Antibody-drug conjugate against CDH17 and use thereof

Abstract

An antibody-drug conjugate and a pharmaceutical composition comprising the antibody-drug conjugate, wherein the antibody is an antibody that binds to CDH17 or an antigen-binding fragment thereof. Use of the antibody-drug conjugate and the composition in preventing and / or treating cancer.

Description

Antibody drug conjugates against CDH17 and uses thereof

[0001] This international patent application claims priority to Chinese patent application No. 202410636042.8, filed May 21, 2024, the entire contents of which are incorporated herein by reference for all purposes. TECHNICAL FIELD

[0002] The present invention relates to antibody drug conjugates against CDH17 and uses thereof, in particular in the treatment of cancer. BACKGROUND

[0003] Cadherin-17 (CDH17) is a new member of the cadherin family, which was first identified from a mouse liver cell cDNA library. It was named Liver-Intestine cadherin (LI-cadherin) because it was expressed only in the liver and small intestine of mice (Berndorff, D. et. al., 1994, Journal of Cell Biology, 125, 1353-1369). Recent studies have shown that it plays an important role in the invasion and metastasis of various tumors.

[0004] The human CDH17 gene is located on chromosome 8q22.1, which encodes a protein belonging to the 7D-cadherin family. CDH17 is often expressed in embryonic, adult intestinal epithelial cells and some pancreatic duct epithelial cells, but there is almost no significant expression in hepatocytes, esophageal epithelial cells and gastric mucosa in healthy people (Gul, I. S. et. al., 2017, Experimental Cell Research, 358, 3-9). Its structure has some homology with classic cadherins, but CDH17 has its unique structure: first, the extracellular region of CDH17 is composed of 7 repeat sequences, which is different from the 5 of non-classical cadherins; secondly, in the EC1 cell adhesion recognition region of the extracellular region, CDH17 contains a segment of AAL sequence instead of the corresponding HAV sequence of classic cadherins; thirdly, the cytoplasmic tail of CDH17 has only 20 amino acid residues, while the cytoplasmic tail of classic cadherins has 150-160 amino acid residues. Studies have shown that structural abnormalities and functional disorders of cadherins are closely related to tumor invasion and metastasis (Pandil, China Medical Frontier, 2012, 7(8): 10-11). Because CDH17 has a short cytoplasmic tail domain, CDH17 cannot interact with the catenin network or the actin cytoskeleton (Kreft, B. et. al., 1997, Journal of Cell Biology, 136, 1109-1121). Therefore, CDH17 is classified as a variant of classic cadherins (Nollet, F. et. al., 2000, Journal of Molecular Biology, 299, 551-572).

[0005] Cadherins play a biological role by mediating calcium-dependent intercellular connections. The ligand of classic cadherins is calcium catenin, including α-chain protein, β-chain protein, γ-chain protein and P120 protein. Cadherins interact with intracellular cytoskeleton through calcium catenin to regulate intercellular adhesion function. CDH17, as a functional Ca 2+Dependent cell adhesion molecules play a role, but CDH17 does not function through tight junctions with the intracellular calpain way of the cell scaffold myosin, but through direct connection with the cell scaffold for its cell adhesion (Marshall, J. F., 2018, Clinical Cancer Research, 24, 253-255). At the same time, studies have found that the expression of CDH17 is an independent prognostic predictor of patient survival (Lee, H. J. et. al., 2010, Gastroenterology, 139, 213-25.e3). The expression of CDH17 has an impact on the prognosis of patients with lymph node-negative gastric cancer, which may reflect the role of the protein in maintaining polarity and normal intercellular adhesion.

[0006] Gastric cancer is one of the more common malignant tumors of the digestive system, with a high incidence and mortality rate (Zuo Tingting et al., China's epidemiology of gastric cancer status [J]. Chinese Journal of Clinical Oncology, 2017, 44(1): 52-58), invasion and metastasis of tumor cells is one of the main reasons for poor prognosis of patients. The decrease in cell adhesion mediated by some adhesion molecules in gastric cancer is a crucial factor in the occurrence of invasion and metastasis. Clinical case data studies have shown that there is an abnormal splice variant of CDH17 in gastric cancer, especially the expression of E-cadherin (CDH17) with exon 8 or exon 9 deletion is dominant in gastric cancer (Becker, K. F. et. al., 1993, Human Molecular Genetics, 2, 803-804). CDH17 has Ca 2+CDH17 has the ability to regulate homotypic cell adhesion and does not depend on cytoskeletal interactions, which indicates that CDH17 plays an important role in tumor metastasis (Ito, R. et. al., 2005, Virchows Archiv, 447, 717-722). CDH17 can serve as a more specific and sensitive marker for gastrointestinal tumors, and CDH17 can provide relevant diagnostic evidence as a marker for judging primary gastrointestinal tumors (Wong, B. W. et. al., 2003, Biochemical and Biophysical Research Communications, 311, 618-624). At the same time, the high expression of CDH17 is closely related to the poor clinicopathological features of gastric cancer, such as positive correlation with histological stage, tumor infiltration and LN metastasis, so the high expression of CDH17 can be an important indicator for predicting the progression and prognosis of gastric cancer (Long, Z. W. et. al., 2015, World Journal of Gastroenterology, 21, 3694-3705; Park, S. S. et. al., 2007, Annals of Surgical Oncology, 14, 94-99; Li, R. et. al., 2017, International Journal of Oncology, 50, 15-22). Cell experiments and animal experiments show that knocking down CDH17 can lead to inactivation of the Wnt signaling pathway, thereby inhibiting the invasive activity of cancer cells (Liu, L. X. et. al, Hepatology, 2010, 51, 358). CDH17 can regulate the Wnt / β-catenin signaling pathway to affect downstream effectors, thereby affecting the proliferation, invasion and apoptosis of gastric cancer cells (Qu, L. P. et. al., 2017, European Review for Medical and Pharmacological Sciences, 21, 1234-1241). In addition, in vitro experimental studies have found that inhibition of CDH17 can reduce the proliferation of gastric cancer cell line MKN28 in vitro and increase its apoptosis, and significantly reduce its tumorigenicity in vivo. In summary, CDH17 is abnormally expressed in gastric cancer tissues and is related to the occurrence, development, invasion, metastasis and poor prognosis of gastric cancer.

[0007] Hepatocellular carcinoma is the most common among liver malignancies and the third most common cause of cancer-related death worldwide (Llovet, J.M. et. al., 2003, Lancet, 362, 1907-1917). CDH17 can be present in the fetal liver and gastrointestinal tract during embryogenesis, but the gene expression is silenced in healthy adult liver and gastric tissue (Lee, N.P. et. al., 2010, Biochimica et Biophysica Acta, 1806, 138-145). Overexpression of CDH17 can be detected in approximately 80% of patients in human hepatocellular carcinoma cell lines (Wang, X.Q. et. al., 2005, Clinical Cancer Research, 11, 483-489). Liu et al. confirmed that overexpression of CDH17 is closely related to advanced tumor stage and tumor infiltration by experimental analysis of 43 patients with liver cancer. Targeting CDH17 can inactivate the Wnt signaling pathway and activate tumor suppressor genes, thereby inhibiting hepatocellular carcinoma tumor growth (Wang, X.Q. et. al., 2005, Clinical Cancer Research, 11, 483-489). If CDH17 is experimentally treated using lentivirus carrying short hairpin RNA (shRNA) against CDH17 in nude mice bearing tumors, the growth of xenograft tumors can be inhibited. This fully demonstrates the possibility of targeting and inhibiting CDH17 in the clinical treatment of hepatocellular carcinoma. Wang et al. (Wang, Y. et. al., 2013, PLoS ONE, 8, e72386) treated hepatocellular carcinoma using a monoclonal antibody (Lic5) against CDH17 antigen, and the results showed that reducing the expression of CDH17 played an important role in the treatment of hepatocellular carcinoma. Targeting CDH17 antibodies can specifically inhibit CDH17, and then inhibit tumor growth by inactivating the Wnt / β-catenin pathway. This confirms that CDH17 can inhibit hepatocellular carcinoma by targeting the Wnt / β-catenin pathway (Qiu, H.B. et. al., 2019, PLoS ONE, 14, Article ID: e0217124). In summary, the expression of CDH17 in liver cancer plays a pro-tumor role and can be used as a target for the treatment of liver cancer.

[0008] Han et al. (Han, Z. et. al., 2017, Chinese Journal of Cellular and Molecular Immunology, 33, 606-610) found that CDH17 was highly expressed in colorectal cancer, and down-regulating the expression of CDH17 gene inhibited the invasion and metastasis of colon cancer cells. Studies have also shown that the expression level of CDH17 is an important prognostic predictor affecting the survival of colorectal cancer patients, and CDH17 has certain clinical application value, for example, as a molecular marker for disease stage classification and evaluation of treatment outcome of colorectal cancer (Kwak, J. M. et. al., 2007, Diseases of the Colon & Rectum, 50, 1873-1880). Studies have also shown that low expression of CDH17 is associated with tumor dedifferentiation, lymphatic vessel invasion, lymph node metastasis and advanced pTNM stage, and is considered an important prognostic factor for colorectal cancer (Ratto, C. et. al., 1998, Diseases of the Colon & Rectum, 41, 1033-1049). Through different cell system experimental observation and analysis, it was found that primary colorectal cancer with low expression of CDH17 might have stronger invasiveness (Takamura, M. et. al., 2004, Cancer Letters, 212, 253-259).

[0009] It has been shown that pancreas, as a component of the gastrointestinal tract, plays a role in the morphological organization of the liver and the intestine. Disruption of CDH17 expression or function also leads to increased tumor cell migration and hyperproliferation during the development of pancreatic tumors (Ivanov, D. B. et. al., 2001, Biochemistry (Moscow), 66, 1174-1186). Takamura et al. (Takamura, M. et. al., 2003, Cancer Science, 94, 425-430) detected strong expression of CDH17 in well-differentiated pancreatic cancer, but not in differentiated areas and poorly differentiated carcinomas. However, it has also been shown that CDH17 is essential for maintaining the tumorigenic activity of pancreatic cancer cells in vitro and for promoting tumor growth in vivo (Kleeff, J. et. al., 2016, Nature Reviews Disease Primers, 2, Article No. 16022). Liu et al. (Liu, X. et. al., 2019, Cancer Letters, 454, 204-214) used siRNA, shRNA, and CRISPR techniques to knock out CDH17 and established the corresponding stable cell lines. Loss-of-function studies were also performed to comprehensively study the potential mechanisms of CDH17 in regulating the occurrence and development of pancreatic cancer. The results obtained from in vitro and in vivo experiments showed that CDH17, as a pro-oncogene, can promote the occurrence and development of pancreatic cancer by regulating cell proliferation and apoptosis signaling pathways.

[0010] The current research results show that CDH17 is closely related to the occurrence of various tumors, and the development of an antibody-drug conjugate targeting CDH17 to kill tumor cells is a possible means of treating cancer. SUMMARY

[0011] The present inventors immunized mice with recombinant CDH17 protein, a gene gun, or a combination thereof, obtained multiple strains of antibodies that recognize human and cynomolgus monkey CDH17 recombinant proteins, and obtained humanized antibodies after humanizing these antibodies. The antibodies of the present application have high binding affinity, good endocytosis activity, and tumor killing effect, and are therefore particularly suitable for use in antibody-drug conjugates (ADC). The antibody-drug conjugate (ADC) formed after the antibody of the present application is coupled with a toxin (such as MMAE or DXD and the like) can effectively kill tumor cells and inhibit tumor growth in mice.

[0012] Accordingly, in one aspect, the present application provides an antibody drug conjugate of Formula (I), or a pharmaceutically acceptable salt, isotopologue, tautomer, stereoisomer, polymorph, hydrate or solvate thereof, or mixtures thereof: y (I)

[0013] wherein,

[0014] T is a targeting moiety, and is an antibody or antigen-binding fragment thereof that binds CDH17;

[0015] L is a linker unit;

[0016] D is a biologically active fragment;

[0017] y is selected from an integer or decimal number from 0.1 to 20.

[0018] In another aspect, the present application provides a pharmaceutical composition comprising the antibody drug conjugate of the present application, and optionally a pharmaceutically acceptable carrier or excipient.

[0019] In another aspect, the present application provides a method for preventing and / or treating a disease in a subject in need thereof, comprising administering to the subject the antibody drug conjugate of the present application, or a pharmaceutically acceptable salt, isotopologue, tautomer, stereoisomer, polymorph, hydrate or solvate thereof, or mixtures thereof.

[0020] In another aspect, the antibody drug conjugate of the present application, or a pharmaceutically acceptable salt, isotopologue, tautomer, stereoisomer, polymorph, hydrate or solvate thereof, or mixtures thereof, for use in preventing and / or treating a disease.

[0021] In another aspect, the present application provides use of the antibody drug conjugate of the present application, or a pharmaceutically acceptable salt, isotopologue, tautomer, stereoisomer, polymorph, hydrate or solvate thereof, or mixtures thereof, in the manufacture of a medicament for preventing and / or treating a disease.

[0022] In a preferred embodiment, the disease is a cancer, for example a cancer associated with CDH17 expression;

[0023] Preferably, the disease is selected from neuroendocrine tumors, gastric cancer, colon cancer, rectal cancer, small intestine cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, esophageal carcinoma, renal cell carcinoma, liver cancer, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, embryonal carcinosarcoma, cervical cancer, uterine cancer, testicular cancer, lung cancer, small cell lung cancer, non-small cell lung cancer, bladder cancer or epithelial carcinoma.

[0024] More preferably, the disease is selected from gastric cancer, liver cancer (e.g. hepatocellular carcinoma), colorectal cancer and pancreatic cancer. BRIEF DESCRIPTION OF DRAWINGS

[0025] Figure 1. Western blot detection of eukaryotic recombinant expressed human and cynomolgus CDH17 protein.

[0026] Figure 2. FACS detection of stable cell line HEK293-hCDH17 and HEK293-cyCDH17 cells.

[0027] Figure 3. FACS detection of stable cell line CHO-hCDH17 cells.

[0028] Figure 4. BLI profile of anti-CDH17 chimeric antibody binding to hCDH17.

[0029] Figure 5. Binding of anti-CDH17 chimeric antibody to tumor cells.

[0030] Figure 6. Endocytosis of anti-CDH17 chimeric antibody.

[0031] Figure 7. BLI profile of anti-CDH17 humanized antibody binding to hCDH17.

[0032] Figure 8A-8B. Binding of anti-CDH17 humanized antibody to tumor cells.

[0033] Figure 9A-9B. Endocytosis of anti-CDH17 humanized antibody.

[0034] Figure 10. Killing of tumor cells SNU-16 by anti-CDH17 chimeric antibody ADC (ADC-1 to ADC-6 and ADC-Reference 1).

[0035] Figure 11. Killing of tumor cells SNU-16 by anti-CDH17 chimeric antibody ADC (ADC-7 to ADC-12 and ADC-Reference 2).

[0036] Figure 12. Killing of tumor cells SNU-16 by anti-CDH17 humanized antibody ADC.

[0037] Figure 13. Killing of tumor cells ASPC-1 by anti-CDH17 humanized antibody ADC.

[0038] Figure 14. Bystander effect of anti-CDH17 humanized antibody ADC.

[0039] Figure 15. Inhibition of tumor growth in vivo by anti-CDH17 humanized antibody ADC in mice.

[0040] Figure 16. Effect of anti-CDH17 humanized antibody ADC on body weight of mice. DETAILED DESCRIPTION

[0041] The above features and advantages of the present application, and additional features and advantages thereof, will be more clearly understood from consideration of the following detailed description when taken in conjunction with the accompanying drawings.

[0042] The embodiments described herein with reference to the drawings are explanatory, illustrative, and for general understanding of the present application. The embodiments should not be interpreted in a limiting manner on the scope of the present application. Identical or similar elements and elements having identical or similar functions are denoted by the same reference numerals throughout the specification.

[0043] In the present application, unless otherwise specified, the scientific and technical terms used herein have meanings commonly understood by a person skilled in the art. Also, the terms related to protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, immunology, and laboratory operation procedures used herein are terms and conventional procedures widely used in the corresponding fields. At the same time, in order to better understand the present application, the definitions and explanations of the related terms are provided below.

[0044] Definitions

[0045] Chemical Definitions

[0046] The definitions of specific functional groups and chemical terms are described in more detail below.

[0047] When a numerical range is listed, each value and sub-range within the range is intended to be included. For example, "C 1-6 "alkyl" includes C1, C2, C3, C4, C5, C6, C 1-6 , C 1-5 , C 1-4 , C 1-3 , C 1-2 , C 2-6 , C 2-5 , C 2-4 , C 2-3 , C 3-6 , C 3-5 , C 3-4 , C 4-6 , C 4-5 and C5-6 alkyl.

[0048] "C 1-10 "alkyl" refers to a straight or branched chain saturated hydrocarbon group having from 1 to 10 carbon atoms. In some embodiments, C 1-8 alkyl, C 1-6 alkyl and C 1-4 alkyl are preferred. C 1-6 Examples of alkyl groups include: methyl (Ci), ethyl (C2), n-propyl (C3), i-propyl (C3), n-butyl (C4), t-butyl (C4), sec-butyl (C4), i-butyl (C4), n-pentyl (C5), 3-pentyl (C5), amyl (C5), neopentyl (C5), 3-methyl-2-butyl (C5), t-amyl (C5), and n-hexyl (C6). The term "C 1-6 alkyl" also includes heteroalkyl groups in which one or more (e.g., 1, 2, 3, or 4) carbon atoms are replaced by a heteroatom (e.g., oxygen, sulfur, nitrogen, boron, silicon, phosphorus). An alkyl group can be optionally substituted by one or more substituents, e.g., by 1 to 5 substituents, 1 to 3 substituents, or 1 substituent. Conventional alkyl abbreviations include: Me (-CH3), Et (-CH2CH3), iPr (-CH(CH3)2), nPr (-CH2CH2CH3), n-Bu (-CH2CH2CH2CH3), or i-Bu (-CH2CH(CH3)2).

[0049] "C 2-10 "alkenyl" refers to a straight or branched chain hydrocarbon group having from 2 to 10 carbon atoms and at least one carbon-carbon double bond. In some embodiments, C 2-6 alkenyl is preferred. C 2-6 Examples of alkenyl groups include: ethenyl (C2), 1-propenyl (C3), 2-propenyl (C3), 1-butenyl (C4), 2-butenyl (C4), butadienyl (C4), pentenyl (C5), pentadienyl (C5), hexenyl (C6), and the like. The term "C 2-6 alkenyl" also includes heteroalkenyl groups in which one or more (e.g., 1, 2, 3, or 4) carbon atoms are replaced by a heteroatom (e.g., oxygen, sulfur, nitrogen, boron, silicon, phosphorus). An alkenyl group can be optionally substituted by one or more substituents, e.g., by 1 to 5 substituents, 1 to 3 substituents, or 1 substituent.

[0050] "C 2-10 "alkynyl" refers to a straight or branched chain hydrocarbon group having from 2 to 10 carbon atoms, at least one carbon-carbon triple bond, and optionally one or more carbon-carbon double bonds. In some embodiments, C 2-6 alkynyl is preferred. C 2-6Examples of alkynyl groups include, but are not limited to: ethynyl (C2), 1- propynyl (C3), 2-propynyl (C3), 1-butynyl (C4), 2-butynyl (C4), pentynyl (C5), hexynyl (C6), and the like. The term "C 2-6 Alkynyl groups also include heteroalkynyl groups wherein one or more (e.g., 1, 2, 3, or 4) carbon atoms are replaced by a heteroatom (e.g., oxygen, sulfur, nitrogen, boron, silicon, phosphorus). Alkynyl groups can be optionally substituted by one or more substituents, e.g., by 1 to 5 substituents, 1 to 3 substituents, or 1 substituent.

[0051] "Halo" or "halogen" means fluoro (F), chloro (CI), bromo (Br), and iodo (I).

[0052] Thus, "C 1-6 Alkyl" means an optionally substituted straight or branched hydrocarbon chain. In some embodiments, C 1-6 Alkyl" means an optionally substituted straight or branched hydrocarbon chain. In some embodiments, C 1-4 Alkyl" means an optionally substituted straight or branched hydrocarbon chain. In some embodiments, C 1-2 Alkyl" means an optionally substituted straight or branched hydrocarbon chain. In some embodiments, C

[0053] "C 1-6 Alkoxy" means an -OR group, wherein R is an alkyl group as defined above. C 1-6 Alkoxy" means an -OR group, wherein R is an alkyl group as defined above. C 1-4 Alkoxy" means an -OR group, wherein R is an alkyl group as defined above. C

[0054] "C 1-10 Alkylene" means a divalent group formed by removing an additional hydrogen from a C 1-10 Alkylene" means a divalent group formed by removing an additional hydrogen from a C 1-8 Alkylene" means a divalent group formed by removing an additional hydrogen from a C 1-6 Alkylene" means a divalent group formed by removing an additional hydrogen from a C 1-4 Alkylene" means a divalent group formed by removing an additional hydrogen from a C 1-2Alkylene is preferred. Unsubstituted alkylene groups include, but are not limited to: methylene (-CH2-), ethylene (-CH2CH2-), propylene (-CH2CH2CH2-), butylene (-CH2CH2CH2CH2-), pentylene (-CH2CH2CH2CH2CH2-), hexylene (-CH2CH2CH2CH2CH2CH2-), and the like. Exemplary substituted alkylene groups, e.g., alkylene groups substituted with one or more alkyl (methyl) groups, include, but are not limited to: substituted methylene (-CH(CH3)-, -C(CH3)2-), substituted ethylene (-CH(CH3)CH2-, -CH2CH(CH3)-, -C(CH3)2CH2-, -CH2C(CH3)2-), substituted propylene (-CH(CH3)CH2CH2-, -CH2CH(CH3)CH2-, -CH2CH2CH(CH3)-, -C(CH3)2CH2CH2-, -CH2C(CH3)2CH2-, -CH2CH2C(CH3)2-), and the like. 2-

[0055] "C 2-10 Alkenylene" refers to a divalent radical formed by the removal of two hydrogens from a C 2-10 alkenyl group, and can be substituted or unsubstituted. In some embodiments, C 2-8 alkenylene, C 4-6 alkenylene, C 1-4 alkenylene, C 2-4 alkenylene is preferred.

[0056] "C 2-10 Alkynylene" refers to a divalent radical formed by the removal of two hydrogens from a C 2-10 alkynyl group, and can be substituted or unsubstituted. In some embodiments, C 2-8 alkynylene, C 4-6 alkynylene, C 1-4 alkynylene, C 2-4 alkynylene is preferred.

[0057] "C 3-10 Cycloalkyl" refers to a non-aromatic hydrocarbon radical of from 3 to 10 ring carbon atoms and zero heteroatoms. In some embodiments, C 5-7 cycloalkyl, C 3-7 cycloalkyl and C 3-5 cycloalkyl is particularly preferred, more preferred C 5-6 ​Cycloalkyl. Cycloalkyl also includes ring systems in which the above cycloalkyl ring is fused with one or more aryl or heteroaryl rings, where the point of attachment is on the cycloalkyl ring, and in such cases the number of carbons refers to the number of carbons in the cycloalkyl ring. Exemplary cycloalkyl groups include, but are not limited to, cyclopropyl (C3), cyclopropenyl (C3), cyclobutyl (C4), cyclobutenyl (C4), cyclopentyl (C5), cyclopentenyl (C5), cyclohexyl (C6), cyclohexenyl (C6), cyclohexadienyl (C6), cycloheptyl (C7), cycloheptenyl (C7), cycloheptadienyl (C7), cycloheptatrienyl (C7), and the like. Cycloalkyl groups can be optionally substituted by one or more substituents, for example, by 1 to 5 substituents, 1 to 3 substituents, or 1 substituent.

[0058] "3-10 membered heterocyclyl" refers to a 3- to 10-membered non-aromatic ring system having ring carbon atoms and 1 to 5 ring heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, sulfur, boron, phosphorus, and silicon. In heterocyclyl groups containing one or more nitrogen atoms, the point of attachment can be a carbon or nitrogen atom, as valence permits. In some embodiments, 4-9 membered heterocyclyl groups are preferred, which are 4- to 9-membered non-aromatic ring systems having ring carbon atoms and 1 to 5 ring heteroatoms; in some embodiments, 5-8 membered heterocyclyl groups are preferred, which are 5- to 8-membered non-aromatic ring systems having ring carbon atoms and 1 to 5 ring heteroatoms; in some embodiments, 3-8 membered heterocyclyl groups are preferred, which are 3- to 8-membered non-aromatic ring systems having ring carbon atoms and 1 to 4 ring heteroatoms; 3-7 membered heterocyclyl groups are preferred, which are 3- to 7-membered non-aromatic ring systems having ring carbon atoms and 1 to 3 ring heteroatoms; 3-5 membered heterocyclyl groups are preferred, which are 3- to 5-membered non-aromatic ring systems having ring carbon atoms and 1 to 2 ring heteroatoms; 4-7 membered heterocyclyl groups are preferred, which are 4- to 7-membered non-aromatic ring systems having ring carbon atoms and 1 to 3 ring heteroatoms; 4-6 membered heterocyclyl groups are preferred, which are 4- to 6-membered non-aromatic ring systems having ring carbon atoms and 1 to 3 ring heteroatoms; 3-5 membered heterocyclyl groups are preferred, which are 3- to 5-membered non-aromatic ring systems having ring carbon atoms and 1 to 3 ring heteroatoms; and 5-6 membered heterocyclyl groups are preferred, which are 5- to 6-membered non-aromatic ring systems having ring carbon atoms and 1 to 3 ring heteroatoms. Heterocyclyl also includes ring systems in which the above heterocyclyl ring is fused with one or more cycloalkyl rings, wherein the point of attachment is on the cycloalkyl ring, or with one or more aryl or heteroaryl rings, wherein the point of attachment is on the heterocyclyl ring; and in such cases the number of ring members continues to refer to the number of ring members in the heterocyclyl ring system. Exemplary 3-membered heterocyclyl groups containing one heteroatom include, but are not limited to: aziridinyl, oxiranyl, thiorenyl. Exemplary 4-membered heterocyclyl groups containing one heteroatom include, but are not limited to: azetidinyl, oxetanyl, and thietanyl. Exemplary 5-membered heterocyclyl groups containing one heteroatom include, but are not limited to: tetrahydrofuranyl, dihydrofuranyl, tetrahydrothiophenyl, dihydrothiophenyl, pyrrolidinyl, dihydropyrrolyl, and pyrrolyl-2,5-dione. Exemplary 5-membered heterocyclyl groups containing two heteroatoms include, but are not limited to: dioxolanyl, oxasulfuranyl, disulfuranyl, and oxazolidin-2-one. Exemplary 5-membered heterocyclyl groups containing three heteroatoms include, but are not limited to: triazolinyl, oxadiazolinyl, and thiadiazolinyl. Exemplary 6-membered heterocyclyl groups containing one heteroatom include, but are not limited to: piperidinyl, tetrahydropyranyl, dihydropyridinyl, and thianyl. Exemplary 6-membered heterocyclyl groups containing two heteroatoms include, but are not limited to: piperazinyl, morpholinyl, dithianyl, dioxanyl.Exemplary 6-membered heterocyclyl groups containing three heteroatoms include, without limitation, hexahydrotriazinanyl. Exemplary 7-membered heterocyclyl groups containing one heteroatom include, without limitation, azepanyl, oxepanyl, and thiepanyl. Exemplary 5-membered heterocyclyl groups fused to a C6aryl ring (also referred to herein as 5,6-bicyclic heterocyclyl groups) include, without limitation, indolinyl, isoindolinyl, dihydrobenzofuranyl, dihydrobenzothienyl, benzoxazolinonyl, and the like. Exemplary 6-membered heterocyclyl groups fused to a C6aryl ring (also referred to herein as 6,6-bicyclic heterocyclyl groups) include, without limitation, tetrahydroquinolinyl, tetrahydroisoquinolinyl, and the like. Heterocyclyl groups can be optionally substituted by one or more substituents, for example, by 1 to 5 substituents, 1 to 3 substituents, or 1 substituent.

[0059] "C 6-10 Aryl" refers to a monocyclic or polycyclic (e.g., bicyclic) 4n+2 aromatic ring system (e.g., having 6 or 10 p electrons shared in a cyclic array) having from 6-10 ring carbon atoms and zero heteroatoms. In some embodiments, the aryl group has six ring carbon atoms ("C6 aryl"; e.g., phenyl). In some embodiments, the aryl group has ten ring carbon atoms ("C10 aryl"; e.g., naphthyl, e.g., 1-naphthyl and 2-naphthyl). Aryl also includes ring systems in which the above-described aryl ring is fused to one or more cycloalkyl or heterocyclyl rings, with the point of attachment being on the aryl ring, in which case the number of carbon atoms designates the number of carbon atoms in the aryl ring system. Aryl groups can be optionally substituted by one or more substituents, for example, by 1 to 5 substituents, 1 to 3 substituents, or 1 substituent. 10 Aryl" refers to a monocyclic or polycyclic (e.g., bicyclic) 4n+2 aromatic ring system (e.g., having 6 or 10 p electrons shared in a cyclic array) having from 6-10 ring carbon atoms and zero heteroatoms. In some embodiments, the aryl group has six ring carbon atoms ("C6 aryl"; e.g., phenyl). In some embodiments, the aryl group has ten ring carbon atoms ("C10 aryl"; e.g., naphthyl, e.g., 1-naphthyl and 2-naphthyl). Aryl also includes ring systems in which the above-described aryl ring is fused to one or more cycloalkyl or heterocyclyl rings, with the point of attachment being on the aryl ring, in which case the number of carbon atoms designates the number of carbon atoms in the aryl ring system. Aryl groups can be optionally substituted by one or more substituents, for example, by 1 to 5 substituents, 1 to 3 substituents, or 1 substituent.

[0060] "5-10 membered heteroaryl" refers to a radical of a 5-10 membered monocyclic or bicyclic 4n+2 aromatic ring system (e.g., having 6, 10, or 14 π electrons shared in a cyclic array) having ring carbon atoms and 1-4 ring heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur. In heteroaryl groups containing one or more nitrogen atoms, the point of attachment can be a carbon or nitrogen atom, as valence permits. Heteroaryl bicyclic systems can include one or more heteroatoms in one or both rings. Heteroaryl also includes ring systems wherein an above-described heteroaryl ring is fused with one or more cycloalkyl or heterocyclyl rings, and the point of attachment is on the heteroaryl ring, in which case the number of carbon atoms indicates the number of carbon atoms in the heteroaryl ring system. In some embodiments, 5-9 membered heteroaryl is preferred, which is a 5-9 membered monocyclic or bicyclic 4n+2 aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms. In other embodiments, 5-6 membered heteroaryl is particularly preferred, which is a 5-6 membered monocyclic or bicyclic 4n+2 aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms. Exemplary 5-membered heteroaryl groups containing one heteroatom include, but are not limited to, pyrrolyl, furanyl, and thiophenyl. Exemplary 5-membered heteroaryl groups containing two heteroatoms include, but are not limited to, imidazolyl, pyrazolyl, oxazolyl, isoxazolyl, thiazolyl, and isothiazolyl. Exemplary 5-membered heteroaryl groups containing three heteroatoms include, but are not limited to, triazolyl, oxadiazolyl (e.g., 1,2,4-oxadiazolyl), and thiadiazolyl. Exemplary 5-membered heteroaryl groups containing four heteroatoms include, but are not limited to, tetrazolyl. Exemplary 6-membered heteroaryl groups containing one heteroatom include, but are not limited to, pyridinyl. Exemplary 6-membered heteroaryl groups containing two heteroatoms include, but are not limited to, pyridazinyl, pyrimidinyl, and pyrazinyl. Exemplary 6-membered heteroaryl groups containing three or four heteroatoms include, but are not limited to, triazinyl and tetrazinyl, respectively. Exemplary 7-membered heteroaryl groups containing one heteroatom include, but are not limited to, azepinyl, oxepinyl, and thiepinyl. Exemplary 5,6-bicyclic heteroaryl groups include, but are not limited to, indolyl, isoindolyl, indazolyl, benzotriazolyl, benzothiophenyl, isobenzothiophenyl, benzofuranyl, benzoisofuranyl, benzoimidazolyl, benzoxazolyl, benzoisoxazolyl, benzoxadiazolyl, benzothiazolyl, benzoisothiazolyl, benzothiadiazolyl, indolizinyl, and purinyl. Exemplary 6,6-bicyclic heteroaryl groups include, but are not limited to, naphthyridinyl, pteridinyl, quinolinyl, isoquinolinyl, cinnolinyl, quinoxalinyl, phthalazinyl, and quinazolinyl. Heteroaryl groups can be optionally substituted with one or more substituents, e.g., with 1 to 5 substituents, 1 to 3 substituents, or 1 substituent.

[0061] "C 3-10 "Cycloalkylene", "3-10 membered heterocyclylene", "C 6-10 "Arylene" and "5-10 membered heteroarylene" refer to divalent radical of a 5-10 membered monocyclic or bicyclic 4n+2 aromatic ring system (e.g., having 6, 10, or 14 π electrons shared in a cyclic array) having ring carbon atoms and 1-4 ring heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur. In heteroaryl groups containing one or more nitrogen atoms, the point of attachment can be a carbon or nitrogen atom, as valence permits. Heteroaryl bicyclic systems can include one or more heteroatoms in one or both rings. Heteroaryl also includes ring systems wherein an above-described heteroaryl ring is fused with one or more cycloalkyl or heterocyclyl rings, and the point of attachment is on the heteroaryl ring, in which case the number of carbon atoms indicates the number of carbon atoms in the heteroaryl ring system. In some embodiments, 5-9 membered heteroaryl is preferred, which is a 5-9 membered monocyclic or bicyclic 4n+2 aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms. In other embodiments, 5-6 membered heteroaryl is particularly preferred, which is a 5-6 membered monocyclic or bicyclic 4n+2 aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms. Exemplary 5-membered heteroaryl groups containing one heteroatom include, but are not limited to, pyrrolyl, furanyl, and thiophenyl. Exemplary 5-membered heteroaryl groups containing two heteroatoms include, but are not limited to, imidazolyl, pyrazolyl, oxazolyl, isoxazolyl, thiazolyl, and isothiazolyl. Exemplary 5-membered heteroaryl groups containing three heteroatoms include, but are not limited to, triazolyl, oxadiazolyl (e.g., 1,2,4-oxadiazolyl), and thiadiazolyl. Exemplary 5-membered heteroaryl groups containing four heteroatoms include, but are not limited to, tetrazolyl. Exemplary 6-membered heteroaryl groups containing one heteroatom include, but are not limited to, pyridinyl. Exemplary 6-membered heteroaryl groups containing two heteroatoms include, but are not limited to, pyridazinyl, pyrimidinyl, and pyrazinyl. Exemplary 6-membered heteroaryl groups containing three or four heteroatoms include, but are not limited to, triazinyl and tetrazinyl, respectively. Exemplary 7-membered heteroaryl groups containing one heteroatom include, but are not limited to, azepinyl, oxepinyl, and thiepinyl. Exemplary 5,6-bicyclic heteroaryl groups include, but are not limited to, indolyl, isoindolyl, indazolyl, benzotriazolyl, benzothiophenyl, isobenzothiophenyl, benzofuranyl, benzoisofuranyl, benzoimidazolyl, benzoxazolyl, benzoisoxazolyl, benzoxadiazolyl, benzothiazolyl, benzoisothiazolyl, benzothiadiazolyl, indolizinyl, and purinyl. Exemplary 6,6-bicyclic heteroaryl groups include, but are not limited to, naphthyridinyl, pteridinyl, quinolinyl, isoquinolinyl, cinnolinyl, quinoxalinyl, phthalazinyl, and quinazolinyl. Heteroaryl groups can be optionally substituted with one or more substituents, e.g., with 1 to 5 substituents, 1 to 3 substituents, or 1 substituent.3-10 cycloalkyl", "3-10 membered heterocyclyl", "C 6-10 aryl" and "5-10 membered heteroaryl" to the parent moiety and can be substituted or unsubstituted. "C 3-10 cycloalkyl", "3-10 membered heterocyclyl", "C 6-10 aryl" and "5-10 membered heteroaryl" are as previously described.

[0062] "Carbonyl", whether used alone or as part of a larger moiety, e.g., amino carbonyl, oxalyl, represents -C(O)-.

[0063] "Oxo" represents =O.

[0064] "Thio" represents =S.

[0065] Alkyl, alkenyl, alkynyl, alkylene, alkenylene, alkynylene, cycloalkyl, heterocyclyl, aryl and heteroaryl groups defined herein are optionally substituted.

[0066] Exemplary substituents on carbon atoms include, but are not limited to: halogen, -CN, -NO2, -N3, -SO2H, -SO3H, -OH, -OR aa , -ON(R bb )2, -N(R bb )2, -N(R bb )3 + X - , -N(OR cc )R bb , -SH, -SR aa , -SSR cc , -C(=O)R aa , -CO2H, -CHO, -C(OR cc )2, -CO2R aa , -OC(=O)R aa , -OCO2R aa , -C(=O)N(R bb )2, -OC(=O)N(R bb )2, -NR bb C(=O)R aa , -NR bb CO2R aa , -NR bb C(=O)N(R bb )2, -C(=NR bb )R aa , -C(=NR bb )OR aa , -OC(=NR bb )R aa, -OC(=NR bb )OR aa , -C(=NR bb )N(R bb )2, -OC(=NR bb )N(R bb )2, -NR bb C(=NR bb )N(R bb )2, -C(=O)NR bb SO2R aa , -NR bb SO2R aa , -SO2N(R bb )2, -SO2R aa , -SO2OR aa , -OSO2R aa , -S(=O)R aa , -OS(=O)R aa , -Si(R aa )3, -OSi(R aa )3, -C(=S)N(R bb )2, -C(=O)SR aa , -C(=S)SR aa , -SC(=S)SR aa , -SC(=O)SR aa , -OC(=O)SR aa , -SC(=O)OR aa , -SC(=O)R aa , -P(=O)2R aa , -OP(=O)2R aa , -P(=O)(R aa )2, -OP(=O)(R aa )2, -OP(=O)(OR cc )2, -P(=O)2N(R bb )2, -OP(=O)2N(R bb )2, -P(=O)(NR bb )2, -OP(=O)(NR bb )2, -NR bb P(=O)(OR cc )2, -NR bb P(=O)(NR bb )2, -P(R cc )2, -P(R cc )3, -OP(R cc )2, -OP(R cc )3, -B(R aa)2, -BR cc )2, -BR aa (OR cc ), alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl, wherein each alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5 R dd groups;

[0067] or two geminal hydrogens on a carbon atom are replaced with a group =0, =S, =NN(R bb )2, =NNR bb C(=0)R aa , =NNR bb C(=0)OR aa , =NNR bb S(=0)2R aa , =NR bb , or =NOR cc ;

[0068] each R aa is independently selected from alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl, or two R aa groups are combined to form a heterocyclyl or heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5 R dd groups;

[0069] each R bb is independently selected from: hydrogen, -OH, -OR aa , -N(R cc )2, -CN, -C(=0)R aa , -C(=0)N(R cc )2, -C02R aa , -S02R aa , -C(=NR cc )OR aa , -C(=NR cc )N(R cc )2, -S02N(R cc )2, -S02R cc , -S02OR cc , -SOR aa , -C(=S)N(R cc )2, -C(=0)SR cc , -C(=S)SR cc , -P(=0)2R aa , -P(=0)(R aa)2, -P(=O)2N(R cc )2, -P(=O)(NR cc )2, alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl, or two R bb groups are combined to form a heterocyclyl or heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5 R dd groups;

[0070] R cc each is independently selected from hydrogen, alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl, or two R cc groups are combined to form a heterocyclyl or heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5 R dd groups;

[0071] R dd each is independently selected from: halogen, -CN, -NO2, -N3, -SO2H, -SO3H, -OH, -OR ee , -ON(R ff )2, -N(R ff )2, -N(R ff )3 + X - , -N(OR ee )R ff , -SH, -SR ee , -SSR ee , -C(=O)R ee , -CO2H, -CO2R ee , -OC(=O)R ee , -OCO2R ee , -C(=O)N(R ff )2, -OC(=O)N(R ff )2, -NR ff C(=O)R ee , -NR ff CO2R ee , -NR ff C(=O)N(R ff )2, -C(=NR ff )OR ee , -OC(=NR ff )R ee , -OC(=NR ff )OR ee , -C(=NR ff )N(Rff )2, -OC(=NR ff )N(R ff )2, -NR ff C(=NR ff )N(R ff )2, -NR ff SO2R ee , -SO2N(R ff )2, -SO2R ee , -SO2OR ee , -OSO2R ee , -S(=O)R ee , -Si(R ee )3, -OSi(R ee )3, -C(=S)N(R ff )2, -C(=O)SR ee , -C(=S)SR ee , -SC(=S)SR ee , -P(=O)2R ee , -P(=O)(R ee )2, -OP(=O)(R ee )2, -OP(=O)(OR ee )2, alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, wherein each alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5 R gg groups, or two geminal R dd substituents can be combined to form =O or =S;

[0072] R ee each is independently selected from alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, aryl, heterocyclyl, and heteroaryl, wherein each alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5 R gg groups;

[0073] R ff each is independently selected from hydrogen, alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl, or two R ff groups combine to form a heterocyclyl or heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5 R gg groups;

[0074] R gg each is independently halogen, -CN, -NO2, -N3, -SO2H, -SO3H, -OH, -OC1-6 alkyl, -ON(C 1-6 alkyl)2, -N(C 1-6 alkyl)2, -N(C 1-6 alkyl)3 + X - , -NH(C 1-6 alkyl)2 + X - , -NH2(C 1-6 alkyl) + X - , -NH3 + X - , -N(OC 1-6 alkyl)(C 1-6 alkyl), -N(OH)(C 1-6 alkyl), -NH(OH), -SH, -SC 1-6 alkyl, -SS(C 1-6 alkyl), -C(=O)(C 1-6 alkyl), -CO2H, -CO2(C 1-6 alkyl), -OC(=O)(C 1-6 alkyl), -OCO2(C 1-6 alkyl), -C(=O)NH2, -C(=O)N(C 1-6 alkyl)2, -OC(=O)NH(C 1- 6alkyl), -NHC(=O)(C 1-6 alkyl), -N(C 1-6 alkyl)C(=O)(C 1-6 alkyl), -NHCO2(C 1-6 alkyl), -NHC(=O)N(C 1-6 alkyl)2, -NHC(=O)NH(C 1-6 alkyl), -NHC(=O)NH2, -C(=NH)O(C 1-6 alkyl), -OC(=NH)(C 1-6 alkyl), -OC(=NH)OC 1-6 alkyl, -C(=NH)N(C 1-6 alkyl)2, -C(=NH)NH(C 1-6 alkyl), -C(=NH)NH2, -OC(=NH)N(C 1-6 alkyl)2, -OC(NH)NH(C 1-6 alkyl), -OC(NH)NH2, -NHC(NH)N(C 1-6 alkyl)2, -NHC(=NH)NH2, -NHSO2(C 1-6 alkyl), -SO2N(C 1-6alkyl)2, -SO2NH(C 1-6 alkyl), -SO2NH2, -SO2C 1-6 alkyl, -SO2OC 1-6 alkyl, -OSO2C 1-6 alkyl, -SOC 1- alkyl, -OSi(C 1-6 alkyl)3, -C(=S)N(C 1-6 alkyl)2, C(=S)NH(C 1-6 alkyl), C(=S)NH2, -C(=O)S(C 1-6 alkyl), -C(=S)SC 1-6 alkyl, -SC(=S)SC 1-6 alkyl, -P(=O)2(C 1-6 alkyl), -P(=O)(C 1-6 alkyl)2, -OP(=O)(C 1-6 alkyl)2, -OP(=O)(OC 1-6 alkyl)2, -C(=O)P(C 1-6 alkyl)2, C 1-6 alkyl, C 1-6 haloalkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C7 cycloalkyl, C6-C 10 aryl, C3-C7 heterocyclyl, C5-C 10 heteroaryl; or two geminal R gg substituents can combine to form =O or =S; wherein X - is a counterion.

