Antibody against CDH17 and use thereof

Abstract

Provided is an antibody against CDH17 and a use thereof, in particular an antibody that binds to CDH17 or an antigen-binding fragment thereof, a nucleic acid comprising a nucleotide sequence encoding the antibody, a vector comprising the nucleic acid, and a host cell comprising the nucleic acid or vector. Also provided are a pharmaceutical composition comprising the antibody, a conjugate, a chimeric antigen receptor, and a treatment method using the antibody.

Description

Antibodies against CDH17 and uses thereof

[0001] This international patent application claims priority to Chinese patent application No. 202410635769.4, 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 antibodies that bind 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 and named Liver-Intestine cadherin (LI-cadherin) because it is expressed only in the liver and small intestine in 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 certain 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, etc. 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 with the cell scaffold myosin way, but through direct connection with the cell scaffold to carry out its cell adhesion effect (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. Using CDH17-targeted antibodies can specifically inhibit CDH17, and thus 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 experimental methods such as siRNA, shRNA, and CRISPR technology to knock out CDH17 and establish the corresponding stable cell lines. Loss-of-function studies were also conducted 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 cancer-promoting gene, can promote the occurrence and development of pancreatic cancer by regulating cell proliferation and apoptosis signaling pathways.

[0010] Current research results show that CDH17 is closely related to the occurrence of various tumors, and developing antibodies 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 (ADCs). 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 disclosure provides an antibody or antigen-binding fragment thereof that binds CDH17, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein:

[0013] (1) the VH comprises HCDR1, HCDR2, HCDR3, each having an amino acid sequence as set forth in SEQ ID NO:5, 6, 7, respectively, and the VL comprises LCDR1, LCDR2, and LCDR3, each having an amino acid sequence as set forth in SEQ ID NO: 10, 11, 12, respectively; or

[0014] (2) the VH comprises HCDR1, HCDR2, HCDR3, each having an amino acid sequence as set forth in SEQ ID NO: 15, 16, 17, respectively, and the VL comprises LCDR1, LCDR2, and LCDR3, each having an amino acid sequence as set forth in SEQ ID NO: 20, 21, 22, respectively; or

[0015] (3) the VH comprises HCDR1 having an amino acid sequence as set forth in SEQ ID NO: 25 or 107, HCDR2 having an amino acid sequence as set forth in SEQ ID NO: 26 or 125, and HCDR3 having an amino acid sequence as set forth in SEQ ID NO: 27, and the VL comprises LCDR1 having an amino acid sequence as set forth in SEQ ID NO: 30 or 120, LCDR2 having an amino acid sequence as set forth in SEQ ID NO: 31, and LCDR3 having an amino acid sequence as set forth in SEQ ID NO: 32; or

[0016] (4) the VH comprises HCDR1, HCDR2, HCDR3, each having an amino acid sequence as set forth in SEQ ID NO: 35, 36, 37, respectively, and the VL comprises LCDR1, LCDR2, and LCDR3, each having an amino acid sequence as set forth in SEQ ID NO: 40, 41, 42, respectively; or

[0017] (5) the VH comprises HCDR1 having an amino acid sequence as set forth in SEQ ID NO: 45, HCDR2 having an amino acid sequence as set forth in SEQ ID NO: 46 or 81, and HCDR3 having an amino acid sequence as set forth in SEQ ID NO: 47, and the VL comprises LCDR1 having an amino acid sequence as set forth in SEQ ID NO: 50 or 90, LCDR2 having an amino acid sequence as set forth in SEQ ID NO: 51, 93 or 96, and LCDR3 having an amino acid sequence as set forth in SEQ ID NO: 52; or

[0018] (6) 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

[0019] (7) 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.

[0020] (1) the VH comprises HCDR1, HCDR2, HCDR3, each having an amino acid sequence as set forth in SEQ ID NOs: 5, 6, 7, respectively, and the VL comprises LCDR1, LCDR2, and LCDR3, each having an amino acid sequence as set forth in SEQ ID NOs: 10, 11, 12, respectively; or

[0021] (2) the VH comprises HCDR1, HCDR2, HCDR3, each having an amino acid sequence as set forth in SEQ ID NOs: 15, 16, 17, respectively, and the VL comprises LCDR1, LCDR2, and LCDR3, each having an amino acid sequence as set forth in SEQ ID NOs: 20, 21, 22, respectively; or

[0022] (3) 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

[0023] (4) 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

[0024] (5) 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

[0025] (6) 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

[0026] (7) 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

[0027] (8) 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

[0028] (9) 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.

