnight at 4°C. After discarding the liquid, 300 μL of 5% nonfat milk (BD, 232100) diluted in PBS was added to each well for blocking and incubation at 37°C for 2 hours. After discarding the blocking solution, the plate was washed three times with PBST buffer (pH 7.4 PBS containing 0.1% Tween-20). Then, 100 μL of the gradient diluted antibody solution was added to each well and incubated at 37°C for 1 hour. After incubation, the plate was washed three times with PBST, and 100 μL of mouse anti-human IgG (H+L) (Jackson ImmunoResearch, 209-035-088, diluted 1:8000) was added to each well and incubated at 37°C for 1 hour. After washing the plate three times with PBST, 100 μL of TMB color developing substrate (KPL, 5120-0077) was added to each well and incubated at room temperature for 10-15 minutes. The reaction was terminated by adding 50 μL of 1 M H2SO4 to each well. The absorbance value at 450 nm was read using a microplate reader, and the binding curve between the antibody and antigen was fitted using software to calculate the EC 50 The binding activity between the antibody and the protein is shown in Table 2.

[0199] [Table 2] The results show that the PD3 antibody in the present application has relatively high binding activity to the hTROP-2 protein.

[0200] Test Example 2: Antibody cell-level binding experiment CHOK1 cells stably transfected with TROP-2 were cultured at 1 × 10 in FACS buffer (2% fetal bovine serum (Gibco, 10099141) pH 7.4 PBS (Sigma, P4417-100TAB)). 6A cell suspension was prepared at 100 μL / well in a 96-well round-bottom plate. After centrifuging and removing the supernatant, 50 μL / well of the target antibody diluted in FACS buffer was added at different concentrations and incubated in a refrigerator at 4°C for 1 hour, protected from light. After washing three times with FACS buffer at 300 g, a working concentration of Alexa Fluor 488 sheep anti-human IgG (H+L) (Invitrogen, A-11013) was added and incubated in a refrigerator at 4°C for 40 minutes, protected from light. After washing three times with FACS buffer at 300 g, the geometric mean fluorescence intensity (MFI) was measured using a BD FACSCanto II flow cytometer to determine the binding EC of the antibody to stably transfected cells expressing TROP-2. 50 The binding activity between the antibody and the cells is shown in FIG. 1 and Table 3.

[0201] [Table 3] The results show that the PD3 antibody in this application has relatively high binding activity to cells expressing TROP-2 protein.

[0202] Test Example 3: Antibody endocytosis experiment The purpose of this experiment was to indirectly reflect the endocytic status of anti-TROP-2 antibodies based on the cell killing of activated DT3C protein when it entered cells. 50 and E max The in vitro endocytic activity of the antibody was evaluated by the following method.

[0203] DT3C is a recombinantly expressed fusion protein composed of diphtheria toxin fragment A (toxin portion only) and group G streptococcus 3C fragment (IgG binding portion). This protein has high affinity for the IgG portion of antibodies, and when endocytosis occurs in antibodies, it enters cells together with the antibody and, under the influence of intracellular furin, releases the toxic DT. DT can inhibit the activity of EF2-ADP-ribosylation, blocking the protein translation process and ultimately causing cell death. DT3C that does not enter cells does not have cell-killing activity. The endocytosis activity of antibodies was evaluated based on the cell-killing status.

[0204] Prepare a cell suspension in fresh cell culture medium containing 20% ​​low-IgG FBS to a cell density of 2 × 10 4 The cells / mL were added to cell culture plates at 50 μL per well and incubated at 37°C in 5% carbon dioxide for 16 hours. DT3C was prepared at a 4x concentration in serum-free medium and filtered through a 0.22 μm microfilter to obtain a sterile solution. Antibody was prepared at a 4x concentration in serum-free medium. 80 μL of DT3C and 80 μL of antibody were mixed uniformly at a 1:1 volume, left at room temperature, and incubated for 30 minutes. 50 μL of the diluted antibody-DT3C mixture was added to 50 μL of cells and incubated in an incubator for 3 days. 50 μL of CTG was added to each well, and the mixture was incubated at room temperature in the dark for 10 minutes. Chemiluminescence was read using a Victor3. The endocytosis activity of the antibodies is shown in Table 4.

[0205] [Table 4] The results show that the PD3 antibody in this application has relatively high endocytosis efficiency.