[0075] Exemplary substituents on a nitrogen atom include, but are not limited to: hydrogen, -OH, -OR aa , -N(R cc )2, -CN, -C(=O)R aa , -C(=O)N(R cc )2, -CO2R aa , -SO2R aa , -C(=NR bb )R aa , -C(=NR cc )OR aa , -C(=NR cc )N(R cc )2, -SO2N(R cc )2, -SO2R cc , -SO2OR cc , -SOR aa , -C(=S)N(R cc )2, -C(=O)SR cc, -C(=S)SR cc , -P(=O)2R aa , -P(=O)(R aa )2, -P(=O)2N(R cc )2, -P(=O)(NR cc )2, alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl, or two R cc groups attached to a nitrogen atom are combined to form a heterocyclyl or heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5 R dd , R aa , R bb , R cc , and R dd are as described above.

[0076] Other Definitions

[0077] As used herein, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “an antibody drug conjugate” includes a plurality of antibody drug conjugates and reference to “an antibody drug conjugate” in some embodiments includes a plurality of antibody drug conjugates, and so forth.

[0078] The term “comprising” and variations thereof as used herein are intended to encompass the presence of stated elements or steps and / or groups of elements or steps, but do not preclude the presence or addition of one or more other elements or steps or groups of elements or steps, unless otherwise stated or indicated by context.

[0079] As used herein, the term “antibody” refers to an immunoglobulin molecule having the ability to specifically bind to a particular antigen. Such molecules are typically comprised of two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain is comprised of a heavy chain variable region (or domain) (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region is comprised of three domains, CH1, CH2 and CH3. Each light chain is comprised of a light chain variable region (or domain) (abbreviated herein as VL) and a light chain constant region. The light chain constant region is comprised of one domain, CL. The variable regions of the antibody heavy and light chains contain the binding domain that interacts with an antigen. The constant regions of the antibodies can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (such as effector cells) and components of the complement system such as Clq (the first component in the classical pathway of complement activation).

[0080] The heavy chain of an immunoglobulin can be divided into three functional regions: an Fd region, a hinge region, and an Fc region (fragment crystallizable). The Fd region comprises the VH and CHI domains and, in combination with the light chain, forms the Fab (antigen-binding fragment). The Fc fragment is responsible for the effector functions of the immunoglobulin, including, for example, complement fixation and binding to cognate Fc receptors on effector cells. The hinge region, found in IgG, IgA, and IgD immunoglobulin classes, acts as a flexible spacer, allowing the Fab portions to move freely in space relative to the Fc region. The hinge domain is structurally diverse, differing in sequence and length between immunoglobulin classes and subclasses.

[0081] According to crystallographic studies, the immunoglobulin hinge region can be further subdivided into three regions structurally and functionally: the upper hinge, the core hinge, and the lower hinge. The upper hinge includes amino acids from the carboxy-terminal end of CHI to the first residue in the hinge that restricts movement, typically the first cysteine residue that forms an interchain disulfide bond between the two heavy chains. The length of the upper hinge region is related to the flexibility of the fragment of the antibody. The core hinge region contains the inter-heavy chain disulfide bonds. The lower hinge region connects the amino-terminal end of the CH2 domain and includes residues in the CH2 domain. Structural and flexibility-allowed conformational changes in the immunoglobulin hinge region polypeptide sequence can influence the effector functions of the Fc portion of the antibody.

[0082] A "light chain variable region" (VL) or "heavy chain variable region" (VH) is composed of "framework" regions separated by three "complementarity determining regions" or "CDRs." The framework regions serve to align the CDRs that specifically bind to an epitope of an antigen. The CDRs include the amino acid residues in the antibody that are primarily responsible for antigen binding. Both the VL and VH domains comprise, from amino-terminus to carboxy-terminus, the following framework (FR) and CDR regions: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. CDR1, CDR2, and CDR3 of a VL domain are also referred to herein as LCDR1, LCDR2, and LCDR3, respectively; and CDR1, CDR2, and CDR3 of a VH domain are also referred to herein as HCDR1, HCDR2, and HCDR3, respectively.

[0083] The amino acid arrangement of each VL domain and VH domain is consistent with any conventional definition of CDRs. Conventional definitions include the Kabat definition (Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, MD, 1987 and 1991), the Chothia definition (Chothia and Lesk, J. Mol. Biol. 196:901-917, 1987; Chothia et al., Nature 342:878-883, 1989); a composite of Chothia and Kabat CDRs, in which CDR-H1 is a composite of Chothia and Kabat CDRs; the AbM definition used by Oxford Molecular's antibody modeling software; and the CONTACT definition of Martin et al. (world wide web bioinfo.org.uk / abs). Kabat provides a widely used numbering convention (the Kabat numbering system) in which corresponding residues between different heavy chains or between different light chains are given the same number. The present disclosure can use CDRs defined according to any of these numbering systems, but preferred embodiments use CDRs defined according to the Kabat definition.

[0084] Based on the amino acid sequences of the constant regions of the heavy chains of the antibodies, immunoglobulin molecules can be assigned to five classes (isotypes): IgA, IgD, IgE, IgG, and IgM, and can be further divided into subclasses, such as IgGl, IgG2, IgG3, IgG4, IgAl, IgA2, and the like. Based on the amino acid sequences of the light chains, the light chains of the antibodies can be assigned to lambda (l) chains and kappa (K) chains.

[0085] As used herein, the term "antibody" shall be construed in its broadest sense and includes monoclonal antibodies (including full-length monoclonal antibodies), antibody fragments, and multi-specific antibodies (e.g., bi-specific antibodies) containing at least two antigen binding regions. Antibodies can contain additional modifications, such as non-naturally occurring amino acids, mutations in the Fc region, and mutations in glycosylation sites. Antibodies also include post-translationally modified antibodies, fusion proteins containing an antigenic determinant of an antibody, and immunoglobulin molecules containing any other modification to the antigen recognition site, so long as the antibodies exhibit the desired biological activity.

[0086] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies. That is, each antibody in the population is identical except for possible naturally occurring mutations that can be present. Monoclonal antibodies are highly specific, being directed against a single antigen. The term "monoclonal antibody" herein is not limited to antibodies produced through hybridoma technology, nor is it intended to be limited to antibodies produced in any particular manner.

[0087] The term "bispecific antibody" is understood in the context of the present application as an antibody having two different antigen binding regions defined by different antibody sequences. This can be understood as binding to different targets, but also includes binding to different epitopes of one target. The term "bispecific antibody" as used herein is to be understood in its broadest sense, including full-length bispecific antibodies and antigen binding fragments thereof. Bispecific antibodies can contain additional modifications, such as non-naturally occurring amino acids, mutations in the Fc region, and mutations of glycosylation sites. Bispecific antibodies also include post-translationally modified antibodies, fusion proteins containing an antigenic determinant of an antibody, and immunoglobulin molecules containing any other modification of the antigen recognition site, as long as these antibodies exhibit the desired biological activity.

[0088] The term "chimeric antibody" generally refers to an antibody in which the variable region is derived from one species and the constant region is derived from another species. Typically, the variable region is derived from an antibody of an experimental animal such as a rodent ("parental antibody") and the constant region is derived from a human antibody, such that the resulting chimeric antibody elicits a reduced likelihood of adverse immune reactions in a human individual as compared to the parental (e.g., mouse-derived) antibody.

[0089] The term "humanized antibody" generally refers to an antibody in which some or all of the amino acids outside the CDR regions of a non-human antibody (e.g., murine antibody) are replaced with corresponding amino acids from a human immunoglobulin. Small additions, deletions, insertions, substitutions or modifications of amino acids in the CDR regions can also be allowed, as long as they retain the ability of the antibody to bind to a particular antigen. A "humanized antibody" retains antigenic specificity similar to that of the original antibody. "Humanized" forms of non-human (e.g., murine) antibodies can minimally contain a chimeric antibody having sequences derived from non-human immunoglobulin. In some cases, CDR region residues in the human immunoglobulin (recipient antibody) are replaced by CDR region residues of a non-human species (donor antibody) such as mouse, rat, rabbit, or non-human primate that have the desired properties, affinities, and / or capabilities. In some cases, FR region residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, a humanized antibody can contain amino acid modifications not found in the recipient antibody or in the donor antibody. These modifications can be made to further improve antibody performance, such as binding affinity.

[0090] As used herein, the term "antigen-binding fragment" of an antibody refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. It has been shown that the antigen binding function of an antibody can be performed by fragments of a full-length antibody.

[0091] Examples of antigen-binding fragments encompassed within the term "antigen-binding portion" of an antibody include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL, and CH1 domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fab' fragment, which is simply a Fab with part of the hinge region; (iv) a Fd fragment consisting of the VH and CH1 domains; (v) a Fd' fragment having VH and CH1 domains and one or more cysteine residues at the C-terminus of the CH1 domain; (vi) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody; (vii) a dAb fragment, which consists of a VH domain; (viii) an isolated complementarity determining region (CDR); and (ix) a nanobody, a heavy chain variable region containing a single variable domain and two constant domains. Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv)). Such single chain antibodies are also intended to be encompassed within the term "antigen-binding portion" of an antibody. Furthermore, this term also includes "linear antibodies", which comprise a pair of tandem Fd segments (VH-CH1-VH-CH1) which, together with complementary light chain polypeptides, form the antigen binding region, as well as modified forms of any of the foregoing fragments that retain antigen binding

[0092] These antigen-binding fragments can be obtained using conventional techniques known to those skilled in the art, and the utility of the fragments is screened in the same manner as for whole antibodies.

[0093] As used herein, the term "binds" or "binds specifically" refers to a non-random binding reaction between two molecules, such as an antibody and its target antigen. The binding specificity of an antibody can be determined based on affinity and / or avidity. Affinity represents the equilibrium constant for dissociation of an antigen from an antibody (KD), a measure of the strength of binding between an antigenic determinant and an antigen binding site of an antibody: the smaller the value of KD, the stronger the binding strength between the antigenic determinant and the antibody. Alternatively, affinity can also be expressed as the affinity constant (KA), which is 1 / KD.

[0094] Affinity is a measure of the strength of binding between an antibody and the associated antigen. Affinity involves both the avidity between the antigenic determinant and the antigen binding site of the antibody and the number of associated binding sites present on the antibody. Typically, an antibody will bind an antigen with a dissociation constant (KD) of 10 -5 M to 10 -12 M or less, and preferably 10 -7 M to 10 -12 M or less, and more preferably 10 -8 M to 10 -12 M, and / or has a binding affinity of at least 10 7 M -1 , preferably at least 10 8 M -1 , more preferably at least 10 9 M -1 , such as at least 10 12 M -1 . It is generally accepted that any K -4 M value greater than 10 D M indicates non-specific binding. Specific binding of an antibody to an antigen or antigenic determinant can be determined in any known suitable manner, including, for example, Scatchard analysis and / or competitive binding assays, such as radioimmunoassays (RIA), enzyme immunoassays (EIA) and sandwich competition assays, and different variations thereof known in the art.

[0095] The term "epitope" refers to a site on an antigen to which an antibody binds. Epitopes can be formed both from contiguous amino acids or noncontiguous amino acids juxtaposed as a result of protein folding. Epitopes formed from contiguous amino acids (also known as linear epitopes) are generally retained upon exposure to denaturing solvents, whereas epitopes formed by tertiary folding (also known as conformational epitopes) are generally lost upon treatment with denaturing solvents. Epitopes usually include at least 3, and more usually at least 5 or 8-10 amino acids in a unique spatial conformation. Epitopes define the minimal binding site of an antibody and are therefore the specific targets of an antibody or antigen binding fragment thereof.

[0096] As used herein, the term "sequence identity" refers to the extent to which two sequences (amino acid) have identical residues in the same positions when aligned. For example, "an amino acid sequence is X% identical to SEQ ID NO: Y" means that the amino acid sequence has X% identity to SEQ ID NO: Y and is set forth as X% of the residues in the amino acid sequence being identical to the residues of the sequence disclosed in SEQ ID NO: Y. Typically, such calculations are performed using a computer program. Exemplary programs for comparing and aligning pairs of sequences include ALIGN, FASTA, gapped BLAST, BLASTP, BLASTN, or GCG.

[0097] Furthermore, in determining the extent of sequence identity between two amino acid sequences, the skilled person can take into account so-called "conservative" amino acid substitutions, which can generally be described as amino acid substitutions in which an amino acid residue is replaced with another amino acid residue having a similar chemical structure that has little or substantially no effect on the function, activity or other biological property of the polypeptide. Such conservative amino acid substitutions are well known in the art.

[0098] Such conservative substitutions are preferably substitutions in which one amino acid is replaced with another amino acid residue in the same group (a) to (e) as follows: (a) small aliphatic, non-polar or weakly polar residues: Ala, Ser, Thr, Pro and Gly; (b) polar, negatively charged residues and their (uncharged) amides: Asp, Asn, Glu and Gin; (c) polar, positively charged residues: His, Arg and Lys; (d) large aliphatic, non-polar residues: Met, Leu, lie, Val and Cys; and (e) aromatic residues: Phe, Tyr and Trp.

[0099] Particularly preferred conservative substitutions are as follows: Ala to Gly or to Ser; Arg to Lys; Asn to Gin or to His; Asp to Glu; Cys to Ser; Gin to Asn; Glu to Asp; Gly to Ala or to Pro; His to Asn or to Gin; lie to Leu or to Val; Leu to lie or to Val; Lys to Arg, to Gin or to Glu; Met to Leu, to Tyr or to lie; Phe to Met, to Leu or to Tyr; Ser to Thr; Thr to Ser; Trp to Tyr; Tyr to Trp; and / or Phe to Val, to lie or to Leu.

[0100] As used herein, unless otherwise indicated, a bivalent structure can be attached to the remainder of the compound in either direction, left to right or right to left. In one embodiment, the bivalent structure is preferably attached to the remainder of the compound in the left to right orientation.

[0101] The term "amino acid residue" means includes any natural or synthetic amino acid residue, not limited to amino acid residues in the group consisting of the 20 naturally occurring amino acids, wherein residue refers to the moiety remaining after removal of water from an amino acid linked by a peptide bond. The 20 naturally occurring amino acid residues are selected from the group consisting of alanine (Ala or A), cysteine (Cys or C), aspartic acid (Asp or D), glutamic acid (Glu or E), phenylalanine (Phe or F), glycine (Gly or G), histidine (His or H), isoleucine (lie or I), lysine (Lys or K), leucine (Leu or L), methionine (Met or M), asparagine (Asn or N), proline (Pro or P), glutamine (Gin or Q), arginine (Arg or R), serine (Ser or S), threonine (Thr or T), valine (Val or V), tryptophan (Trp or W), and tyrosine (Tyr or Y) residues.

[0102] The term "antibody conjugate" generally refers to an antibody linked to another chemical moiety. The chemical moiety can be a cytotoxic drug, an immunostimulatory molecule, and a detectable label. The drug can be, for example, a microtubule inhibitor, an antibiotic, a DNA synthesis inhibitor, a topoisomerase inhibitor, an RNA polymerase II inhibitor, and an RNA spliceosome inhibitor. The terms "antibody-drug conjugate," "antibody conjugate," and "ADC" can be used interchangeably.

[0103] The term "pharmaceutically acceptable" means that which the carrier or excipient is compatible with the other ingredients of the composition and not deleterious to the recipient thereof, and / or such carrier or excipient is approved or can be used in a pharmaceutical composition intended for parenteral administration to humans.

[0104] As used herein, the terms "treat," "treatment," "therapy," and the like, refer to the application of an agent or performance of a procedure for the purpose of effecting an outcome. The outcomes can be prophylactic, in terms of completely or partially preventing a disease or symptom thereof, and / or therapeutic, in terms of partially or completely curing a disease and / or symptoms of a disease. As used herein, "treatment" can include treatment of a disease or condition (e.g., an inflammatory disease) in a mammal, particularly in a human, and includes: (a) preventing the disease or symptom of the disease from occurring in an individual which can be predisposed to the disease but has not yet been diagnosed as having it; (b) inhibiting the disease, i.e., arresting its development; and (c) relieving the disease, i.e., causing regression of the disease. Treatment can refer to any successful indication in the treatment or amelioration or prevention of cancer, including any objective or subjective parameter, such as reduction of symptoms; alleviation of disease symptoms or making the disease condition more tolerable to the patient; slowing in rate of disease progression or degeneration; or improving quality of life. Treatment or amelioration of symptoms is based on one or more objective or subjective parameters; including physician-based measures. Thus, the term "treatment" includes the administration of an antibody or composition or conjugate disclosed herein to prevent or delay, alleviate or ameliorate a symptom or condition associated with a disease (e.g., an inflammatory disease). The term "therapeutic effect" refers to the reduction, elimination or prevention of a disease, a symptom of a disease or a side effect of a disease in a subject.

[0105] As used herein, the term "effective amount" refers to an amount of a drug administered to a subject to treat a disease sufficient to effect treatment of the disease.

[0106] As used herein, the term "subject" refers to any mammalian subject in which diagnosis, treatment or therapy is desired. "Mammalian" for purposes of treatment refers to any animal classified as a mammal, including humans, domestic and farm animals, and laboratory and sport or pet animals, such as dogs, horses, cats, cows, sheep, goats, pigs, mice, rats, rabbits, guinea pigs, monkeys, etc.

[0107] "Stereoisomers" refer to compounds which have the same chemical constitution, but differ in the arrangement of atoms or groups in space. Stereoisomers include enantiomers, diastereomers, conformational isomers (rotamers), geometric isomers (cis / trans), atropisomers, and the like.

[0108] "Chiral" refers to a molecule which has the property of non-superimposability with its mirror image; while "achiral" refers to a molecule which is superimposable with its mirror image.

[0109] "Enantiomers" refer to two isomers of a compound which are nonsuperimposable mirror images of one another.

[0110] "Diastereomers" refers to two or more stereoisomers of a compound which are not mirror images of one another and which have different physical properties. Mixtures of diastereomers can be separated by high resolution analytical procedures such as electrophoresis and chromatography, e.g., HPLC.

[0111] The stereochemical definitions and rules as used herein generally follow S. P. Parker, Ed., McGraw-Hill Dictionary of Chemical Terms (1984) McGraw-Hill Book Company, New York; and Eliel, E. and Wilen, S., "Stereochemistry of Organic Compounds", John Wiley & Sons, Inc., New York, 1994.

[0112] Many organic compounds exist in optically active forms, i.e., they have the ability to rotate the plane of plane-polarized light. In describing an optically active compound, the prefixes D and L or R and S are used to denote the absolute configuration of the molecule about its chiral center(s). The prefixes d and 1 or (+) and (-) are employed to designate the sign of the rotation of plane-polarized light by the compound; (-) or 1 meaning that the compound is levorotatory. A compound, which is (+) or d is dextrorotatory. A particular stereoisomer is an enantiomer, and a mixture of such isomers is called an enantiomeric mixture. A 50:50 mixture of enantiomers is referred to as a racemic mixture or racemate.

[0113] Any asymmetric atom (e.g., carbon, etc.) of a compound disclosed herein can exist in the racemic or enantiomeric enriched form, e.g., the (R)-, (S)-, or (R,S)-configurational form. In certain embodiments, each asymmetric atom is in at least a 50% enantiomeric excess of the (R)- or (S)- configuration, at least a 60% enantiomeric excess, at least a 70% enantiomeric excess, at least an 80% enantiomeric excess, at least a 90% enantiomeric excess, at least a 95% enantiomeric excess, or at least a 99% enantiomeric excess.

[0114] Depending on the choice of starting materials and methods, the compounds of the application can be present in the form of one or more of possible isomers, such as racemates and mixtures of diastereomers, depending on the number of asymmetric carbon atoms. The optically active (R)- or (S)-isomers can be prepared using chiral synthons or chiral reagents, or by resolution of the racemic mixtures using conventional techniques. If the compounds contain a double bond, the substituents can be in the E or Z configuration; if the compounds contain a disubstituted cycloalkyl ring, the substituents on the cycloalkyl ring can be in the cis- or trans-configuration.

[0115] Any mixture of stereoisomers can be separated into the individual isomers by conventional techniques, such as HPLC or fractional crystallization, and any enantiomers can be converted into the individual isomers by the application of known techniques, such as those described in Jacques, et al., Enantiomers, Racemates and Resolutions (Wiley Interscience, New York, 1981); Principles of Asymmetric Synthesis (2nd Ed. Robert E. Gawley, Jeffrey Aubé, Elsevier, Oxford, UK, 2012); Eliel, E. L. Stereochemistry of Carbon Compounds (McGraw-Hill, NY, 1962); Wilen, S. H. Tables of Resolving Agents and Optical Resolutions p. 268 (E. L. Eliel, Ed., Univ. of Notre Dame Press, Notre Dame, IN 1972); Chiral Separation Techniques: A Practical Approach (Subramanian, G. Ed., Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim, Germany, 2007).

[0116] Any racemate of an end product or intermediate can be separated by the application of known techniques, such as those described in Jacques, et al., Enantiomers, Racemates and Resolutions (Wiley Interscience, New York, 1981); Principles of Asymmetric Synthesis (2nd Ed. Robert E. Gawley, Jeffrey Aubé, Elsevier, Oxford, UK, 2012); Eliel, E. L. Stereochemistry of Carbon Compounds (McGraw-Hill, NY, 1962); Wilen, S. H. Tables of Resolving Agents and Optical Resolutions p. 268 (E. L. Eliel, Ed., Univ. of Notre Dame Press, Notre Dame, IN 1972); Chiral Separation Techniques: A Practical Approach (Subramanian, G. Ed., Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim, Germany, 2007).

[0117] The present application includes tautomers, which are isomers of a functional group resulting from the movement of a single atom between two positions in a molecule. A compound exists in different tautomeric forms, and one said compound is not limited to any particular tautomer, but is intended to encompass all tautomeric forms.

[0118] The compounds of the present application can include one or more asymmetric centers and can thus occur as individual enantiomers, diastereomers and / or geometric isomers, for example, as racemic mixtures, scalemic mixtures, or as individual non-racemic mixtures, and it is intended to embrace all such isomeric forms. The compounds of the present application can be separated into their individual enantiomers and / or geometric isomers by the application of well-known mass spectrometry, chiral column chromatography, chiral phase HPLC, and the like. The compounds of the present application can be prepared in and isolated as racemic, scalemic or non-racemic mixtures of enantiomers and / or geometric isomers, and all such isomeric forms are intended to be included within the scope of the present application. The compounds of the present application can be separated into their individual enantiomers and / or geometric isomers by the application of well-known mass spectrometry, chiral column chromatography, chiral phase HPLC, and the like. The compounds of the present application can be prepared in and isolated as racemic, scalemic or non-racemic mixtures of enantiomers and / or geometric isomers, and all such isomeric forms are intended to be included within the scope of the present application.

[0119] Those skilled in the art will appreciate that organic compounds can form complexes with solvents, often in the process of their formation or their recovery. These complexes are known as "solvates". When the solvent is water, the solvate is known as a "hydrate". The present application encompasses all solvates of the compounds of the present application.

[0120] The term "solvate" refers to a form of a compound or salt thereof in combination with a solvent, typically formed by a solvolysis reaction. This physical association can include hydrogen bonding. Conventional solvents include water, methanol, ethanol, acetic acid, DMSO, THF, diethyl ether and the like. The compounds described herein can be prepared, for example, in crystalline form, and can be solvated. Suitable solvates include pharmaceutically acceptable solvates and further include stoichiometric solvates and non-stoichiometric solvates. In some cases, the solvate will be capable of isolation, for example, when one or more solvent molecules are incorporated into the crystal lattice of the crystalline solid. "Solvate" includes both solution-phase solvates and isolatable solvates. Representative solvates include hydrates, ethanolates, and methanolates.

[0121] The term "hydrate" refers to a compound in combination with water. Typically, the number of water molecules in a hydrate of a compound is stoichiometrically defined. Thus, a hydrate of a compound can be represented, for example, by the general formula R x H2O, where R is the compound and x is a number greater than zero. A given compound can form more than one type of hydrate, including, for example, monohydrates (x is 1), lower hydrates (x is a number greater than zero and less than one, e.g., a hemihydrate (R 0.5 H2O)), and polyhydrates (x is a number greater than one, e.g., dihydrates (R 2 H2O) and hexahydrates (R 6 H2O)).

[0122] The compounds of this invention can be in amorphous or crystalline forms (polymorphs). Furthermore, the compounds of this invention can exist in one or more crystalline forms. Therefore, this invention encompasses all amorphous or crystalline forms of the compounds of this invention within its scope. The term "polymorph" refers to the crystalline form of a compound (or its salts, hydrates, or solvates) with a particular crystal packing arrangement. All polymorphs have the same elemental composition. Different crystalline forms typically have different X-ray diffraction patterns, infrared spectra, melting points, densities, hardness, crystal shapes, photoelectric properties, stability, and solubility. Recrystallization solvents, crystallization rates, storage temperatures, and other factors can lead to the dominance of one crystalline form. Various polymorphs of the compounds can be prepared by crystallization under different conditions.

[0123] The present invention also includes isotopically labeled compounds (isotopic variants) that are equivalent to those described in formula (I), but in which one or more atoms are replaced by atoms with atomic masses or mass numbers different from those commonly found in nature. Examples of isotopes that can be introduced into the compounds of the present invention include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, and chlorine, respectively, for example... 2 H, 3 H, 13 C 11 C 14 C 15 N、 18 O、 17 O、 31 P, 32 P, 35 S, 18 F and 36 Cl. Other isotopes of the present invention containing the aforementioned isotopes and / or other atoms, their prodrugs, and pharmaceutically acceptable salts of said compounds or said prodrugs are all within the scope of this invention. Certain isotope-labeled compounds of the present invention, for example, those incorporating radioactive isotopes (e.g.,...) 3 H and 14 Those in category C) can be used for drug and / or substrate tissue distribution determination. Tritium, i.e. 3 H and carbon-14, i.e. 14 Carbon isotopes are particularly preferred because they are easy to prepare and detect. Subsequently, they are replaced by heavier isotopes, such as deuterium, i.e., 2 H, because higher metabolic stability can provide therapeutic benefits, such as prolonged in vivo half-life or reduced dosage requirements, may be preferred in some cases. Isotope-labeled compounds of formula (I) of the present invention and their prodrugs can generally be prepared by using readily available isotope-labeled reagents instead of non-isotope-labeled reagents when performing the processes described below and / or the techniques disclosed in the examples and preparation examples.

[0124] As described in this invention, the compounds of this invention may optionally be substituted with one or more substituents, such as the general formula compounds above, or as the specific examples in the embodiments, and the class of compounds included in this invention.

[0125] "Pharmaceutical acceptable" means that, within the bounds of reliable medical judgment, it is suitable for contact with the tissues of humans and lower animals without excessive toxicity, irritation, allergic reactions, etc., and that the benefits / risks are proportionate to a reasonable ratio.

[0126] As used in this invention, "pharmaceutically acceptable salts" refers to the organic and inorganic salts of the compounds of this invention. Pharmaceutically acceptable salts are well-known in the field, as described in the literature: SMBerge et al., describe pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences, 1977, 66: 1-19. Salts formed from pharmaceutically acceptable non-toxic acids include, but are not limited to, inorganic acid salts formed by reactions with amino groups, such as hydrochlorides, hydrobromic acids, phosphates, sulfates, and perchlorates, and organic acid salts such as acetates, oxalates, maleates, tartrates, citrates, succinates, malonates, or these salts obtained by other methods described in the literature, such as ion exchange. Other pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, cyclopentylpropionate, digluconate, dodecyl sulfate, ethanesulfonate, formate, transbutenedioic acid, gluconate, glycerophosphate, gluconate, hemisulfate, heptaate, hexanoate, hydroiodate, 2-hydroxy-ethanesulfonate, lacturonate, lactate, laurate, lauryl sulfate, malate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, palmitate, pyruvate, pectinate, persulfate, 3-phenylpropionate, picrate, pentanoate, propionate, stearate, thiocyanate, p-toluenesulfonate, undecanoate, valerate, etc. Salts obtained by means of appropriate bases include alkali metals, alkaline earth metals, ammonium, and N+(C1-C4 alkyl)4 salts. This invention also envisions the formation of quaternary ammonium salts from any compound containing an N-group. Water-soluble or oil-soluble or dispersed products can be obtained via quaternization. Alkali metal or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, etc. Pharmaceutically acceptable salts further include suitable, non-toxic ammonium, quaternary ammonium salts, and amine cations resistant to the formation of equilibrium ions, such as halides, hydroxides, carboxylates, sulfates, phosphates, nitrates, C1-8 sulfonates, and aromatic sulfonates.

[0127] Certain embodiments of the application are now described in greater detail with reference to the accompanying structures and chemical formulas. The application intends to encompass all alternatives, modifications and equivalents thereof which are included within the scope of the application as defined by the claims. One skilled in the art will recognize many methods and materials as being suitable for use in practicing the application. The present application is in no way limited to the methods and materials described herein. In the event that one or more of the incorporated references contradicts the prior description, including definitions of terms, usage of

[0128] It is further recognized that certain of the present features are described in the context of separate embodiments for clarity, but can also be provided in combination in a single embodiment. Conversely, various features of the present application are described in the context of a single embodiment for brevity, but can also be provided separately or in any appropriate subcombination.

[0129] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. All patents and publications referred to in this application are incorporated herein by reference in their entirety.

[0130] Unless defined otherwise, all terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. For purposes of the present application, the term "consisting essentially of shall mean excluding other elements of like kind. Structure-activity relationship is intended to encompass the broad disclosure as contained herein, and can be used in connection with any and all compounds described herein.

[0131] Anti-CDH17 antibodies

[0132] The present disclosure provides an antibody or antigen-binding fragment thereof that binds CDH17, the antibody or antigen-binding fragment thereof comprising a heavy chain variable region (VH) and a light chain variable region (VL).

[0133] In some embodiments, the VH comprises HCDR1, HCDR2, HCDR3, having the amino acid sequences set forth in SEQ ID NOs: 5, 6, 7, respectively, and the VL comprises LCDR1, LCDR2, and LCDR3, having the amino acid sequences set forth in SEQ ID NOs: 10, 11, 12, respectively.

[0134] In some embodiments, the VH comprises HCDR1, HCDR2, HCDR3, having the amino acid sequences set forth in SEQ ID NOs: 15, 16, 17, respectively, and the VL comprises LCDR1, LCDR2, and LCDR3, having the amino acid sequences set forth in SEQ ID NOs: 20, 21, 22, respectively.

[0135] In some embodiments, the VH comprises a HCDR1 having an amino acid sequence as set forth in SEQ ID NO: 25 or 107, a HCDR2 having an amino acid sequence as set forth in SEQ ID NO: 26 or 125, and a HCDR3 having an amino acid sequence as set forth in SEQ ID NO: 27, and the VL comprises a LCDR1 having an amino acid sequence as set forth in SEQ ID NO: 30 or 120, a LCDR2 having an amino acid sequence as set forth in SEQ ID NO: 31, and a LCDR3 having an amino acid sequence as set forth in SEQ ID NO: 32. In some embodiments, the VH comprises a HCDR1 having an amino acid sequence as set forth in SEQ ID NO: 25 or 107, a HCDR2 having an amino acid sequence as set forth in SEQ ID NO: 26, and a HCDR3 having an amino acid sequence as set forth in SEQ ID NO: 27, and the VL comprises a LCDR1, a LCDR2, and a LCDR3, respectively, having an amino acid sequence as set forth in SEQ ID NO: 30, 31, 32. In some embodiments, the VH comprises a HCDR1 having an amino acid sequence as set forth in SEQ ID NO: 25 or 107, a HCDR2 having an amino acid sequence as set forth in SEQ ID NO: 125, and a HCDR3 having an amino acid sequence as set forth in SEQ ID NO: 27, and the VL comprises a LCDR1, a LCDR2, and a LCDR3, respectively, having an amino acid sequence as set forth in SEQ ID NO: 30, 31, 32. In some embodiments, the VH comprises a HCDR1 having an amino acid sequence as set forth in SEQ ID NO: 25 or 107, a HCDR2 having an amino acid sequence as set forth in SEQ ID NO: 26, and a HCDR3 having an amino acid sequence as set forth in SEQ ID NO: 27, and the VL comprises a LCDR1, a LCDR2, and a LCDR3, respectively, having an amino acid sequence as set forth in SEQ ID NO: 120, 31, 32. In some embodiments, the VH comprises a HCDR1 having an amino acid sequence as set forth in SEQ ID NO: 25 or 107, a HCDR2 having an amino acid sequence as set forth in SEQ ID NO: 125, and a HCDR3 having an amino acid sequence as set forth in SEQ ID NO: 27, and the VL comprises a LCDR1, a LCDR2, and a LCDR3, respectively, having an amino acid sequence as set forth in SEQ ID NO: 120, 31, 32. In preferred embodiments, the VH comprises a HCDR1, a HCDR2, a HCDR3, respectively, having an amino acid sequence as set forth in SEQ ID NO: 25, 26, 27, and the VL comprises a LCDR1, a LCDR2, and a LCDR3, respectively, having an amino acid sequence as set forth in SEQ ID NO: 30, 31, 32.In another preferred embodiment, the VH comprises HCDR1, HCDR2, HCDR3, having the amino acid sequences set forth in SEQ ID NOs: 25, 125, 27, respectively, and the VL comprises LCDR1, LCDR2, and LCDR3, having the amino acid sequences set forth in SEQ ID NOs: 120, 31, 32, respectively.

[0136] In some embodiments, the VH comprises HCDR1, HCDR2, HCDR3, having the amino acid sequences set forth in SEQ ID NOs: 35, 36, 37, respectively, and the VL comprises LCDR1, LCDR2, and LCDR3, having the amino acid sequences set forth in SEQ ID NOs: 40, 41, 42, respectively.

[0137] In some embodiments, the VH comprises a HCDR1 having an amino acid sequence as set forth in SEQ ID NO: 45, a HCDR2 having an amino acid sequence as set forth in SEQ ID NO: 46 or 81, and a HCDR3 having an amino acid sequence as set forth in SEQ ID NO: 47, and the VL comprises a LCDR1 having an amino acid sequence as set forth in SEQ ID NO: 50 or 90, a LCDR2 having an amino acid sequence as set forth in SEQ ID NO: 51, 93, or 96, and a LCDR3 having an amino acid sequence as set forth in SEQ ID NO: 52. In some embodiments, the VH comprises a HCDR1 having an amino acid sequence as set forth in SEQ ID NO: 45, a HCDR2 having an amino acid sequence as set forth in SEQ ID NO: 46 or 81, and a HCDR3 having an amino acid sequence as set forth in SEQ ID NO: 47, and the VL comprises a LCDR1, a LCDR2, and a LCDR3 having an amino acid sequence as set forth in SEQ ID NO: 50, 51, 52, respectively. In some embodiments, the VH comprises a HCDR1 having an amino acid sequence as set forth in SEQ ID NO: 45, a HCDR2 having an amino acid sequence as set forth in SEQ ID NO: 46 or 81, and a HCDR3 having an amino acid sequence as set forth in SEQ ID NO: 47, and the VL comprises a LCDR1, a LCDR2, and a LCDR3 having an amino acid sequence as set forth in SEQ ID NO: 90, 51, 52, respectively. In some embodiments, the VH comprises a HCDR1 having an amino acid sequence as set forth in SEQ ID NO: 45, a HCDR2 having an amino acid sequence as set forth in SEQ ID NO: 46 or 81, and a HCDR3 having an amino acid sequence as set forth in SEQ ID NO: 47, and the VL comprises a LCDR1, a LCDR2, and a LCDR3 having an amino acid sequence as set forth in SEQ ID NO: 50, 93, 52, respectively. In some embodiments, the VH comprises a HCDR1 having an amino acid sequence as set forth in SEQ ID NO: 45, a HCDR2 having an amino acid sequence as set forth in SEQ ID NO: 46 or 81, and a HCDR3 having an amino acid sequence as set forth in SEQ ID NO: 47, and the VL comprises a LCDR1, a LCDR2, and a LCDR3 having an amino acid sequence as set forth in SEQ ID NO: 50, 96, 52, respectively.In some embodiments, the VH comprises a HCDR1 having an amino acid sequence as set forth in SEQ ID NO: 45, a HCDR2 having an amino acid sequence as set forth in SEQ ID NO: 46 or 81, and a HCDR3 having an amino acid sequence as set forth in SEQ ID NO: 47, and the VL comprises a LCDR1, a LCDR2, and a LCDR3 having an amino acid sequence as set forth in SEQ ID NO: 90, 93, 52, respectively. In some embodiments, the VH comprises a HCDR1 having an amino acid sequence as set forth in SEQ ID NO: 45, a HCDR2 having an amino acid sequence as set forth in SEQ ID NO: 46 or 81, and a HCDR3 having an amino acid sequence as set forth in SEQ ID NO: 47, and the VL comprises a LCDR1, a LCDR2, and a LCDR3 having an amino acid sequence as set forth in SEQ ID NO: 90, 96, 52, respectively. In a preferred embodiment, the VH comprises a HCDR1, a HCDR2, a HCDR3 having an amino acid sequence as set forth in SEQ ID NO: 45, 46, 47, respectively, and the VL comprises a LCDR1, a LCDR2, and a LCDR3 having an amino acid sequence as set forth in SEQ ID NO: 50, 51, 52, respectively. In another preferred embodiment, the VH comprises a HCDR1, a HCDR2, a HCDR3 having an amino acid sequence as set forth in SEQ ID NO: 45, 46, 47, respectively, and the VL comprises a LCDR1, a LCDR2, and a LCDR3 having an amino acid sequence as set forth in SEQ ID NO: 90, 51, 52, respectively.

[0138] In some embodiments, the VH comprises a HCDR1, a HCDR2, a HCDR3 having an amino acid sequence as set forth in SEQ ID NO: 55, 56, 57, respectively, and the VL comprises a LCDR1, a LCDR2, and a LCDR3 having an amino acid sequence as set forth in SEQ ID NO: 60, 61, 62, respectively.