[0206] Test Example 4: Measurement of antibody affinity The affinity of antibodies for TROP-2 was detected using a capture antibody. The capture antibody was bound to a Protein A (Cat. # 29127556, GE) biosensor chip conjugated with an anti-human IgG antibody (Cat. # BR-1008-39, Lot. # 10260416, GE). The antigen hTROP-2 was then flowed over the chip surface, and the binding and dissociation curves were obtained by detecting the response signal in real time using a Biacore T200 instrument. After each cycle of dissociation was completed, the chip was regenerated by washing with regeneration buffer Glycine 1.5 (Cat. # BR100354, GE) or 3 M MgCl2 (from the Human Antibody Capture Kit, Cat. # BR100839, GE). After the experiment was completed, the data was fitted with the (1:1) Langmuir model using GE Biacore T200 Evaluation version 3.0 software to obtain affinity values. The affinity between antibodies and proteins is shown in Table 5.

[0207] [Table 5] The results show that the affinity of the PD3 antibody in this application to hTROP-2 is relatively high.

[0208] Test Example 5: ADC molecular cellular activity experiment The cells used in this experiment are as follows: FaDu(++++) was purchased from ATCC, product number: HTB-43 TM HCC827(+++) was purchased from ATCC, product number: CRL-2868. Colo205(++) was purchased from the Chinese Academy of Sciences Cell Bank, product number: TCHu102. DMS53(++) was purchased from ATCC, product number: CRL-2062. TM SK-OV-3(+) was purchased from ATCC, product number: HTB-77. CHO-K1(-) was purchased from ATCC, product number: CCL-61. TMThe "+" indicates the level of TROP-2 expression in the cells, with a larger "+" indicating a higher level of TROP-2 expression. A "-" indicates no TROP-2 expression.

[0209] A cell suspension was prepared in fresh cell culture medium containing 10% FBS at a density of 3703 cells / mL. 135 μL per well was added to a 96-well cell culture plate and incubated at 37°C under 5% carbon dioxide for 16 hours. ADC samples were prepared in PBS to a concentration of 5 μM. This initial concentration was diluted five-fold with PBS to give eight different concentrations. 15 μL of the above ADC solution was added to each well. The plates were incubated at 37°C under 5% carbon dioxide for 6 days. 70 μL of CTG was added to each well, and the plates were incubated at room temperature for 10 minutes in the dark. Chemiluminescence was measured using a Victor3 scanner, and data were analyzed using GraphPad Prism software.

[0210] The results showed that ADC-1 had a relatively strong cell-killing effect, and the killing effect was positively correlated with the expression level of TROP-2 on the surface of tumor cells. [Table 6-1] A second parallel comparative experiment was conducted using the above method, and the following results were obtained. [Table 6-2]

[0211] Test Example 6: Bystander killing activity experiment BxPC3 (human pancreatic cancer cells, ATCC, CRL-1687) and MiaPaCa2 cells (human pancreatic cancer cells, biocytogen, B-HCL-014) were cultured in RPMI1640 + 10% FBS and DMEM / high glucose + 10% FBS, respectively. The cells were digested with pancreatin, neutralized with fresh medium, centrifuged at 1000 rpm for 3 minutes, the supernatant was discarded, and the cells were resuspended in RPMI1640 + 10% FBS. After cell counting, the BxPC3 cell density was adjusted to 6 × 10 4The cell density of MiaPaCa2-luc was adjusted to 1.5 × 10 cells / mL. 4 The concentration was adjusted to cells / mL. 500 μL of BxPC3 cells and 500 μL of MiaPaCa2-luc cells were added to each well of plate 1 of a 12-well plate. 500 μL of MiaPaCa2-luc cells and 500 μL of RPMI1640 medium containing 10% FBS serum were added to plate 2 of a 12-well plate. The plates were cultured at 37°C under 5% carbon dioxide for 24 hours.

[0212] ADC samples were prepared at a 40x concentration (0.2 μM) in a neutral solution. 25 μL of each sample was added to the corresponding wells of a 12-well plate. A solvent control group was also included. The cells were cultured for 6 days at 37°C under 5% carbon dioxide. The cells in the 12-well plate were digested with pancreatin, neutralized with fresh medium, centrifuged at 1000 rpm for 3 minutes, discarded the supernatant, and resuspended in 1 mL of FACS buffer (PBS + 2.5% FBS). 20 μL of cells were taken, 20 μL of trypan blue was added, and counted. The cells in plate 1 were centrifuged at 1000 rpm for 3 minutes, discarded the supernatant, and resuspended in 100 μL of FACS buffer. 2 μL of TROP-2 (EGP-1) monoclonal antibody (MR54) was added and incubated on ice for 30 minutes. The cells were centrifuged at 2000 rpm at 4°C for 1 minute, the supernatant was discarded, and 150 μL of FACS buffer was added to resuspend the cells. Detection was performed using BD FACSVerse. Data was analyzed using Flowjo 7.6. See Figure 2 for the results of the bystander killing experiment.

[0213] The results show that the ADC-1 in this disclosure has a clear bystander killing effect: although the ADC does not kill TROP-2-negative MiaPaCa2 cells, after mixing TROP-2-expressing BxPC3 cells with negative MiaPaCa2 cells, ADC-1 also has a killing effect on the TROP-2-negative cells.

[0214] Biological evaluation of in vivo activity Test Example 7: Fadu cell CDX mouse model in vivo drug efficacy evaluation Fadu cells (3 × 10 6 ) were inoculated subcutaneously into the right rib cage of Balb / c nude mice, and 10 days after inoculation, the tumor volume reached approximately 245 mm 3 After reaching the target weight, mice with oversized or undersized tumors were removed, and the mice were randomly divided into 5 groups, each with 8 mice, according to tumor volume. The ADC was injected intraperitoneally twice, on days 0 and 8, at a dose of 10 g / 0.1 mL based on body weight, for a total dose of 1 mg / kg. Tumor volume and body weight were measured twice a week, and data were recorded for a total of 21 days.

[0215] Data were recorded using Excel statistical software. Mean values ​​were calculated as avg, SD values ​​as stdev, and SEM values ​​as stdev / sqrt (number of animals in each group). Data were plotted using GraphPad Prism software, and statistical analysis was performed using two-way ANOVA or one-way ANOVA. Tumor volume (V) calculation formula: V = 1 / 2 × L 長 ×L 短 2 In the relative tumor growth rate T / C (%) = (T-T0) / (C-C0) × 100, T and C are the tumor volumes of the treatment and control groups at the end of the experiment, and T0 and C0 are the tumor volumes at the start of the experiment. Tumor inhibition rate TGI(%)=1- T / C(%).

[0216] The results are shown in Table 7 and Figure 3. At a dose of 1 mpk, ADC-1 has a relatively strong tumor-inhibitory effect on FaDu-transplanted tumors. [Table 7]

[0217] Test Example 8: Evaluation of in vivo drug efficacy in SKOV3 cell CDX mouse model SKOV3 cells (5 x 10 6 ) were inoculated subcutaneously into the right rib cage of Balb / c nude mice, and 23 days after inoculation, the tumor volume reached approximately 180 mm 3After reaching the target weight, mice with oversized or undersized tumors were removed, and the mice were randomly divided into 5 groups, each with 8 mice, according to tumor volume. The ADC was administered intraperitoneally twice, at a dose of 10 g / 0.1 mL depending on body weight. The dose is shown in the table below. Tumor volume and body weight were measured twice weekly and recorded. Data were recorded using Excel statistical software. The mean was calculated as avg, SD as standard deviation, and SEM as standard deviation / square root of square root (number of animals in each group). Data were plotted using GraphPad Prism software, and statistical analysis was performed using two-way or one-way ANOVA. Tumor volume (V) calculation formula: V = 1 / 2 × L 長 ×L 短 2 In the relative tumor growth rate T / C (%) = (T-T0) / (C-C0) × 100, T and C are the tumor volumes of the treatment and control groups at the end of the experiment, and T0 and C0 are the tumor volumes at the start of the experiment. Tumor inhibition rate TGI(%)=1- T / C(%).