[0139] In some embodiments, the VH comprises a HCDR1, a HCDR2, a HCDR3 having an amino acid sequence as set forth in SEQ ID NO: 65, 66, 67, respectively, and the VL comprises a LCDR1, a LCDR2, and a LCDR3 having an amino acid sequence as set forth in SEQ ID NO: 70, 71, 72, respectively.

[0140] In some embodiments, the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 3, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 8.

[0141] In some embodiments, the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 13, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 18.

[0142] In some embodiments, the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to any one selected from SEQ ID NO: 23, 99, 101, 103, 105, 108, 110, 112, 114, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to any one selected from SEQ ID NO: 28, 116, 118, 121, 123. In some embodiments, the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to any one selected from SEQ ID NO: 23, 101, 103, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to any one selected from SEQ ID NO: 28, 118, 123. In preferred embodiments, the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 23, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 28. In preferred embodiments, the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 101, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 118. In preferred embodiments, the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 101, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 123.In preferred embodiments, the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 103, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 118. In preferred embodiments, the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 103, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 123.

[0143] In some embodiments, the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 33, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 38.

[0144] In some embodiments, the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to any one of SEQ ID NOs: 43, 73, 75, 77, 79, 82, 84, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to any one of SEQ ID NOs: 48, 86, 88, 91, 94, 97.

[0145] In some embodiments, the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to any one selected from SEQ ID NO: 43, 77, 82, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to any one selected from SEQ ID NO: 48, 86, 88. In preferred embodiments, the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 43, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 48. In preferred embodiments, the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 77, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 86. In preferred embodiments, the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 82, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 88.

[0146] In some embodiments, the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 53, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 58.

[0147] In some embodiments, the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 63, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 68.

[0148] In some embodiments, the VH comprises a functional variant of an amino acid sequence as disclosed herein above by insertion, deletion and / or substitution of one or more amino acids thereof, provided that the functional variant retains the ability to bind to CDH17. In some embodiments, the VL comprises a functional variant of an amino acid sequence as disclosed herein above by insertion, deletion and / or substitution of one or more amino acids thereof, provided that the functional variant retains the ability to bind to CDH17.

[0149] A functional variant comprises or consists of an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% sequence identity to the amino acid sequence of the parent polypeptide.

[0150] In the context of a functional variant, the number of inserted, deleted and / or substituted amino acids preferably does not exceed 40%, more preferably does not exceed 35%, more preferably is between 1 and 33%, more preferably is between 5 and 30%, more preferably is between 10 and 25%, more preferably is between 15 and 20% of the total number of amino acids in the parent amino acid sequence. For example, the number of inserted, deleted and / or substituted amino acids can be between 1 and 20, preferably between 1 and 10, more preferably between 1 and 7, still more preferably between 1 and 5, most preferably between 1 and 2. In preferred embodiments, the number of inserted, deleted and / or substituted amino acids is 1, 2, 3, 4, 5, 6 or 7.

[0151] In some embodiments, the insertion, deletion and / or substitution can be made in a framework (FR) region, e.g., in FR1, FR2, FR3 and / or FR4.

[0152] In some embodiments, substitution of one or more amino acids can be conservative substitution of one or more amino acids. Such conservative substitutions preferably are substitutions of one amino acid for another within the same group (a) to (e) below: (a) small aliphatic, nonpolar or weakly polar residues: Ala, Ser, Thr, Pro, and Gly; (b) polar, negatively charged residues and their (uncharged) amides: Asp, Asn, Glu, and Gin; (c) polar, positively charged residues: His, Arg, and Lys; (d) large aliphatic, nonpolar residues: Met, Leu, lie, Val, and Cys; and (e) aromatic residues: Phe, Tyr, and Trp.

[0153] Particularly preferred conservative substitutions are as follows: Ala to Gly or to Ser; Arg to Lys; Asn to Gin or to His; Asp to Glu; Cys to Ser; Gin to Asn; Glu to Asp; Gly to Ala or to Pro; His to Asn or to Gin; lie to Leu or to Val; Leu to lie or to Val; Lys to Arg, to Gin, or to Glu; Met to Leu, to Tyr, or to lie; Phe to Met, to Leu, or to Tyr; Ser to Thr; Thr to Ser; Trp to Tyr; Tyr to Trp; and / or Phe to Val, to lie, or to Leu.

[0154] In preferred embodiments, the VH comprises the amino acid sequence set forth in SEQ ID NO: 3 and the VL comprises the amino acid sequence set forth in SEQ ID NO: 8.

[0155] In other preferred embodiments, the VH comprises the amino acid sequence set forth in SEQ ID NO: 13 and the VL comprises the amino acid sequence set forth in SEQ ID NO: 18.

[0156] In other preferred embodiments, the VH comprises the amino acid sequence set forth in SEQ ID NO: 23 and the VL comprises the amino acid sequence set forth in SEQ ID NO: 28.

[0157] In other preferred embodiments, the VH comprises the amino acid sequence set forth in SEQ ID NO: 33 and the VL comprises the amino acid sequence set forth in SEQ ID NO: 38.

[0158] In other preferred embodiments, the VH comprises the amino acid sequence set forth in SEQ ID NO: 43 and the VL comprises the amino acid sequence set forth in SEQ ID NO: 48.

[0159] In other preferred embodiments, the VH comprises an amino acid sequence as set forth in SEQ ID NO: 53, and the VL comprises an amino acid sequence as set forth in SEQ ID NO: 58.

[0160] In other preferred embodiments, the VH comprises an amino acid sequence as set forth in SEQ ID NO: 63, and the VL comprises an amino acid sequence as set forth in SEQ ID NO: 68.

[0161] In other preferred embodiments, the VH comprises an amino acid sequence as set forth in SEQ ID NO: 101, and the VL comprises an amino acid sequence as set forth in SEQ ID NO: 118.

[0162] In other preferred embodiments, the VH comprises an amino acid sequence as set forth in SEQ ID NO: 101, and the VL comprises an amino acid sequence as set forth in SEQ ID NO: 123.

[0163] In other preferred embodiments, the VH comprises an amino acid sequence as set forth in SEQ ID NO: 103, and the VL comprises an amino acid sequence as set forth in SEQ ID NO: 118.

[0164] In other preferred embodiments, the VH comprises an amino acid sequence as set forth in SEQ ID NO: 103, and the VL comprises an amino acid sequence as set forth in SEQ ID NO: 123.

[0165] In other preferred embodiments, the VH comprises an amino acid sequence as set forth in SEQ ID NO: 77, and the VL comprises an amino acid sequence as set forth in SEQ ID NO: 86.

[0166] In other preferred embodiments, the VH comprises an amino acid sequence as set forth in SEQ ID NO: 82, and the VL comprises an amino acid sequence as set forth in SEQ ID NO: 88.

[0167] In some embodiments, the antibody is a murine antibody, a chimeric antibody, or a humanized antibody. In preferred embodiments, the antibody is a humanized antibody.

[0168] Based on the amino acid sequences of the constant regions of the heavy chains, immunoglobulin molecules can be assigned to five classes (isotypes): IgA, IgD, IgE, IgG, and IgM, and can be further divided into subclasses, e.g., IgGl, IgG2, IgG3, IgG4, IgAl, IgA2, etc. Based on the amino acid sequences of the light chains, the light chains of antibodies can be assigned to lambda (l) chains and kappa (K) chains. The antibodies disclosed herein can be of any of the above classes or subclasses.

[0169] In some embodiments, the antibody is of an isotype selected from the group consisting of IgG, IgA, IgM, IgE, and IgD. In some embodiments, the antibody is of a subclass selected from the group consisting of IgGl, IgG2, IgG3, and IgG4. In preferred embodiments, the antibody is an IgGl antibody.

[0170] The antibodies disclosed herein can be intact antibodies or antigen-binding fragments thereof. In some embodiments, the antibody comprises an immunoglobulin heavy chain (HC) and an immunoglobulin light chain (LC), wherein the HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 126 or SEQ ID NO: 130, and the LC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 128 or SEQ ID NO: 132.

[0171] In some embodiments, the HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 126, and the LC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 128.

[0172] In some embodiments, the HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 130, and the LC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 132.

[0173] In some embodiments, the HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 134, and the LC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 136.

[0174] In some embodiments, the HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 134, and the LC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 138.

[0175] In some embodiments, the HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 140, and the LC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 136.

[0176] In some embodiments, the HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 140, and the LC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 138.

[0177] In some embodiments, the HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 142, and the LC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 143.

[0178] In preferred embodiments, the HC comprises an amino acid sequence as set forth in SEQ ID NO: 126, and the LC comprises an amino acid sequence as set forth in SEQ ID NO: 128.

[0179] In other preferred embodiments, the HC comprises an amino acid sequence as set forth in SEQ ID NO: 130, and the LC comprises an amino acid sequence as set forth in SEQ ID NO: 132.

[0180] In other preferred embodiments, the HC comprises an amino acid sequence as set forth in SEQ ID NO: 134, and the LC comprises an amino acid sequence as set forth in SEQ ID NO: 136.

[0181] In other preferred embodiments, the HC comprises an amino acid sequence as set forth in SEQ ID NO: 134, and the LC comprises an amino acid sequence as set forth in SEQ ID NO: 138.

[0182] In other preferred embodiments, the HC comprises an amino acid sequence as set forth in SEQ ID NO: 140, and the LC comprises an amino acid sequence as set forth in SEQ ID NO: 136.

[0183] In other preferred embodiments, the HC comprises an amino acid sequence as set forth in SEQ ID NO: 140, and the LC comprises an amino acid sequence as set forth in SEQ ID NO: 138.

[0184] In other preferred embodiments, the HC comprises an amino acid sequence as set forth in SEQ ID NO: 142, and the LC comprises an amino acid sequence as set forth in SEQ ID NO: 143.

[0185] The antigen-binding fragment can be any fragment of an antibody that retains the ability to specifically bind to IL-13. Examples of antigen-binding fragments include, but are not limited to: Fab fragments; F(ab')2 fragments; Fab' fragments; Fd fragments; Fd' fragments; Fv fragments; scFv fragments; dAb fragments; isolated complementarity determining regions (CDRs); nanobodies; linear antibodies composed of a pair of tandem Fd segments (VH-CH1-VH-CH1), and modified forms of any of the foregoing fragments that retain antigen binding activity.

[0186] In some embodiments, the antigen-binding fragment is selected from the group consisting of Fab, Fab', F(ab')2, Fv, scFv, and ds-scFv. In preferred embodiments, the antigen-binding fragment is Fab. In another preferred embodiment, the antigen-binding fragment is Fv. In another preferred embodiment, the antigen-binding fragment is scFv.

[0187] In some embodiments, the antibody is a monoclonal antibody, a bispecific antibody, or a multispecific antibody. In some embodiments, the antibody is a bispecific antibody further comprising a second antigen binding region that binds to a second antigen. In some embodiments, the second antigen is a tumor associated antigen or an immune cell antigen.

[0188] A number of tumor associated antigens have been identified in the art that are associated with particular cancers. In some embodiments, a tumor associated antigen is an antigen that can elicit a significant tumor specific immune response. Some of these antigens are encoded by normal cells, but are not necessarily expressed by normal cells. These antigens can be characterized as antigens that are normally silent (i.e., not expressed) in normal cells, antigens that are expressed only at certain stages of differentiation, and antigens that are expressed over time such as embryonic and fetal antigens. Other cancer cell antigens are encoded by mutated cellular genes such as oncogenes (e.g., activated ras oncogenes), suppressor genes (e.g., P53 mutants), and fusion proteins resulting from internal deletions or chromosomal translocations. Still other cancer antigens can be encoded by viral genes such as those carried by RNA and DNA tumor viruses. Numerous other tumor associated antigens and antibodies against them are known and / or commercially available, and can also be prepared by those skilled in the art.

[0189] Examples of tumor associated antigens include, but are not limited to, 5T4, alphafetoprotein, CA-125, carcinoembryonic antigen, CD19, CD20, CD22, CD23, CD30, CD33, CD40, CD56, CD79, CD78, CD123, CD138, c-Met, CSPG4, IgM, AXL, EGFR, EGFRvIII, epithelial tumor antigen, ERBB2, FLT3, folate binding protein, GD2, GD3, HIV-1 envelope glycoprotein gp41, HIV-1 envelope glycoprotein gpl20, melanoma-associated antigen, MUC-1, mutated p53, mutated ras, ROR1, GPC3, VEGFR2, and combinations thereof.

[0190] In some embodiments, the second antigen is a T cell antigen. In some embodiments, the T cell antigen is selected from the group consisting of a T cell receptor (TCR), CD3, CD4, CD8, CD16, CD25, CD28, CD38, CD44, CD62L, CD69, ICOS, 41-BB (CD137), and NKG2D, or any combination thereof. In some embodiments, the T cell antigen is CD3, and the second antigen binding region binds to any of the gamma, delta, epsilon, zeta, and eta chains of CD3.

[0191] In some embodiments, the antibodies of the application bind to human and cynomolgus CDH17. In some embodiments, the antibodies of the application bind to CDH17-positive tumor cells with an EC50 in the nM range, e.g., about 0.5-2.0 nM. In some embodiments, the antibodies of the application are endocytosed by CDH17-positive tumor cells with an IC50 of less than 1 nM, e.g., 0.01-0.30 nM. In some embodiments, the antibodies of the application are capable of inhibiting the Wnt / beta-catenin signaling pathway.

[0192] The antibodies disclosed herein can comprise an Fc region. The Fc region can be of any isotype, including but not limited to IgGl, IgG2, IgG3, and IgG4, and can comprise one or more mutations or modifications. In one embodiment, the Fc region is or is derived from an IgGl or IgG4 isotype, optionally with one or more mutations or modifications. In one embodiment, the Fc region is a human IgGl Fc.

[0193] In some embodiments, the Fc region has reduced effector function, e.g., reduced ADCC, ADCP, CDC, and / or Clq, FcyRI, FcyRII, or FcyRIIIA binding. For example, the Fc region can be of an IgGl isotype, or of a non-IgGl type, e.g., IgG2, IgG3, or IgG4, which has been mutated such that the ability to mediate effector function is reduced or even eliminated. Such mutations have been described, e.g., in Dall’Acqua WF, et al., J Immunol. 177(2): 1129-1138 (2006) and Hezareh M, J Virol.; 75(24): 12161-12168 (2001). For example, the Fc region can comprise an amino acid sequence with one or more of the following amino acid substitutions compared to the wild-type sequence: E233P, L234A, L234F, L235A, L235E, G237A, N297A, N297D, P331S, and P329G. In a preferred embodiment the Fc region comprises L234A and L235A (LA mutations).

[0194] In one embodiment, the Fc region comprises a mutation that removes the Asn-linked glycosylation acceptor site or is otherwise manipulated to alter the glycosylation properties. For example, in an IgGl Fc region, the N297Q mutation can be used to remove the Asn-linked glycosylation site. Thus, in a specific embodiment, the Fc region comprises an IgGl sequence with the N297Q mutation.

[0195] In a further embodiment, the Fc region is glycoengineered to reduce fucose and thus enhance ADCC, for example by the addition of a compound to the culture medium during antibody production, as described in US2009317869 or as described in van Berkel et al. (2010) Biotechnol. Bioeng. 105:350, or by the use of FUT8 knockout cells, as described in Yamane-Ohnuki et al. (2004) Biotechnol. Bioeng 87:614. Alternatively, the ADCC can be optimized using the methods described in Shields et al. (2002) J. Biol. Chem. 277: 26733. Natsume et al. (2009) Cancer Sci. 100:2411. Alternatively, the Fc region can be engineered to increase the affinity for FcγRIIB, as described in

[0196] In other embodiments, the Fc region has an extended serum half-life. For examples of altering (e.g., decreasing or increasing) the in vivo half-life of an antibody, see, e.g., International Publication Nos. WO 02 / 060919, WO 98 / 23289, and WO 97 / 34631; and U.S. Patent Nos. 5,869,046; 6,121,022; 6,277,375; and 6,165,745, all of which are incorporated herein by reference in their entireties. In some embodiments, one, two, or more amino acid mutations (e.g., substitutions, deletions, or insertions) are introduced into an IgG constant domain or FcRn binding fragment thereof (preferably an Fc or hinge region-Fc domain fragment) to decrease the in vivo half-life of the antibody. In other embodiments, one, two, or more amino acid mutations (e.g., substitutions, deletions, or insertions) are introduced into an IgG constant domain or FcRn binding fragment thereof (preferably an Fc or hinge region-Fc domain fragment) to increase the in vivo half-life of the antibody. In a particular embodiment, the antibody can have one or more amino acid mutations (e.g., substitutions) in the second constant (CH2) domain (residues 231-340 of human IgGl) and / or the third constant (CH3) domain (residues 341-447 of human IgGl) (numbered according to the EU numbering system). In a particular embodiment, the constant region of an antibody IgGl described herein comprises a substitution of methionine (M) to tyrosine (Y) at position 252, a substitution of serine (S) to threonine (T) at position 254; and a substitution of threonine (T) to glutamic acid (E) at position 256 (numbered according to the EU numbering system). See U.S. Patent No. 7,658,921, which is incorporated herein by reference in its entirety. Such mutant IgGs, termed “YTE mutants,” have been demonstrated to exhibit a four-fold increase in half-life compared to the wild-type version of the same antibody (see Dall Acqua WF et al. (2006) J Biol Chem 281:23514-24, which is incorporated herein by reference in its entirety). In some embodiments, the Fc region comprises M252Y, S254T, and T256E (YTE mutations).

[0197] Antibody drug conjugates (ADCs)

[0198] In another aspect, the present application provides an antibody drug conjugate comprising an antibody or antigen binding fragment thereof disclosed herein and a chemical moiety conjugated to the antibody or antigen binding fragment thereof.

[0199] In the context of this disclosure, "antibody-drug conjugate," "antibody-drug conjugate," or "antibody conjugate" refers to an antibody or antibody fragment (such as an antigen-binding fragment) covalently linked to a chemical moiety. The chemical moiety may be selected from cytotoxic drugs, immunostimulatory molecules, and detectable markers, such as drugs, toxins, therapeutic agents, detectable markers, proteins, nucleic acids, lipids, nanoparticles, carbohydrates, or recombinant viruses. When an antibody-drug conjugate contains an antibody linked to a drug (e.g., a cytotoxic agent), the conjugate is generally referred to as an "antibody-drug conjugate" or "ADC."

[0200] The terms "coupling," "conjugation," or "linking" can refer to the process by which two polypeptides become a single, continuous polypeptide molecule. In one embodiment, an antibody is linked to a chemical moiety. In another embodiment, the antibody linked to the chemical moiety is further linked to a lipid or other molecule to a protein or peptide to increase its half-life in vivo. Linking can be performed chemically or recombinantly. In one embodiment, the linking is chemical, wherein a reaction between the antibody moiety and the chemical moiety produces a covalent bond formed between the two molecules to form a single molecule. A peptide linker (short peptide sequence) may optionally be included between the antibody and the chemical moiety.

[0201] Chemical motifs can be linked to the antibodies of the present invention in any number of ways known to those skilled in the art. In this document, the number of chemical motifs linked to a single antibody of the present invention can be expressed as “DAR”. For example, in some embodiments, “DAR” represents the number of linker-payloads linked to a single antibody of the present invention.

[0202] Covalent and non-covalent attachment methods can be used. The procedure for attaching the chemical moiety to the antibody varies depending on the chemical structure of the chemical moiety. Peptides typically contain multiple functional groups; such as carboxylic acid (COOH), free amine (-NH2), or thiol (-SH) moieties, which can be used to react with suitable functional groups on the antibody to result in the binding of the chemical moiety. Alternatively, the antibody is derivatized to expose or attach additional reactive functional groups. Derivatization can involve attaching any of many known linker molecules. The linker can be any molecule used to link the antibody to the chemical moiety. The linker is capable of forming a covalent bond with both the antibody and the chemical moiety. Suitable linkers are well known to those skilled in the art and include, but are not limited to, straight-chain or branched-chain carbon linkers, heterocyclic carbon linkers, or peptide linkers. In the case where the antibody and the chemical moiety are peptides, the linker can be attached to the constituent amino acid (e.g., via a disulfide bond to cysteine) or to the α-carbon amino and carboxyl groups of the terminal amino acid via their side groups.

[0203] In certain cases, it is desirable to release the chemical moiety from the antibody when the antibody conjugate reaches its target site. Thus, in these cases, the antibody conjugate will comprise a cleavable linkage that is cleavable near the target site.

[0204] The conditions experienced by the enzymatic activity or antibody conjugate within the target cell or near the target site can prompt cleavage of the linker to release the chemical moiety from the antibody.

[0205] Given the large number of methods reported for attaching various radiodiagnostic compounds, radiotherapeutic compounds, markers (such as enzymes or fluorescent molecules), drugs, toxins, and other agents to antibodies, one of skill in the art will be able to determine a suitable method for attaching a given agent to an antibody or other polypeptide.

[0206] In one embodiment, the present application provides an antibody drug conjugate of Formula (I), or a pharmaceutically acceptable salt, isotopic variant, tautomer, stereoisomer, polymorph, hydrate, or solvate thereof, or mixtures thereof: y (I)

[0207] wherein,

[0208] T is a targeting moiety, and is an antibody or antigen-binding fragment thereof that binds CDH17;

[0209] L is a linker unit;

[0210] D is a biologically active fragment;

[0211] y is selected from an integer or decimal number from 0.1 to 20.

[0212] T

[0213] In one embodiment, T is a targeting moiety, and is an antibody or antigen-binding fragment thereof that binds CDH17.

[0214] In one embodiment, the antibody or antigen-binding fragment thereof that binds CDH17 is as defined herein.

[0215] In one specific embodiment, T is mAb 1; in another specific embodiment, T is mAb 2; in another specific embodiment, T is mAb 3; in another specific embodiment, T is mAb 4; in another specific embodiment, T is mAb 5; in another specific embodiment, T is mAb 6; in another specific embodiment, T is mAb 7; in another specific embodiment, T is mAb 3-H2K2; in another specific embodiment, T is mAb 3-H3K2; in another specific embodiment, T is mAb 3-H3K4; in another specific embodiment, T is mAb 5-H3K1; in another specific embodiment, T is mAb 5-H5K2.

[0216] L

[0217] In one specific embodiment, L comprises a cleavable linker or a non-cleavable linker.

[0218] In one specific embodiment, L comprises a cleavable linker, which comprises an acid-labile linker, a hydrophilic linker, a protease-sensitive linker, a photo-labile linker, a hydrazone linker, a dimethyl linker, or a disulfide-containing linker.

[0219] In one specific embodiment, the L comprises or is derived from a fragment of mc(6-maleimidocaproyl), Val-Cit(valine-citrulline), p-amino-benzyloxy carbonyl (PABC), SPDB (N-succinimidyl-4-(2-pyridyldithio)-butyrate), sulfo-SPDB (N-succinimidyl-4-(2-pyridyldithio)-2-sulfo-butyrate), beta-glucuronic acid, dimethyl ethyl amine (DMEA), Val-Cit-PABC, mc-Val-Cit-PABC, CL2A, mal-PEG8-Val-Ala-PABC, mc-VC-PABC-DMEA, GGFG (glycine-glycine-phenylalanine-glycine), mc-GGFG- aminomethyl, AcBut (4-(4-acetylphenoxy)-butyric acid), dimethyl hydrazide (3-methyl-) 3- mercaptobutane hydrazide), AcBut-dimethyl hydrazide, or SMCC (N-succinimidyl-4-(N- maleimidomethyl) cyclohexane-carboxylate).

[0220] In one specific embodiment, L is -L1-L2-L3-.

[0221] In one more specific embodiment, L is In another more specific embodiment, L is In another more specific embodiment, L is In another, more specific implementation, L is In another, more specific implementation, L is In another, more specific implementation, L is In another, more specific implementation, L is In another, more specific implementation, L is In another, more specific implementation, L is In another, more specific implementation, L is In another, more specific implementation, L is In another, more specific implementation, L is In another, more specific implementation, L is In another, more specific implementation, L is

[0222] L1

[0223] In one specific implementation, L1 is -L 1a -L 1b -L 1c -L 1d -L 1e -

[0224] In one specific implementation plan, L 1a for In another specific implementation scheme, L 1a for In another specific implementation scheme, L 1a for In another specific implementation scheme, L 1a for

[0225] In a more specific implementation scheme, L 1a for L represents 1a The binding site with the antibody; in another, more specific embodiment, L 1a for L represents 1a The binding site with the antibody; in another, more specific embodiment, L 1a for L represents 1a The binding site with the antibody; in another, more specific embodiment, L 1a for L represents 1a The binding site with the antibody.

[0226] In one specific implementation plan, L 1b C 1-10 Alkylene; in another embodiment, L 1b C 1-6 Alkylene; in another embodiment, L 1b C 2-10 alkenyl; in another embodiment, L 1b C 2-6 alkenyl; in another embodiment, L 1b C 2- 10 etymynyl; in another embodiment, L 1b C 2-6 alkenyl; in another embodiment, L 1b C 2-6 Idemyne ​​group; in another embodiment, the aforementioned C 1-10 Alkylene, C 1-6 Alkylene, C 2-10 imidene group, C 2-6 imidene group, C 2-10 etyne and C 2-6 Each CH2 group in the ethynyl group is not substituted; in another embodiment, the aforementioned C 1-10 Alkylene, C 2-10 imide and C 2-10 Each CH2 group in the ethynyl group is optionally surrounded by one, two, or three R groups. x replace.

[0227] In a specific implementation plan, each R x and R x 'Independently selected from H, halogen, C 1-6 Alkyl or C 1-6 Haloalkyl; or, two R atoms on any same or different carbon atoms. x Can be connected to form C 3-10 Cycloalkylene or 3-10 membered heterocyclic alkylene, preferably C 3-7 Cycloalkylene or 3-7 membered heterocyclic alkylene, more preferably C 3-5 Cycloalkylene.

[0228] In one specific implementation plan, L 1c For chemical bonds; in another embodiment, L 1c For -C(O)-; in another embodiment, L 1c For -C(O)NH-; in another embodiment, L 1c It is -NHC(O)-.

[0229] In one specific implementation plan, L1d For chemical bonds; in another embodiment, L 1d C 1-8 Alkylene; in another embodiment, L 1d C 1-6 Alkylene; in another embodiment, L 1d -(CH2CH2O) n -C 1-4 alkylene-; in another embodiment, L 1d C 1-8 Alkylene or C 1-6 Alkylene, the C 1-8 Alkylene and C 1-6 Alkylene is optionally labeled -NHC(O)-(CH2CH2O) w -C 1-4 Alkyl substitution; in another embodiment, L 1d C 1-8 Alkylene or C 1-6 Alkylene, the C 1-8 Alkylene and C 1-6 Alkylene is optionally prefixed with -C(O)NH-(CH2CH2O) w -C 1-4 Alkyl substitution.

[0230] In a specific implementation plan, n is selected from 1, 2, 3, 4, 5, 6, 7 or 8.

[0231] In a specific implementation plan, w is selected from 1, 2, 3, 4, 5, 6, 7 or 8.

[0232] In one specific implementation plan, L 1e For -C(O)-; in another embodiment, L 1e For -NHC(O)-; in another embodiment, L 1e It is -C(O)-NHC(O)-.

[0233] L2

[0234] In one embodiment, L2 is a chemical bond; in another embodiment, L2 is selected from a divalent peptide group comprising 2 to 8 optionally substituted natural or non-natural amino acid residues.

[0235] In one embodiment, L2 is selected from the group consisting of a divalent peptidyl group comprising 2 to 5 optionally substituted natural amino acid residues or non-natural amino acid residues; in another embodiment, L2 is selected from the group consisting of gly-gly, gly-gly-gly, gly-gly-gly-gly, val-gly-gly, val-cit-gly, val-gln-gly, val-glu-gly, phe-lys-gly, leu-lys-gly, gly-val-lys-gly, val-lys-gly-gly, val-lys-gly, val-lys-ala, val-lys-leu, leu-leu-gly, gly-gly-phe-gly, gly-gly-phe-gly-gly, val-gly, val-cit, or val-lys-β-ala.

[0236] In one embodiment, L2 is selected from the group consisting of a divalent peptidyl group comprising 2 to 5 optionally substituted natural amino acid residues or non-natural amino acid residues; in another embodiment, L2 is selected from the group consisting of gly-gly, gly-gly-gly, gly-gly-gly-gly, val-gly-gly, val-cit-gly, val-gln-gly, val-glu-gly, phe-lys-gly, leu-lys-gly, gly-val-lys-gly, val-lys-gly-gly, val-lys-gly, val-lys-ala, val-lys-leu, leu-leu-gly, gly-gly-phe-gly, gly-gly-phe-gly-gly, val-gly, val-cit, or val-lys-β-ala.

[0237] In one embodiment, L2 is selected from the group consisting of a divalent peptidyl group comprising 2 to 5 optionally substituted natural amino acid residues or non-natural amino acid residues; in another embodiment, L2 is selected from the group consisting of gly-gly, gly-gly-gly, gly-gly-gly-gly, val-gly-gly, val-cit-gly, val-gln-gly, val-glu-gly, phe-lys-gly, leu-lys-gly, gly-val-lys-gly, val-lys-gly-gly, val-lys-gly, val-lys-ala, val-lys-leu, leu-leu-gly, gly-gly-phe-gly, gly-gly-phe-gly-gly, val-gly, val-cit, or val-lys-β-ala.

[0238] In one embodiment, L2 is selected from the group consisting of a divalent peptidyl group comprising 2 to 5 optionally substituted natural amino acid residues or non-natural amino acid residues; in another embodiment, L2 is selected from the group consisting of gly-gly, gly-gly-gly, gly-gly-gly-gly, val-gly-gly, val-cit-gly, val-gln-gly, val-glu-gly, phe-lys-gly, leu-lys-gly, gly-val-lys-gly, val-lys-gly-gly, val-lys-gly, val-lys-ala, val-lys-leu, leu-leu-gly, gly-gly-phe-gly, gly-gly-phe-gly-gly, val-gly, val-cit, or val-lys-β-ala. y substituted with 1, 2, 3, 4, or 5 R

[0239] In one embodiment, each R y is independently selected from H, halogen, C 1-6 alkyl, C 1-6 alkoxy, C 1-6 haloalkyl, or C 1- 6haloalkoxy.

[0240] L3

[0241] In one embodiment, L3 is an optional spacer, which can or can not be substituted; in one embodiment, L3 is -NH-CH2-(AM); in one embodiment, L3 is In one embodiment, L3 is an optional spacer, which can or can not be substituted; in one embodiment, L3 is -NH-CH2-(AM); in one embodiment, L3 is

[0242] In a particular embodiment, L3 is unsubstituted; in another particular embodiment, L3 is optionally substituted with 1, 2, or 3 R z substituted.

[0243] In a particular embodiment, each R z is independently selected from H, halogen, C 1-6 alkyl, C 1-6 alkoxy, C 1-6 haloalkyl, or C 1- 6haloalkoxy.

[0244] D

[0245] In one embodiment, D is selected from the group consisting of: metal complexes; antibiotics; DNA topoisomerase inhibitors; microtubulin inhibitors and microtubule polymerization inhibitors; DNA synthesis inhibitors; RNA polymerase II inhibitors; RNA splicesome inhibitors; agents acting on structural proteins; tumor signaling pathway inhibitors; proteasome inhibitors; histone deacetylase inhibitors; tumor neovascularization inhibitors; cyclin inhibitors; serine / threonine kinase inhibitors, tyrosine kinase inhibitors, aspartate kinase inhibitors, or histidine kinase inhibitors; and other active substances that inhibit tumor cell growth, promote tumor cell apoptosis or necrosis.

[0246] In one embodiment, D is selected from the group consisting of microtubulin inhibitors and microtubule polymerization inhibitors, antibiotics, DNA synthesis inhibitors, topoisomerase I inhibitors, RNA polymerase II inhibitors, and RNA splicesome inhibitors.

[0247] In one embodiment, the D is selected from the group consisting of microtubulin inhibitors and microtubule polymerization inhibitors, for example auristatins, maytansinoids, tubulysins, cryptophycins, or rhizoxin.

[0248] In one embodiment, the D is an antibiotic, for example calicheamicin, doxorubicin, and anthracycline antibiotics;

[0249] In one embodiment, the D is selected from the group consisting of DNA synthesis inhibitors, for example duocarmycins, PBDs (pyrroloridine dioxanes), or IGNs (indolinobenzodiazepines).

[0250] In one embodiment, the D is selected from the group consisting of DNA topoisomerase I inhibitors, for example camptothecin or a camptothecin derivative.

[0251] In one embodiment, the D is selected from the group consisting of RNA polymerase II inhibitors, for example an andromedol.

[0252] In one embodiment, D is selected from RNA splicesome inhibitor drugs, such as splicetastatins and telatinastatins;

[0253] In one embodiment, D is selected from camptothecin or a camptothecin derivative, such as hydroxycamptothecin, 9-aminocamptothecin, SN-38, exatecan, Dxd, irinotecan, topotecan, or other derivatives.

[0254] In one embodiment, D is selected from auristatin drugs, such as MMAE or MMAF.

[0255] In one embodiment, D is selected from maytansinoid drugs, such as DM1, DM2, DM3, or DM4.

[0256] In one embodiment, D is selected from a compound of Formula (D-I), Formula (D-II), Formula (D-III), Formula (D-IV), or Formula (D-V), or a pharmaceutically acceptable salt, isotopologue, tautomer, stereoisomer, racemate, polymorph, hydrate, or solvate thereof, or mixtures thereof:

[0257] In one embodiment, each of the compounds of Formula (D-I), Formula (D-II), Formula (D-III), Formula (D-IV), and Formula (D-V) is optionally substituted with 1, 2, 3, 4, or 5 substituents selected from:

[0258] deuterium, halogen, C 1-6 alkyl, C 1-6 haloalkyl, C 2-6 alkenyl, C 2-6 alkynyl, C 3-8 cycloalkyl, 3-8 membered heterocyclyl, C 6-10 aryl or 5-10 membered heteroaryl.

[0259] In one specific embodiment, D is In another specific embodiment, D is In another specific embodiment, D is In another specific embodiment, D is

[0260] *1, *2, and *3 and q

[0261] In one embodiment, *1 is a chiral center selected from (S) or (R) absolute configuration, or a mixture thereof.

[0262] In one embodiment, *2 is a chiral center selected from (S) or (R) absolute configuration, or a mixture thereof.

[0263] In one embodiment, *3 is a chiral center selected from the (S) or (R) absolute configuration, or a mixture thereof; in a preferred embodiment, *3 is the (S) absolute configuration.

[0264] In one embodiment, q is 0, 1, 2, 3 or 4, preferably 1, 2 or 3;

[0265] L D1 , L D2 , L D3 and L D4

[0266] In one embodiment, L D1 is a chemical bond; in another embodiment, L D1 is -NH-; in another embodiment, L D1 is -O-; in another embodiment, L D1 is -C(O)-; in another embodiment, L D1 is -NHC(O)-; in another embodiment, L D1 is -C(O)NH-.

[0267] In one embodiment, L D2 is -NH-; in another embodiment, L D2 is -O-; in another embodiment, L D2 is -C(O)-; in another embodiment, L D2 is -NHC(O)-; in another embodiment, L D2 is -C(O)NH-.

[0268] In one embodiment, L D3 is a chemical bond; in another embodiment, L D3 is C 1-10 alkylene, for example C 1-6 alkylene; in another embodiment, L D3 is C 1-10 haloalkylene, for example C 1-6 haloalkylene; in another embodiment, L D3 is C 2-10 alkenylene, for example C 2-6 alkenylene; in another embodiment, L D3 is C 2-10 alkynylene, for example C 2-6 alkynylene.

[0269] In one embodiment, L D3 is unsubstituted; in another embodiment, L D3 is optionally substituted by 1, 2 or 3 RD3 replace.

[0270] In one implementation, L D4 For -NH-; in another embodiment, L D4 For -O-; in another implementation, L D4 For -C(O)-; in another embodiment, L D4 For -NHC(O)-; in another embodiment, L D4 It is -C(O)NH-.

[0271] R D1 R D2 R D3 R D4 R D5 and R D6

[0272] In one implementation, R D1 For H; in another implementation, R D1 For halogen; in another embodiment, R D1 C 1-6 Alkyl, such as Me; in another embodiment, R D1 C 1-6 Halogenated alkyl; in another embodiment, R D1 C 1-6 Alkyl group.

[0273] In one implementation, R D2 For H; in another implementation, R D2 For example, halogen, such as F; in another embodiment, R D2 C 1-6 Alkyl; in another embodiment, R D2 C 1-6 Halogenated alkyl; in another embodiment, R D2 C 1-6 Alkyl group.

[0274] In one implementation, R D1 R D2 Together with the carbon atoms they are attached to, they form C 3-7 cycloalkyl or 3-7 membered heterocyclic group; in another embodiment, R D1 R D2 Together with the carbon atoms they are attached to, they form 5-6 membered heterocyclic groups.

[0275] In one implementation, R D6 For H; in another implementation, R D6 For halogen; in another embodiment, RD6 C; 1-6 alkyl; in another embodiment, R D6 C; 1-6 haloalkyl; in another embodiment, R D6 C; 1-6 alkoxy.

[0276] in one embodiment, R D1 , R D6 and the carbon atom to which they are attached together form C 3-7 cycloalkyl or 3-7 membered heterocyclyl; in another embodiment, R D1 , R D6 and the carbon atom to which they are attached together form C 5-6 cycloalkyl.

[0277] in one embodiment, R D3 is H; in another embodiment, R D3 is halo; in another embodiment, R D3 C; 1-6 alkyl; in another embodiment, R D3 C; 1-6 haloalkyl; in another embodiment, R D3 C; 1-6 alkoxy; in another embodiment, R D3 C; 1-6 haloalkoxy; in another embodiment, R D3 C; 3-7 cycloalkyl; in another embodiment, R D3 3-7 membered heterocyclyl.

[0278] in one embodiment, R D4 is H; in another embodiment, R D4 is halo; in another embodiment, R D4 C; 1-6 alkyl; in another embodiment, R D4 C; 1-6 haloalkyl; in another embodiment, R D4 C; 1-6 alkoxy; in another embodiment, R D4 C; 1-6 haloalkoxy; in another embodiment, R D4 C; 3-7 cycloalkyl; in another embodiment, R D4 3-7 membered heterocyclyl.

[0279] in one embodiment, R D3 and RD4 is not simultaneously H.

[0280] In one embodiment, R D3 , R D4 and the carbon atom to which they are attached together form a C 3-7 cycloalkyl or 3-7 membered heterocyclyl; in another embodiment, R D3 , R D4 and the carbon atom to which they are attached together form a C 3-5 cycloalkyl.

[0281] In one embodiment, R D5 is halo; in another embodiment, R D5 is C 1-6 alkyl; in another embodiment, R D5 is C 1-6 haloalkyl; in another embodiment, R D5 is C 3-10 cycloalkyl; in another embodiment, R D5 is 3-10 membered heterocyclyl; in another embodiment, R D5 is C 6-10 aryl; in another embodiment, R D5 is 5-10 membered heteroaryl; in another embodiment, R D5 is -C 1-6 alkylene-C 3-10 cycloalkyl, preferably -C 1-4 alkylene-C 3-5 cycloalkyl; in another embodiment, R D5 is -C 1-6 alkylene-3-10 membered heterocyclyl, preferably -C 1-4 alkylene-3-5 membered heterocyclyl; in another embodiment, R D5 is -C 1-6 alkylene-C 6-10 aryl; in another embodiment, R D5 is -C 1-6 alkylene-5-10 membered heteroaryl.

[0282] y

[0283] In one embodiment, y is selected from an integer or decimal number from about 0.1 to about 20; in another embodiment, y is selected from an integer or decimal number from about 1 to about 10; in another embodiment, y is selected from an integer or decimal number from about 2 to about 8.

[0284] In one specific embodiment, y is about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15; in another specific embodiment, y is about 5.5, 6.5, 7.5, 8.5, 9.5; in another specific embodiment, y is about 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, or 6.0; in another specific embodiment, y is about 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, or 9.0.

[0285] Any of the technical solutions in any of the above specific embodiments or any combination thereof can be combined with any of the technical solutions in other specific embodiments or any combination thereof. For example, any of the technical solutions of T or any combination thereof can be combined with any of the technical solutions of y, L1, L2, L3, L 1a 1b 1c 1d 1e D1 D2 D3 D4 D1 D2 D3 D4 D5 D6 The present application is intended to include all combinations of these technical solutions, which are not listed one by one due to the length of the article.

[0286] In a more specific embodiment, the present application relates to the antibody drug conjugate of formula (I) above, wherein the antibody or antigen binding fragment thereof that binds CDH17 comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein:

[0287] (1) the VH comprises HCDR1, HCDR2, HCDR3 having the amino acid sequences of SEQ ID NOs: 15, 16, 17, respectively, and the VL comprises LCDR1, LCDR2, and LCDR3 having the amino acid sequences of SEQ ID NOs: 20, 21, 22, respectively; or ​​​​​​​​​​​​​​

[0288] (2) the VH comprises HCDR1, HCDR2, HCDR3, each having an amino acid sequence as set forth in SEQ ID NOs: 25, 26, 27, respectively, and the VL comprises LCDR1, LCDR2, and LCDR3, each having an amino acid sequence as set forth in SEQ ID NOs: 30, 31, 32, respectively; or

[0289] (3) the VH comprises HCDR1, HCDR2, HCDR3, each having an amino acid sequence as set forth in SEQ ID NOs: 35, 36, 37, respectively, and the VL comprises LCDR1, LCDR2, and LCDR3, each having an amino acid sequence as set forth in SEQ ID NOs: 40, 41, 42, respectively; or

[0290] (4) the VH comprises HCDR1, HCDR2, HCDR3, each having an amino acid sequence as set forth in SEQ ID NOs: 45, 46, 47, respectively, and the VL comprises LCDR1, LCDR2, and LCDR3, each having an amino acid sequence as set forth in SEQ ID NOs: 50, 51, 52, respectively; or

[0291] (5) the VH comprises HCDR1, HCDR2, HCDR3, each having an amino acid sequence as set forth in SEQ ID NOs: 55, 56, 57, respectively, and the VL comprises LCDR1, LCDR2, and LCDR3, each having an amino acid sequence as set forth in SEQ ID NOs: 60, 61, 62, respectively; or

[0292] (6) the VH comprises HCDR1, HCDR2, HCDR3, each having an amino acid sequence as set forth in SEQ ID NOs: 65, 66, 67, respectively, and the VL comprises LCDR1, LCDR2, and LCDR3, each having an amino acid sequence as set forth in SEQ ID NOs: 70, 71, 72, respectively; or

[0293] (7) the VH comprises HCDR1, HCDR2, HCDR3, each having an amino acid sequence as set forth in SEQ ID NOs: 25, 125, 27, respectively, and the VL comprises LCDR1, LCDR2, and LCDR3, each having an amino acid sequence as set forth in SEQ ID NOs: 120, 31, 32, respectively; or

[0294] (8) the VH comprises HCDR1, HCDR2, HCDR3, each having an amino acid sequence as set forth in SEQ ID NOs: 45, 46, 47, respectively, and the VL comprises LCDR1, LCDR2, and LCDR3, each having an amino acid sequence as set forth in SEQ ID NOs: 90, 51, 52, respectively.

[0295] In a more particular embodiment, the application relates to the above antibody drug conjugate of formula (I), wherein:

[0296] (1) the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 13, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 18; or

[0297] (2) the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 23, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 28; or

[0298] (3) the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 33, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 38; or

[0299] (4) the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 43, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 48; or

[0300] (5) the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 53, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 58; or

[0301] (6) the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 63, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 68; or

[0302] (7) the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 101, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 118; or

[0303] (8) the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 101, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 123; or

[0304] (9) the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 103, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 118; or

[0305] (10) the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 103, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 123; or

[0306] (11) the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 77, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 86; or

[0307] (12) the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 82, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 88.

[0308] In one specific embodiment:

[0309] (1) the VH comprises the amino acid sequence set forth in SEQ ID NO: 13, and the VL comprises the amino acid sequence set forth in SEQ ID NO: 18; or

[0310] (2) the VH comprises the amino acid sequence set forth in SEQ ID NO: 23, and the VL comprises the amino acid sequence set forth in SEQ ID NO: 28; or

[0311] (3) the VH comprises the amino acid sequence set forth in SEQ ID NO: 33, and the VL comprises the amino acid sequence set forth in SEQ ID NO: 38; or

[0312] (4) the VH comprises the amino acid sequence set forth in SEQ ID NO: 43, and the VL comprises the amino acid sequence set forth in SEQ ID NO: 48; or

[0313] (5) the VH comprises the amino acid sequence set forth in SEQ ID NO: 53, and the VL comprises the amino acid sequence set forth in SEQ ID NO: 58; or

[0314] (6) the VH comprises the amino acid sequence set forth in SEQ ID NO: 63, and the VL comprises the amino acid sequence set forth in SEQ ID NO: 68; or

[0315] (7) the VH comprises the amino acid sequence set forth in SEQ ID NO: 101, and the VL comprises the amino acid sequence set forth in SEQ ID NO: 118; or

[0316] (8) the VH comprises an amino acid sequence as set forth in SEQ ID NO: 101, and the VL comprises an amino acid sequence as set forth in SEQ ID NO: 123; or

[0317] (9) the VH comprises an amino acid sequence as set forth in SEQ ID NO: 103, and the VL comprises an amino acid sequence as set forth in SEQ ID NO: 118; or

[0318] (10) the VH comprises an amino acid sequence as set forth in SEQ ID NO: 103, and the VL comprises an amino acid sequence as set forth in SEQ ID NO: 123; or

[0319] (11) the VH comprises an amino acid sequence as set forth in SEQ ID NO: 77, and the VL comprises an amino acid sequence as set forth in SEQ ID NO: 86; or

[0320] (12) the VH comprises an amino acid sequence as set forth in SEQ ID NO: 82, and the VL comprises an amino acid sequence as set forth in SEQ ID NO: 88.

[0321] In a more specific embodiment, the present application relates to the above antibody drug conjugate of formula (I), wherein the antibody is a murine antibody, a chimeric antibody or a humanized antibody.

[0322] In a more specific embodiment, the present application relates to the above antibody drug conjugate of formula (I), wherein the antibody belongs to an isotype selected from the group consisting of IgG, IgA, IgM, IgE and IgD.

[0323] In a more specific embodiment, the present application relates to the above antibody drug conjugate of formula (I), wherein the antibody belongs to a subtype selected from the group consisting of IgGl, IgG2, IgG3 and IgG4.

[0324] In a more specific embodiment, the present application relates to the above antibody drug conjugate of formula (I), wherein the antibody comprises a heavy chain (HC) and a light chain (LC), wherein:

[0325] (1) the HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity with SEQ ID NO: 126, and the LC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity with SEQ ID NO: 128; or

[0326] (2) the HC comprises an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 130, and the LC comprises an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 132; or

[0327] (3) the HC comprises an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 134, and the LC comprises an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 136; or

[0328] (4) the HC comprises an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 134, and the LC comprises an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 138; or

[0329] (5) the HC comprises an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 140, and the LC comprises an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 136; or

[0330] (6) the HC comprises an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 140, and the LC comprises an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 138; or

[0331] (7) the HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 142, and the LC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 143.

[0332] In a more specific embodiment, the present application relates to the above antibody drug conjugate of Formula (I), wherein the antibody comprises a heavy chain (HC) and a light chain (LC), wherein:

[0333] (1) the HC comprises an amino acid sequence as set forth in SEQ ID NO: 126, and the LC comprises an amino acid sequence as set forth in SEQ ID NO: 128; or

[0334] (2) the HC comprises an amino acid sequence as set forth in SEQ ID NO: 130, and the LC comprises an amino acid sequence as set forth in SEQ ID NO: 132; or

[0335] (3) the HC comprises an amino acid sequence as set forth in SEQ ID NO: 134, and the LC comprises an amino acid sequence as set forth in SEQ ID NO: 136; or

[0336] (4) the HC comprises an amino acid sequence as set forth in SEQ ID NO: 134, and the LC comprises an amino acid sequence as set forth in SEQ ID NO: 138; or

[0337] (5) the HC comprises an amino acid sequence as set forth in SEQ ID NO: 140, and the LC comprises an amino acid sequence as set forth in SEQ ID NO: 136; or

[0338] (6) the HC comprises an amino acid sequence as set forth in SEQ ID NO: 140, and the LC comprises an amino acid sequence as set forth in SEQ ID NO: 138; or

[0339] (7) the HC comprises an amino acid sequence as set forth in SEQ ID NO: 142, and the LC comprises an amino acid sequence as set forth in SEQ ID NO: 143.

[0340] In a more specific embodiment, the present application relates to the above antibody drug conjugate of Formula (I), wherein the antigen binding fragment is selected from the group consisting of Fab, Fab’, F(ab’)2, Fv, scFv, and ds-scFv.

[0341] In a more particular embodiment, the application relates to the above antibody drug conjugate of formula (I), wherein the antibody is a monoclonal antibody, a bispecific antibody or a multispecific antibody.

[0342] In a more particular embodiment, the application relates to the above antibody drug conjugate of formula (I), wherein the antibody is a bispecific antibody, further comprising a second antigen binding region binding to a second antigen.

[0343] In a more particular embodiment, the application relates to the above antibody drug conjugate of formula (I), wherein,

[0344] L comprises a cleavable linker or a non-cleavable linker;

[0345] Preferably, the cleavable linker comprises an acid-labile linker, a hydrophilic linker, a protease-sensitive linker, a photolabile linker, a hydrazone linker, a dimethyl linker or a disulfide-containing linker.

[0346] In a particular embodiment, wherein,

[0347] L comprises or is derived from a fragment of mc(6-maleimidocaproyl), Val-Cit(valine-citrulline), p-amino-benzyloxycarbonyl (PABC), SPDB (N-succinimidyl-4-(2-pyridyldithio)-butyrate), sulfo-SPDB (N-succinimidyl-4-(2-pyridyldithio)-2-sulfo-butyrate), beta-glucuronide, dimethyl ethyl amine (DMEA), Val-Cit-PABC, mc-Val-Cit-PABC, CL2A, mal-PEG8-Val-Ala-PABC, mc-VC-PABC-DMEA, GGFG (glycine-glycine-phenylalanine-glycine), mc-GGFG- aminomethyl, AcBut (4-(4-acetylphenoxy)-butyric acid), dimethyl hydrazide (3-methyl-) 3- mercaptobutane hydrazide), AcBut-dimethyl hydrazide or SMCC (N-succinimidyl-4-(N- maleimidomethyl) cyclohexane-carboxylate).

[0348] In a more particular embodiment, the application relates to the above antibody drug conjugate of formula (I), wherein,

[0349] L is -L1-L2-L3-;

[0350] L1 is -L 1a -L 1b -L 1c -L 1d -L 1e -;

[0351] L 1a is selected from

[0352] L 1b is selected from C 1-10 alkylene, C 2-10 alkenylene, or C 2-10 alkynylene, wherein each CH2in said C 1-10 alkylene, C 2-10 alkenylene, and C 2-10 alkynylene is optionally substituted with 1, 2, or 3 R x ;

[0353] each R x is independently selected from H, halogen, C 1-6 alkyl, or C 1-6 haloalkyl; or, two R x on any identical or different carbon atom can be linked to form a C 3-10 cycloalkylene, or 3-10 membered heterocyclylene;

[0354] L 1c is selected from a bond, -C(O)-, -C(O)NH-, or -NHC(O)-;

[0355] L 1d is selected from a bond, C 1-8 alkylene, or -(CH2CH2O) n -C 1-4 alkylene-; said C 1-8 alkylene is optionally substituted with -NHC(O)-(CH2CH2O) w -C 1-4 alkyl, or -C(O)NH-(CH2CH2O) w -C 1-4 alkyl;

[0356] n and w are independently selected from 1, 2, 3, 4, 5, 6, 7, or 8;

[0357] L 1e is selected from -C(O)-, -NHC(O)-, or -C(O)-NHC(O)-;

[0358] L2is a bond or is selected from a divalent peptidyl group comprising 2 to 8 optionally substituted natural amino acid residues or non-natural amino acid residues,

[0359] each amino acid residue in said L2is optionally substituted with 1, 2, 3, 4, or 5 R y ;

[0360] each R y is independently selected from H, halogen, C1-6 Alkyl, C 1-6 Alkoxy, C 1-6 Halogenated alkyl or C 1-6 Halogenated alkoxy groups;

[0361] L3 can be an optional substituted or unsubstituted spacer, for example, L3 is selected from -NH-CH2-(AM).

[0362] L3 can be optionally controlled by one, two, or three Rs. z Replace, each R z Independently selected from H, halogen, C 1-6 Alkyl, C 1-6 Alkoxy, C 1-6 Halogenated alkyl or C 1-6 Halogenated alkoxy groups.

[0363] In a more specific embodiment, the present invention relates to antibody-drug conjugates of formula (I) above, wherein,

[0364] L is -L1-L2-L3-;

[0365] L1 is -L 1a -L 1b -L 1c -L 1d -L 1e -;

[0366] L 1a Selected from L represents 1a Binding site with antibody;

[0367] L 1b Selected from C 1-6 Alkylene, C 2-6 imide or C 2-6 Idemynyl group, in which C 1-6 Alkylene, C 2-6 imide and C 2-6 Each CH2 group in the ethynyl group is optionally surrounded by one or two R groups. x replace;

[0368] Each R x Independently selected from H, halogen, or C 1-6 Alkyl group; or, any two R atoms on the same or different carbon atoms. x Can be connected to form C 3- 7-membered cycloalkyl or 3-7-membered heterocyclic group, preferably C 3-7 Cycloalkylene, more preferably C10, 3-5 Cycloalkylene;

[0369] L1c is selected from a chemical bond, -C(O)-, -C(O)NH- or -NHC(O)-, preferably a chemical bond or -C(O)NH-;

[0370] L 1d is selected from a chemical bond or C 1-6 alkylene; said C 1-6 alkylene is optionally substituted with -NHC(O)-(CH2CH2O) w -C 1-4 alkyl or -C(O)NH-(CH2CH2O) w -C 1-4 alkyl;

[0371] w is selected from 2, 3 or 4, preferably 3;

[0372] L 1e is selected from -C(O)-, -NHC(O)- or -C(O)-NHC(O)-, preferably a chemical bond or -C(O)-;

[0373] L2is selected from a bivalent peptidic moiety comprising 2 to 5 optionally substituted natural or unnatural amino acid residues, each amino acid residue in said L2is optionally substituted with 1, 2 or 3 R y substituents;

[0374] each R y is independently selected from H, halogen, C 1-6 alkyl or C 1-6 haloalkyl;

[0375] L3is an optionally substituted or unsubstituted spacer, for example L3is selected from -NH-CH2-(AM) or

[0376] L3is optionally substituted with 1, 2 or 3 R z substituents, each R z is independently selected from H, halogen or C 1-6 alkyl.

[0377] In a more specific embodiment, the application relates to the antibody drug conjugate of formula (I) above, wherein,

[0378] L2is selected from a divalent peptidyl group comprising 2 to 5 optionally substituted natural or unnatural amino acid residues, selected from gly-gly, gly-gly-gly, gly-gly-gly-gly, val-gly-gly, val-cit-gly, val-gln-gly, val-glu-gly, phe-lys-gly, leu-lys-gly, gly-val-lys-gly, val-lys-gly-gly, val-lys-gly, val-lys-ala, val-lys-leu, leu-leu-gly, gly-gly-phe-gly, gly-gly-phe-gly-gly, val-gly, val-cit or val-lys-β-ala;

[0379] each amino acid residue in said L2is optionally substituted with 1, 2 or 3 R y ;

[0380] each R y is independently selected from H, halogen, C 1-6 alkyl or C 1-6 haloalkyl;

[0381] Preferably,

[0382] L2is selected from a divalent peptidyl group comprising 2 to 5 optionally substituted natural or unnatural amino acid residues, said amino acids being selected from glycine, phenylalanine, alanine, valine, citrulline or lysine;

[0383] each amino acid residue in said L2is optionally substituted with 1, 2 or 3 R y ;

[0384] each R y is independently selected from H, halogen or C 1-6 alkyl;

[0385] More preferably, L2is -gly-gly-phe-gly- or -val-cit-.

[0386] In a more specific embodiment, the application relates to the antibody drug conjugate of formula (I) above, wherein L is selected from the following structures:

[0387] In a more specific embodiment, the application relates to the antibody drug conjugate of formula (I) above, wherein,

[0388] L is selected from the following structures:

[0389] In a more specific embodiment, the application relates to an antibody drug conjugate of formula (I) as described above, wherein

[0390] D is selected from the group consisting of metal complexes; antibiotics; DNA topoisomerase inhibitors; microtubulin inhibitors and microtubule polymerization inhibitors; DNA synthesis inhibitors; RNA polymerase II inhibitors; RNA splicesome inhibitors; agents acting on structural proteins; tumor signaling pathway inhibitors; proteasome inhibitors; histone deacetylase inhibitors; tumor neovascularization inhibitors; cyclin inhibitors; serine / threonine kinase inhibitors, tyrosine kinase inhibitors, aspartate kinase inhibitors or histidine kinase inhibitors; and other active substances that inhibit the growth of tumor cells, promote apoptosis or necrosis of tumor cells;

[0391] Preferably,

[0392] D is selected from the group consisting of microtubulin inhibitors and microtubule polymerization inhibitors, antibiotics, DNA synthesis inhibitors, topoisomerase I inhibitors, RNA polymerase II inhibitors and RNA splicesome inhibitors;

[0393] More preferably,

[0394] Said D is selected from the group consisting of microtubulin inhibitors and microtubule polymerization inhibitors, such as auristatins, maytansinoids, tubulysins, cryptophycins or rhizoxin;

[0395] Alternatively, said D is an antibiotic, such as a calicheamicin, an anthracycline and an anthracycline antibiotic;

[0396] Alternatively, said D is selected from the group consisting of DNA synthesis inhibitors, such as duocarmycins, PBDs (pyrroloridine dioxanes) or IGNs (indolinobenzodiazepines);

[0397] Alternatively, said D is selected from the group consisting of DNA topoisomerase I inhibitors, such as camptothecin or a camptothecin derivative;

[0398] Alternatively, said D is selected from the group consisting of RNA polymerase II inhibitors, such as an andrographolide;

[0399] Alternatively, said D is selected from the group consisting of RNA splicesome inhibitors, such as splicetastatins and telatinib;

[0400] Preferably, D is selected from the group consisting of camptothecin or a camptothecin derivative, such as hydroxy camptothecin, 9-amino camptothecin, SN-38, exatecan, Dxd, irinotecan, topotecan or other derivatives;

[0401] Preferably, D is selected from an auristatin, such as MMAE or MMAF;

[0402] Preferably, D is selected from a maytansinoid, such as DM1, DM2, DM3 or DM4.

[0403] In a more specific embodiment, the present application relates to an antibody drug conjugate of formula (I) as described above, wherein,

[0404] D is selected from an auristatin, such as MMAE or MMAF;

[0405] or D is selected from a compound of formula (D-I) or formula (D-II), or a pharmaceutically acceptable salt, isotopic variant, tautomer, stereoisomer, racemic, polymorph, hydrate or solvate thereof, or mixtures thereof:

[0406] *1, *2 and *3 are chiral centers independently selected from (S) or (R) absolute configuration, or mixtures thereof; preferably *3 is (S) absolute configuration;

[0407] L D1 is selected from a chemical bond, -NH-, -O-, -C(O)-, -NHC(O)- or -C(O)NH-;

[0408] q is 0, 1, 2, 3 or 4, preferably 1, 2 or 3;

[0409] L D2 is selected from -NH-, -O-, -C(O)-, -NHC(O)- or -C(O)NH-;

[0410] L D3 is selected from a chemical bond, C 1-10 alkylene, C 1-10 haloalkylene, C 2-10 alkenylene or C 2-10 alkynylene, said L D3 is optionally substituted with 1, 2 or 3 R D3 ;

[0411] L D4 is selected from -NH-, -O-, -C(O)-, -NHC(O)- or -C(O)NH-;

[0412] R D1 , R D2 and R D6 are independently selected from H, halogen, C 1-6 alkyl, C 1-6 haloalkyl or C 1-6 alkoxy; or, R D1 , RD2 together with the carbon atom to which they are attached form C 3-7 cycloalkyl or 3-7 membered heterocyclyl, preferably 5-6 membered heterocyclyl; or, R D1 , R D6 together with the carbon atom to which they are attached form C 3-7 cycloalkyl or 3-7 membered heterocyclyl, preferably C 5-6 cycloalkyl;

[0413] each R D3 and R D4 are independently selected from H, halogen, C 1-6 alkyl, C 1-6 haloalkyl, C 1-6 alkoxy, C 1-6 haloalkoxy, C 3-7 cycloalkyl or 3-7 membered heterocyclyl; or, R D3 , R D4 together with the carbon atom to which they are attached form C 3-7 cycloalkyl or 3-7 membered heterocyclyl, preferably C 3-5 cycloalkyl;

[0414] R D5 is selected from halogen, C 1-6 alkyl, C 1-6 haloalkyl, C 3-10 cycloalkyl, 3-10 membered heterocyclyl, C 6-10 aryl, 5-10 membered heteroaryl, -C 1-6 alkylene-C 3-10 cycloalkyl, -C 1-6 alkylene-3-10 membered heterocyclyl, -C 1-6 alkylene-C 6-10 aryl or -C 1-6 alkylene-5-10 membered heteroaryl, preferably -C 1-6 alkylene-C 3-10 cycloalkyl;

[0415] the compounds of formula (D-I) and (D-II) are each optionally substituted with 1, 2, 3, 4, or 5 substituents selected from:

[0416] deuterium, halogen, C 1-6 alkyl, C 1-6 haloalkyl, C 2-6 alkenyl, C 2-6 alkynyl, C 3-8 cycloalkyl, 3-8 membered heterocyclyl, C 6-10 aryl or 5-10 membered heteroaryl.

[0417] In a more specific embodiment, the present application relates to the antibody drug conjugate of formula (I) as described above, wherein

[0418] D is selected from an auristatin, such as MMAE or MMAF;

[0419] Alternatively, D is selected from a compound of formula (D-III) or (D-IV), or a pharmaceutically acceptable salt, isotopologue, tautomer, stereoisomer, racemate, polymorph, hydrate or solvate thereof, or mixtures thereof:

[0420] *1 and *2 are chiral centers independently selected from (S) or (R) absolute configuration, or mixtures thereof;

[0421] L D1 is selected from a chemical bond, -NH-, -O-, -C(O)-, -NHC(O)- or -C(O)NH-;

[0422] q is 0, 1, 2, 3 or 4, preferably 1, 2 or 3;

[0423] L D2 is selected from -NH-, -O-, -C(O)-, -NHC(O)- or -C(O)NH-;

[0424] L D3 is selected from a chemical bond, C 1-10 alkylene, C 1-10 haloalkylene, C 2-10 alkenylene or C 2-10 alkynylene, said L D3 is optionally substituted with 1, 2 or 3 R D3 ;

[0425] L D4 is selected from -NH-, -O-, -C(O)-, -NHC(O)- or -C(O)NH-;

[0426] R D1 , R D2 and R D6 are independently selected from H, halogen, C 1-6 alkyl, C 1-6 haloalkyl or C 1-6 alkoxy; or, R D1 , R D2 and the carbon atom to which they are attached form together a C 3-7 cycloalkyl or 3-7 membered heterocyclyl, preferably 5-6 membered heterocyclyl; or, R D1 , R D6 and the carbon atom to which they are attached form together a C 3-7cycloalkyl or 3-7 membered heterocyclyl, preferably C 5-6 cycloalkyl;

[0427] each R D3 and R D4 is independently selected from H, halogen, C 1-6 alkyl, C 1-6 haloalkyl or C 1-6 alkoxy; or, R D3 , R D4 and the carbon atom to which they are attached form C 3-7 cycloalkyl or 3-7 membered heterocyclyl, preferably C 3-5 cycloalkyl;

[0428] R D5 is selected from C 1-6 alkyl, C 3-10 cycloalkyl, 3-10 membered heterocyclyl, C 6-10 aryl, 5-10 membered heteroaryl, -C 1-6 alkylene-C 3-10 cycloalkyl, -C 1-6 alkylene-3-10 membered heterocyclyl, -C 1-6 alkylene-C 6-10 aryl or -C 1-6 alkylene-5-10 membered heteroaryl, preferably -C 1-6 alkylene-C 3-10 cycloalkyl.

[0429] In a more specific embodiment, the present application relates to the antibody drug conjugate of formula (I) above, wherein,

[0430] D is selected from an auristatin, such as MMAE or MMAF;

[0431] or, D is selected from a compound of formula (D-III), or a pharmaceutically acceptable salt, isotopologue, tautomer, stereoisomer, racemate, polymorph, hydrate or solvate thereof, or mixtures thereof:

[0432] *1 and *2 are chiral centers independently selected from (S) or (R) absolute configuration, or mixtures thereof;

[0433] L D1 is selected from -NHC(O)- or -C(O)NH-;

[0434] q is 1, 2 or 3, preferably 1;

[0435] L D2 is selected from -NH- or -O-;

[0436] R D1H, halogen, C 1-6 alkyl or C 1-6 haloalkyl, preferably C 1-6 alkyl, e.g. Me;

[0437] R D2 H, halogen, C 1-6 alkyl or C 1-6 haloalkyl, preferably halogen, e.g. F;

[0438] R D1 , R D2 and the carbon atom to which they are attached together form a C 3-7 cycloalkyl or 3-7 membered heterocyclyl, preferably 5-6 membered heterocyclyl;

[0439] each R D3 and R D4 are independently selected from H, halogen, C 1-6 alkyl, C 1-6 haloalkyl or C 1-6 alkoxy; or, R D3 , R D4 and the carbon atom to which they are attached together form a C 3-7 cycloalkyl or 3-7 membered heterocyclyl, preferably C 3-5 cycloalkyl;

[0440] R D5 -C 1-4 alkylene-C 3-5 cycloalkyl or -C 1-4 alkylene-3-5 membered heterocyclyl, preferably -C 1-4 alkylene-C 3-5 cycloalkyl.

[0441] In a more specific embodiment, the present application relates to the antibody drug conjugate of formula (I) above, wherein,

[0442] D is selected from an auristatin, e.g. MMAE or MMAF;

[0443] or, D is selected from a compound of formula (D-V), or a pharmaceutically acceptable salt, isotopologue, tautomer, stereoisomer, racemate, polymorph, hydrate or solvate thereof, or mixtures thereof:

[0444] *2 is a chiral center independently selected from (S) or (R) absolute configuration, or a mixture thereof;

[0445] q is 1, 2 or 3, preferably 1;

[0446] R D1 H, halogen, C1-6 alkyl or C 1-6 haloalkyl, preferably C 1-6 alkyl, for example Me;

[0447] R D2 is selected from H, halogen, C 1-6 alkyl or C 1-6 haloalkyl, preferably halogen, for example F;

[0448] or, R D1 , R D2 and the carbon atom to which they are attached together form a C 3-7 cycloalkyl or 3-7 membered heterocyclyl, preferably 5-6 membered heterocyclyl;

[0449] each R D3 and R D4 are independently selected from H, halogen, C 1-6 alkyl, C 1-6 haloalkyl or C 1-6 alkoxy, preferably R D3 and R D4 are not simultaneously H; or, R D3 , R D4 and the carbon atom to which they are attached together form a C 3-7 cycloalkyl or 3-7 membered heterocyclyl, preferably C 3-5 cycloalkyl;

[0450] R D5 is selected from -C 1-4 alkylene-C 3-5 cycloalkyl or -C 1-4 alkylene-3-5 membered heterocyclyl, preferably -C 1-4 alkylene-C 3-5 cycloalkyl; more preferably R D5 is selected from -methylene-cyclopropyl or -methylene-cyclobutyl.

[0451] In a more specific embodiment, the present application relates to the antibody drug conjugate of formula (I) above, wherein D is selected from the following compounds, or a pharmaceutically acceptable salt, isotopologue, tautomer, stereoisomer, racemate, polymorph, hydrate or solvate thereof, or mixtures thereof:

[0452] Preferably, D is selected from the following compounds, or a pharmaceutically acceptable salt, isotopologue, tautomer, stereoisomer, racemate, polymorph, hydrate or solvate thereof, or mixtures thereof:

[0453] In a more specific embodiment, the present application relates to the antibody drug conjugate of the above formula (I), wherein

[0454] y is an integer or decimal number selected from 0.1 to 20, preferably an integer or decimal number selected from 1 to 10, more preferably an integer or decimal number selected from 2 to 8.

[0455] In a more specific embodiment, the present application relates to the antibody drug conjugate of the above formula (I) having the structure of formula (I-1) or (I-2):

[0456] wherein,

[0457] T, D and y are as defined herein;

[0458] each m is selected from 1, 2, 3, 4 or 5;

[0459] each w is selected from 1, 2, 3, 4, 5, 6, 7 or 8;

[0460] each R x and R x are independently selected from H, halogen or C 1-6 alkyl; or, two R x on any identical or different carbon atom can be linked to form a C 3-7 cycloalkylene or 3-7 membered heterocyclylene, preferably a C 3-7 cycloalkylene, more preferably a C 3-5 cycloalkylene;

[0461] each R y is independently selected from H, halogen, C 1-6 alkyl, C 1-6 alkoxy, C 1-6 haloalkyl or C 1-6 haloalkoxy.

[0462] In a more specific embodiment, the present application relates to the antibody drug conjugate of the above formula (I), wherein the antibody drug conjugate is selected from the following structures:

[0463] wherein,

[0464] T, D and y are as defined herein;

[0465] preferably,

[0466] D is selected from an auristatin, a camptothecin or a camptothecin derivative, such as MMAE, MMAF, hydroxycamptothecin, 9-aminocamptothecin, SN-38, exatecan, Dxd, irinotecan, topotecan, or a compound of Formula (D-I), Formula (D-II), Formula (D-III), Formula (D-IV), or Formula (D-V), wherein the variables are as defined herein;

[0467] y is selected from an integer or a decimal number from 1 to 10.

[0468] In a more specific embodiment, the present application relates to the antibody drug conjugate of formula (I) above, wherein the antibody drug conjugate is selected from the following structures:

[0469] T and y are as defined herein;

[0470] Preferably, y is selected from an integer or a decimal number from 1 to 10.

[0471] In a more specific embodiment, the present application relates to the antibody drug conjugate of formula (I) above, wherein the antibody drug conjugate is selected from the following structures:

[0472] wherein,

[0473] the mAb2 comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises an amino acid sequence as set forth in SEQ ID NO: 13, and the VL of the mAb2 comprises an amino acid sequence as set forth in SEQ ID NO: 18;

[0474] the mAb3 comprises a heavy chain variable region (VH) and a light chain variable region (VL), the VH comprising an amino acid sequence as set forth in SEQ ID NO: 23, and the VL comprising an amino acid sequence as set forth in SEQ ID NO: 28;

[0475] the mAb4 comprises a heavy chain variable region (VH) and a light chain variable region (VL), the VH comprising an amino acid sequence as set forth in SEQ ID NO: 33, and the VL comprising an amino acid sequence as set forth in SEQ ID NO: 38;

[0476] The mAb5 comprises a heavy chain variable region (VH) comprising an amino acid sequence as set forth in SEQ ID NO: 43, and a light chain variable region (VL) comprising an amino acid sequence as set forth in SEQ ID NO: 48;

[0477] The mAb6 comprises a heavy chain variable region (VH) comprising an amino acid sequence as set forth in SEQ ID NO: 53, and a light chain variable region (VL) comprising an amino acid sequence as set forth in SEQ ID NO: 58;

[0478] The mAb7 comprises a heavy chain variable region (VH) comprising an amino acid sequence as set forth in SEQ ID NO: 63, and a light chain variable region (VL) comprising an amino acid sequence as set forth in SEQ ID NO: 68;

[0479] The mAb3 H2K2 comprises a heavy chain variable region (VH) comprising an amino acid sequence as set forth in SEQ ID NO: 101, and a light chain variable region (VL) comprising an amino acid sequence as set forth in SEQ ID NO: 118;

[0480] The mAb3 H2K4 comprises a heavy chain variable region (VH) comprising an amino acid sequence as set forth in SEQ ID NO: 101, and a light chain variable region (VL) comprising an amino acid sequence as set forth in SEQ ID NO: 123;

[0481] The mAb3 H3K2 comprises a heavy chain variable region (VH) comprising an amino acid sequence as set forth in SEQ ID NO: 103, and a light chain variable region (VL) comprising an amino acid sequence as set forth in SEQ ID NO: 118;

[0482] The mAb3 H3K4 comprises a heavy chain variable region (VH) comprising an amino acid sequence as set forth in SEQ ID NO: 103, and a light chain variable region (VL) comprising an amino acid sequence as set forth in SEQ ID NO: 123;

[0483] The mAb5 H3K1 comprises a heavy chain variable region (VH) comprising an amino acid sequence as set forth in SEQ ID NO: 77, and a light chain variable region (VL) comprising an amino acid sequence as set forth in SEQ ID NO: 86;

[0484] The mAb5 H5K2 comprises a heavy chain variable region (VH) and a light chain variable region (VL), the VH comprising an amino acid sequence as set forth in SEQ ID NO: 82, and the VL comprising an amino acid sequence as set forth in SEQ ID NO: 88;

[0485] The TORL-3-600 comprises a heavy chain (HC) and a light chain (LC), the HC comprising an amino acid sequence as set forth in SEQ ID NO: 142, and the LC comprising an amino acid sequence as set forth in SEQ ID NO: 143;

[0486] Preferably,

[0487] The mAb5 H3K1 comprises a heavy chain (HC) and a light chain (LC), the HC comprising an amino acid sequence as set forth in SEQ ID NO: 126, and the LC comprising an amino acid sequence as set forth in SEQ ID NO: 128;

[0488] The mAb5 H5K2 comprises a heavy chain (HC) and a light chain (LC), the HC comprising an amino acid sequence as set forth in SEQ ID NO: 130, and the LC comprising an amino acid sequence as set forth in SEQ ID NO: 132;

[0489] The mAb3 H2K2 comprises a heavy chain (HC) and a light chain (LC), the HC comprising an amino acid sequence as set forth in SEQ ID NO: 134, and the LC comprising an amino acid sequence as set forth in SEQ ID NO: 136;

[0490] The mAb3 H2K4 comprises a heavy chain (HC) and a light chain (LC), the HC comprising an amino acid sequence as set forth in SEQ ID NO: 134, and the LC comprising an amino acid sequence as set forth in SEQ ID NO: 138;

[0491] The mAb3 H3K2 comprises a heavy chain (HC) and a light chain (LC), the HC comprising an amino acid sequence as set forth in SEQ ID NO: 140, and the LC comprising an amino acid sequence as set forth in SEQ ID NO: 136;

[0492] The mAb3 H3K4 comprises a heavy chain (HC) and a light chain (LC), the HC comprising an amino acid sequence as set forth in SEQ ID NO: 140, and the LC comprising an amino acid sequence as set forth in SEQ ID NO: 138;

[0493] The TORL-3-600 comprises a heavy chain (HC) and a light chain (LC), the HC comprises an amino acid sequence as set forth in SEQ ID NO: 142, and the LC comprises an amino acid sequence as set forth in SEQ ID NO: 143;

[0494] More preferably,

[0495] The -L-D structure is linked to a cysteine in the antibody or antigen-binding fragment thereof that binds CDH17.

[0496] Pharmaceutical compositions

[0497] The present application provides pharmaceutical compositions comprising an antibody drug conjugate disclosed herein, and a pharmaceutically acceptable carrier or excipient.

[0498] The antibody drug conjugates of the present application (also referred to herein as "active compounds") can be incorporated into a pharmaceutical composition suitable for administration. Such compositions typically comprise an antibody drug conjugate, and a pharmaceutically acceptable carrier. As used herein, the term "pharmaceutically acceptable carrier" is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Preferred examples of such carriers or excipients include, but are not limited to, water, saline, Ringer's solutions, dextrose solutions, and 5% human serum albumin. Liposomes and non-aqueous vehicles such as fixed oils can also be used. Such media and agents for use in

[0499] In some embodiments, the pharmaceutical composition further comprises a second therapeutic agent. In some embodiments, the second therapeutic agent is selected from the group consisting of an antibody, a chemotherapeutic agent, and a small molecule drug.

[0500] The pharmaceutical compositions of the present application can be formulated to be compatible with their intended route of administration. Examples of routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (i.e., topical), transmucosal, and rectal administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetates, citrates or phosphates; and agents for the adjustment of tonicity such as sodium chloride or dextrose. The pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampules, disposable syringes or multiple dose vials made of glass or plastic.

[0501] Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (if water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringeability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol and liquid polyethylene glycol, and the like), and suitable mixtures thereof. Proper fluidity can be maintained, for example, by the use of coating such as lecithin, by the maintenance of required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by the inclusion of agents which delay absorption, for example, aluminum monostearate and gelatin. TM (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringeability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol and liquid polyethylene glycol, and the like), and suitable mixtures thereof. Proper fluidity can be maintained, for example, by the use of coating such as lecithin, by the maintenance of required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by the inclusion of agents which delay absorption, for example, aluminum monostearate and gelatin.

[0502] Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in the appropriate solvent with one or more of the ingredients enumerated above, as required, followed by filter sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, methods of preparation are vacuum drying and freeze drying which yields a powder of the active ingredient plus any additional desired ingredient from the previously sterile-filtered solution thereof.

[0503] Oral compositions generally include an inert diluent or an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules. Oral compositions can also be prepared using a fluid carrier, for example, a fluid carrier that is used as a mouthwash, wherein the compound in the fluid carrier is applied orally and swished and expectorated or swallowed. Pharmaceutically compatible binding agents, and / or adjuvant materials can be included as part of the composition. The tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.

[0504] For administration by inhalation, the compounds are delivered in the form of an aerosol spray from a pressurized container or a nebulizer or a dispenser containing a suitable propellant, e.g., a gas such as carbon dioxide.

[0505] Systemic administration can also be by way of transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. For transdermal administration, the active compound is formulated into a

[0506] The active compounds can also be prepared in the form of suppositories (e.g., using conventional suppository

[0507] In one embodiment, the active compound is prepared with an organic carrier for the purpose of making it more acceptable to the body, which can protect the compound from being rapidly eliminated from the body. Controlled release preparations are included. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for making such formulations are apparent to those skilled in the art. The compositions can also contain other pharmaceutically acceptable additives, such as wetting agents, preservatives, sweetening, flavoring, or coloring agents.

[0508] The present disclosure provides therapeutic compositions comprising the antibody drug conjugates of the present disclosure. The therapeutic compositions according to the present disclosure will be administered with suitable carriers, excipients and other agents that are incorporated into formulations to provide improved transfer, delivery, tolerance, etc. Many suitable formulations can be found in the formulary of all pharmaceutical chemists: Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, PA. These formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, vesicles (cationic or anionic) such as LIPOFECTIN TM ), DNA conjugates, anhydrous absorption pastes, oil-in- water and water-in-oil emulsions, polyethylene glycol emulsions (polyethylene glycol of various molecular weights), semi-solid gels, and semi-solid mixtures with polyethylene glycol. See also, Powell et al. "Compendium of excipients for parenteral formulations" PDA (1998) J Pharm Sci Technol 52:238-311.

[0509] Therapeutic methods and uses

[0510] The present disclosure provides a method for treating a disease in a subject, comprising administering to the subject an effective amount of the antibody drug conjugate disclosed herein or the pharmaceutical composition disclosed herein.

[0511] The present disclosure also provides use of the antibody drug conjugate disclosed herein or the pharmaceutical composition disclosed herein in the manufacture of a medicament for treating a disease in a subject.

[0512] The present disclosure also provides the antibody drug conjugate disclosed herein or the pharmaceutical composition disclosed herein for use in treating a disease in a subject.

[0513] The present disclosure also provides a method for preventing and / or treating a disease in a subject in need thereof, comprising administering to the subject the antibody drug conjugate disclosed herein, or a pharmaceutically acceptable salt, isotopic variant, tautomer, stereoisomer, polymorph, hydrate, or solvate thereof, or a mixture thereof.

[0514] In some embodiments, the method further comprises administering to the subject a second therapeutic agent, preferably the second therapeutic agent is selected from the group consisting of a chemotherapeutic agent, a monoclonal antibody drug, a bi- / multi-specific antibody drug, a recombinant protein drug, a nucleotide drug (including siRNA and antisense oligonucleotide), a small molecule drug, an immunomodulatory drug, and a cell therapy drug.

[0515] The present disclosure also provides an antibody drug conjugate disclosed herein, or a pharmaceutically acceptable salt, isotopic variant, tautomer, stereoisomer, polymorph, hydrate, or solvate thereof, or a mixture thereof, for use in the prevention and / or treatment of a disease.

[0516] In some embodiments, the disease is a cancer, for example a cancer associated with CDH17 expression or a CDH17-positive cancer. In more preferred embodiments, the disease is selected from a neuroendocrine tumor, gastric cancer, colon cancer, rectal cancer, small intestine cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, esophageal carcinoma, renal cell carcinoma, liver cancer, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, embryonal carcinosarcoma, cervical cancer, uterine cancer, testicular cancer, lung cancer, small cell lung cancer, non-small cell lung cancer, bladder cancer, or epithelial carcinoma. In preferred embodiments, the cancer is selected from gastric cancer, liver cancer (e.g., hepatocellular carcinoma), colorectal cancer, and pancreatic cancer.

[0517] In some embodiments, the dose administered to a subject can vary with the embodiment, the drug used, the method of administration, and the site and subject to be treated. However, the dose should be sufficient to provide a therapeutic response. A clinician can determine the effective amount of the antibody drug conjugate or composition to be administered to a human or other subject to treat a medical condition. The precise amount required can depend on many factors such as the activity of the drug conjugate and the route of administration.