[0218] The results are shown in Table 8 and Figure 4. At different doses, ADC-1 has a relatively strong tumor-inhibiting effect on SKOV3 xenograft tumors, and the tumor-inhibiting effect is dose-dependent. The effect is dose-dependent. [Table 8]

[0219] Test Example 9: In vivo drug efficacy evaluation in Colo205 cell CDX mouse model Colo205 cells (5 × 10 6 ) were inoculated subcutaneously into the right rib cage of Balb / c nude mice, and 10 days after inoculation, the tumor volume reached approximately 245 mm 3 After reaching the target weight, mice with oversized or undersized tumors were removed, and the mice were randomly divided into 6 groups with 8 mice per group according to tumor volume. The ADC was injected intraperitoneally twice, on days 0 (D0) and 10, at a dose of 10 mg / kg, based on body weight. Tumor volume and body weight were measured twice weekly, and data were recorded for a total of 28 days (D28).

[0220] Data were recorded using Excel statistical software. Mean values ​​were calculated as avg, SD values ​​as stdev, and SEM values ​​as stdev / sqrt (number of animals in each group). Data were plotted using GraphPad Prism software, and statistical analysis was performed using two-way ANOVA or one-way ANOVA. Tumor volume (V) calculation formula: V = 1 / 2 × L 長 ×L 短 2 In the relative tumor growth rate T / C (%) = (T-T0) / (C-C0) × 100, T and C are the tumor volumes of the treatment and control groups at the end of the experiment, and T0 and C0 are the tumor volumes at the start of the experiment. Tumor inhibition rate TGI(%)=1- T / C(%).

[0221] The results are shown in Table 9 and Figure 5. At a dose of 1 mpk, ADC-9 and ADC-7 conjugated with compound 9-A have relatively strong tumor inhibitory effects on Colo205 xenograft tumors. [Table 9]

Claims

1. A ligand-drug conjugate represented by the general formula (Pc-LYD) or a pharmaceutically acceptable salt thereof, 【Chemistry 1】 Among them, Y is -O-(CR a R b ) m -CR 1 R 2 -C(O)-, R a and R b are the same or different and each independently represent a hydrogen atom, a deuterium atom, a halogen atom, and C 1-6 selected from alkyl groups, R 1 is C 3-6 is a cycloalkyl group, R 2 is a hydrogen atom, m is 0 or 1; n is 1 to 10, and n is a decimal or an integer; L is a linker unit, Pc is an anti-TROP-2 antibody or an antigen-binding fragment thereof; The anti-TROP-2 antibody or antigen-binding fragment thereof is a ligand-drug conjugate represented by the general formula (Pc-LYD), or a pharmaceutically acceptable salt thereof, which comprises a heavy chain variable region represented by SEQ ID NO: 3 and a light chain variable region represented by SEQ ID NO:

4.

2. The ligand-drug conjugate represented by the general formula (Pc-LYD) or a pharmaceutically acceptable salt thereof according to claim 1, wherein the anti-TROP-2 antibody is a humanized antibody.

3. 3. The ligand-drug conjugate represented by the general formula (Pc-LYD) or a pharmaceutically acceptable salt thereof according to claim 1 or 2, wherein the anti-TROP-2 antibody or antigen-binding fragment thereof comprises an antibody heavy chain constant region and a light chain constant region, the heavy chain constant region being selected from the constant regions of human IgG1, IgG2, IgG3, and IgG4, and the light chain constant region being selected from the constant regions of human antibody κ and λ chains.

4. The ligand-drug conjugate represented by the general formula (Pc-LYD) or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 3, wherein the anti-TROP-2 antibody comprises a heavy chain constant region represented by SEQ ID NO: 11 and a light chain constant region represented by SEQ ID NO:

12.

5. The ligand-drug conjugate represented by the general formula (Pc-LYD) or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 4, wherein the anti-TROP-2 antibody comprises a heavy chain represented by SEQ ID NO: 13 and a light chain represented by SEQ ID NO:

14.

6. A ligand-drug conjugate represented by the general formula (Pc-LYD) according to any one of claims 1 to 5, or a pharmaceutically acceptable salt thereof, wherein n is 2 to 10 and n is a decimal number or an integer.

7. 7. A ligand-drug conjugate represented by the general formula (Pc-LYD) according to claim 6, or a pharmaceutically acceptable salt thereof, wherein n is 4 to 8 and n is a decimal number or an integer.