[0518] The antibody drug conjugate or composition described herein can be administered to a mammal in a single dose or in a series of sub-doses over a suitable period of time, for example, as needed, once a day, every half week, every week, every two weeks, every half month, every two months, every half year, or every year. A dosage unit comprising an effective amount of the antibody drug conjugate or composition can be administered as a single daily dose, or the total daily dose can be administered in two, three, four, or more divided doses administered daily as needed.

[0519] A suitable mode of administration can be selected by a physician. The route of administration can be parenteral administration, for example, administration by injection, nasal administration, pulmonary administration, or transdermal administration. Systemic or local administration can be performed by intravenous injection, intramuscular injection, intraperitoneal injection, subcutaneous injection. In some embodiments, the antibody drug conjugate or composition is selected for parenteral delivery, for inhalation, or for delivery through the digestive tract, for example, orally. The dose and method of administration can vary depending on the weight, age, condition, etc. of the subject, and can be appropriately selected.

[0520] In some embodiments, the methods further comprise administering a second therapeutic agent to the subject. In some embodiments, the antibody drug conjugate or pharmaceutical composition disclosed herein is used in combination with a second therapeutic agent. In some embodiments, the second therapeutic agent is selected from the group consisting of an antibody, a chemotherapeutic agent, and a small molecule drug. In certain embodiments, the antibody drug conjugate or composition disclosed herein is administered prior to, substantially simultaneously with, or following administration of the second therapeutic agent.

[0521] Kit / administration device

[0522] The present disclosure provides a kit or administration device comprising an antibody drug conjugate disclosed herein or a pharmaceutical composition disclosed herein.

[0523] In some embodiments, the kit or administration device comprises one or more containers with one or more of the components of a pharmaceutical composition described herein, such as an antibody drug conjugate disclosed herein.

[0524] In particular embodiments, the kit comprises a first container comprising a pharmaceutical conjugate disclosed herein. In particular embodiments, the kit comprises a first container which is a vial containing the pharmaceutical conjugate as a lyophilized sterile powder under vacuum, and the kit further comprises a second container containing a pharmaceutically acceptable fluid.

[0525] In particular embodiments, provided herein is an injection device comprising a pharmaceutical conjugate. In particular embodiments, the injection device comprises a pharmaceutical conjugate in a sterile solution. In particular embodiments, the injection device is a syringe.

[0526] In one embodiment, the kit includes instructional materials which disclose the manner in which the pharmaceutical conjugate of the disclosure is to be used. The instructional materials can be written, electronic (e.g., computer readable medium), or visual (e.g., video files) and can take the form of an instruction manual, package insert, audio or video presentation, or internet site. The kit can further include additional components to facilitate the use of the kit for which it is designed. Thus, for example, the kit can additionally contain tools (e.g., enzyme substrates for enzymatic labels, filter sets for detecting fluorescent labels, appropriate secondary labels such as a second antibody, etc.) for detecting the label. The kit can also include buffers and other reagents commonly used to practice the particular method for which the kit is designed. Such kits and suitable contents are well known to those of skill in the art.

[0527] Examples

[0528] The following examples are given for the purpose of illustrating various embodiments of the application and are not meant to limit the present application in any way. This example, as well as the methods described herein, are presently representative of preferred embodiments, are exemplary, and are not intended as limitations on the scope of the application. Changes therein and other uses will occur to those skilled in the art. Those skilled in the art will readily understand that the application can be practiced with materials and reagents other than those disclosed in the examples without departing from the spirit and scope of the application.

[0529] The following reference antibodies are used herein:

[0530] TORL-3-600: CDH17 monoclonal antibody, its preparation method is described in WO2023107558A1;

[0531] Example 1, antigen preparation

[0532] 1.1 Preparation of CDH17 recombinant protein

[0533] The nucleic acid sequence encoding the antigen protein with his and Fc tags was integrated into a mammalian cell expression vector, and after bacterial infection, lysis, plasmid extraction and washing, the plasmid expressing the antigen protein was obtained. P11 was subcultured in HEK293F cells to a suitable number of passages, and the complex formed by PEI and plasmid was added dropwise to 293 cells subcultured at a cell density of 1.5 x 10 6 cells / mL the day before, shaking the bottle body at the same time, or mixing the cells after adding part of the complex, and then repeating the addition, to avoid excessive local concentration of the complex. The 293 cells were placed in a cell incubator at 37°C, 120 rpm, 5% CO2, and cultured, with day 0 being the day of transfection. On days 1 and 3, 5% OPM-293 Profeed of the volume of cells on the day of transfection was added. On day 4 after transfection, the cell viability after transfection was detected using a Vi-cell. When the cell viability decreased to about 70%, the fermentation broth was collected, filtered and purified to obtain the antigen protein, which was subjected to western blot detection. The results are shown in Figure 1, and human CDH17 (hCDH17) recombinant protein and cynomolgus monkey CDH17 (cyCDH17) recombinant protein were obtained. The amino acid sequences of human and cynomolgus monkey CDH17 proteins are as follows:

[0534] Human CDH17 (SEQ ID NO. 1)

[0535] Cynomolgus monkey CDH17 (SEQ ID NO. 2)

[0536] 1.2 Construction of HEK293-hCDH17 and HEK293-cyCDH17 stable cell lines

[0537] The full-length human or cynomolgus monkey CDH17 gene was cloned into an expression vector containing a puromycin resistance gene using molecular cloning technology, resulting in plasmids plenti-hCDH17-IRES-p or plenti-cyCDH17-IRES-p. HEK293 cells were then transfected with these expression vectors. After entering the host cells, the target gene underwent reverse transcription and integrated into the genome, thereby expressing the human or cynomolgus monkey CDH17 gene. Cells were continuously screened for 30 days using a puromycin-containing selection medium to obtain cell populations containing HEK293-hCDH17 or HEK293-cyCDH17 positive cell lines (HEK293-hCDH17mix pool or HEK293-cyCDH17mix pool). Next, subcloning was performed using either the HEK293-hCDH17 mix pool or the HEK293-cyCDH17 mix pool. Cells were seeded at 1 cell / well in 96-well cell culture plates and cultured at 37°C with 5% CO2 for 2 weeks. Single-clone cells were then expanded to 24-well plates. After cell growth, single clones were screened and validated using anti-human or anti-cynomolgus monkey CDH17 antibodies. As shown in Figure 2, the expression of human or anti-cynomolgus monkey CDH17 in HEK293 cells was significantly increased, indicating successful construction of stable cell lines.

[0538] 1.3 Construction of a stable CHO-hCDH17 cell line

[0539] The full-length hCDH17 gene was cloned into an expression vector containing a puromycin resistance gene using molecular cloning technology, resulting in plenti- Plasmids were transfected into CHO cells using electroporation. After the target gene entered the host cell, it underwent reverse transcription and integrated into the genome, thereby expressing the hCDH17 gene. Cell lines expressing the hCDH17 gene were obtained through cell line selection with the addition of puromycin. Selection was continued for 2 weeks with 8 μg / mL puromycin-containing medium to obtain a cell population containing CHO-hCDH17 positive cells (CHO-hCDH17mix pool). Next, CHO-hCDH17mix pools were subcloned, and seeded at 1 cell / well in 96-well cell culture plates. After culturing at 37°C in a 5% CO2 incubator for 10–15 days, single-clone cells were expanded to 24-well plates. Once cells had grown sufficiently, single clones were screened and validated using anti-hCDH17 antibody. As shown in Figure 3, the CHO-hCDH17 stable cell line was successfully constructed.

[0540] Example 2: Generation of anti-CDH17 chimeric antibody

[0541] 2.1 Mouse immunization and antibody screening

[0542] Mouse immunization

[0543] Select 4-6 week old female Balb / C mice (Beijing Weitong Lihua), and use the following four groups of immunization strategies for immunization.

[0544] Group 1: Immunization with gene gun and antigen combination. One week before the first immunization, the mice were shocked with mFlt3L plasmid, 3 μg of plasmid per mouse per gene gun immunization, a total of 1 time. Subsequently, each mouse was immunized with 3 μg of plasmid per gene gun, once a week, a total of 4 times. One week later, immunization was performed with hCDH17 protein, 15 μg per mouse, a total of 1 time.

[0545] Group 2: Cell immunization. The first immunization was performed with CHO-hCDH17 cells for subcutaneous and footpad immunization, with Sigma adjuvant system as the adjuvant, 1×10 6 cells per mouse, a total of 1 time. Two weeks later, CHO-hCDH17 cells were used for footpad immunization, with Sigma adjuvant system as the adjuvant, 1×10 6 cells per mouse, a total of 3 times.

[0546] Group 3: Antigen immunization. The first immunization was performed with hCDH17 protein for subcutaneous and footpad immunization, with TiterMax as the adjuvant, 50 μg per mouse, a total of 1 time. Two weeks later, hCDH17 protein was used for footpad immunization, with Sigma adjuvant system as the adjuvant, 15 μg per mouse, a total of 3 times.

[0547] Group 4: Antigen immunization. The first immunization was performed with hCDH17 protein for subcutaneous and footpad immunization, with CFA as the adjuvant, 50 μg per mouse, a total of 1 time. Two weeks later, hCDH17 protein was used for footpad immunization, with Sigma adjuvant system as the adjuvant, 15 μg per mouse, a total of 3 times.

[0548] The antibody titers of each group were detected by ELISA and FACS, ELISA detected the binding of antibodies at the antigen level, FACS detected the binding of antibodies at the cell level, and the antibody titers met the requirements for fusion and screening.

[0549] Antibody screening

[0550] The first group was fused twice. A total of 845 positive clones were screened in the primary screening, and 739 positive clones were screened in the retest. A total of 53 384-well plates were screened in the first fusion, 1210 clones that combined with hCDH17 were screened by ELISA, and 257 clones that combined with HEK293-hCDH17 and HEK293-cyCDH17 were screened by FACS. A total of 217 positive clones were obtained by cell level binding, and the supernatant of 217 clones was used to detect endocytosis and killing. A total of 63 384-well plates were screened in the second fusion, 1749 clones that combined with hCDH17 were screened by ELISA, and 588 clones that combined with HEK293-hCDH17 and HEK293-cyCDH17 were screened by FACS. A total of 522 positive clones were obtained by cell level binding.

[0551] The second group screened a total of 7 96-well plates selected by clonepix, 10 clones that combined with hCDH17 were screened by ELISA, and 7 clones that combined with HEK293-hCDH17 and HEK293-cyCDH17 were screened by FACS. A total of 7 positive clones were obtained by cell level binding.

[0552] The third group screened a total of 62 384-well plates, 939 clones that combined with hCDH17 were screened by ELISA, and 139 clones that combined with HEK293-hCDH17 and HEK293-cyCDH17 were screened by FACS. A total of 33 positive clones were obtained by cell level binding.

[0553] The fourth group screened a total of 29 384-well plates, 394 clones that combined with hCDH17 were screened by ELISA, and 59 clones that combined with HEK293-hCDH17 and HEK293-cyCDH17 were screened by FACS. The supernatant of 59 clones was used to detect endocytosis and killing.

[0554] Based on the cell binding, endocytosis and killing activity of the four groups of clones, 7 clones with better endocytosis and killing were finally selected. The ELISA binding activity and FACS binding activity of the 7 clones are shown in Table 1 and Table 2, respectively.

[0555] Table 1. ELISA binding activity of supernatant culture of different hybridoma clones to human or cynomolgus CDH17 protein

[0556] Table 2. FACS binding activity of supernatant culture of different hybridoma clones to HEK293 surface human or cynomolgus CDH17 protein

[0557] 2.2 Determination of antibody sequence

[0558] According to the results of hybridoma screening, the positive monoclonal cells were centrifuged at 1000 rpm, the cells were collected, and total RNA was extracted with Trizol. The first strand cDNA was synthesized, and the variable region DNA sequence corresponding to the hybridoma cells was amplified using the first strand cDNA as the subsequent template. In a 50 μL reaction system, 1 μL of cDNA, 5 μL of 10× PCR buffer, 1 μL of forward and reverse primers, 1 μL of dNTP, 1 μL of 25 mmol MgCl2, 39 μL of H2O, and 1 μL of Taq enzyme were added. The pre-denaturation was performed at 95°C for 10 minutes, and then the temperature cycle was entered for PCR amplification. The reaction conditions were denaturation at 94°C for 1 minute, annealing at 58°C for 1 minute, extension at 72°C for 15 seconds, a total of 30 cycles, and then 72°C for 10 minutes. According to the results of phage screening, the variable region sequence of the positive clone was amplified. After sequencing, the variable region sequences of the heavy chain and light chain of the candidate positive clone were obtained.

[0559] Table 3 Anti-CDH17 murine antibody heavy chain variable region (VH) and light chain variable region (VL) sequences

[0560] The heavy chain / light chain variable region sequences of the anti-CDH17 murine antibody are as follows (wherein the CDR sequences are defined according to the Kabat definition rule): mAb1

[0561] The amino acid sequence of the heavy chain VH of mAb1 is shown in SEQ ID NO. 3, the encoding nucleic acid is shown in SEQ ID NO. 4, and the CDR1, CDR2, and CDR3 are shown in SEQ ID NO. 5, 6, and 7, respectively.

[0562] Nucleotide sequence

[0563] HCDR1: SYWIH (SEQ ID NO. 5)

[0564] HCDR2: RIYPGTGISYYNENFKG (SEQ ID NO. 6)

[0565] HCDR3: SSVGWGVLSAMDY (SEQ ID NO. 7)

[0566] The amino acid sequence of the light chain VL of mAb1 is shown in SEQ ID NO. 8, the encoding nucleic acid is shown in SEQ ID NO. 9, and the CDR1, CDR2, and CDR3 are shown in SEQ ID NO. 10, 11, and 12, respectively.

[0567] Nucleotide sequence

[0568] LCDR1: KASQGVDFDGYSYMN (SEQ ID NO. 10)

[0569] LCDR2: AASNLES (SEQ ID NO. 11)

[0570] LCDR3: QQTNEDPPT (SEQ ID NO. 12)

[0571] mAb2

[0572] The amino acid sequence of the heavy chain VH of mAb2 is shown in SEQ ID NO. 13, the encoding nucleic acid is shown in SEQ ID NO. 14, and the CDR1, CDR2 and CDR3 are shown in SEQ ID NO. 15, 16, 17, respectively.

[0573] Nucleotide sequence

[0574] HCDR1: TYGMS (SEQ ID NO. 15)

[0575] HCDR2: SISRNGNTYYPDSVKG (SEQ ID NO. 16)

[0576] HCDR3: GHFGSSYNAMDY (SEQ ID NO. 17)

[0577] The amino acid sequence of the light chain VL of mAb2 is shown in SEQ ID NO. 18, the encoding nucleic acid is shown in SEQ ID NO. 19, and the CDR1, CDR2 and CDR3 are shown in SEQ ID NO. 20, 21, 22, respectively.

[0578] Nucleotide sequence

[0579] LCDR1: KASQDVSTAVA (SEQ ID NO. 20)

[0580] LCDR2: SASYRYS (SEQ ID NO. 21)

[0581] LCDR3: QQHYITPT (SEQ ID NO. 22)

[0582] mAb3

[0583] The amino acid sequence of the heavy chain VH of mAb3 is shown in SEQ ID NO. 23, the encoding nucleic acid is shown in SEQ ID NO. 24, and the CDR1, CDR2 and CDR3 are shown in SEQ ID NO. 25, 26, 27, respectively.

[0584] Nucleotide sequence

[0585] HCDR1: SGYYWN (SEQ ID NO. 35)

[0586] HCDR2: YITYDGSNNYNPSLKN (SEQ ID NO. 36)

[0587] HCDR3: SLQIYYYGTSEYLDY (SEQ ID NO. 27)

[0588] The amino acid sequence of the light chain VL of mAb3 is shown in SEQ ID NO. 28, the encoding nucleic acid is shown in SEQ ID NO. 29, and the CDR1, CDR2 and CDR3 are shown in SEQ ID NO. 30, 31, 32, respectively.

[0589] Nucleotide sequence

[0590] LCDR1: RASESVDDYGFSFMN (SEQ ID NO. 30)

[0591] LCDR2: AASNQGS (SEQ ID NO. 31)

[0592] LCDR3: QQSKEVPFT (SEQ ID NO. 32)

[0593] mAb4

[0594] The amino acid sequence of the heavy chain VH of mAb4 is shown in SEQ ID NO. 33, the encoding nucleic acid is shown in SEQ ID NO. 34, and the CDR1, CDR2 and CDR3 are shown in SEQ ID NO. 35, 36, 37, respectively.

[0595] Nucleotide sequence

[0596] HCDR1: SGYYWN (SEQ ID NO. 35)

[0597] HCDR2: YITYDGSNNYNPSLKN (SEQ ID NO. 36)

[0598] HCDR3: EGENGIGYFDV (SEQ ID NO. 37)

[0599] The amino acid sequence of the VL of the light chain of mAb4 is shown in SEQ ID NO. 38, the encoding nucleic acid is shown in SEQ ID NO. 39, and the CDR1, CDR2 and CDR3 thereof are shown in SEQ ID NO. 40, 41, 42, respectively.

[0600] Nucleotide sequence

[0601] LCDR1: SASSSVSSIY (SEQ ID NO. 40)

[0602] LCDR2: LTSNLAS (SEQ ID NO. 41)

[0603] LCDR3: QQWSFYPYT (SEQ ID NO. 42)

[0604] mAb5

[0605] The amino acid sequence of the VL of the light chain of mAb5 is shown in SEQ ID NO. 48, the encoding nucleic acid is shown in SEQ ID NO. 49, and the CDR1, CDR2 and CDR3 thereof are shown in SEQ ID NO. 50, 51, 52, respectively.

[0606] Nucleotide sequence

[0607] HCDR1: DYYIH (SEQ ID NO. 45)

[0608] HCDR2: WIYPGSGNLKYNEKFKG (SEQ ID NO. 46)

[0609] HCDR3: GNLLGF (SEQ ID NO. 47)

[0610] The amino acid sequence of the VL of the light chain of mAb5 is shown in SEQ ID NO. 48, the encoding nucleic acid is shown in SEQ ID NO. 49, and the CDR1, CDR2 and CDR3 thereof are shown in SEQ ID NO. 50, 51, 52, respectively.

[0611] Nucleotide sequence

[0612] LCDR1: KASQDVGTAVA (SEQ ID NO. 50)

[0613] LCDR2: WASTRHT (SEQ ID NO. 51)

[0614] LCDR3: QHYSTYPYT (SEQ ID NO. 52)

[0615] mAb6

[0616] The amino acid sequence of the heavy chain VH of mAb6 is set forth in SEQ ID NO. 53, the encoding nucleic acid is set forth in SEQ ID NO. 54, and the CDR1, CDR2, and CDR3 are set forth in SEQ ID NO. 55, 56, 57, respectively.

[0617] Nucleotide sequence

[0618] HCDR1: NYWMN (SEQ ID NO. 55)

[0619] HCDR2: EIRMKSHDYGTHYAESVKG (SEQ ID NO. 56)

[0620] HCDR3: GDGYSALDY (SEQ ID NO. 57)

[0621] The amino acid sequence of the light chain VL of mAb6 is set forth in SEQ ID NO. 58, the encoding nucleic acid is set forth in SEQ ID NO. 59, and the CDR1, CDR2, and CDR3 are set forth in SEQ ID NO. 60, 61, 62, respectively.

[0622] Nucleotide sequence

[0623] LCDR1: RASKSISKYLA (SEQ ID NO. 60)

[0624] LCDR2: SGSTLQS (SEQ ID NO. 61)

[0625] LCDR3: QQHNEYPYT (SEQ ID NO. 62)

[0626] mAb7

[0627] The amino acid sequence of the heavy chain VH of mAb7 is set forth in SEQ ID NO. 63, the encoding nucleic acid is set forth in SEQ ID NO. 64, and the CDR1, CDR2, and CDR3 are set forth in SEQ ID NO. 65, 66, 67, respectively.

[0628] Nucleotide sequence

[0629] HCDR1: TYWIN (SEQ ID NO. 65)

[0630] HCDR2: NIYPSNSYTNYNQKFKD (SEQ ID NO. 66)

[0631] HCDR3: HYGSSYWYFDV (SEQ ID NO. 67)

[0632] The amino acid sequence of the VL of the mAb7 light chain is shown in SEQ ID NO. 68, the encoding nucleic acid is shown in SEQ ID NO. 69, and the CDR1, CDR2 and CDR3 are shown in SEQ ID NO. 70, 71, 72, respectively.

[0633] Nucleotide sequence

[0634] LCDR1: KASENVETYVS (SEQ ID NO. 70)

[0635] LCDR2: GASNRYT (SEQ ID NO. 71)

[0636] LCDR3: GQSYTYPFT (SEQ ID NO. 72)

[0637] Example 3, Characterization of Anti-CDH17 Chimeric Antibody

[0638] The above heavy and light chain variable region sequence fragments were PCR amplified, the heavy chain variable region was cloned into a vector containing human heavy chain constant region to express the complete IgGl heavy chain in mammalian cells. Similarly, the light chain variable region was cloned into a vector containing human light chain constant region to express the complete kappa light chain in mammalian cells. After sequencing correctly, it was transfected into HEK293.6E mammalian cells, IgGl was expressed and secreted into the culture medium, the supernatant was collected and combined, filtered and purified. IgG was purified by Protein A chromatography, the eluted protein was ultrafiltrated and concentrated, the concentration of IgG was determined by spectrophotometry, and the purity of IgG was analyzed by size exclusion chromatography.

[0639] 3.1 Detection of the affinity of anti-CDH17 chimeric antibody to hCDH17

[0640] Using Bio-Layer Interferometry (BLI / gator prime), candidate chimeric antibodies were immobilized with Protein A probe, then the probe was transferred to gradient-diluted hCDH17 avicHis antigen, by detecting the signal change on the probe, through kinetic fitting, the affinity of antibody and antigen was obtained, as shown in Figure 4 and Table 4. The results showed that each chimeric antibody could bind to hCDH17.

[0641] Table 4. Affinity of anti-CDH17 chimeric antibodies

[0642] 3.2 Binding of anti-CDH17 chimeric antibodies to tumor cells

[0643] Using SNU-16 as target cells, the cell density was adjusted to 1x10 7 cells / mL with blocking solution, and the sample was prepared in ice bath for 30 minutes. The antibody was diluted to 30 μg / mL with flow buffer, and 5-fold gradient dilution was prepared. The cell density was diluted to 5x10 5 cells / mL with flow buffer, 100 μL was inoculated into a U-shaped 96-well plate; centrifuged to discard the supernatant, and 100 μL of diluted antibody was added to each well, and the cells were incubated on ice for 60 minutes.

[0644] After incubation, the cells were washed with 200 μL of flow buffer for 3 times, 50 μL of AF647-Goat Anti-human IgG, Fc (1:300 dilution) was added to each well to label the surface of the test cells, and the cells were incubated on ice for 25 minutes. Then 50 μL of PI staining solution (1:100 dilution) was added to each well, and the incubation was continued for 5 minutes. After washing once, PBS was added to resuspend the cells, and the mean fluorescence intensity of AF647 of SNU-16 single living cells was detected by flow cytometry. The determination results were recorded, and the antibody concentration was taken as the abscissa, and the mean fluorescence intensity was taken as the ordinate. The four-parameter regression calculation method was used for analysis, and the EC 50 of the sample was obtained, as shown in Figure 5 and Table 5. The irrelevant antibody anti-KLH antibody was used as a control, and the results showed that all antibody clones could bind to SNU-16 cells with high activity, and the EC 50 was in the order of nM, even less than 1 nM.

[0645] Table 5. Binding activity of anti-CDH17 chimeric antibodies to tumor cells

[0646] 3.3 Endocytosis activity of anti-CDH17 chimeric antibodies

[0647] Using SNU-16 as target cells, the cell density was adjusted to 1x10 5 ~ 5x105 100 μL / well to a 96-well flat-bottom plate.

[0648] The purified chimeric antibodies and DT3C were diluted with RPMI-1640 basal medium to a concentration of 3.33 μg / mL, and the antibodies and DT3C were mixed in equal volumes, incubated at 37°C for 30 min, and then diluted by 4-fold gradient dilution. 100 μL of cells were added to each well (at this time, the actual working concentration of the antibodies was 2.5 μg / mL, the 4-fold dilution, the molar ratio of the antibodies to DT3C was 1:2, and the FBS concentration was 10%), and the culture was continued for 72 h. The cells were stained with CCK-8 reagent at a concentration of 10%, and the absorbance at 450 nm was read. The relative viability was calculated, and the antibody concentration was used as the abscissa, and the relative viability was used as the ordinate. The IC50values of the samples were calculated by four-parameter regression analysis. 50 As shown in FIG. 6 and Table 6. The irrelevant antibody anti-KLH antibody was used as a control. The results showed that all the antibody clones had high endocytosis activity, and the IC50values were all less than 1 nM. 50 As shown in FIG. 6 and Table 6. The irrelevant antibody anti-KLH antibody was used as a control. The results showed that all the antibody clones had high endocytosis activity, and the IC50values were all less than 1 nM.

[0649] Table 6. Endocytosis activity of anti-CDH17 chimeric antibodies

[0650] Example 4, Humanization of anti-CDH17 chimeric antibodies

[0651] Using mAb3 and mAb5 as candidate molecules, the sequence of the murine antibody mAb3 was aligned with the sequence of the human antibody germ line, and the key amino acid sequences with good homology, maintaining the core of the antibody structure (Upper hydrophobic core), were found to be completely identical, and the human germ line light chain gene IMGT_Hvk1-39 or IMGT_Hvk3-11, IGKJ2*01 and the human germ line heavy chain gene IMGT_hVH1-46 or IMGT_hVH3-23, IGHJ4*01, were used for murine antibody CDR grafting. Homology modeling was performed using a computer to analyze the CDR region and its surrounding framework amino acid sequence, avoiding the concentration of molecular surface charge or hydrophobic region. A total of 8 heavy chain variants hmAb3-H1-H8 and 4 light chain variants hmAb3-K1-K4 were designed. The sequence of the murine antibody mAb5 was aligned with the sequence of the human antibody germ line, and the human germ line light chain gene IMGT_hVK1-39, IGKJ2*01 and the human germ line heavy chain gene IMGT_Hvh1-46, IGHJ4*01 were determined for murine antibody CDR grafting. A total of 6 heavy chain variants mAb5-H1-H6 and 5 light chain variants mAb5-K1-K5 were designed.

[0652] After the light chain variable region and the heavy chain variable region were synthesized in whole sequence respectively, they were cloned into eukaryotic expression vectors containing the antibody kappa chain constant region Ckappa or human IgG1 constant region CH1-CH3. After the light chain and heavy chain plasmids were combined and paired, they were transfected into CHO-S cells, which were expressed at 37°C for 2 days. The culture supernatant was collected, and the affinity and biological activity were detected.

[0653] The sequences of the heavy chain variable region and the light chain variable region of the anti-CDH17 humanized antibody are shown in Table 7 and Table 8 respectively.

[0654] Table 7. Sequence of the heavy chain variable region of the anti-CDH17 humanized antibody

[0655] Table 8. Sequence of the light chain variable region of the anti-CDH17 humanized antibody

[0656] The sequences of the heavy chain / light chain variable region of the anti-CDH17 humanized antibody are as follows:

[0657] The amino acid sequence of mAb5 H1 is shown as SEQ ID NO. 73, the encoding nucleic acid is shown as SEQ ID NO. 74, and the CDR1, CDR2 and CDR3 are shown as SEQ ID NO. 45, 46 and 47 respectively.

[0658] Nucleotide sequence

[0659] The amino acid sequence of mAb5 H2 is shown as SEQ ID NO. 75, the encoding nucleic acid is shown as SEQ ID NO. 76, and the CDR1, CDR2 and CDR3 are shown as SEQ ID NO. 45, 46 and 47 respectively.

[0660] Nucleotide sequence

[0661] The amino acid sequence of mAb5 H3 is shown as SEQ ID NO. 77, the encoding nucleic acid is shown as SEQ ID NO. 78, and the CDR1, CDR2 and CDR3 are shown as SEQ ID NO. 45, 46 and 47 respectively.

[0662] Nucleotide sequence

[0663] The amino acid sequence of mAb5 H4 is shown as SEQ ID NO. 79, the encoding nucleic acid is shown as SEQ ID NO. 80, and the CDR1, CDR2 and CDR3 are shown as SEQ ID NO. 45, SEQ ID NO. 81 (YIYPGSGNLKYNEKFKG), SEQ ID NO. 47, respectively.

[0664] Nucleotide sequence

[0665] The amino acid sequence of mAb5 H5 is shown as SEQ ID NO. 82, the encoding nucleic acid is shown as SEQ ID NO. 83, and the CDR1, CDR2 and CDR3 are shown as SEQ ID NO. 45, 46, 47, respectively.

[0666] Nucleotide sequence

[0667] The amino acid sequence of mAb5 H6 is shown as SEQ ID NO. 84, the encoding nucleic acid is shown as SEQ ID NO. 85, and the CDR1, CDR2 and CDR3 are shown as SEQ ID NO. 45, 81, 47, respectively.

[0668] Nucleotide sequence

[0669] The amino acid sequence of mAb5 K1 is shown as SEQ ID NO. 86, the encoding nucleic acid is shown as SEQ ID NO. 87, and the CDR1, CDR2 and CDR3 are shown as SEQ ID NO. 50, 51, 52, respectively.

[0670] Nucleotide sequence

[0671] The amino acid sequence of mAb5 K2 is shown as SEQ ID NO. 88, the encoding nucleic acid is shown as SEQ ID NO. 89, and the CDR1, CDR2 and CDR3 are shown as SEQ ID NO. 90 (RASQDVGTAVA), 51, 52, respectively.

[0672] Nucleotide sequence

[0673] The amino acid sequence of mAb 5 K3 is shown in SEQ ID NO. 91, its encoding nucleic acid is shown in SEQ ID NO. 92, its CDR1, CDR2 and CDR3 are shown in SEQ ID NO. 90, SEQ ID NO. 93 (WASTRHS), SEQ ID NO. 52, respectively.

[0674] Nucleotide sequence

[0675] The amino acid sequence of mAb 5 K4 is shown in SEQ ID NO. 94, its encoding nucleic acid is shown in SEQ ID NO. 95, its CDR1, CDR2 and CDR3 are shown in SEQ ID NO. 90, SEQ ID NO. 96 (YASTRHS), SEQ ID NO. 52, respectively.

[0676] Nucleotide sequence

[0677] The amino acid sequence of mAb 5 K5 is shown in SEQ ID NO. 97, its encoding nucleic acid is shown in SEQ ID NO. 98, its CDR1, CDR2 and CDR3 are shown in SEQ ID NO. 90, 96, 52, respectively.

[0678] Nucleotide sequence

[0679] The amino acid sequence of mAb 3 H1 is shown in SEQ ID NO. 99, its encoding nucleic acid is shown in SEQ ID NO. 100, its CDR1, CDR2 and CDR3 are shown in SEQ ID NO. 25, 26, 27, respectively.

[0680] Nucleotide sequence

[0681] The amino acid sequence of mAb 3 H2 is shown in SEQ ID NO. 101, its encoding nucleic acid is shown in SEQ ID NO. 102, its CDR1, CDR2 and CDR3 are shown in SEQ ID NO. 25, SEQ ID NO. 125 (AIYPGDGVTRYSQKFKD), SEQ ID NO. 27, respectively.

[0682] Nucleotide sequence

[0683] The amino acid sequence of mAb3 H3 is shown as SEQ ID NO. 103, the encoding nucleic acid is shown as SEQ ID NO. 104, and the CDR1, CDR2 and CDR3 are shown as SEQ ID NO. 25, 125, 27, respectively.

[0684] Nucleotide sequence

[0685] The amino acid sequence of mAb3 H4 is shown as SEQ ID NO. 105, the encoding nucleic acid is shown as SEQ ID NO. 106, and the CDR1, CDR2 and CDR3 are shown as SEQ ID NO. 107 (SYYMQ), 125, 27, respectively.

[0686] Nucleotide sequence

[0687] The amino acid sequence of mAb3 H5 is shown as SEQ ID NO. 108, the encoding nucleic acid is shown as SEQ ID NO. 109, and the CDR1, CDR2 and CDR3 are shown as SEQ ID NO. 25, 26, 27, respectively.

[0688] Nucleotide sequence

[0689] The amino acid sequence of mAb3 H6 is shown as SEQ ID NO. 110, the encoding nucleic acid is shown as SEQ ID NO. 111, and the CDR1, CDR2 and CDR3 are shown as SEQ ID NO. 25, 26, 27, respectively.

[0690] Nucleotide sequence

[0691] The amino acid sequence of mAb3 H7 is shown as SEQ ID NO. 112, the encoding nucleic acid is shown as SEQ ID NO. 113, and the CDR1, CDR2 and CDR3 are shown as SEQ ID NO. 25, 26, 27, respectively.

[0692] Nucleotide sequence

[0693] The amino acid sequence of mAb3 H8 is shown as SEQ ID NO. 114, the encoding nucleic acid is shown as SEQ ID NO. 115, and the CDR1, CDR2 and CDR3 are shown as SEQ ID NO. 107, 26, 27, respectively.

[0694] Nucleotide sequence

[0695] The amino acid sequence of mAb3 K1 is shown as SEQ ID NO. 116, the encoding nucleic acid is shown as SEQ ID NO. 117, and the CDR1, CDR2 and CDR3 are shown as SEQ ID NO. 30, 31, 32, respectively.

[0696] Nucleotide sequence

[0697] The amino acid sequence of mAb3 K2 is shown as SEQ ID NO. 118, the encoding nucleic acid is shown as SEQ ID NO. 119, and the CDR1, CDR2 and CDR3 are shown as SEQ ID NO. 120 (RASESVDDYGFSFLN), 31, 32, respectively.

[0698] Nucleotide sequence

[0699] The amino acid sequence of mAb3 K3 is shown as SEQ ID NO. 121, the encoding nucleic acid is shown as SEQ ID NO. 122, and the CDR1, CDR2 and CDR3 are shown as SEQ ID NO. 30, 31, 32, respectively.

[0700] Nucleotide sequence

[0701] The amino acid sequence of mAb3 K4 is shown as SEQ ID NO. 123, the encoding nucleic acid is shown as SEQ ID NO. 124, and the CDR1, CDR2 and CDR3 are shown as SEQ ID NO. 120, 31, 32, respectively.

[0702] Nucleotide sequence

[0703] The heavy chain amino acid sequence of anti-CDH17 humanized antibody MAB5 H3K1 is shown as SEQ ID NO. 126, the encoding nucleic acid is shown as SEQ ID NO. 127, and the light chain amino acid sequence is shown as SEQ ID NO. 128, the encoding nucleic acid is shown as SEQ ID NO. 129.

[0704] Heavy chain amino acid sequence (SEQ ID NO. 126)

[0705] Heavy chain nucleotide sequence (SEQ ID NO. 127)

[0706] Light chain amino acid sequence (SEQ ID NO. 128)

[0707] Light chain nucleotide sequence (SEQ ID NO. 129)

[0708] The heavy chain amino acid sequence of anti-CDH17 humanized antibody MAB5 H5K2 is set forth in SEQ ID NO. 130, and the encoding nucleic acid is set forth in SEQ ID NO. 131; the light chain amino acid sequence is set forth in SEQ ID NO. 132, and the encoding nucleic acid is set forth in SEQ ID NO. 133.

[0709] Heavy chain amino acid sequence (SEQ ID NO. 130)

[0710] Heavy chain nucleotide sequence (SEQ ID NO. 131)

[0711] Light chain amino acid sequence (SEQ ID NO. 132)

[0712] Light chain nucleotide sequence (SEQ ID NO. 133)

[0713] The heavy chain amino acid sequence of anti-CDH17 humanized antibody MAB3 H2K2 is set forth in SEQ ID NO. 134, and the encoding nucleic acid is set forth in SEQ ID NO. 135; the light chain amino acid sequence is set forth in SEQ ID NO. 136, and the encoding nucleic acid is set forth in SEQ ID NO. 137.

[0714] Heavy chain amino acid sequence (SEQ ID NO. 134)

[0715] Heavy chain nucleotide sequence (SEQ ID NO. 135)

[0716] Light chain amino acid sequence (SEQ ID NO. 136)

[0717] Light chain nucleotide sequence (SEQ ID NO. 137)

[0718] The heavy chain amino acid sequence of anti-CDH17 humanized antibody MAB3 H2K4 is set forth in SEQ ID NO. 134, and the encoding nucleic acid is set forth in SEQ ID NO. 135; the light chain amino acid sequence is set forth in SEQ ID NO. 138, and the encoding nucleic acid is set forth in SEQ ID NO. 139.

[0719] Heavy chain amino acid sequence (SEQ ID NO. 134)

[0720] Heavy chain nucleotide sequence (SEQ ID NO. 135)

[0721] Light chain amino acid sequence (SEQ ID NO. 138)

[0722] Light chain nucleotide sequence (SEQ ID NO. 139)

[0723] The heavy chain amino acid sequence of anti-CDH17 humanized antibody MAB3 H2K4 is set forth in SEQ ID NO. 134, and the encoding nucleic acid is set forth in SEQ ID NO. 135; the light chain amino acid sequence is set forth in SEQ ID NO. 138, and the encoding nucleic acid is set forth in SEQ ID NO. 139.

[0724] Heavy chain amino acid sequence (SEQ ID NO. 140)

[0725] Heavy chain nucleotide sequence (SEQ ID NO. 141)

[0726] Light chain amino acid sequence (SEQ ID NO. 136)

[0727] Light chain nucleotide sequence (SEQ ID NO. 137)

[0728] The heavy chain amino acid sequence of anti-CDH17 humanized antibody MAB3 H2K4 is set forth in SEQ ID NO. 134, and the encoding nucleic acid is set forth in SEQ ID NO. 135; the light chain amino acid sequence is set forth in SEQ ID NO. 138, and the encoding nucleic acid is set forth in SEQ ID NO. 139.

[0729] Heavy chain amino acid sequence (SEQ ID NO. 140)

[0730] Heavy chain nucleotide sequence (SEQ ID NO. 141)

[0731] Light chain amino acid sequence (SEQ ID NO. 138)

[0732] Light chain nucleotide sequence (SEQ ID NO. 139)

[0733] TORL-3-600Mab is an antibody targeting CDH17, prepared with reference to patent WO2023107558A1, the heavy chain amino acid sequence of which is shown as SEQ ID NO. 142, and the encoding nucleic acid thereof is shown as SEQ ID NO. 143; the light chain amino acid sequence is shown as SEQ ID NO. 144, and the encoding nucleic acid thereof is shown as SEQ ID NO. 145.

[0734] Heavy chain amino acid sequence (SEQ ID NO. 142)

[0735] Heavy chain nucleotide sequence (SEQ ID NO. 143)

[0736] Light chain amino acid sequence (SEQ ID NO. 144)

[0737] Light chain nucleotide sequence (SEQ ID NO. 145)

[0738] Example 5, Affinity characterization of anti-CDH17 humanized antibodies

[0739] 5.1 Affinity of anti-CDH17 humanized antibodies to hCDH17

[0740] Using the Bio-Layer Interferometry technology (BLI / gator prime), anti-CDH17 humanized antibody molecules in the transient supernatant were immobilized with Protein A probe, and then the probe was transferred to gradient-diluted hCDH17 avi cHis antigen, and by detecting the signal change on the probe, the affinity of the antibody to the antigen was obtained by kinetic fitting, as shown in Figure 7 and Table 9. The results showed that the affinity of the humanized antibody did not change significantly compared with the parent.

[0741] Table 9 Affinity of anti-CDH17 humanized antibodies

[0742] 5.2 Binding of anti-CDH17 humanized antibody to tumor cells

[0743] Using SNU-16 as the target cell, the cell density was adjusted to 1×10⁶ cells per ml using blocking solution. 7 Incubate cells on ice for 30 minutes, preparing samples during this time. Dilute antibody to 30 μg / ml with flow cytometry buffer, performing 5-fold serial dilutions. Add flow cytometry buffer to dilute cell density to 5 × 10⁶ cells / ml. 5 100 μl of each cell was seeded into a U-shaped 96-well plate; after centrifugation and discarding the supernatant, 100 μl of diluted antibody was added to each well, and the cells were incubated on ice for 60 minutes.

[0744] After incubation, the cells were washed three times with 200 μl of flow cytometry buffer. 50 μl of AF647-Goat Anti-human IgG,Fc (1:300 dilution) labeled on the cell surface was added to each well. After incubation on ice for 25 minutes, 50 μl of PI staining solution (1:100 dilution) was added to each well, and incubation continued for 5 minutes. After washing once, the cells were resuspended in PBS. The mean fluorescence intensity of AF647 in single viable SNU-16 cells was detected by flow cytometry. The results are shown in Figure 8A and Table 10, respectively. Using the unrelated anti-KLH antibody as a control, the binding activities of mAb5 H3K1 and mAb5 H5K2 did not change significantly compared to the parent cell; the binding activities of four humanized antibodies, including mAb3 H2K4, were slightly higher than those of the parent cell.