8. The linker unit -L- is -L 1 -L 2 -L 3 -L 4 - and L 1 -(succinimide-3-yl-N)-WC(O)- or -CH 2 -C(O)-NR 3 -WC(O)-, where W is C 1-8 is an alkyl group, L 2 is -NR 4 (CH 2 CH 2 O) pCH 2 CH 2 C(O)-, -NR 4 (CH 2 CH 2 O) pCH 2 C(O)-, -S(CH 2 ) pC(O)— and a chemical bond, where p is an integer from 1 to 20; L 3 is a peptide residue consisting of 2 to 7 amino acid residues, wherein the amino acid residues are selected from amino acid residues formed by amino acids selected from phenylalanine, glycine, valine, lysine, citrulline, serine, glutamic acid, and aspartic acid; L 4 is -NR 5 (CR 6 R 7 ) t -, -C(O)NR 5 -, -C(O)NR 5 (CH 2 ) t - and a chemical bond, wherein t is an integer from 1 to 6; R 3 , R 4 and R 5 are identical or different and each independently represent a hydrogen atom, C 1-6 Alkyl group, C 1-6 Halogenated alkyl groups, deuterated C 1-6 Alkyl groups and C 1-6 hydroxyalkyl groups, R 6 and R 7 are the same or different and each independently represent a hydrogen atom, a halogen, or C 1-6 Alkyl group, C 1-6 Halogenated alkyl groups, deuterated C 1-6 Alkyl groups and C 1-6 hydroxyalkyl groups, A ligand-drug complex represented by the general formula (Pc-LYD) according to any one of claims 1 to 7, or a pharmaceutically acceptable salt thereof.

9. The linker unit -L- is -L 1 -L 2 -L 3 -L 4 - and L 1 teeth 【Chemistry 2】 and s 1 is an integer between 2 and 8, L 2 is a chemical bond, L 3 is a tetrapeptide residue, L 4 is -NR 5 (CR 6 R 7 ) t - and R 5 , R 6 or R 7 are the same or different and each independently represent a hydrogen atom or C 1-6 alkyl group, t is 1 or 2, Among them, the above L 1 The end is connected to the Pc and L 4 The end is connected to the Y. A ligand-drug complex represented by the general formula (Pc-LYD) according to any one of claims 1 to 8, or a pharmaceutically acceptable salt thereof.

10. L 3 A ligand-drug complex represented by the general formula (Pc-LYD) according to claim 9, or a pharmaceutically acceptable salt thereof, wherein Pc-LYD is a tetrapeptide residue of GGFG.

11. -L- is 【Transformation 3】 That is, A ligand-drug complex represented by the general formula (Pc-LYD) according to any one of claims 1 to 10, or a pharmaceutically acceptable salt thereof.

12. General formula (Pc-L a -YD) or a pharmaceutically acceptable salt thereof, 【Chemistry 4】 Among them, Pc, n, m, W, L 2 , L 3 , R 1 , R 2 , R 5 , R 6 and R 7 is as defined in claim 8, A ligand-drug complex represented by the general formula (Pc-LYD) according to any one of claims 1 to 8, or a pharmaceutically acceptable salt thereof.

13. General formula (Pc-L b -YD) or a pharmaceutically acceptable salt thereof, 【Transformation 5】 Among them, s 1 is an integer between 2 and 8, Pc, R 1 , R 2 , R 5 ~R 7 , m and n are as defined in any one of claims 8 to 10; A ligand-drug complex represented by the general formula (Pc-LYD) according to any one of claims 1 to 10 and 12, or a pharmaceutically acceptable salt thereof.

14. The ligand-drug conjugate is 【Transformation 6】 Selected from wherein Pc and n are as defined in any one of claims 1 to 13; A ligand-drug complex represented by the general formula (Pc-LYD) according to any one of claims 1 to 13, or a pharmaceutically acceptable salt thereof.

15. The ligand-drug conjugate is 【Transformation 7】 and Among them, n is 1 to 8, and n is a decimal or an integer; Pc is an anti-TROP-2 antibody, which comprises a heavy chain represented by SEQ ID NO: 13 and a light chain represented by SEQ ID NO: 14; A ligand-drug complex represented by the general formula (Pc-LYD) according to any one of claims 1 to 14, or a pharmaceutically acceptable salt thereof.

16. 16. A ligand-drug complex represented by the general formula (Pc-LYD) according to claim 15, or a pharmaceutically acceptable salt thereof, wherein n is an integer or decimal number of 4 to 6.

17. An anti-TROP-2 antibody or antigen-binding fragment thereof, comprising a heavy chain variable region and a light chain variable region, wherein the amino acid sequence of the heavy chain variable region is represented by SEQ ID NO: 3 and the amino acid sequence of the light chain variable region is represented by SEQ ID NO:

4.

18. The anti-TROP-2 antibody or antigen-binding fragment thereof according to claim 17, wherein the anti-TROP-2 antibody is a humanized antibody.

19. The anti-TROP-2 antibody or antigen-binding fragment thereof according to claim 17 or 18, comprising an antibody heavy chain constant region and a light chain constant region, wherein the heavy chain constant region is selected from the constant regions of human IgG1, IgG2, IgG3, and IgG4, and the light chain constant region is selected from the constant regions of human antibody κ and λ chains.

20. The anti-TROP-2 antibody or its antigen-binding fragment according to claim 19, wherein the anti-TROP-2 antibody comprises a heavy chain constant region represented by SEQ ID NO: 11 and a light chain constant region represented by SEQ ID NO:

12.

21. The anti-TROP-2 antibody or its antigen-binding fragment according to any one of claims 17 to 20, wherein the anti-TROP-2 antibody comprises a heavy chain represented by SEQ ID NO: 13 and a light chain represented by SEQ ID NO:

14.

22. A nucleic acid molecule encoding the anti-TROP-2 antibody or antigen-binding fragment thereof of any one of claims 17 to 21.

23. 23. A host cell comprising the nucleic acid molecule of claim 22.

24. General formula (Pc-L a 1. A method for preparing a ligand-drug conjugate represented by formula (I) or a pharmaceutically acceptable salt thereof, comprising the steps of: 【Transformation 8】 After reducing Pc, the general formula (L a -YD) and the compound represented by the general formula (Pc-L a obtaining a compound represented by the formula: Among them, Pc is the anti-TROP-2 antibody or antigen-binding fragment thereof according to any one of claims 17 to 21; n, m, W, L 2 , L 3 , R 1 , R 2 , R 5 , R 6 and R 7 is as defined in claim 12, method.

25. An antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 16, or an anti-TROP-2 antibody or an antigen-binding fragment thereof according to any one of claims 17 to 21; and one or more pharmaceutically acceptable excipients, diluents, or vectors, Pharmaceutical compositions.

26. A drug for treating and / or preventing tumors and cancers, comprising the antibody-drug conjugate or pharmaceutically acceptable salt thereof according to any one of claims 1 to 16, the anti-TROP-2 antibody or antigen-binding fragment thereof according to any one of claims 17 to 21, or the pharmaceutical composition according to claim 25, wherein the tumors and cancers are selected from the group consisting of head and neck squamous cell carcinoma, head and neck cancer, brain cancer, glioma, glioblastoma multiforme, neuroblastoma, central nervous system cancer, neuroendocrine tumor, pharyngeal cancer, pharyngeal squamous cell carcinoma, oral squamous cell carcinoma, nasopharyngeal carcinoma, The drug is for the following cancers: esophageal cancer, thyroid cancer, malignant pleural mesothelioma, lung cancer, breast cancer, liver cancer, hepatobiliary cancer, pancreatic cancer, stomach cancer, gastrointestinal cancer, intestinal cancer, colon cancer, colorectal cancer, kidney cancer, clear cell renal cell carcinoma, ovarian cancer, endometrial cancer, cervical cancer, bladder cancer, prostate cancer, testicular cancer, skin cancer, melanoma, leukemia, lymphoma, bone cancer, chondrosarcoma, myeloma, multiple myeloma, myelodysplastic syndrome, Krueckenberg tumor, myeloproliferative neoplasm, squamous cell carcinoma, Ewing sarcoma, urothelial cancer, or Merkel cell carcinoma.

27. The drug described in claim 26, wherein the lymphoma is selected from Hodgkin's lymphoma, non-Hodgkin's lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, primary mediastinal large B-cell lymphoma, mantle cell lymphoma, small lymphocytic lymphoma, T-cell / histiocyte-rich large B-cell lymphoma, and lymphoplasmacytic lymphoma; the lung cancer is selected from non-small cell lung cancer and small cell lung cancer; and the leukemia is selected from chronic myeloid leukemia, acute myeloid leukemia, lymphocytic leukemia, lymphoblastic leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, and myeloid cell leukemia.

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