[0745] Using SNU-16 and ASPC-1 cells as target cells, the binding activities of mAb5 H3K1, the control antibody TORL-3-600, and Anti-KLH were detected using the same method as before. The results are shown in Figure 8B and Table 11. The binding plateau of mAb5 H3K1 was higher than that of the control antibody TORL-3-600, indicating that the cell binding activity of mAb5 H3K1 was superior to that of TORL-3-600.

[0746] Table 10. Binding activity of anti-CDH17 humanized antibodies to tumor cells. Note: " / " indicates that no curve was fitted.

[0747] Table 11 Binding activity of anti-CDH17 humanized antibodies to different tumor cells Note: " / " indicates that no curve was fitted.

[0748] 5.3 Internalization activity of anti-CDH17 humanized antibody with tumor cells

[0749] Using SNU-16 as the target cell, the cell density was adjusted to 1 x 102 cells per ml using RPMI-1640 complete medium containing 20% ​​FBS (Low IgG). 5Cells, 100 μl were plated in 96-well flat-bottom plates.

[0750] The purified hybridoma antibody and DT3C were diluted with RPMI-1640 basic medium to a concentration of 40 mg / ml, and the antibody and DT3C were mixed in equal volumes, incubated at 37°C for 30 min, and then diluted by 4-fold gradient dilution, and 100 μl of the diluted solution was added to each well (at this time, the actual working concentration of the antibody was 10 μg / ml, the molar ratio of the antibody to DT3C was 1:2, and the FBS concentration was 10%), and the culture was continued for 72 h. The cells were stained with CCK-8 reagent at a concentration of 10%, and the absorbance at 450 nm was read. The relative viability was calculated, and the antibody concentration was taken as the abscissa, and the relative viability was taken as the ordinate. The IC50 of the sample was calculated by four-parameter regression analysis. 50 The results are shown in FIG. 9A and Table 12, respectively. The endocytosis activity of the humanized antibodies was not significantly changed from that of the parent antibody.

[0751] The endocytosis activity of mAb5-H3K1, control antibody TORL-3-600, and Anti-KLH was detected using SNU-16 as the target cells. The SNU-16 cells were cultured in RPMI 1640 complete medium at 37°C and 5% CO2, and the cells were collected by centrifugation at 4°C and 300 x g for 10 min. The cell density was adjusted to 1 x 10 5 The cells were inoculated in 96-well cell culture plates at 100 μl per well, and the culture was continued at 37°C and 5% CO2 overnight.

[0752] The prepared mAb5-H3K1, TORL-3-600, and Anti-KLH were mixed with gradient-diluted DT3C in equal volumes and incubated at 37°C for 30 min. The cells were added at 100 μl per well, and the culture was continued at 37°C and 5% CO2 for 3 days. CCK-8 color developing solution was added at 20 μl per well, and the culture was continued at 37°C and 5% CO2 for 2 h. The optical absorbance was measured at a wavelength of 450 nm using an enzyme marker, and the experimental results were recorded. The target cell wells to which 100 μl of reaction buffer was added were used as the control wells, and the target cell wells to which 200 μl of reaction buffer was added were used as the blank wells. The endocytosis activity of mAb5-H3K1 was superior to that of TORL-3-600, as shown in FIG. 9B and Table 13.

[0753] Table 12 Endocytosis activity of anti-CDH17 humanized antibodies Note: " / " represents that the curve was not fitted.

[0754] Table 13 Endocytosis activity of anti-CDH17 humanized antibodies Note: " / " represents that the curve was not fitted.

[0755] Example 6, Compound Example

[0756] The structure of the compound is determined by nuclear magnetic resonance (NMR) or mass spectrometry (MS). The chemical shift δ is given in units of 10 -6 (ppm). The NMR is measured by a Bruker nuclear magnetic instrument, and the determination solvent is deuterated dimethyl sulfoxide (DMSO-d6), deuterated chloroform (CDCl3), and deuterated methanol (CD3OD), and the internal standard is tetramethylsilane (TMS).

[0757] The LCMS is measured by: Agilent 1260 Infinity II (ESI) mass spectrometer, Waters UPLC H Class plus (ESI), or Shimadzu LCMS-2020 (ESI).

[0758] The high-performance liquid chromatography (HPLC) analysis uses Agilent 1260 or Shimadzu LC-20AD.

[0759] The preparative high-performance liquid chromatography (pre-HPLC) uses GILSON GX-281 or Agilent 1260 Infinity II preparative liquid.

[0760] The chiral preparation uses critical fluid chromatography (SFC), and the instrument uses Shimadzu LC-30Adsf or Shimadzu LC-20AD.

[0761] The thin layer chromatography silica gel plate uses the GF254 acrylic adhesive silica gel plate of Anhui Liangchen Silicon Source Material Co., Ltd., and the silica gel plate used in the thin layer chromatography (TLC) adopts a specification of 0.2mm silica gel plate, and the thin layer chromatography separation and purification product adopts a specification of 0.5mm silica gel plate.

[0762] The column chromatography generally uses 200-300 mesh silica gel of Anhui Liangchen Silicon Source Material Co., Ltd. as the carrier.

[0763] The determination of the average inhibition rate and IC 50 value of the kinase uses SpectraMax i3X enzyme marker (MD company, USA).

[0764] The known starting materials of the present disclosure can be synthesized or purchased from companies such as Bide Pharmaceutical, Leyan, Shaoyuan Chemical Technology, and Anning Chemicals, etc. according to methods known in the art.

[0765] In the following examples, the reactions are carried out under an argon atmosphere or a nitrogen atmosphere unless otherwise specified.

[0766] The argon atmosphere or nitrogen atmosphere refers to that the reaction bottle is connected to an argon or nitrogen balloon with a volume of about 1L.

[0767] Hydrogen atmosphere means that the reaction bottle is connected to a hydrogen balloon of about 1 L volume.

[0768] The hydrogenation reaction is usually performed by vacuuming and filling hydrogen repeatedly for 3 times.

[0769] Oxygen atmosphere means that the reaction bottle is connected to an oxygen balloon of about 1 L volume.

[0770] In the following examples, unless otherwise specified, the solution means an aqueous solution, and the reaction temperature is room temperature, i.e. 20-30°C.

[0771] In the examples, the reaction progress is monitored by thin layer chromatography (TLC), and the developing agent used in the reaction, the eluent used in the column chromatography for purifying the compound, and the developing agent system of thin layer chromatography include: A: dichloromethane / methanol system, B: petroleum ether / ethyl acetate system, and the volume ratio of the solvents is adjusted according to the polarity of the compound, and a small amount of basic or acidic reagent such as triethylamine and acetic acid can also be added for adjustment.

[0772] Example 6-1: Preparation of compound 1 and isomers 1-A, 1-B, 1-C and 1-D thereof

[0773] N-((1R,9S)-9-(cyclopropylmethyl)-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 1-A

[0774] N-((1S,9S)-9-(cyclopropylmethyl)-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 1-B

[0775] N-((1S,9R)-9-(cyclopropylmethyl)-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 1-C

[0776] N-((1R,9R)-9-(cyclopropylmethyl)-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] quinoline-1-yl)-2-hydroxyacetamide 1-D

[0777] First step

[0778] 2-(6-cyano-5-oxo-2,3-dihydro-5H-spiro[indolizine-1,2'-[1,3]dioxolane]-7-yl)-3- cyclopropylpropanoic acid ethyl ester 1b

[0779] Dissolve 1a (1.01 g, 3.31 mmol, prepared by the method disclosed in patent application “WO2019238046A1” page 28, example 30) in 15 mL of acetonitrile, add bromomethylcyclopropane (894.93 mg, 6.63 mmol) and potassium carbonate (916.16 mg, 6.63 mmol), stir at 80°C for 13 hours. Add 10 mL of water, extract the diluted reaction solution with ethyl acetate (15 mL x 2), wash the organic phase with saturated sodium chloride solution (10 mL x 2), then dry over anhydrous sodium sulfate, filter, concentrate the filtrate by reduced pressure distillation, purify the obtained residue by silica gel column chromatography with the developing system A to obtain the title product 1b (1.12 g, yield: 92%) in the form of a yellow solid.

[0780] MS m / z (ESI): 359.1 [M+1].

[0781] Second step

[0782] 3-cyclopropyl-2-(6-formyl-5-oxo-2,3-dihydro-5H-spiro[indolizine-1,2'-[1,3]dioxolane]-7- yl)propanoic acid ethyl ester 1c

[0783] Dissolve 1b (1.12 g, 3.05 mmol) in a mixed solvent of 5 mL water, 5 mL acetonitrile and 5 mL formic acid, protect under nitrogen, add Raney nickel (261.72 mg), replace with hydrogen three times, stir the reaction solution under hydrogen (15 Psi) at 60 °C for 4 hours. Filter the reaction solution with diatomite, wash the filter cake with dichloromethane (50 mL x 3), wash the filtrate with hydrochloric acid aqueous solution (4 M, 20 mL) and then with sodium carbonate aqueous solution (12 M, 50 mL), dry the organic phase with anhydrous sodium sulfate, filter, concentrate the filtrate by distillation under reduced pressure, and purify the obtained residue by reverse phase liquid chromatography (separation conditions: column: 120 g Flash Coulmn Welch Ultimate XB_C18 20-40 μm; mobile phase: A-water: B-acetonitrile, gradient elution, flow rate: 85 mL / min, instrument: ISCO) to obtain the title product 1c (680 mg, yield: 60%) in the form of a yellow solid.

[0784] MS m / z (ESI): 362.1 [M+1].

[0785] Third step

[0786] 4-(cyclopropylmethyl)-1,4,7,8-tetrahydro-3H,10H-spiro[pyrano[3,4-f]indolizine-6,2'- [1,3]dioxolane]-3,10-dione 1d

[0787] Dissolve 1c (680 mg, 1.85 mmol) in 10 mL dichloromethane, protect under nitrogen, cool to 0 °C with an ice water bath, add sodium borohydride (108.02 mg, 2.86 mmol) in batches, stir the reaction solution at 0 °C for 30 minutes. Add acetic acid (133.15 mg, 2.22 mmol) dropwise at 25 °C, generate gas, continue to stir for 2 hours. Add 30 mL water dropwise at 15 °C, generate gas, stir at 15 °C for 1.5 hours. Wash the reaction solution with water (50 mL), add p-toluenesulfonic acid monohydrate (35.15 mg, 184.78 μmol) to the washed organic phase at 15 °C, stir at 15 °C for 12 hours. Add 50 mL water, extract the reaction solution with dichloromethane (45 mL x 3), dry the organic phase with anhydrous sodium sulfate, filter, concentrate the filtrate by distillation under reduced pressure, and purify the obtained residue by silica gel column chromatography with developing system B to obtain the title product 1d (200 mg, yield: 32%) in the form of a yellow oil.

[0788] MS m / z (ESI): 318.1 [M+1].

[0789] Fourth step

[0790] 4-(cyclopropylmethyl)-4-hydroxy-7,8-dihydro-lH-pyrano[3,4- f]indolizine-3,6,10(4H)-trione If

[0791] Dissolve 1d (202.13 mg, 598.74 μmol) in 0.5 mL of methanol, cool to 0 °C in an ice water bath, add potassium carbonate (82.75 mg, 598.74 μmol) in portions, stir at 0 °C under oxygen bubbling (15 psi) for 5 h. Pour the reaction into 10 mL of saturated aqueous ammonium chloride, remove the methanol by distillation under reduced pressure, extract the reaction with dichloromethane (20 mL x 3), dry the organic phase over anhydrous sodium sulfate, filter, and concentrate the filtrate by distillation under reduced pressure to give the crude title product 1e (140 mg) as a yellow oil. Use the product without purification in the next reaction.

[0792] MS m / z (ESI): 334.1 [M + 1].

[0793] Fifth Step

[0794] 4-(cyclopropylmethyl)-4-hydroxy-7,8-dihydro-lH-pyrano[3,4- f]indolizine-3,6,10(4H)-trione If

[0795] Dissolve 1e (64.52 mg, 180.00 μmol) in 0.5 mL of a mixture of trifluoroacetic acid and 0.125 mL of water, stir at 25 °C for 1 h. Concentrate the reaction under reduced pressure, and obtain the crude title product If (40 mg) as a yellow solid without purification. Use the product without purification in the next reaction.

[0796] MS m / z (ESI): 290.1 [M + 1].

[0797] Sixth Step

[0798] N-(9-(cyclopropylmethyl)-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo- 2,3,9,10,13,15-hexahydro-lH, 12H-benzo[de]pyrano[3',4':6,7]indolizino[l,2- b]quinolin-l-yl)acetamide Ih

[0799] Dissolve 1f (40 mg, 128.59 μmol) and 1g (38.62 mg, 154.31 μmol, prepared by the method disclosed in Example 5-1, page 61 of the patent application "CN111065621A") and 4-methylbenzenesulfonic acid pyridine (6.46 mg, 25.72 μmol) in 0.5 mL of toluene, protect under nitrogen, stir at 130 °C for 2 hours. Filter the reaction solution with diatomite, add 10 mL of water to the filtrate, extract with dichloromethane (8 mL x 3), dry the organic phase with anhydrous sodium sulfate, filter, and concentrate the filtrate under reduced pressure. Purify the obtained residue by preparative thin layer chromatography with developing system A to obtain the title product 1h (15 mg, yield: 23%) in the form of a brown solid.

[0800] MS m / z (ESI): 504.2 [M+1].

[0801] Seventh step

[0802] 1-Amino-9-(cyclopropylmethyl)-5-fluoro-9-hydroxy-4-methyl-1,2,3,9,12,15-hexahydro- 10H,13H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-10,13-dione methanesulfonate 1i

[0803] Dissolve 1h (50 mg, 96.5 μmol) in 1 mL of ethylene glycol dimethyl ether, add 0.5 mL of methyl sulfonic acid and 0.5 mL of water, stir at 85 °C for 18 hours. Pour the reaction solution into 10 mL of water, adjust the pH to 7-8 with saturated sodium bicarbonate solution, extract with dichloromethane (10 mL x 5), dry the organic phase with anhydrous sodium sulfate, filter, and concentrate the filtrate under reduced pressure to obtain the title product 1i (40 mg) in the form of a brown solid. The product is used directly in the next reaction without purification.

[0804] MS m / z (ESI): 462.3 [M+1].

[0805] Eighth step

[0806] N-((1R,9S)-9-(cyclopropylmethyl)-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 1-A

[0807] N-((1S,9S)-9-(cyclopropylmethyl)-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 1-B

[0808] N-((1S,9R)-9-(cyclopropylmethyl)-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 1-C

[0809] N-((1R,9R)-9-(cyclopropylmethyl)-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 1-D

[0810] The methanesulfonic acid salt of 1i (35 mg, 62.2 μmol) and glycolic acid (5.7 mg, 74.6 μmol) were dissolved in 1.5 mL of N,N-dimethylformamide, then 1-hydroxybenzotriazole (12.6 mg, 93.2 μmol), N,N-diisopropylethylamine (16.1 mg, 124 μmol) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (17.9 mg, 93.2 μmol) were added successively, and stirred at 25 °C for 1 hour. 20 mL of water was added to the reaction solution, and the diluted reaction solution was extracted with dichloromethane (10 mL x 5), the organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure, and the obtained residue was purified by supercritical fluid chromatography to obtain four single configuration title products 1-A, 1-B, 1-C and 1-D:

[0811] Single configuration compound one of compound 1 (labeled as 1-B, 1.02 mg)

[0812] SFC analysis: retention time 1.472 min, purity: 98% (chromatographic column: Chiralpak IC-3 50 x 4.6 mm I.D., 3 μm, mobile phase: A-carbon dioxide, B-methanol and acetonitrile (0.05% diethylamine), isocratic elution: B: 60%, flow rate: 3 mL / min, instrument: Shimadzu LC-30AD sf).

[0813] MS m / z (ESI): 520.5 [M+1].

[0814] 1 H NMR (400 MHz, CD3OD) δ 7.71 - 7.61 (m, 2H), 5.73 - 5.66 (m, 1H), 5.63 - 5.56 (m, 1H), 5.40 - 5.32 (m, 2H), 5.12 - 5.06 (m, 1H), 4.28 - 4.22 (m, 1H), 4.18 - 4.12 (m, 1H), 3.32 - 3.32 (m, 1H), 3.20 - 3.11 (m, 1H), 2.42 (s, 3H), 2.39 - 2.30 (m, 1H), 2.29 - 2.19 (m, 1H), 1.97 - 1.89 (m, 1H), 1.88 - 1.80 (m, 1H), 0.94 - 0.86 (m, 1H), 0.51 - 0.38 (m, 2H), 0.14 - 0.07 (m, 1H), 0.04 - -0.01 (m, 1H).

[0815] Single configuration compound two of compound 1 (labeled 1-B, 1.02 mg)

[0816] SFC analysis: Retention time 2.075 min, purity: 93%. (Chromatography column: Chiralpak AS-3 50 x 4.6 mm I.D., 3 pm, mobile phase: A-Carbon dioxide, B-Methanol (0.05% diethylamine), Gradient elution: B%: 5%-40%, Flow rate: 3 mL / min, Instrument: Shimadzu LC-30AD sf).

[0817] MS m / z (ESI): 520.4 [M+1].

[0818] 1 H NMR (400 MHz, CD3OD) δ 7.66 - 7.53 (m, 2H), 5.70 - 5.63 (m, 1H), 5.60 - 5.54 (m, 1H), 5.41 - 5.31 (m, 2H), 5.22 - 5.14 (m, 1H), 4.34 - 4.23 (m, 1H), 4.22 - 4.11 (m, 1H), 3.43 - 3.36 (m, 1H), 3.22 - 3.13 (m, 1H), 2.43 - 2.37 (m, 3H), 2.35 - 2.26 (m, 2H), 1.95 - 1.80 (m, 2H), 0.93 - 0.86 (m, 1H), 0.55 - 0.35 (m, 2H), 0.17 - 0.08 (m, 1H), 0.07 - -0.03 (m, 1H).

[0819] Single configuration compound three of compound 1 (1.21 mg)

[0820] SFC analysis: Retention time 0.464 min, purity: 98%. (Column: Chiralcel OD-3 50 x 4.6 mm I.D., 3 pm, Mobile phase: A - Carbon dioxide, B - Methanol (0.05% Diethylamine), Gradient elution: B%: 5% - 40%, Flow rate: 3 mL / min, Instrument: Shimadzu LC-30AD sf).

[0821] MS m / z (ESI): 520.1 [M+1].

[0822] 1 H NMR (400 MHz, CD3OD) δ 7.71 - 7.64 (m, 2H), 5.73 - 5.67 (m, 1H), 5.62 - 5.56 (m, 1H), 5.43 - 5.40 (m, 1H), 5.38 - 5.35 (m, 1H), 5.18 - 5.16 (m, 1H), 4.28 - 4.21 (m, 1H), 4.18 - 4.12 (m, 1H), 3.50 - 3.45 (m, 1H), 3.20 - 3.12 (m, 1H), 2.49 - 2.40 (m, 3H), 2.36 - 2.23 (m, 2H), 1.96 - 1.82 (m, 2H), 0.93 - 0.88 (m, 1H), 0.52 - 0.39 (m, 2H), 0.15 - 0.08 (m, 1H), 0.06 - -0.02 (m, 1H).

[0823] Single configuration compound four of compound 1 (0.27 mg)

[0824] SFC analysis: Retention time 0.464 min, purity: 98%. (Column: Chiralcel OD-3 50 x 4.6 mm I.D., 3 pm, Mobile phase: A - Carbon dioxide, B - Methanol (0.05% Diethylamine), Gradient elution: B%: 5% - 40%, Flow rate: 3 mL / min, Instrument: Shimadzu LC-30AD sf).

[0825] MS m / z (ESI): 520.1 [M+1].

[0826] 1H NMR (400 MHz, CD3OD) δ 7.71 - 7.62 (m, 2H), 5.73 - 5.67 (m, 1H), 5.62 - 5.56 (m, 1H), 5.41 - 5.35 (m, 2H), 5.23 - 5.20 (m, 1H), 4.33 - 4.23 (m, 1H), 4.21 - 4.12 (m, 1H), 3.38 - 3.36 (m, 1H), 3.22 - 3.13 (m, 1H), 2.44 (s, 3H), 2.37 - 2.28 (m, 2H), 1.96 - 1.83 (m, 2H), 0.91 - 0.86 (m, 1H), 0.50 - 0.38 (m, 2H), 0.14 - 0.09 (m, 1H), 0.06 - -0.01 (m, 1H).

[0827] Examples 6-2 to 6-6

[0828] The following compounds were prepared according to the procedure of Reference Example 6-1, selecting the corresponding starting materials:

[0829] Example 6-2: Preparation of Compound 2 and its isomers 2-A and 2-B

[0830] (S)-N-((1S,9S)-9-(Cyclopropylmethyl)-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-hydroxypropanamide 2-A

[0831] (S)-N-((1R,9S)-9-(Cyclopropylmethyl)-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-hydroxypropanamide 2-B

[0832] First step

[0833] (S)-4-(Cyclopropylmethyl)-4-hydroxy-1,4,7,8-tetrahydro-3H,10H-spiro[pyrano[3,4- f]indolizine-6,2'-[1,3]dioxolane]-3,10-dione 1e-1

[0834] (R)-4-(Cyclopropylmethyl)-4-hydroxy-1,4,7,8-tetrahydro-3H,10H-spiro[pyrano[3,4- f]indolizine-6,2'-[1,3]dioxolane]-3,10-dione 1e-2

[0835] The le (1.30 g, 3.88 mmol) was separated by SFC (Separation condition: Column: DAICEL CHIRALPAK AS 250 mm x 50 mm, 10 pm; Mobile phase: A-Carbon dioxide: B-Methanol (0.1% NH3-H2O), isocratic elution: B: 20%, flow rate: 120 mL / min, Instrument: Shimadzu LC-30ADsf) to give the title product le-1 (301 mg, yield: 22.1%) as a yellow solid, and the title product le-2 (285 mg, yield: 19.4%) as a yellow solid.

[0836] Single configuration compound le-1

[0837] SFC analysis: Retention time 1.392 min. (Column: Chiralpak AS-3 50 x 4.6 mm I.D., 3 pm, Mobile phase: A-Carbon dioxide, B-Methanol (0.05% diethylamine), Gradient elution: B%: 5%-40%, flow rate: 3 mL / min, Instrument: Shimadzu LC-30ADsf).

[0838] MS m / z (ESI): 334.0 [M+1].

[0839] Single configuration compound le-2

[0840] SFC analysis: Retention time 1.762 min. (Column: Chiralpak AS-3 50 x 4.6 mm I.D., 3 pm, Mobile phase: A-Carbon dioxide, B-Methanol (0.05% diethylamine), Gradient elution: B%: 5%-40%, flow rate: 3 mL / min, Instrument: Shimadzu LC-30ADsf).

[0841] MS m / z (ESI): 333.9 [M+1].

[0842] Second step

[0843] (S)-4-(cyclopropylmethyl)-4-hydroxy-7,8-dihydro-lH-pyrano[3,4-f]indolizine-3,6,10(4H)-trione If-1

[0844] The le-1 (301 mg, 857 pmol) was dissolved in 2 mL of a mixture solution of trifluoroacetic acid and 0.5 mL of water, stirred at 25 °C for 4 hours. The reaction solution was concentrated under reduced pressure, and the obtained residue gave the title product If-1 (209 mg) as a yellow solid without purification, and the product was directly used in the next step reaction without purification.

[0845] MS m / z (ESI): 290.1 [M+1].

[0846] Third Step

[0847] N-((9S)-9-(Cyclopropylmethyl)-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo- 2,3,9,10,13,15-hexahydro-lH, 12H-benzo[de]pyrano[3',4':6,7]indolizino[l,2- b]quinolin-l-yl)acetamide 1h-1

[0848] Dissolve 1f-1 (50.0 mg, 159 μmol) with 1g (48.0 mg, 192 μmol), 4-methylbenzenesulfonic acid pyridine (8.0 mg, 31.8 μmol) in 2 mL of toluene, protect with nitrogen, stir at 120 °C for 16 hours. Filter the reaction solution with diatomite, add 20 mL of water to the filtrate, extract with dichloromethane (15 mL x 3), dry the organic phase with anhydrous sodium sulfate, filter, concentrate the filtrate under reduced pressure, purify the obtained residue with preparative thin layer chromatography with developing system A to obtain the title product 1h-1 (190 mg, yield: 56%) in the form of a yellow solid.

[0849] MS m / z (ESI): 504.1 [M+1].

[0850] Fourth Step

[0851] (9S)-l-Amino-9-(cyclopropylmethyl)-5-fluoro-9-hydroxy-4-methyl-l,2,3,9,12,15- hexahydro-10H, 13H-benzo[de]pyrano[3',4':6,7]indolizino[l,2-b]quinoline-10,13- dione 1i-1

[0852] Dissolve 1h-1 (60.0 mg, 111 μmol) in 4.5 mL of ethyleneglycol dimethyl ether, add 1.5 mL of methylsulfonic acid and 1.5 mL of water, stir at 85 °C for 16 hours. Add the reaction solution dropwise to 10 mL of water with stirring, extract with dichloromethane (10 mL x 5), wash the organic phase with 25 mL of 0.05 M aqueous hydrochloric acid, filter the aqueous phase, combine all the extracted aqueous phases, adjust the pH of the aqueous phase to 7-8 with saturated potassium bicarbonate solution at 0 °C, extract with a mixed solvent (dichloromethane / methanol: 20 / 3, 15 mL x 5), dry the organic phase with anhydrous sodium sulfate, filter, concentrate the filtrate under reduced pressure to obtain the crude title product 1i-1 (22 mg) in the form of a brown solid, which was used directly in the next reaction without purification.

[0853] MS m / z (ESI): 462.1 [M+1].

[0854] Fifth Step

[0855] (S)-N-((1S,9S)-9-(cyclopropylmethyl)-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo- 2,3,9,10,13,15-hexahydro-lH, 12H-benzo[de]pyrano[3',4':6,7]indolizino[l,2-b] quinoline-l-yl)-2-hydroxypropanamide 2-A

[0856] (S)-N-((1R,9S)-9-(cyclopropylmethyl)-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo- 2,3,9,10,13,15-hexahydro-lH, 12H-benzo[de]pyrano[3',4':6,7]indolizino[l,2-b] quinoline-l-yl)-2-hydroxypropanamide 2-B

[0857] The mesylate salt of li-1 (22.0 mg, 47.7 μmol) and (2S)-2-hydroxypropanoic acid (6.4 mg, 71.5 μmol) were dissolved in 1.5 mL of N,N-dimethylformamide, then 1-hydroxybenzotriazole (9.7 mg, 71.5 μmol), N,N-diisopropylethylamine (18.5 mg, 143 μmol) and l-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (13.7 mg, 71.5 μmol) were added successively, stirred at 25 °C for 4 hours. Toluene (4 mL x 2) was added to the reaction solution, which was concentrated under reduced pressure at 25 °C, the obtained residue was diluted with N,N-dimethylformamide to 1.5 mL, the diluted solution was purified by pre-HPLC (column: Phenomenex luna C18 150 x 25 mm x 10 μm; mobile phase: A-water (0.225% formic acid), B-acetonitrile; gradient elution: B%: 26%-56%), to obtain a mixture (10 mg), which was further purified by supercritical fluid chromatography to obtain two single configuration title products 2-A and 2-B:

[0858] Single configuration compound 2-A of compound 2 (5.55 mg, yield: 20%, off-white solid)

[0859] SFC analysis: retention time 1.381 min, purity: 92%. (column: Chiralcel OJ-3 50 x 4.6 mm I.D., 3 μm, mobile phase: A-carbon dioxide, B-methanol (0.05% diethylamine), gradient elution: B%: 5%-40%, flow rate: 3 mL / min, instrument: Shimadzu LC-30AD sf).

[0860] MS m / z (ESI): 534.1 [M+1].

[0861] 1H NMR (400 MHz, CD3OD) δ 7.61 (s, 1H), 7.53 - 7.49 (m, 1H), 5.65 - 5.54 (m, 2H), 5.36 - 5.29 (m, 1H), 5.25 - 5.18 (m, 1H), 4.81 - 4.77 (m, 1H), 4.41 - 4.33 (m, 1H), 3.28 - 3.23 (m, 1H), 3.15 - 3.05 (m, 1H), 2.36 - 2.33 (m, 3H), 2.33 - 2.27 (m, 1H), 2.17 - 2.10 (m, 1H), 1.95 - 1.87 (m, 1H), 1.85 - 1.77 (m, 1H), 1.47 - 1.44 (m, 3H), 0.91 - 0.87 (m, 1H), 0.51 - 0.35 (m, 2H), 0.15 - 0.06 (m, 1H), 0.04 - -0.05 (m, 1H).

[0862] Single configuration compound 2-B of compound 2 (4.06 mg, yield: 16%, yellow solid)

[0863] SFC analysis: Retention time 1.299 min, purity: 97%. (Chromatographic column: Chiralcel OJ-3 50x4.6 mm I.D., 3 pm, mobile phase: A-Carbon dioxide, B-Methanol (0.05% Diethylamine), Gradient elution: B%: 5%-40%, Flow rate: 3 mL / min, Instrument: Shimadzu LC-30AD sf).

[0864] MS m / z (ESI): 534.1 [M+1].

[0865] 1 H NMR (400 MHz, CD3OD) δ 7.61 (s, 1H), 7.57 - 7.51 (m, 1H), 5.65 - 5.52 (m, 2H), 5.42 - 5.30 (m, 2H), 5.12 - 5.02 (m, 1H), 4.38 - 4.30 (m, 1H), 3.29 - 3.24 (m, 1H), 3.20 - 3.07 (m, 1H), 2.43 - 2.37 (m, 3H), 2.35 - 2.26 (m, 2H), 1.97 - 1.80 (m, 2H), 1.64 - 1.58 (m, 3H), 0.92 - 0.83 (m, 1H), 0.51 - 0.36 (m, 2H), 0.16 - 0.07 (m, 1H), 0.06 - -0.02 (m, 1H).

[0866] Example 6-3: Preparation of compound 3 and its isomers 3-A and 3-B

[0867] (R)-N-((1S,9S)-9-(cyclopropylmethyl)-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo- 2,3,9,10,13,15-hexahydro-lH, 12H-benzo[de]pyrano[3',4':6,7]indolizino[l,2-b] quinolin-l-yl)-2-hydroxypropanamide 3-A

[0868] (R)-N-((1R,9S)-9-(cyclopropylmethyl)-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo- 2,3,9,10,13,15-hexahydro-lH, 12H-benzo[de]pyrano[3',4':6,7]indolizino[l,2-b] quinolin-l-yl)-2-hydroxypropanamide 3-B

[0869] First step

[0870] (R)-N-((1S,9S)-9-(cyclopropylmethyl)-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo- 2,3,9,10,13,15-hexahydro-lH, 12H-benzo[de]pyrano[3',4':6,7]indolizino[l,2-b] quinolin-l-yl)-2-hydroxypropanamide 3-A

[0871] (R)-N-((1R,9S)-9-(cyclopropylmethyl)-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo- 2,3,9,10,13,15-hexahydro-lH, 12H-benzo[de]pyrano[3',4':6,7]indolizino[l,2-b] quinolin-l-yl)-2-hydroxypropanamide 3-B

[0872] Dissolve li-1 (40.0 mg, 80.8 μmol) and (2R)-2-hydroxypropanoic acid (10.9 mg, 121 μmol) in 1.5 mL of N,N-dimethylformamide, then add 1-hydroxybenzotriazole (16.4 mg, 121 μmol), N,N-diisopropylethylamine (31.3 mg, 242 μmol) and l-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (23.2 mg, 121 μmol) successively, stir at 25 °C for 4 hours. Add toluene (4 mL x 2) to the reaction solution, concentrate under reduced pressure, dilute the obtained residue with N,N-dimethylformamide to 1.5 mL, and purify the diluted solution by pre-HPLC (column: Xtimate C18 150 x 40 mm x 10 μm; mobile phase: A-water (0.225% formic acid), B-acetonitrile; gradient elution: B%: 23%-53%), and purify the obtained mixture by supercritical fluid chromatography to give two single configuration title products 3-A and 3-B:

[0873] Single configuration compound 3-A of compound 3 (6.11 mg, yield: 14%, off-white solid)

[0874] SFC analysis: Retention time 1.413 min, purity: 97%. (Column: Chiralcel OJ-3 50x4.6 mm I.D., 3 pm, Mobile phase: A-Carbon dioxide, B-Methanol (0.05% Diethylamine), Gradient elution: B%: 5-40%, Flow rate: 3 mL / min, Instrument: Shimadzu LC-30ADsf).

[0875] MS m / z (ESI): 534.3 [M+1].

[0876] 1 H NMR (400 MHz, CD3OD) δ 7.61 (s, 1H), 7.54 - 7.48 (m, 1H), 5.70 - 5.62 (m, 1H), 5.59 - 5.51 (m, 1H), 5.38 - 5.30 (m, 1H), 5.29 - 5.21 (m, 1H), 4.83 - 4.78 (m, 1H), 4.33 - 4.25 (m, 1H), 3.30 - 3.23 (m, 1H), 3.20 - 3.09 (m, 1H), 2.40 - 2.36 (m, 3H), 2.35 - 2.31 (m, 1H), 2.25 - 2.14 (m, 1H), 1.95 - 1.88 (m, 1H), 1.86 - 1.79 (m, 1H), 1.60 - 1.54 (m, 3H), 0.95 - 0.82 (m, 1H), 0.52 - 0.35 (m, 2H), 0.15 - 0.07 (m, 1H), 0.04 - -0.04 (m, 1H).

[0877] Single configuration compound 3-B of compound 3 (2.78 mg, yield: 6%, off-white solid)

[0878] SFC analysis: Retention time 2.013 min, purity: 97%. (Column: Chiralcel OD-3 50x4.6 mm I.D., 3 pm, Mobile phase: A-Carbon dioxide, B-Methanol (0.05% Diethylamine), Gradient elution: B%: 5-40%, Flow rate: 3 mL / min, Instrument: Shimadzu LC-30ADsf).

[0879] MS m / z (ESI): 534.1 [M+1].

[0880] 1H NMR (400 MHz, DMSO-d6) δ 8.42 - 8.36 (m, 1H), 7.81 - 7.75 (m, 1H), 7.37 (s, 1H), 6.57 (s, 1H), 5.58 - 5.49 (m, 2H), 5.41 (s, 2H), 5.26 - 5.12 (m, 2H), 4.17 - 4.09 (m, 1H), 3.19 - 3.14 (m, 1H), 2.42 - 2.36 (m, 3H), 2.23 - 2.10 (m, 2H), 1.90 - 1.81 (m, 1H), 1.79 - 1.71 (m, 1H), 1.32 - 1.27 (m, 3H), 0.85 - 0.77 (m, 1H), 0.39 - 0.27 (m, 2H), 0.10 - 0.02 (m, 1H), -0.03 - -0.11 (m, 1H).

[0881] Example 6-4: Preparation of compound 4 and its isomers 4-A and 4-B

[0882] (S)-2-cyclopropyl-N-((1S,9S)-9-(cyclopropylmethyl)-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 4-A

[0883] (S)-2-cyclopropyl-N-((1R,9S)-9-(cyclopropylmethyl)-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 4-B

[0884] First step

[0885] The mesylate salt of 1i-1 (37.0 mg, 78.5 μmol) and (2S)-2-cyclopropyl-2- hydroxyacetic acid (9.59 mg, 78.5 μmol) were dissolved in 1 mL of N,N- dimethylformamide, then 1-hydroxybenzotriazole (15.9 mg, 118 μmol), N,N- diisopropylethylamine (30.4 mg, 236 μmol) and 1-(3-dimethylaminopropyl)-3- ethylcarbodiimide hydrochloride (22.6 mg, 118 μmol) were added in turn, and stirred at 25 °C for 12 hours. To the reaction solution was added ethyl acetate (10 mL), washed with water (5 mL x 2), the organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The obtained residue was purified by pre-HPLC (column: Phenomenex luna C18 150 x 25 mm x 10 μm; mobile phase: A-water (0.225% formic acid), B-acetonitrile; gradient elution: B%: 31%-61%), to obtain a mixture in the form of a yellow solid (15 mg, yield 31.2%), which was separated by preparative SFC (column: REGIS (s,s) WHELK-O1 250 x 30 mm, 5 μm; mobile phase: A-carbon dioxide (isopropanol), B-acetonitrile; isocratic elution: B: 65%), to obtain two single configuration title products 4-A and 4-B:

[0886] Single configuration compound 4-A of compound 4 (7.0 mg, yield: 50.2%, yellow solid)

[0887] SFC analysis: retention time 0.761 min, purity: 98%. (column: (S,S) WHELK-O1 50 x 4.6 mm I.D., 3.5 μm, mobile phase: A-carbon dioxide, B-isopropanol and acetonitrile (0.05% diethylamine), isocratic elution: B: 65%, flow rate: 3 mL / min, instrument: Shimadzu LC-30AD sf).

[0888] MS m / z (ESI): 560.5 [M+1].

[0889] 1H NMR (400 MHz, CD3OD) δ 7.67 (s, 1H), 7.64-7.60 (m, 1H), 5.68-5.62 (m, 1H), 5.61-5.55 (m, 1H), 5.41-5.37 (m, 1H), 5.36-5.33 (m, 1H), 5.06-5.00 (m, 1H), 3.87-3.84 (m, 1H), 3.21-3.11 (m, 2H), 2.43-2.40 (m, 3H), 2.37-2.31 (m, 1H), 2.25-2.18 (m, 1H), 1.96-1.89 (m, 1H), 1.87-1.80 (m, 1H), 1.32-1.28 (m, 1H), 0.92-0.87 (m, 1H), 0.61-0.55 (m, 2H), 0.53-0.45 (m, 3H), 0.44-0.38 (m, 1H), 0.14-0.08 (m, 1H), 0.04- -0.03 (m, 1H).

[0890] Single configuration compound 4-B of compound 4 (6.0 mg, yield: 41.9%, yellow solid)

[0891] SFC analysis: Retention time 1.486 min, purity: 96%. (Chromatographic column: (S, S) WHELK-01 50 x 4.6 mm I.D., 3.5 pm, Mobile phase: A-Carbon dioxide, B-Isopropyl alcohol and Acetonitrile (0.05% Diethylamine), Isocratic elution: B: 65%, Flow rate: 3 mL / min, Instrument: Shimadzu LC-30AD sf).

[0892] MS m / z (ESI): 560.3 [M+1].

[0893] 1H NMR (400 MHz, CD3OD) δ 7.64 (s, 1H), 7.61 - 7.57 (m, 1H), 5.67 - 5.62 (m, 1H), 5.59 - 5.52 (m, 1H), 5.46 - 5.39 (m, 1H), 5.38 - 5.32 (m, 1H), 5.21 - 5.14 (m, 1H), 3.72 - 3.68 (m, 1H), 3.22 - 3.11 (m, 2H), 2.42 - 2.39 (m, 3H), 2.34 - 2.28 (m, 2H), 1.96 - 1.89 (m, 1H), 1.87 - 1.81 (m, 1H), 1.42 - 1.35 (m, 1H), 0.91 - 0.85 (m, 1H), 0.69 - 0.58 (m, 3H), 0.56 - 0.51 (m, 1H), 0.48 - 0.40 (m, 2H), 0.15 - 0.07 (m, 1H), 0.06 - -0.02 (m, 1H).

[0894] Example 6-5: Preparation of compound 5 and its isomers 5-A and 5-B

[0895] (R)-2-cyclopropyl-N-((1S,9S)-9-(cyclopropylmethyl)-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 5-A

[0896] (R)-2-cyclopropyl-N-((1R,9S)-9-(cyclopropylmethyl)-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 5-B

[0897] First step

[0898] The mesylate salt of 1i-1 (25.0 mg, 42.3 pmol) and (2R)-2-cyclopropyl-2- hydroxyacetic acid (5.89 mg, 50.7 pmol) were dissolved in 1.5 mL of N,N- dimethylformamide, then 1-hydroxybenzotriazole (8.57 mg, 63.4 pmol), N,N- diisopropylethylamine (16.4 mg, 127 pmol) and 1-(3-dimethylaminopropyl)-3- ethylcarbodiimide hydrochloride (12.2 mg, 63.4 pmol) were added in turn, stirred at 20 °C for 4 hours. Toluene (4 mL x 2) was added to the reaction solution, concentrated under reduced pressure at 25 °C, the obtained residue was diluted with N,N- dimethylformamide to 1.5 mL, and the diluted solution was purified by pre-HPLC (column: Phenomenex luna C18 150 x 25 mm x 10 pm; mobile phase: A-water (0.225% formic acid), B-acetonitrile; gradient elution: B%: 28%-58%), to obtain two single configuration title products 5-A and 5-B:

[0899] Single configuration compound 5-A of compound 5 (4.95 mg, yield: 20%, off-white solid)

[0900] SFC analysis: retention time 2.115 minutes, purity: 97%. (column: Chiralpak IC-3 50 x 4.6 mm I.D., 3 pm, mobile phase: A-carbon dioxide, B-methanol and acetonitrile (0.05% diethylamine), isocratic elution: B: 50%, flow rate: 3 mL / min, instrument: Shimadzu LC-30AD sf).

[0901] MS m / z (ESI): 560.1 [M+1].

[0902] 1 H NMR (400 MHz, CD3OD) d 7.70 - 7.64 (m, 2H), 5.70 - 5.65 (m, 1H), 5.60 - 5.54 (m, 1H), 5.46 - 5.38 (m, 1H), 5.37 - 5.32 (m, 1H), 5.23 - 5.17 (m, 1H), 3.72 - 3.67 (m, 1H), 3.36 - 3.34 (m, 1H), 3.23 - 3.17 (m, 1H), 2.48 - 2.42 (m, 3H), 2.37 - 2.31 (m, 1H), 2.31 - 2.25 (m, 1H), 1.96 - 1.89 (m, 1H), 1.88 - 1.80 (m, 1H), 1.34 - 1.31 (m, 1H), 0.92 - 0.89 (m, 1H), 0.68 - 0.42 (m, 6H), 0.15 - 0.07 (m, 1H), 0.05 - -0.02 (m, 1H).

[0903] Single configuration compound 5-B of compound 5 (1.95 mg, yield: 8%, white solid)

[0904] SFC analysis: Retention time 1.179 min, purity: 97%. (Chromatographic column: Chiralpak IC-3 50 x 4.6 mm I.D., 3 pm, Mobile phase: A-Carbon dioxide, B-Methanol and Acetonitrile (0.05% Diethylamine), Isocratic elution: B: 50%, Flow rate: 3 mL / min, Instrument: Shimadzu LC-30AD sf).

[0905] MS m / z (ESI): 560.1 [M+1].

[0906] 1 H NMR (400 MHz, CD3OD) δ 7.69 (s, 1H), 7.69 - 7.62 (m, 1H), 5.69 - 5.63 (m, 1H), 5.61 - 5.55 (m, 1H), 5.49 - 5.42 (m, 1H), 5.40 - 5.34 (m, 1H), 5.24 - 5.16 (m, 1H), 3.91 - 3.84 (m, 1H), 3.38 - 3.36 (m, 1H), 3.24 - 3.16 (m, 1H), 2.44 (s, 3H), 2.40 - 2.23 (m, 2H), 1.97 - 1.80 (m, 2H), 1.29 - 1.21 (m, 1H), 0.95 - 0.85 (m, 1H), 0.64 - 0.36 (m, 6H), 0.17 - 0.07 (m, 1H), 0.07 - -0.02 (m, 1H).

[0907] Example 6-6: Preparation of compound 6

[0908] First step

[0909] (1S,9S)-1-amino-9-(cyclopropylmethyl)-5-fluoro-9-hydroxy-4-methyl 1,2,3,9,12,15- hexahydro-10H,13H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-10,13-dione 1i-1-1

[0910] (1R,9S)-1-amino-9-(cyclopropylmethyl)-5-fluoro-9-hydroxy-4-methyl 1,2,3,9,12,15- hexahydro-10H,13H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-10,13-dione 1i-1-2

[0911] The 1i-1 (550 mg, 1.10 mmol) was separated by SFC (Separation condition: Column: DAICEL CHIRALPAK IC 250 mm x 50 mm, 10 pm; Mobile phase: A - n- hexane: B - Ethanol, Isocratic elution: B: 45%,) to get the title product 1i-1-1 (260 mg, yield: 41%) as yellow solid, the title product 1i-1-2 (290 mg, yield: 46%) as yellow solid.

[0912] Single configuration compound 1i-1-1

[0913] SFC analysis: Retention time 3.039 min. (Column: Chiralpak IC-3 50 x 4.6 mm I.D., 3 pm, Mobile phase: A - n-hexane (0.05% isopropylamine), B - Ethanol and Acetonitrile (0.05% isopropylamine), Isocratic elution: B: 45%, Flow rate: 1 mL / min, Instrument: Shimadzu LC-20AD).

[0914] MS m / z (ESI): 462.3 [M+1].

[0915] Single configuration compound 1i-1-2

[0916] SFC analysis: Retention time 4.951 min. (Column: Chiralpak IC-3 50 x 4.6 mm I.D., 3 pm, Mobile phase: A - n-hexane (0.05% isopropylamine), B - Ethanol and Acetonitrile (0.05% isopropylamine), Isocratic elution: B: 45%, Flow rate: 1 mL / min, Instrument: Shimadzu LC-20AD).

[0917] MS m / z (ESI): 462.3 [M+1].

[0918] Second step

[0919] Dissolve 1i-1-1 (13 mg, 25.35 μmol), 1-hydroxycyclopropanecarboxylic acid (4 mg, 38.03 μmol) and 2-(7-azobenzotriazol)-N,N,N',N'-tetramethyluronium hexafluorophosphate (12 mg, 30.42 μmol) in 1 mL of N,N-dimethylformamide, add N,N-diisopropylethylamine (10 mg, 76.06 μmol), stir at 25 °C for 1 hour. Purify the reaction solution by pre-HPLC (column: InfinityLab Poroshell 120 SB-C18 21.2 x 150 mm, 4 μm; mobile phase: A-water (0.1% formic acid), B-acetonitrile; gradient elution: B%: 20%-80%), to obtain the title product 6 (2.25 mg, yield: 16%) in the form of a white solid.

[0920] MS m / z (ESI): 546.3 [M+1].

[0921] 1 H NMR (400 MHz, DMSO-d6) δ 8.66 - 8.58 (m, 1H), 7.80 - 7.74 (m, 1H), 7.41 - 7.34 (m, 1H), 6.62 - 6.58 (m, 1H), 6.37 - 6.29 (m, 1H), 5.61 - 5.53 (m, 1H), 5.48 - 5.37 (m, 2H), 5.33 - 5.24 (m, 1H), 5.12 - 5.04 (m, 1H), 3.28 - 3.22 (m, 1H), 3.17 - 3.06 (m, 1H), 2.44 - 2.33 (m, 3H), 2.28 - 2.15 (m, 2H), 1.86 - 1.73 (m, 2H), 1.27 - 1.15 (m, 2H), 1.00 - 0.89 (m, 2H), 0.87 - 0.75 (m, 1H), 0.40 - 0.25 (m, 2H), 0.08 - 0.03 (m, 1H), -0.06 - -0.14 (m, 1H).

[0922] Examples 6-7 to 6-13

[0923] The following compounds were prepared according to the method of Reference Example 6-1, selecting the corresponding starting materials:

[0924] Example 6-24: Preparation of compound 24

[0925] (S)-N-((8-ethyl-4-fluoro-8-hydroxy-9,12-dioxo-2,3,8,9,12,14-hexahydro-1H,11H- cyclopenta[f]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-15-yl)methyl)-2-hydroxyacetamide 24

[0926] First step

[0927] 4-bromo-7-fluoro-5-nitro-2,3-dihydro-1H-indene 24b

[0928] Dissolve 24a (17 g, 79.05 mmol, prepared by the method disclosed in patent application “WO2021093820 A1” Example 9, page 31) in 200 mL trifluoroacetic acid, protect with nitrogen, cool to 0 °C with ice water bath, slowly add nitric acid (20.45 g, 292.08 mmol, content 90%) dropwise to the reaction solution, stir at 0 °C for 10 minutes, remove the ice water bath, stir at 25 °C for 30 minutes. Add 100 mL water, extract the diluted reaction solution with ethyl acetate (500 mL x 3), wash the organic phase with water (500 mL x 3), dry over anhydrous sodium sulfate, filter, concentrate the filtrate by reduced pressure distillation, purify the obtained residue by silica gel column chromatography with developing system B, to obtain the title product 24b (8.96 g, yield: 44%) in the form of colorless oil.

[0929] 1 H NMR (400 MHz, CDCl3) δ 7.45 (d, J = 8.0 Hz, 1H), 3.05-3.16 (m, 4H), 2.18-2.27 (m, 2H).

[0930] Second step

[0931] 4-bromo-7-fluoro-2,3-dihydro-1H-inden-5-amine 24c

[0932] Dissolve 24b (8.96 g, 34.61 mmol) in a mixture solution of 100 mL ethanol and 20 mL water, add iron powder (5.80 g, 103.82 mmol) and ammonium chloride (5.55 g, 103.82 mmol) successively, protect with nitrogen, stir the reaction solution at 80 °C for 1 hour. Filter the reaction solution, concentrate the filtrate by reduced pressure distillation, to obtain the crude title product 24c (7.2 g) in the form of white solid. The product is directly used in the next step reaction without purification.

[0933] MS m / z (ESI): 229.9 [M+1].

[0934] Third step

[0935] tert-butyl (4-bromo-7-fluoro-2,3-dihydro-1H-inden-5-yl)(tert-butoxycarbonyl)carbamate 24d

[0936] Dissolve 24c (7.2 g, 31.29 mmol) in 50 mL of tetrahydrofuran, add di-tert-butyl dicarbonate (27.32 g, 125.18 mmol), 4-dimethylaminopyridine (38.23 mg, 312.94 μmol) and triethylamine (12.67 g, 125.18 mmol), stir the reaction solution at 25 °C for 16 hours. Concentrate the reaction solution by distillation under reduced pressure, purify the obtained residue by silica gel column chromatography with developing system B to obtain the title product 24d (9.2 g, yield: 68%) as a white solid.

[0937] MS m / z (ESI): 317.9 [M+1-56-56].

[0938] Fourth step

[0939] tert-butyl (tert-butoxycarbonyl)(7-fluoro-4-vinyl-2,3-dihydro-1H-inden-5-yl)carbamate 24e

[0940] Dissolve 24d (1.5 g, 4.38 mmol) and vinylboronic acid pinacol ester (810.20 mg, 5.26 mmol) in a mixture solution of 6 mL of 1,4-dioxane and 2 mL of water, protect under nitrogen, add potassium phosphate tribasic (2.79 g, 13.15 mmol) and methane sulfonic acid (2-dicyclohexylphosphino-2',4',6'-tri-isopropyl-1,1'- biphenyl) (2'-amino-1,1'-biphenyl-2-yl)palladium(II) (371.07 mg, 438.38 μmol) in sequence, stir the reaction solution at 95 °C for 10 hours under nitrogen protection. Add 20 mL of water, dilute the reaction solution with ethyl acetate (10 mL x 3), wash the organic phase with saturated sodium chloride solution (5 mL x 2), dry over anhydrous sodium sulfate, filter, concentrate the filtrate by distillation under reduced pressure, purify the obtained residue by silica gel column chromatography with developing system B to obtain the title product 24e (350 mg, yield: 26%) as a colorless oil.

[0941] MS m / z (ESI): 222.1 [M+1-100-56].

[0942] Fifth step

[0943] tert-butyl (tert-butoxycarbonyl)(7-fluoro-4-formyl-2,3-dihydro-1H-inden-5-yl)carbamate 24f

[0944] Dissolve 24e (755 mg, 2.00 mmol) in a mixture of 1.5 mL of dioxane and 1.5 mL of water, add sodium periodate (1.29 g, 6.01 mmol) and potassium osmate dihydrate (73.8 mg, 200 μmol), stir at 25 °C for 5 hours. Filter the reaction solution, concentrate the filtrate by distillation under reduced pressure, and purify the obtained residue by silica gel column chromatography with the developing system B to obtain the title product 24f (538 mg, yield: 71%) in the form of a yellow solid. MS m / z (ESI): 180.2 [M+1-100-100].

[0945] Sixth step

[0946] (S)-8-ethyl-4-fluoro-8-hydroxy-1,2,3,8,11,14-hexahydro-9H,12H- cyclopenta[f]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-9,12-dione 24h

[0947] Dissolve 24f (538 mg, 1.42 mmol) and 24g (i.e. the compound 22c described above) (375 mg, 1.42 mmol, prepared by the method disclosed in patent application "WO2019238046 A1", page 28, example 30) in 10 mL of ethanol, add concentrated hydrochloric acid (12 M, 1.58 mL), stir at 80 °C for 2 hours. Concentrate the reaction solution by distillation under reduced pressure to remove ethanol, add 10 mL of water, and filter to obtain the filter cake to obtain the crude title product 24h (500 mg) in the form of a yellow solid. The product is used directly in the next step without purification.

[0948] MS m / z (ESI): 407.2 [M+1].

[0949] Seventh step

[0950] (S)-8-ethyl-4-fluoro-8-hydroxy-9,12-dioxo-2,3,8,9,12,14-hexahydro-1H,11H- cyclopenta[f]pyrano[3',4':6,7]indolizino[1,2-b]quinoline 6-oxide 24i

[0951] Dissolve 24h (500 mg, 1.21 mmol) in 70 mL of acetic acid, add hydrogen peroxide (16.9 g, 149 mmol, content 30%) under nitrogen protection, stir at 75 °C for 3 hours. LCMS shows that the starting material 24h remains, add hydrogen peroxide (16.5 g, 145 mmol, content 30%) again, stir at 75 °C for 4 hours. Pour the reaction solution into 100 mL of water, filter to obtain the filter cake to obtain the crude title product 24i (400 mg) in the form of a yellow solid. The product is used directly in the next step without purification.

[0952] MS m / z (ESI): 423.2 [M+1].

[0953] Eighth Step

[0954] (S)-15-chloro-8-ethyl-4-fluoro-8-hydroxy-1,2,3,8,11,14-hexahydro-9H,12H- cyclopenta[f]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-9,12-dione 24j

[0955] Dissolve 24i (100 mg, 142 μmol) in 2 mL of N,N-dimethylformamide, protect under nitrogen, cool to 0 °C in an ice-water bath, add oxalyl chloride (45.0 mg, 355 μmol), stir at 25 °C for 1 h. Pour the reaction into 10 mL of water, filter to obtain the filter cake, and obtain the crude title product 24j (70 mg) as a brown solid. Use the product directly in the next step without purification.

[0956] MS m / z (ESI): 441.2 [M+1].

[0957] Ninth Step

[0958] (S)-((8-ethyl-4-fluoro-8-hydroxy-9,12-dioxo-2,3,8,9,12,14-hexahydro-1H,11H- cyclopenta[f]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-15-yl)methyl)carbamic acid tert-butyl ester 24k

[0959] Dissolve 24j (20.0 mg, 26.3 μmol) and potassium ((tert-butoxycarbonylamino)methyl)trifluoroborate (14.0 mg, 59.2 μmol) in 1 mL of a mixture of dioxane and 0.2 mL of water, protect under nitrogen, add 1,1'- bis(diphenylphosphino)ferrocene palladium dichloride (2.89 mg, 3.95 μmol) and potassium carbonate (25.1 mg, 118 μmol), stir at 95 °C under nitrogen for 1 h. Pour the reaction into aqueous hydrochloric acid (1 M, 2 mL), concentrate the filtrate by distillation under reduced pressure, and obtain the crude title product 24k (20 mg) as a brown oil. Use the product directly in the next step without purification.

[0960] MS m / z (ESI): 436.2 [M+1-100].

[0961] Tenth Step

[0962] (S)-15-(aminomethyl)-8-ethyl-4-fluoro-8-hydroxy-1,2,3,8,11,14-hexahydro- 9H,12H-cyclopenta[f]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-9,12-dione 24l

[0963] Dissolve 24k (1.00 mg, 1.87 μmol) in 0.6 mL of dichloromethane, add hydrochloric acid in dioxane (4 M, 0.2 mL), stir at 25 °C for 1 h. Add 1 mL of water, extract the diluted reaction solution with ethyl acetate (1 mL x 3), filter the aqueous phase, concentrate the filtrate by distillation under reduced pressure to obtain the crude title product 24l (0.5 mg) as a yellow solid. The product is used directly in the next step without purification.

[0964] MS m / z (ESI): 436.1 [M+1].

[0965] Tenth step

[0966] (S)-N-((8-ethyl-4-fluoro-8-hydroxy-9,12-dioxo-2,3,8,9,12,14-hexahydro-1H,11H- cyclopenta[f]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-15-yl)methyl)-2-hydroxyacetamide 24

[0967] Dissolve 24l (13.3 mg, 22.9 μmol) and 2-hydroxyacetic acid (2.10 mg, 27.5 μmol) in 1 mL of dichloromethane, add N,N-diisopropylethylamine (8.90 mg, 68.9 μmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (6.60 mg, 34.4 μmol) and 1-hydroxybenzotriazole (4.65 mg, 34.4 μmol), stir at 25 °C for 30 min. Concentrate the reaction solution by distillation under reduced pressure, add 2 mL of water, extract with ethyl acetate (1 mL x 3), wash the organic phase with saturated sodium chloride solution (1 mL), dry the organic phase over anhydrous sodium sulfate, filter, concentrate the filtrate by distillation under reduced pressure, purify the obtained residue by preparative HPLC (separation condition: column: Welch ultimate C18 150 x 25 mm x 7 μm, mobile phase: A-water (containing 0.225% formic acid), B-acetonitrile, gradient elution, flow rate: 25 mL / min, instrument: GILSON GX-281) to obtain the title product 24 (0.98 mg, yield: 8%) as a yellow solid.

[0968] MS m / z (ESI): 494.1 [M+1].

[0969] 1H NMR (400 MHz, CD3OD) δ 7.71 - 7.66 (m, 1H), 7.63 (s, 1H), 5.61 - 5.56 (m, 1H), 5.50 - 5.48 (m, 1H), 5.43 - 5.35 (m, 3H), 5.18 - 5.09 (m, 3H), 3.73 - 3.65 (m, 2H), 3.20 - 3.12 (m, 2H), 2.39 - 2.29 (m, 2H), 2.02 - 1.93 (m, 2H), 1.05 - 0.94 (m, 3H).

[0970] Example 6-25: Preparation of compound 25

[0971] (S)-N-(((S)-8-ethyl-4-fluoro-8-hydroxy-9,12-dioxo-2,3,8,9,12,14-hexahydro-1H,11H- cyclopenta[f]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-15-yl)methyl)-2-hydroxypropanamide 25

[0972] Dissolve 24l (22.3 mg, 45.1 μmol) and (2R)-2-hydroxypropanoic acid (4.07 mg, 45.2 μmol) in 0.5 mL of dichloromethane, add N,N-diisopropylethylamine (17.5 mg, 135 μmol), 1-(3- dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (10.4 mg, 54.2 μmol) and 1- hydroxybenzotriazole (7.33 mg, 54.2 μmol), stir at 25 °C for 3 hours. Concentrate the reaction solution by distillation under reduced pressure, purify the resulting residue by pre-HPLC (separation condition: column: Phenomenex luna C18 250 x 50 mm x 15 μm, mobile phase: A-water (0.225% formic acid), B-acetonitrile, gradient elution, B%: 23%-53%), to obtain the title product 25 (0.49 mg, yield: 2%) as a yellow gum. SFC analysis: retention time 0.931 minute, purity: 97%. (column: Chiralcel OD-3 50 x 4.6 mm I.D., 3 μm, mobile phase: A-carbon dioxide, B-isopropyl alcohol and acetonitrile (0.05% diethylamine), isocratic elution: B: 40%, flow rate: 3 mL / min, instrument: Shimadzu LC-30AD sf).

[0973] MS m / z (ESI): 508.1 [M+1].

[0974] 1H NMR (400 MHz, CD3OD) δ 7.70 (d, J = 9.6 Hz, 1H), 7.63 (s, 1H), 5.63 - 5.56 (m, 1H), 5.43 - 5.36 (m, 3H), 5.15 - 5.10 (m, 2H), 4.25 - 4.18 (m, 1H), 3.71 - 3.66 (m, 2H), 3.19 - 3.13 (m, 2H), 2.39 - 2.31 (m, 2H), 2.01 - 1.93 (m, 2H), 1.43 - 1.38 (m, 3H), 1.04 - 0.98 (m, 3H).

[0975] Example 6-26: Preparation of compound 26

[0976] (R)-N-(((S)-8-ethyl-4-fluoro-8-hydroxy-9,12-dioxo-2,3,8,9,12,14-hexahydro-1H,11H- cyclopenta[f]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-15-yl)methyl)-2-hydroxypropanamide 26

[0977] Dissolve 24l (22.3 mg, 45.2 μmol) and (2S)-2-hydroxypropanoic acid (4.07 mg, 45.1 μmol) in 0.5 mL of dichloromethane, add N,N-diisopropylethylamine (17.5 mg, 135 μmol), 1-(3- dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (10.4 mg, 54.2 μmol) and 1- hydroxybenzotriazole (7.33 mg, 54.2 μmol), stir at 25 °C for 3 hours. Concentrate the reaction solution by distillation under reduced pressure, purify the resulting residue by pre-HPLC (separation conditions: column: Welch ultimate C18 150 x 25 mm x 7 μm, mobile phase: A - water (0.225% formic acid), B - acetonitrile, gradient elution, B%: 20% - 50%), to obtain the title product 26 (1.09 mg, yield: 5%) as a yellow gum. SFC analysis: retention time 0.745 min, purity: 98%. (column: Chiralcel OD-3 50 x 4.6 mm I.D., 3 μm, mobile phase: A - carbon dioxide, B - isopropanol and acetonitrile (0.05% diethylamine), isocratic elution: B: 40%, flow rate: 3 mL / min, instrument: Shimadzu LC-30AD sf).

[0978] MS m / z (ESI): 508.1 [M + 1].

[0979] 1H NMR (400 MHz, CD3OD) δ 7.72 (d, J = 9.4 Hz, 1H), 7.66 (s, 1H), 5.63 - 5.59 (m, 1H), 5.45 - 5.39 (m, 3H), 5.17 - 5.14 (m, 2H), 4.26 - 4.22 (m, 1H), 3.74 - 3.68 (m, 2H), 3.21 - 3.16 (m, 2H), 2.40 - 2.35 (m, 2H), 2.01 - 1.95 (m, 2H), 1.44 - 1.39 (m, 3H), 1.06 - 1.00 (m, 3H).

[0980] Example 6-27: Preparation of compound 27

[0981] (S)-2-cyclopropyl-N-(((S)-8-ethyl-4-fluoro-8-hydroxy-9,12-dioxo-2,3,8,9,12,14- hexahydro-lH,l lH-cyclopenta[f]pyrano[3',4':6,7]indolizino[l,2-b]quinolin-15-yl)methyl)- 2-hydroxyacetamide 27

[0982] Dissolve 24l (25.0 mg, 50.5 μmol) and (2S)-2-cyclopropyl-2-hydroxyacetic acid (8.80 mg, 75.8 μmol) in 1 mL of dichloromethane, add N,N-diisopropylethylamine (26.1 mg, 202 μmol), l-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (14.5 mg, 75.8 μmol) and l-hydroxybenzotriazole (10.2 mg, 75.8 μmol), stir at 25 °C for 30 min. Concentrate the reaction by distillation under reduced pressure, purify the resulting residue by pre-HPLC (separation conditions: column: Welch ultimate C18 150 x 25 mm x 7 μm, mobile phase: A - water (0.225% formic acid), B - acetonitrile, gradient elution, flow rate: 25 mL / min) to give the title product 27 (2.15 mg, yield: 8%) as a brown oil. SFC analysis: retention time 1.131 min, purity: 97%. (column: Chiralcel OD-3 50 x 4.6 mm I.D., 3 μm; mobile phase: A - carbon dioxide: B - ethanol (0.05% diethylamine), isocratic elution: B: 40%, flow rate: 3 mL / min, instrument: Shimadzu LC-30AD sf).

[0983] MS m / z (ESI): 534.3 [M + 1].

[0984] 1H NMR (400 MHz, CD3OD) δ 7.72-7.66 (m, 1H), 7.63 (s, 1H), 5.61-5.55 (m, 1H), 5.45-5.34 (m, 3H), 5.12 (s, 2H), 3.75-3.64 (m, 3H), 3.19-3.10 (m, 2H), 2.40-2.30 (m, 2H), 2.02-1.91 (m, 2H), 1.21-1.14 (m, 1H), 1.06-0.93 (m, 3H), 0.57-0.40 (m, 4H).

[0985] Example 6-28: Preparation of compound 28

[0986] (R)-2-cyclopropyl-N-(((S)-8-ethyl-4-fluoro-8-hydroxy-9,12-dioxo-2,3,8,9,12,14- hexahydro-lH,l lH-cyclopenta[f]pyrano[3',4':6,7]indolizino[l,2-b]quinolin-15-yl)methyl)- 2-hydroxyacetamide 28

[0987] Dissolve 24l (25.0 mg, 50.5 μmol) and (2R)-2-cyclopropyl-2-hydroxyacetic acid (8.80 mg, 75.8 μmol) in 1 mL of dichloromethane, add N,N-diisopropylethylamine (26.1 mg, 202 μmol), l-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (14.5 mg, 75.8 μmol) and l-hydroxybenzotriazole (10.2 mg, 75.8 μmol), stir at 25 °C for 30 min. Concentrate the reaction solution by distillation under reduced pressure, purify the resulting residue by pre-HPLC (separation condition: column: Phenomenex luna C18 150 x 25 mm x 10 μm, mobile phase: A-water (0.225% formic acid), B-acetonitrile, gradient elution) to give the title product 28 (2.77 mg, yield: 10%) as a brown oil. SFC analysis: retention time 0.898 min, purity: 99%. (column: Chiralcel OD-3 50 x 4.6 mm I.D., 3 μm; mobile phase: A-carbon dioxide: B-ethanol (0.05% diethylamine), isocratic elution: B: 40%, flow rate: 3 mL / min, instrument: Shimadzu LC-30AD sf).

[0988] MS m / z (ESI): 534.4 [M+l].

[0989] 1H NMR (400 MHz, CD3OD) δ 7.75-7.71 (m, 1H), 7.66 (s, 1H), 5.63-5.59 (m, 1H), 5.47-5.43 (m, 2H), 5.40-5.37 (m, 1H), 5.16-5.14 (m, 2H), 3.75-3.66 (m, 3H), 3.21-3.15 (m, 2H), 2.41-2.32 (m, 2H), 2.04-1.95 (m, 2H), 1.21-1.15 (m, 1H), 1.07-1.00 (m, 3H), 0.56-0.42 (m, 4H).

[0990] Example 6-34: Preparation of compound 34

[0991] (S)-N-((8-(cyclopropylmethyl)-4-fluoro-8-hydroxy-9,12-dioxo-2,3,8,9,12,14- hexahydro-lH,l lH-cyclopenta[f]pyrano[3',4':6,7]indolizino[l,2-b]quinolin-15- yl)methyl)-2-hydroxyacetamide 34

[0992] First step

[0993] Dissolve 24f (500 mg, 1.32 mmol) and le-l (578 mg, 1.73 mmol) in 20 mL of ethanol, add concentrated hydrochloric acid (12 M, 5.02 mL), stir at 80 °C for 16 hours. Concentrate the reaction solution by distillation under reduced pressure, add 10 mL of water, precipitate the solid, and collect the solid by filtration to obtain the title product 34a (496 mg) as a yellow solid. The product is used directly in the next step without purification.

[0994] MS m / z (ESI): 433.1 [M+l].

[0995] Second step

[0996] Dissolve 34a (596 mg, 1.38 mmol) in 36 mL of acetic acid, add hydrogen peroxide (3.86 g, 34.0 mmol, content 30%), stir at 75 °C for 3 hours. Add 100 mL of water to the reaction solution, precipitate the solid, and collect the solid by filtration to obtain the title product 34b (500 mg) as a yellow solid. The product is used directly in the next step without purification.

[0997] MS m / z (ESI): 449.0 [M+l].

[0998] Third step

[0999] Dissolve 34b (490 mg, 915 μmol) in 30 mL of N,N-dimethylformamide, protect under nitrogen, and cool to 0 °C in an ice-water bath. Add oxalyl chloride (465 mg, 3.66 mmol) and stir at 0 °C for 1 h. Pour the reaction into 100 mL of water and collect the solid by filtration to give the title product 34c (500 mg) as a yellow solid. Use the product directly in the next step without purification.

[1000] MS m / z (ESI): 467.1 [M+1].

[1001] Fourth Step

[1002] Dissolve 34c (200 mg, 428 μmol) in 5 mL of a mixture of dioxane and 0.5 mL of water, and add potassium N-tert-butyi carbonylmethyl trifluoroborate (203 mg, 856 μmol), 1,1-bis(diphenylphosphino)ferrocene palladium dichloride (31.3 mg, 42.8 μmol), and potassium phosphate tribasic (272 mg, 1.29 mmol). Protect under nitrogen and stir at 95 °C for 5 h. Concentrate the reaction under reduced pressure and collect the title product 34d (250 mg) as a brown solid. Use the product directly in the next step without purification.

[1003] MS m / z (ESI): 562.3 [M+1].

[1004] Fifth Step

[1005] Dissolve 34d (240 mg, 427 μmol) in 5 mL of aqueous hydrochloric acid (6 M) and stir at 40 °C for 10 h. Add 5 mL of water to the reaction, extract with dichloromethane (10 mL x 3), and lyophilize the aqueous phase to give the title product 34e as a hydrochloride salt (200 mg) as a yellow solid. Use the product directly in the next step without purification.

[1006] MS m / z (ESI): 462.3 [M+1].

[1007] Sixth Step

[1008] Dissolve 34e hydrochloride (20.0 mg, 40.1 μmol) and glycolic acid (4.6 mg, 60.2 μmol) in 2 mL of N,N-dimethylformamide, add N,N-diisopropylethylamine (15.5 mg, 120 μmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (11.5 mg, 60.2 μmol) and 1-hydroxybenzotriazole (8.1 mg, 60.2 μmol), stir at 25 °C for 2 hours. Add 1 mL of water to the reaction solution, extract with dichloromethane (3 mL x 3), dry the organic phase over anhydrous sodium sulfate, filter, concentrate the filtrate by distillation under reduced pressure, purify the obtained residue by pre-HPLC (separation conditions: column: Phenomenex Luna C18 150 x 25 mm x 10 μm, mobile phase: A-water (0.225% formic acid), B-acetonitrile, gradient elution, B%: 26%-56%), to obtain the title product 34 (2.51 mg, yield: 12%) as a yellow solid.

[1009] MS m / z (ESI): 520.3 [M+1]. 1 H NMR (400 MHz, CD3OD) δ 7.78 - 7.62 (m, 2H), 5.64 - 5.55 (m, 1H), 5.47 - 5.32 (m, 3H), 5.20 - 5.10 (m, 2H), 4.15 - 4.04 (m, 2H), 3.74 - 3.63 (m, 2H), 3.21 - 3.10 (m, 2H), 2.40 - 2.29 (m, 2H), 1.96 - 1.82 (m, 2H), 0.94 - 0.86 (m, 1H), 0.51 - 0.38 (m, 2H), 0.15 - -0.02 (m, 2H).

[1010] Example 6-35: Preparation of compound 35

[1011] (S)-N-((4-(Cyclopropylmethyl)-8-fluoro-4-hydroxy-9-methyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-11-yl)methyl)-2-hydroxyacetamide 35

[1012] First step

[1013] Dissolve 2-amino-4-fluoro-5-methylbenzaldehyde hydrochloride (138 mg, 686 μmol) and 1f-1 (200 mg, 686 μmol) in 35 mL of toluene, add 4-methylbenzenesulfonic acid pyridine (103 mg, 412 μmol) and o-methylphenol (596 mg, 5.52 mmol), stir at 135 °C for 3 hours. Concentrate the reaction solution by distillation under reduced pressure, purify the obtained residue by silica gel column chromatography with developing system A to obtain the title product 35a (213 mg, yield: 73%) in the form of yellow solid.

[1014] MS m / z (ESI): 407.2 [M+1].

[1015] Second step

[1016] Dissolve 35a (250 mg, 479 μmol) in 12 mL of acetic acid, add hydrogen peroxide (4.41 g, 38.9 mmol, content 30%), stir at 70 °C for 1.5 hours. Quench the reaction solution with 20 mL of sodium thiosulfate at 0 °C, extract with ethyl acetate (25 mL x 2), wash the organic phase with saturated sodium chloride solution (25 mL x 2), dry the organic phase with anhydrous sodium sulfate, filter, concentrate the filtrate by distillation under reduced pressure, purify the obtained residue by silica gel column chromatography with developing system A to obtain the title product 35b (120 mg, yield: 55%) in the form of yellow solid.

[1017] MS m / z (ESI): 423.2 [M+1].

[1018] Third step

[1019] Dissolve 35b (120 mg, 263 μmol) in 2 mL of N,N-dimethylformamide, protect with nitrogen, cool to 0 °C with ice water bath, add oxalyl chloride (167 mg, 1.32 mmol), stir at 0 °C for 1 hour. Pour the reaction solution into 10 mL of water, precipitate the solid, collect the solid by filtration, dry to obtain the title product 35c (115 mg) in the form of yellow solid. The product is directly used in the next step without purification.

[1020] MS m / z (ESI): 441.1 [M+1].

[1021] Fourth step

[1022] Dissolve 35c (90.0 mg, 167 μmol) in 3 mL of a mixture solvent of dioxane and 0.2 mL of water, add potassium N-tert-butyloxy carbonylmethyl trifluoroborate (198 mg, 838 μmol), 1,1-bis(diphenylphosphino)ferrocene palladium dichloride (24.5 mg, 33.5 μmol) and cesium carbonate (163 mg, 503 μmol), stir at 105 °C for 5 hours under nitrogen protection. Add 5 mL of water to the reaction solution, extract with ethyl acetate (8 mL x 3), dry the organic phase with anhydrous sodium sulfate, filter, concentrate the filtrate by distillation under reduced pressure, and obtain the title product 35d (110 mg) in the form of a yellow solid. The product is directly used in the next step without purification.

[1023] MS m / z (ESI): 536.1 [M+1].

[1024] Fifth step

[1025] Dissolve 35d (110 mg, 205 μmol) in 3 mL of aqueous hydrochloric acid (6 M), stir at 40 °C for 10 hours. Add 5 mL of water to the reaction solution, extract with dichloromethane (10 mL x 3), and freeze dry the aqueous phase to obtain the hydrochloride salt of the title product 35e (70 mg) in the form of a yellow solid. The product is directly used in the next step without purification.

[1026] MS m / z (ESI): 436.0 [M+1].

[1027] Sixth step

[1028] Dissolve the hydrochloride salt of 35e (30.0 mg, 68.8 μmol) and glycolic acid (5.76 mg, 75.7 μmol) in 2 mL of N,N-dimethylformamide, add N,N-diisopropylethylamine (26.7 mg, 206 μmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (19.8 mg, 103 μmol) and 1-hydroxybenzotriazole (13.9 mg, 103 μmol), and stir at 25 °C for 1.5 hours. Add 2 mL of water to the reaction solution, extract with dichloromethane (5 mL x 3), dry the organic phase with anhydrous sodium sulfate, filter, concentrate the filtrate by distillation under reduced pressure, and purify the obtained residue by pre-HPLC (separation condition: column: Phenomenex Luna C18 150 x 25 mm x 10 μm, mobile phase: A-water (0.225% formic acid), B-acetonitrile, gradient elution, B%: 19%-49%), to obtain the title product 35 (1.63 mg, yield: 5%) in the form of a yellow solid.

[1029] MS m / z (ESI): 494.3 [M+1].

[1030] 1 H NMR (400 MHz, CD3OD) δ 8.34 - 8.29 (m, 1H), 7.83 - 7.77 (m, 1H), 7.73 - 7.70 (m, 1H), 5.64 - 5.58 (m, 1H), 5.56 - 5.49 (m, 2H), 5.44 - 5.37 (m, 1H), 5.07 - 5.01 (m, 2H), 4.06 - 3.99 (m, 2H), 2.62 - 2.51 (m, 3H), 1.95 - 1.83 (m, 2H), 0.93 - 0.88 (m, 1H), 0.50 - 0.38 (m, 2H), 0.15 - 0.08 (m, 1H), 0.07 - -0.01 (m, 1H).

[1031] Example 6-36: Preparation of compound 36

[1032] (S)-N-(((S)-4-(Cyclopropylmethyl)-8-fluoro-4-hydroxy-9-methyl-3,14-dioxo-3,4,12,14- tetrahydro-lH-pyrano[3',4':6,7]indolizino[l,2-b]quinolin-ll-yl)methyl)-2- hydroxypropanamide 36

[1033] (S)-2-Hydroxypropanoic acid (2.1 mg, 23.3 μmol) was dissolved in 0.5 mL of N,N- dimethylformamide, followed by the addition of 1H-benzotriazol-l-yloxytripyrrolidinophosphonium hexafluorophosphate (13.1 mg, 25.2 μmol), N,N-diisopropylethylamine (14.9 mg, 155 μmol) and 35e (10.2 mg, 23.4 μmol) in sequence, stirred at 25 °C for 0.5 hours. The reaction solution was concentrated by distillation under reduced pressure, and the obtained residue was purified by pre-HPLC (separation condition: column: InfinityLab Poroshell 120 SB-C18 21.2 x 150 mm, 4 um, mobile phase: A-water (0.1% formic acid), B-acetonitrile, gradient elution, B%: 10%-70%), to give the title product 36 (5.3 mg, yield: 44.56%) as a white solid.

[1034] MS m / z (ESI): 508.4 [M+l].

[1035] 1H NMR (400 MHz, DMSO-d6) δ 8.71 (t, J = 6.0 Hz, 1H), 8.42 (d, J = 8.3 Hz, 1H), 7.89 (d, J = 10.8 Hz, 1H), 7.38 (s, 1H), 6.58 (s, 1H), 5.61 (d, J = 4.9 Hz, 1H), 5.49 (d, J = 2.1 Hz, 2H), 5.43 (d, J = 2.6 Hz, 2H), 4.92 - 4.75 (m, 2H), 4.05 - 3.94 (m, 1H), 3.09 - 2.95 (m, 4H), 1.89 - 1.75 (m, 2H), 1.75 - 1.68 (m, 4H), 1.20 (d, J = 6.8 Hz, 3H), 0.86 - 0.76 (m, 1H), 0.41 - 0.25 (m, 2H), 0.11 - 0.02 (m, 1H), -0.04 - -0.13 (m, 1H).

[1036] Example 6-37: Preparation of compound 37

[1037] (R)-N-(((S)-4-(cyclopropylmethyl)-8-fluoro-4-hydroxy-9-methyl-3,14-dioxo-3,4,12,14- tetrahydro-lH-pyrano[3',4':6,7]indolizino[l,2-b]quinolin-ll-yl)methyl)-2-hydroxypropanamide 37

[1038] (R)-2-hydroxypropanoic acid (2.1 mg, 23.3 μmol) was dissolved in 0.5 mL of N,N- dimethylformamide, followed by the addition of 1H-benzotriazol-l-yloxytripyrrolidinophosphonium hexafluorophosphate (13.1 mg, 25.2 μmol), N,N-diisopropylethylamine (14.9 mg, 155 μmol) and 35e (10.2 mg, 23.4 μmol) successively, stirred at 25 °C for 0.5 hours. The reaction solution was concentrated by distillation under reduced pressure, and the obtained residue was purified by pre-HPLC (separation condition: column: InfinityLab Poroshell 120 SB-C18 21.2 x 150 mm, 4 um, mobile phase: A-water (0.1% formic acid), B-acetonitrile, gradient elution, B%: 10%-70%), to give the title product 37 (3.6 mg, yield: 30.27%) as a white solid.

[1039] MS m / z (ESI): 508.4 [M+l].

[1040] 1H NMR (400 MHz, DMSO-d6) δ 8.71 (s, 1H), 8.42 (d, J = 8.2 Hz, 1H), 7.89 (d, J = 10.8 Hz, 1H), 7.38 (s, 1H), 6.59 (s, 1H), 5.61 (d, J = 4.9 Hz, 1H), 5.49 (s, 2H), 5.43 (d, J = 3.0 Hz, 2H), 4.91 - 4.75 (m, 2H), 4.04 - 3.96 (m, 1H), 3.03 - 3.00 (m, 4H), 1.89 - 1.77 (m, 2H), 1.76 - 1.73 (m, 4H), 1.19 (d, J = 6.8 Hz, 3H), 0.85 - 0.76 (m, 1H), 0.40 - 0.25 (m, 2H), 0.12 - 0.03 (m, 1H), -0.04 - -0.13 (m, 1H).

[1041] Example 7. Preparation of conjugate intermediate linker-drug

[1042] Example 7-1: Preparation of LD-1

[1043] 2-(((S)-10-benzyl-l-(((lS,9S)-9-(cyclopropylmethyl)-5-fluoro-9-hydroxy-4- methyl-10, 13-dioxo-2, 3, 9, 10, 13, 15-hexahydro-lH, 12H-benzo[de]pyrano[3',4':6,7] indolizino[l,2-b]quinolin-l-yl)amino)-l,6,9,12,15,18-hexaoxo-3-oxa-5,8,11,14,17- pentaazahenicosan-21-yl)oxy)-5-(2,5-dioxo-2,5-dihydro-lH-pyrrol-l-yl)-N 1 , N 3 bis(2,5,8,11,14,17,20,23-o xoapentacosan-25-yl)isophthalamide LD-1

[1044] First

[1045] Step

[1046] 2-(4-(tert-butoxy)-4-oxobutoxy)-5-nitroisophthalic acid dimethyl ester LD-1b

[1047] LD-1a (5 g, 19.55 mmol, prepared using the method disclosed in the literature "Journal of Physical Organic Chemistry, 2010, vol. 16, #10, p. 682-690"), tert-butyl 4-hydroxybutanoate (3.76 g, 23.47 mmol) and triphenylphosphine (7.69 g, 29.33 mmol) were dissolved in 40 mL of a mixture solvent of N,N-dimethylformamide and 120 mL of tetrahydrofuran, protected by nitrogen, and cooled to 0°C with an ice water bath. Diisopropyl azodicarboxylate (5.93 g, 29.33 mmol) was slowly added dropwise to the reaction solution, and stirred at 25°C for 20 hours. 150 mL of water was added, and the diluted reaction solution was extracted with ethyl acetate (200 mL x 3). The organic phase was dried with anhydrous sodium sulfate, filtered, and the filtrate was concentrated by reduced pressure distillation. The obtained residue was purified by reverse phase high performance liquid chromatography (separation conditions: column: I.D. 100 mm x H 300 mm Welch Ultimate XB_C18 20-40 μm, mobile phase: A-water, B-acetonitrile, gradient elution, flow rate: 200 mL / min, instrument: Agela Astra), to obtain the title product LD-1b (4.8 g, yield: 61%) as a yellow solid.

[1048] MS m / z (ESI): 420.2 [M+23].

[1049] Second step

[1050] 2-(4-(tert-butoxy)-4-oxobutoxy)-5-nitroisophthalic acid LD-1c

[1051] LD-1b (1.5 g, 3.71 mmol) was dissolved in 15 mL of a mixture solvent of water and 15 mL of tetrahydrofuran, and lithium hydroxide monohydrate (311.10 mg, 7.41 mmol) was added. The mixture was stirred at 25°C for 2 hours. 1N hydrochloric acid solution was added to the reaction solution to adjust the pH to 2-3, and extracted with ethyl acetate (40 mL x 3). The organic phase was dried with anhydrous sodium sulfate, filtered, and the filtrate was concentrated by reduced pressure distillation, to obtain the crude title product LD-1c (1.49 g) as a yellow solid. The product was used directly in the next reaction without purification.

[1052] MS m / z (ESI): 392.1 [M+23].

[1053] Third step

[1054] tert-Butyl 4-(2,6-bis((2,5,8,11,14,17,20,23-oxy-pentacosyl)carbamoyl)-4-nitrophenoxy)butyrate LD-1d

[1055] LD-1c (400 mg, 933.60 pmol) was dissolved in 5 mL of N,N-dimethylformamide, 1-hydroxybenzotriazole (378.45 mg, 2.80 mmol), 1-(3-dimethylaminopropyl)-3- ethylcarbodiimide hydrochloride (536.92 mg, 2.80 mmol), N,N-diisopropylethylamine (723.97 mg, 5.60 mmol) and 3,6,9,12,15,18,21,24-octaoxa-pentacosan-1-amine (895.03 mg, 2.33 mmol) were added, and stirred at 25 °C for 2 h. 10 mL of water was added, extracted with dichloromethane (10 mL x 3), the organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated by distillation under reduced pressure. The obtained residue was purified by silica gel column chromatography with developing system B to obtain the title product LD-1d (515 mg, yield: 50%) in the form of yellow oil.

[1056] MS m / z (ESI): 1117.7 [M+18].

[1057] Fourth step

[1058] 4-(2,6-bis((2,5,8,11,14,17,20,23-octaoxa-pentacosan-25-yl)carbamoyl)-4- aminophenoxy)butanoic acid tert-butyl ester LD-1e

[1059] LD-1d (515 mg, 466.74 pmol) was dissolved in a mixed solvent of 20 mL of ethanol and 10 mL of water, iron powder (130.32 mg, 2.33 mmol) and ammonium chloride (249.66 mg, 4.67 mmol) were added, and stirred at 80 °C for 16 h. The reaction solution was filtered with diatomite, 10 mL of water was added to the filtrate, and the diluted filtrate was extracted with dichloromethane (10 mL x 3). The organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated by distillation under reduced pressure. The obtained residue was purified by silica gel column chromatography with developing system A to obtain the title product LD-1e (435 mg, yield: 87%) in the form of yellow oil.

[1060] MS m / z (ESI): 1070.8 [M+1].

[1061] Fifth step

[1062] 4-(2,6-bis((2,5,8,11,14,17,20,23-octaoxa-pentacosan-25-yl)carbamoyl)-4-(2,5- dioxo-2,5-dihydro-1H-pyrrol-1-yl)phenoxy)butanoic acid tert-butyl ester LD-1f

[1063] LD-1e (380 mg, 352.47 pmol) was dissolved in 6 mL of dioxane, stirred at 25 °C for 1 h, TLC monitored the reaction was completed, then added ammonium persulfate (160.87 mg, 704.93 pmol) and dimethyl sulfoxide (55.08 mg, 704.93 pmol) to the reaction solution, stirred at 100 °C for 1 h. The reaction solution was concentrated by distillation under reduced pressure, the obtained residue was purified by preparative HPLC (separation conditions: column: Welch Xtimate C18 150 x 25 mm x 5 pm; mobile phase: A-water (0.1% trifluoroacetic acid), B-acetonitrile, gradient elution, B%: 25%-55%, flow rate 25 mL / min) to give the title product LD-1f (91 mg, yield: 21%) as a yellow oil.

[1064] MS m / z (ESI): 1167.8 [M+18].

[1065] Sixth step

[1066] 4-(2,6-bis((2,5,8,11,14,17,20,23-o xoabicycoctacosan-25-yl)carbamoyl)-4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1- yl)phenoxy)butanoic acid LD-1g

[1067] LD-1f (2 mg, 1.74 pmol) was dissolved in 0.5 mL of dichloromethane, added trifluoroacetic acid (0.1 mL), stirred at 25 °C for 1 h. The reaction solution was concentrated by distillation under reduced pressure to give the crude title product LD-1g (1.5 mg) as a colorless oil, which was used directly in the next step without purification.

[1068] MS m / z (ESI): 1111.8 [M+18].

[1069] Seventh step

[1070] (9H-fluoren-9-yl)methyl ((S)-10-benzyl-1-(((1S,9S)-9-(cyclopropylmethyl)-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- pento xo-3-oxa-5,8,11,14-tetraazahexadec-16-yl)carbamate LD-1i

[1071] LD-1h (68.7 mg, 94.7 μmol, prepared by the method disclosed in patent application “CN113402584 A” page 9, example LD1067-L1) and 1i-1-1 (5...

Claims

An antibody drug conjugate of Formula (I), or a pharmaceutically acceptable salt, isotopologue, tautomer, stereoisomer, polymorph, hydrate, or solvate thereof, or a mixture thereof: T—[L—D] y (I) wherein, T is a targeting moiety, and is an antibody or antigen-binding fragment thereof that binds CDH17; L is a linker unit; D is a biologically active moiety; y is selected from an integer or decimal number from 0.1 to 20. The antibody drug conjugate of claim 1, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein: (1) the VH comprises HCDR1, HCDR2, HCDR3 having the amino acid sequences set forth in SEQ ID NOs: 15, 16, 17, respectively, and the VL comprises LCDR1, LCDR2, and LCDR3 having the amino acid sequences set forth in SEQ ID NOs: 20, 21, 22, respectively; or (2) the VH comprises HCDR1, HCDR2, HCDR3 having the amino acid sequences set forth in SEQ ID NOs: 25, 26, 27, respectively, and the VL comprises LCDR1, LCDR2, and LCDR3 having the amino acid sequences set forth in SEQ ID NOs: 30, 31, 32, respectively; or (3) the VH comprises HCDR1, HCDR2, HCDR3 having the amino acid sequences set forth in SEQ ID NOs: 35, 36, 37, respectively, and the VL comprises LCDR1, LCDR2, and LCDR3 having the amino acid sequences set forth in SEQ ID NOs: 40, 41, 42, respectively; or (4) the VH comprises HCDR1, HCDR2, HCDR3 having the amino acid sequences set forth in SEQ ID NOs: 45, 46, 47, respectively, and the VL comprises LCDR1, LCDR2, and LCDR3 having the amino acid sequences set forth in SEQ ID NOs: 50, 51, 52, respectively; or (5) the VH comprises HCDR1, HCDR2, HCDR3 having the amino acid sequences set forth in SEQ ID NOs: 55, 56, 57, respectively, and the VL comprises LCDR1, LCDR2, and LCDR3 having the amino acid sequences set forth in SEQ ID NOs: 60, 61, 62, respectively; or (6) the VH comprises HCDR1, HCDR2, HCDR3 having the amino acid sequences set forth in SEQ ID NOs: 65, 66, 67, respectively, and the VL comprises LCDR1, LCDR2, and LCDR3 having the amino acid sequences set forth in SEQ ID NOs: 70, 71, 72, respectively; or (7) the VH comprises HCDR1, HCDR2, HCDR3 having the amino acid sequences set forth in SEQ ID NOs: 25, 125, 27, respectively, and the VL comprises LCDR1, LCDR2, and LCDR3 having the amino acid sequences set forth in SEQ ID NOs: 120, 31, 32, respectively; or (8) the VH comprises HCDR1, HCDR2, HCDR3, respectively, having an amino acid sequence as set forth in SEQ ID NOs: 45, 46, 47, and the VL comprises LCDR1, LCDR2, and LCDR3, respectively, having an amino acid sequence as set forth in SEQ ID NOs: 90, 51, 52. The antibody drug conjugate of claim 2, wherein: (1) the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 13, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 18; or (2) the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 23, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 28; or (3) the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 33, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 38; or (4) the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 43, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 48; or (5) the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 53, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 58; or (6) the VH comprises an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 63, and the VL comprises an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 68; or (7) the VH comprises an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 101, and the VL comprises an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 118; or (8) the VH comprises an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 101, and the VL comprises an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 123; or (9) the VH comprises an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 103, and the VL comprises an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 118; or (10) the VH comprises an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 103, and the VL comprises an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 123; or (11) the VH comprises an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 77, and the VL comprises an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 86; or (12) the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 82, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:

88. The antibody drug conjugate of claim 3, wherein: (1) the VH comprises an amino acid sequence as set forth in SEQ ID NO: 13, and the VL comprises an amino acid sequence as set forth in SEQ ID NO: 18; or (2) the VH comprises an amino acid sequence as set forth in SEQ ID NO: 23, and the VL comprises an amino acid sequence as set forth in SEQ ID NO: 28; or (3) the VH comprises an amino acid sequence as set forth in SEQ ID NO: 33, and the VL comprises an amino acid sequence as set forth in SEQ ID NO: 38; or (4) the VH comprises an amino acid sequence as set forth in SEQ ID NO: 43, and the VL comprises an amino acid sequence as set forth in SEQ ID NO: 48; or (5) the VH comprises an amino acid sequence as set forth in SEQ ID NO: 53, and the VL comprises an amino acid sequence as set forth in SEQ ID NO: 58; or (6) the VH comprises an amino acid sequence as set forth in SEQ ID NO: 63, and the VL comprises an amino acid sequence as set forth in SEQ ID NO: 68; or (7) the VH comprises an amino acid sequence as set forth in SEQ ID NO: 101, and the VL comprises an amino acid sequence as set forth in SEQ ID NO: 118; or (8) the VH comprises an amino acid sequence as set forth in SEQ ID NO: 101, and the VL comprises an amino acid sequence as set forth in SEQ ID NO: 123; or (9) the VH comprises an amino acid sequence as set forth in SEQ ID NO: 103, and the VL comprises an amino acid sequence as set forth in SEQ ID NO: 118; or (10) the VH comprises an amino acid sequence as set forth in SEQ ID NO: 103, and the VL comprises an amino acid sequence as set forth in SEQ ID NO: 123; or (11) the VH comprises an amino acid sequence as set forth in SEQ ID NO: 77, and the VL comprises an amino acid sequence as set forth in SEQ ID NO: 86; or (12) the VH comprises an amino acid sequence as set forth in SEQ ID NO: 82, and the VL comprises an amino acid sequence as set forth in SEQ ID NO:

88. The antibody drug conjugate of any one of claims 1-4, wherein the antibody is a murine antibody, a chimeric antibody, or a humanized antibody. The antibody drug conjugate of any one of claims 1-5, wherein the antibody belongs to an isotype selected from the group consisting of IgG, IgA, IgM, IgE, and IgD. The antibody drug conjugate of any one of claims 1-6, wherein the antibody belongs to a subtype selected from the group consisting of IgGl, IgG2, IgG3, and IgG4. The antibody drug conjugate of any one of claims 1-7, wherein the antibody comprises a heavy chain (HC) and a light chain (LC), wherein: (1) the HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 126, and the LC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 128; or (2) the HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 130, and the LC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 132; or (3) the HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 134, and the LC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 136; or (4) the HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 134, and the LC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 138; or (5) the HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 140, and the LC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 136; or (6) the HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 140, and the LC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 138; Preferably, (1) the HC comprises an amino acid sequence as set forth in SEQ ID NO: 126, and the LC comprises an amino acid sequence as set forth in SEQ ID NO: 128; or (2) the HC comprises an amino acid sequence as set forth in SEQ ID NO: 130, and the LC comprises an amino acid sequence as set forth in SEQ ID NO: 132; or (3) the HC comprises an amino acid sequence as set forth in SEQ ID NO: 134, and the LC comprises an amino acid sequence as set forth in SEQ ID NO: 136; or (4) the HC comprises an amino acid sequence as set forth in SEQ ID NO: 134, and the LC comprises an amino acid sequence as set forth in SEQ ID NO: 138; or (5) the HC comprises an amino acid sequence as set forth in SEQ ID NO: 140, and the LC comprises an amino acid sequence as set forth in SEQ ID NO: 136; or (6) the HC comprises an amino acid sequence as set forth in SEQ ID NO: 140, and the LC comprises an amino acid sequence as set forth in SEQ ID NO:

138. The antibody drug conjugate according to any one of claims 1-8, wherein the antigen binding fragment is selected from the group consisting of Fab, Fab', F(ab')2, Fv, scFv, and ds-scFv. The antibody drug conjugate according to any one of claims 1-8, wherein the antibody is a monoclonal antibody, a bispecific antibody, or a multispecific antibody. Preferably, wherein the antibody is a bispecific antibody, which further comprises a second antigen binding region that binds to a second antigen. The antibody drug conjugate according to any one of claims 1-10, wherein, the L comprises a cleavable linker or a non-cleavable linker; Preferably, the cleavable linker comprises an acid-labile linker, a hydrophilic linker, a protease-sensitive linker, a photo-labile linker, a hydrazone linker, a dimethyl linker, or a disulfide-containing linker. The antibody drug conjugate according to any one of claims 1-11, wherein, the L comprises a cleavable linker or a non-cleavable linker; L comprises or is derived from a fragment of mc(6-maleimidocaproyl), Val-Cit(valine-citrulline), p-amino-benzyloxycarbonyl (PABC), SPDB (N-succinimidyl-4-(2- pyridyldithio)-butyrate), sulfo-SPDB (N-succinimidyl-4-(2-pyridyldithio)-2-sulfo- butyrate), beta-glucuronic acid, dimethyl ethyl amine (DMEA), Val-Cit-PABC, mc- Val-Cit-PABC, CL2A, mal-PEG8-Val-Ala-PABC, mc-VC-PABC-DMEA, GGFG (glycine- glycine-phenylalanine-glycine), mc-GGFG-aminomethyl, AcBut (4-(4-acetyloxy)- butyric acid), dimethyl hydrazide (3-methyl-) 3-mercaptobutane hydrazide), AcBut- dimethyl hydrazide, or SMCC (N-succinimidyl-4-(N-maleimidomethyl) cyclohexane- carboxylate). The antibody drug conjugate of any one of claims 1-12, wherein, L is -L1-L2-L3-; L1is -L 1a -L 1b -L 1c -L 1d -L 1e -; L 1a selected from L 1b selected from C 1-10 alkylene, C 2-10 alkenylene or C 2-10 alkynylene, wherein each CH2in said C 1-10 alkylene, C 2-10 alkenylene and C 2-10 alkynylene is optionally substituted with 1, 2 or 3 R x ; each R x is independently selected from H, halogen, C 1-6 alkyl or C 1-6 haloalkyl; or, two R x may be joined to form a C 3-10 cycloalkylene or 3-10 membered heterocyclylene; L 1c is selected from a chemical bond, -C(O)-, -C(O)NH-, or -NHC(O)-; L 1d selected from a chemical bond, C 1-8 alkylene or -(CH2CH20) n -C 1-4 alkylene; said C 1-8 alkylene is optionally substituted with -NHC(O)-(CH2CH20) w -C 1-4 alkyl or -C(O)NH-(CH2CH20) w -C 1-4 alkyl; n and w are independently selected from 1, 2, 3, 4, 5, 6, 7, or 8; L 1e is selected from -C(O)-, -NHC(O)- or -C(O)-NHC(O)-; L2is a chemical bond or a divalent peptidic moiety comprising 2 to 8 optionally substituted natural or unnatural amino acid residues, each amino acid residue in said L2 is optionally substituted with 1, 2, 3, 4, or 5 R y substituents; each R is independently selected from H, halogen, C y is independently selected from H, halogen, C 1-6 alkyl, C 1-6 alkoxy, C 1-6 haloalkyl or C 1-6 haloalkoxy; L3 is an optional substituted or unsubstituted spacer, for example L3 is selected from -NH-CH2-(AM), (PABC) or (PAB); L3is optionally substituted with 1, 2, or 3 R z substituents, each R z is independently selected from H, halo, C 1-6 alkyl, C 1-6 alkoxy, C 1-6 haloalkyl, or C 1-6 haloalkoxy; Preferably, L is -L1-L2-L3-; L1is -L 1a -L 1b -L 1c -L 1d -L 1e -; L 1a selected from represents L 1a linkage site for attachment of an antibody; L 1b selected from C 1-6 alkylene, C 2-6 alkenylene or C 2-6 alkynylene, wherein C 1-6 alkylene, C 2-6 alkenylene and C 2-6 alkynylene each CH2is optionally substituted with 1 or 2 R x ; each R is independently selected from H, halogen, or C x is independently selected from H, halogen, or C 1-6 alkyl; or, two R groups on any identical or different carbon atom can be joined to form a C x may be joined to form a C 3- 7 cycloalkylene or 3-7 membered heterocyclyl, preferably C 3-7 cycloalkylene, more preferably C 3-5 cycloalkylene; L 1c is selected from a chemical bond, -C(O)-, -C(O)NH-, or -NHC(O)-, preferably a chemical bond or -C(O)NH-; L 1d is selected from a chemical bond or C 1-6 alkylene; said C 1-6 alkylene is optionally substituted with -NHC(O)-(CH2CH2O) w -C 1-4 alkyl or -C(O)NH-(CH2CH2O) w -C 1-4 alkyl; w is selected from 2, 3, or 4, preferably 3; L 1e is selected from -C(O)-, -NHC(O)- or -C(O)-NHC(O)-, preferably a chemical bond or -C(O)-; L2is selected from a divalent peptidyl group comprising 2 to 5 optionally substituted natural amino acid residues or non-natural amino acid residues, each amino acid residue in L2is optionally substituted with 1, 2, or 3 R y substituents; each R is independently selected from H, halogen, C y is independently selected from H, halogen, C 1-6 alkyl or C 1-6 haloalkyl; L3 is an optional substituted or unsubstituted spacer, for example L3 is selected from -NH-CH2-(AM) or (PABC); L3is optionally substituted with 1, 2, or 3 R z substituents, each R z is independently selected from H, halo, or C 1-6 alkyl. The antibody drug conjugate of any one of claims 1-13, wherein, L2is a divalent peptidic moiety comprising 2 to 5 optionally substituted natural or unnatural amino acid residues, selected from gly-gly, gly-gly-gly, gly-gly-gly-gly, val-gly-gly, val-cit-gly, val-gln-gly, val-glu-gly, phe-lys-gly, leu-lys-gly, gly-val-lys-gly, val-lys-gly-gly, val-lys-gly, val-lys-ala, val-lys-leu, leu-leu-gly, gly-gly-phe-gly, gly-gly-phe-gly-gly, val-gly, val-cit, or val-lys-beta-ala; each amino acid residue in said L2 is optionally substituted with 1, 2, or 3 R y substituents; each R is independently selected from H, halogen, C y is independently selected from H, halogen, C 1-6 alkyl or C 1-6 haloalkyl; Preferably, L2is a divalent peptidic moiety comprising 2 to 5 optionally substituted natural or unnatural amino acid residues, the amino acids being selected from glycine, phenylalanine, alanine, valine, citrulline, or lysine; each amino acid residue in said L2 is optionally substituted with 1, 2, or 3 R y substituents; each R is independently selected from H, halogen, or C1-C6alkyl; y R is independently selected from H, halogen, or C1-C6alkyl; 1-6 alkyl; More preferably, L2is -gly-gly-phe-gly- or -val-cit-. The antibody drug conjugate of any one of claims 1-14, wherein, L is selected from the following structures: (mc-GGFG-AM), (mc-GGFG-AM), (mc-GGFG-AM), (mc-val-cit-PABC), (mc-val-cit-pabc), (mc-val-cit-PABC), Preferably, L is selected from the following structures: (mc-GGFG-AM), (mc-GGFG-AM), (mc-GGFG-AM), (mc-val-cit-PABC), (mc-val-cit-PABC), (mc-val-cit-PABC), The antibody drug conjugate of any one of claims 1-15, wherein, D is selected from the group consisting of metal complexes; antibiotics; DNA topoisomerase inhibitors; tubulin inhibitors and microtubule polymerization inhibitors; DNA synthesis inhibitors; RNA polymerase II inhibitors; RNA splicesome inhibitors; agents acting on structural proteins; tumor signaling pathway inhibitors; proteasome inhibitors; histone deacetylase inhibitors; tumor neovascularization inhibitors; cyclin inhibitors; serine / threonine kinase inhibitors, tyrosine kinase inhibitors, aspartate kinase inhibitors or histidine kinase inhibitors; and other active substances that inhibit the growth of tumor cells, promote apoptosis or necrosis of tumor cells; Preferably, D is selected from the group consisting of tubulin inhibitors and microtubule polymerization inhibitors, antibiotics, DNA synthesis inhibitors, topoisomerase I inhibitors, RNA polymerase II inhibitors and RNA splicesome inhibitors; More preferably, D is selected from the group consisting of tubulin inhibitors and microtubule polymerization inhibitors, such as auristatins, maytansinoids, tubulysins, cryptophycins or rhizoxin; Alternatively, D is an antibiotic, such as a calicheamicin, an anthracycline and an anthracycline antibiotic; Alternatively, D is selected from the group consisting of DNA synthesis inhibitors, such as duocarmycins, PBDs (pyrrolodinoindoles) or IGNs (indolinobenzodiazepines); Alternatively, D is selected from the group consisting of DNA topoisomerase I inhibitors, such as camptothecin or a camptothecin derivative; Alternatively, D is selected from the group consisting of RNA polymerase II inhibitors, such as an alpha-amanitin; Alternatively, D is selected from the group consisting of RNA splicesome inhibitors, such as splicetastatins and telatinastatins; Preferably, D is selected from the group consisting of camptothecin or a camptothecin derivative, such as hydroxy camptothecin, 9-amino camptothecin, SN-38, exatecan, Dxd, irinotecan, topotecan or other derivatives; Preferably, D is selected from the group consisting of auristatin drugs, such as MMAE or MMAF; Preferably, D is selected from the group consisting of maytansinoid drugs, such as DM1, DM2, DM3 or DM4. The antibody drug conjugate of any one of claims 1 to 16, wherein D is selected from the group consisting of auristatin drugs, such as MMAE or MMAF; Alternatively, D is selected from a compound of Formula (D-I) or Formula (D-II), or a pharmaceutically acceptable salt, isotopologues, tautomer, stereoisomer, racemate, polymorph, hydrate, or solvate thereof, or mixtures thereof: *1, *2 and *3 are chiral centers independently selected from the group consisting of (S) or (R) absolute configuration, or a mixture thereof, preferably *3 is in (S) absolute configuration; L D1 is selected from a chemical bond, -NH-, -0-, -C(O)-, -NHC(O)-, or -C(0)NH-; q is 0, 1, 2, 3 or 4, preferably 1, 2 or 3; L D2 is selected from -NH-, -0-, -C(O)-, -NHC(O)-, or -C(0)NH-; L D3 is selected from a chemical bond, C 1-10 alkylene, C 1-10 haloalkylene, C 2-10 alkenylene, or C 2-10 alkynylene, said L D3 is optionally substituted with 1, 2, or 3 R D3 groups; L D4 is selected from -NH-, -0-, -C(O)-, -NHC(O)-, or -C(0)NH-; R D1 , R D2 and R D6 are independently selected from H, halogen, C 1-6 alkyl, C 1-6 haloalkyl or C 1-6 alkoxy; or, R D1 , R D2 and the carbon atom to which they are attached form a C 3-7 cycloalkyl or 3-7 membered heterocyclyl, preferably a 5-6 membered heterocyclyl; or, R D1 , R D6 and the carbon atom to which they are attached form a C 3-7 cycloalkyl or 3-7 membered heterocyclyl, preferably a C 5-6 cycloalkyl; each R D3 and R D4 is independently selected from H, halogen, C 1-6 alkyl, C 1-6 haloalkyl, C 1-6 alkoxy, C 1-6 haloalkoxy, C 3-7 cycloalkyl or 3-7 membered heterocyclyl; or, R D3 , R D4 and the carbon atom to which they are attached form a C 3-7 cycloalkyl or 3-7 membered heterocyclyl, preferably C 3-5 cycloalkyl; R D5 selected from halogen, C 1-6 alkyl, C 1-6 haloalkyl, C 3-10 cycloalkyl, 3-10 membered heterocyclyl, C 6-10 aryl, 5-10 membered heteroaryl, -C 1-6 alkylene-C 3-10 cycloalkyl, -C 1-6 alkylene-3-10 membered heterocyclyl, -C 1-6 alkylene-C 6-10 aryl or -C 1-6 alkylene-5-10 membered heteroaryl, preferably -C 1-6 alkylene-C 3-10 cycloalkyl; each of the compounds of formula (D-I) and (D-II) is optionally substituted with 1, 2, 3, 4 or 5 substituents selected from the group consisting of: deuterium, halogen, C 1-6 alkyl, C 1-6 haloalkyl, C 2-6 alkenyl, C 2-6 alkynyl, C 3-8 cycloalkyl, 3-8 membered heterocyclyl, C 6-10 aryl or 5-10 membered heteroaryl. Preferably, D is selected from the group consisting of auristatin drugs, such as MMAE or MMAF; Alternatively, D is selected from a compound of Formula (D-III) or Formula (D-IV), or a pharmaceutically acceptable salt, isotopologue, tautomer, stereoisomer, racemic, polymorph, hydrate, or solvate thereof, or mixtures thereof: *1 and *2 are chiral centers independently selected from the group consisting of (S) or (R) absolute configuration, or a mixture thereof; L D1 is selected from a chemical bond, -NH-, -0-, -C(O)-, -NHC(O)-, or -C(0)NH-; q is 0, 1, 2, 3 or 4, preferably 1, 2 or 3; L D2 is selected from -NH-, -0-, -C(O)-, -NHC(O)-, or -C(0)NH-; L D3 is selected from a chemical bond, C 1-10 alkylene, C 1-10 haloalkylene, C 2-10 alkenylene, or C 2-10 alkynylene, said L D3 is optionally substituted with 1, 2, or 3 R D3 substituents; L D4 is selected from -NH-, -0-, -C(O)-, -NHC(O)-, or -C(0)NH-; R D1 , R D2 and R D6 are independently selected from H, halogen, C 1-6 alkyl, C 1-6 haloalkyl or C 1-6 alkoxy; or, R D1 , R D2 and the carbon atom to which they are attached form a C 3-7 cycloalkyl or 3-7 membered heterocyclyl, preferably a 5-6 membered heterocyclyl; or, R D1 , R D6 and the carbon atom to which they are attached form a C 3-7 cycloalkyl or 3-7 membered heterocyclyl, preferably a C 5-6 cycloalkyl; each R D3 and R D4 is independently selected from H, halogen, C 1-6 alkyl, C 1-6 haloalkyl or C 1-6 alkoxy; or, R D3 , R D4 and the carbon atom to which they are attached form a C 3-7 cycloalkyl or 3-7 membered heterocyclyl, preferably C 3-5 cycloalkyl; R D5 selected from C 1-6 alkyl, C 3-10 cycloalkyl, 3-10 membered heterocyclyl, C 6-10 aryl, 5-10 membered heteroaryl, -C 1-6 alkylene-C 3-10 cycloalkyl, -C 1-6 alkylene-3-10 membered heterocyclyl, -C 1-6 alkylene-C 6-10 aryl or -C 1-6 alkylene-5-10 membered heteroaryl, preferably -C 1-6 alkylene-C 3-10 cycloalkyl. The antibody drug conjugate of any one of claims 1 to 17, wherein D is selected from the group consisting of auristatin drugs, such as MMAE or MMAF; Alternatively, D is selected from a compound of Formula (D-III), or a pharmaceutically acceptable salt, isotopologue, tautomer, stereoisomer, racemic, polymorph, hydrate, or solvate thereof, or mixtures thereof: *1 and *2 are chiral centers independently selected from (S) or (R) absolute configuration, or a mixture thereof; L D1 is selected from -NHC(O)- or -C(O)NH-; q is 1, 2 or 3, preferably 1; L D2 is selected from -NH- or -O-; R D1 selected from H, halogen, C 1-6 alkyl or C 1-6 haloalkyl, preferably C 1-6 alkyl, for example Me; R D2 selected from H, halogen, C 1-6 alkyl or C 1-6 haloalkyl, preferably halogen, e.g. F; or R D1 or R D2 and the carbon atom to which they are attached form a C 3-7 cycloalkyl or 3-7 membered heterocyclyl, preferably 5-6 membered heterocyclyl; each R D3 and R D4 is independently selected from H, halogen, C 1-6 alkyl, C 1-6 haloalkyl or C 1-6 alkoxy; or, R D3 , R D4 and the carbon atom to which they are attached form a C 3-7 cycloalkyl or 3-7 membered heterocyclyl, preferably C 3-5 cycloalkyl; R D5 selected from -C 1-4 alkylene-C 3-5 cycloalkyl or -C 1-4 alkylene-3- to 5-membered heterocyclyl, preferably -C 1-4 alkylene-C 3-5 cycloalkyl; Preferably, D is selected from an auristatin, e.g., MMAE or MMAF; Alternatively, D is selected from a compound of Formula (D-V), or a pharmaceutically acceptable salt, isotopologue, tautomer, stereoisomer, racemic, polymorph, hydrate, or solvate thereof, or mixtures thereof: *2 is a chiral center independently selected from (S) or (R) absolute configuration, or a mixture thereof; q is 1, 2 or 3, preferably 1; R D1 selected from H, halogen, C 1-6 alkyl or C 1-6 haloalkyl, preferably C 1-6 alkyl, for example Me; R D2 selected from H, halogen, C 1-6 alkyl or C 1-6 haloalkyl, preferably halogen, e.g. F; or R D1 , R D2 and the carbon atom to which they are attached form a C 3-7 cycloalkyl or 3-7 membered heterocyclyl, preferably 5-6 membered heterocyclyl; each R D3 and R D4 is independently selected from H, halogen, C 1-6 alkyl, C 1-6 haloalkyl or C 1-6 alkoxy, preferably R D3 and R D4 are not simultaneously H; or, R D3 , R D4 and the carbon atom to which they are attached form a C 3-7 cycloalkyl or 3-7 membered heterocyclyl, preferably C 3-5 cycloalkyl; R D5 selected from -C 1-4 alkylene-C 3-5 cycloalkyl or -C 1-4 alkylene-3- to 5-membered heterocyclyl, preferably -C 1-4 alkylene-C 3-5 cycloalkyl. The antibody drug conjugate of any one of claims 1-18, wherein, D is selected from the following compounds, or a pharmaceutically acceptable salt, isotopologues, tautomer, stereoisomer, racemate, polymorph, hydrate, or solvate thereof, or mixtures thereof: (MMAE), (Dxd), Preferably, D is selected from the following compounds, or a pharmaceutically acceptable salt, isotopic variant, tautomer, stereoisomer, racemic, polymorph, hydrate, or solvate thereof, or mixtures thereof: (MMAE), (Dxd), The antibody drug conjugate of any one of claims 1-19, wherein y is an integer or decimal number selected from 0.1 to 20, preferably an integer or decimal number selected from 1 to 10, more preferably an integer or decimal number selected from 2 to 8. The antibody drug conjugate of any one of claims 1-20, having the structure of formula (I-l) or (I-2): wherein, T, D and y are as defined in any one of claims 1-20; each m is selected from 1, 2, 3, 4 or 5; each w is selected from 1, 2, 3, 4, 5, 6, 7 or 8; each R x and R x ' is independently selected from H, halogen or C 1-6 alkyl; or, two R x groups on any identical or different carbon atom can be joined to form a C 3-7 cycloalkylene or 3-7 membered heterocyclyl, preferably C 3-7 cycloalkylene, more preferably C 3-5 cycloalkylene; each R is independently selected from H, halogen, C y independently selected from H, halogen, C 1-6 alkyl, C 1-6 alkoxy, C 1-6 haloalkyl or C 1-6 haloalkoxy. The antibody drug conjugate of any one of claims 1-21, wherein, The antibody drug conjugate is selected from the group consisting of the following structures: wherein, T, D and y are as defined in any one of claims 1-20; Preferably, D is selected from an auristatin, camptothecin or a camptothecin derivative, e.g., MMAE, MMAF, hydroxycamptothecin, 9-aminocamptothecin, SN-38, exatecan, Dxd, irinotecan, topotecan or a compound of Formula (D-I), Formula (D-II), Formula (D-III), Formula (D-IV) or Formula (D-V), wherein the variables are as defined in any one of claims 17-18; y is an integer or decimal number selected from 1 to 10. The antibody drug conjugate of any one of claims 1-22, wherein, The antibody drug conjugate is selected from the group consisting of the following structures: T and y are as defined in any one of claims 1-20; Preferably, y is an integer or decimal number selected from 1 to 10. The antibody drug conjugate of claims 1-23, wherein, The antibody drug conjugate is selected from the group consisting of the following structures: wherein, the mAb2 comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises an amino acid sequence as set forth in SEQ ID NO: 13, and the VL of the mAb2 comprises an amino acid sequence as set forth in SEQ ID NO: 18; the mAb3 comprises a heavy chain variable region (VH) and a light chain variable region (VL), the VH comprising an amino acid sequence as set forth in SEQ ID NO: 23, and the VL comprising an amino acid sequence as set forth in SEQ ID NO: 28; the mAb4 comprises a heavy chain variable region (VH) and a light chain variable region (VL), the VH comprising an amino acid sequence as set forth in SEQ ID NO: 33, and the VL comprising an amino acid sequence as set forth in SEQ ID NO: 38; the mAb5 comprises a heavy chain variable region (VH) and a light chain variable region (VL), the VH comprising an amino acid sequence as set forth in SEQ ID NO: 43, and the VL comprising an amino acid sequence as set forth in SEQ ID NO: 48; the mAb6 comprises a heavy chain variable region (VH) and a light chain variable region (VL), the VH comprising an amino acid sequence as set forth in SEQ ID NO: 53, and the VL comprising an amino acid sequence as set forth in SEQ ID NO: 58; the mAb7 comprises a heavy chain variable region (VH) and a light chain variable region (VL), the VH comprising an amino acid sequence as set forth in SEQ ID NO: 63, and the VL comprising an amino acid sequence as set forth in SEQ ID NO: 68; The mAb3 H2K2 comprises a heavy chain variable region (VH) comprising an amino acid sequence as set forth in SEQ ID NO: 101 and a light chain variable region (VL) comprising an amino acid sequence as set forth in SEQ ID NO: 118; The mAb3 H2K4 comprises a heavy chain variable region (VH) comprising an amino acid sequence as set forth in SEQ ID NO: 101 and a light chain variable region (VL) comprising an amino acid sequence as set forth in SEQ ID NO: 123; The mAb3 H3K2 comprises a heavy chain variable region (VH) comprising an amino acid sequence as set forth in SEQ ID NO: 103 and a light chain variable region (VL) comprising an amino acid sequence as set forth in SEQ ID NO: 118; The mAb3 H3K4 comprises a heavy chain variable region (VH) comprising an amino acid sequence as set forth in SEQ ID NO: 103 and a light chain variable region (VL) comprising an amino acid sequence as set forth in SEQ ID NO: 123; The mAb5 H3K1 comprises a heavy chain variable region (VH) comprising an amino acid sequence as set forth in SEQ ID NO: 77 and a light chain variable region (VL) comprising an amino acid sequence as set forth in SEQ ID NO: 86; The mAb5 H5K2 comprises a heavy chain variable region (VH) comprising an amino acid sequence as set forth in SEQ ID NO: 82 and a light chain variable region (VL) comprising an amino acid sequence as set forth in SEQ ID NO: 88; The TORL-3-600 comprises a heavy chain (HC) comprising an amino acid sequence as set forth in SEQ ID NO: 142 and a light chain (LC) comprising an amino acid sequence as set forth in SEQ ID NO: 143; Preferably, The mAb5 H3K1 comprises a heavy chain (HC) comprising an amino acid sequence as set forth in SEQ ID NO: 126 and a light chain (LC) comprising an amino acid sequence as set forth in SEQ ID NO: 128; The mAb5 H5K2 comprises a heavy chain (HC) comprising an amino acid sequence as set forth in SEQ ID NO: 130 and a light chain (LC) comprising an amino acid sequence as set forth in SEQ ID NO: 132; The mAb3 H2K2 comprises a heavy chain (HC) comprising an amino acid sequence as set forth in SEQ ID NO: 134 and a light chain (LC) comprising an amino acid sequence as set forth in SEQ ID NO: 136; The mAb3 H2K4 comprises a heavy chain (HC) comprising an amino acid sequence as set forth in SEQ ID NO: 134 and a light chain (LC) comprising an amino acid sequence as set forth in SEQ ID NO: 138; the mAb3 H3K2 comprises a heavy chain (HC) comprising an amino acid sequence as set forth in SEQ ID NO: 140, and a light chain (LC) comprising an amino acid sequence as set forth in SEQ ID NO: 136; the mAb3 H3K4 comprises a heavy chain (HC) comprising an amino acid sequence as set forth in SEQ ID NO: 140, and a light chain (LC) comprising an amino acid sequence as set forth in SEQ ID NO: 138; the TORL-3-600 comprises a heavy chain (HC) comprising an amino acid sequence as set forth in SEQ ID NO: 142, and a light chain (LC) comprising an amino acid sequence as set forth in SEQ ID NO: 143; more preferably, the -L-D structure is linked to a cysteine in the antibody or antigen-binding fragment thereof that binds CDH17. A pharmaceutical composition comprising the antibody drug conjugate of any one of claims 1-24, and optionally a pharmaceutically acceptable carrier or excipient. The pharmaceutical composition of claim 25, wherein the composition further comprises a second therapeutic agent, preferably the second therapeutic agent is selected from the group consisting of a chemotherapeutic agent, a monoclonal antibody drug, a bi- / multi-specific antibody drug, a recombinant protein drug, a nucleotide drug (including siRNA and antisense oligonucleotide), a small molecule drug, an immunomodulatory drug, and a cell therapy drug. Use of the antibody drug conjugate of any one of claims 1-24, or a pharmaceutically acceptable salt, isotopic variant, tautomer, stereoisomer, polymorph, hydrate, or solvate thereof, or a mixture thereof, in the manufacture of a medicament for preventing and / or treating a disease. Use according to claim 27, wherein The disease is cancer, for example a cancer associated with CDH17 expression; Preferably, the disease is selected from the group consisting of a neuroendocrine tumor, a gastric cancer, a colon cancer, a rectal cancer, a small intestine cancer, a pancreatic cancer, a breast cancer, an ovarian cancer, a prostate cancer, a squamous cell carcinoma, a basal cell carcinoma, an adenocarcinoma, a sweat gland carcinoma, a sebaceous gland carcinoma, a papillary carcinoma, a papillary adenocarcinoma, a cystadenocarcinoma, a medullary carcinoma, a bronchogenic carcinoma, an esophageal carcinoma, a renal cell carcinoma, a hepatoma, a bile duct carcinoma, a choriocarcinoma, a seminoma, an embryonal carcinoma, a Wilms' tumor, a cervical cancer, a uterine cancer, a testicular cancer, a lung cancer, a small cell lung cancer, a non-small cell lung cancer, a bladder carcinoma, or an epithelial carcinoma; More preferably, the disease is selected from the group consisting of a gastric cancer, a liver cancer (e.g., hepatocellular carcinoma), a colorectal cancer, and a pancreatic cancer.

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