[0029] In some embodiments, (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: 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; or

[0030] (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: 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

[0031] (3) the VH comprises an amino acid sequence with 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 NOs: 23, 99, 101, 103, 105, 108, 110, 112, 114, and the VL comprises an amino acid sequence with 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 NOs: 28, 116, 118, 121, 123; or

[0032] (4) the VH comprises an amino acid sequence with 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 with 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

[0033] (5) the VH comprises an amino acid sequence with 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 NOs: 43, 73, 75, 77, 79, 82, 84, and the VL comprises an amino acid sequence with 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 NOs: 48, 86, 88, 91, 94, 97; or

[0034] (6) the VH comprises an amino acid sequence with 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 with 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

[0035] (7) the VH comprises an amino acid sequence with 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 with 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.

[0036] In some embodiments, (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: 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; or

[0037] (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: 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

[0038] (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: 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

[0039] (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: 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

[0040] (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: 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

[0041] (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: 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

[0042] (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: 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

[0043] (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: 118; or

[0044] (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: 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

[0045] (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: 118; or

[0046] (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: 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

[0047] (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: 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

[0048] (13) 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.

[0049] In some embodiments, (1) 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; or

[0050] (2) 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

[0051] (3) 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

[0052] (4) 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

[0053] (5) 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

[0054] (6) 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

[0055] (7) 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

[0056] (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: 118; or

[0057] (9) 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

[0058] (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: 118; or

[0059] (11) 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

[0060] (12) 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

[0061] (13) 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.

[0062] In some embodiments, the antibody is a murine antibody, a chimeric antibody, or a humanized antibody.

[0063] In some embodiments, the antibody belongs to an isotype selected from the group consisting of IgG, IgA, IgM, IgE, and IgD.

[0064] In some embodiments, the antibody belongs to a subtype selected from the group consisting of IgGl, IgG2, IgG3, and IgG4.

[0065] In some embodiments, the antibody comprises a heavy chain (HC) and a light chain (LC), wherein:

[0066] (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 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; or

[0067] (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 to SEQ ID NO: 134 or 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 or SEQ ID NO: 138.

[0068] In some embodiments:

[0069] (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 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; or

[0070] (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 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; or

[0071] (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 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; or

[0072] (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 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; or

[0073] (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 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; or

[0074] (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.

[0075] In some embodiments:

[0076] (1) the HC comprises the amino acid sequence set forth in SEQ ID NO: 126, and the LC comprises the amino acid sequence set forth in SEQ ID NO: 128; or

[0077] (2) the HC comprises the amino acid sequence set forth in SEQ ID NO: 130, and the LC comprises the amino acid sequence set forth in SEQ ID NO: 132; or

[0078] (3) the HC comprises the amino acid sequence set forth in SEQ ID NO: 134, and the LC comprises the amino acid sequence set forth in SEQ ID NO: 136; or

[0079] (4) the HC comprises the amino acid sequence set forth in SEQ ID NO: 134, and the LC comprises the amino acid sequence set forth in SEQ ID NO: 138; or

[0080] (5) the HC comprises the amino acid sequence set forth in SEQ ID NO: 140, and the LC comprises the amino acid sequence set forth in SEQ ID NO: 136; or

[0081] (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.

[0082] In some embodiments, the antigen binding fragment is selected from the group consisting of a Fab, a Fab’, a F(ab’)2, a Fv, a scFv, and a ds-scFv.

[0083] In some embodiments, the antibody is a monoclonal antibody, a bispecific antibody, or a multispecific antibody.

[0084] In some embodiments, the antibody is a bispecific antibody further comprising a second antigen binding region that binds to a second antigen.

[0085] In another aspect, the present disclosure provides a nucleic acid comprising a nucleotide sequence encoding an antibody or antigen binding fragment thereof disclosed herein.

[0086] In yet another aspect, the present disclosure provides a vector comprising a nucleic acid disclosed herein.

[0087] In another aspect, the present disclosure provides a host cell comprising a nucleic acid or a vector disclosed herein.

[0088] In another aspect, the present disclosure provides a pharmaceutical composition comprising (i) an antibody or antigen binding fragment thereof disclosed herein; and (ii) a pharmaceutically acceptable carrier or excipient.

[0089] In some embodiments, the pharmaceutical composition further comprises a second therapeutic agent.

[0090] In some embodiments, the second therapeutic agent is selected from the group consisting of an antibody, a chemotherapeutic agent, and a small molecule drug.

[0091] In another aspect, the present disclosure provides a conjugate comprising an antibody or antigen binding fragment thereof disclosed herein, and a chemical moiety conjugated thereto.

[0092] In some embodiments, the chemical moiety is selected from the group consisting of a therapeutic agent, a detectable moiety, and an immunostimulatory molecule.

[0093] In yet another aspect, the present disclosure provides a chimeric antigen receptor (CAR) comprising an antibody or antigen binding fragment thereof disclosed herein.

[0094] In another aspect, the present disclosure provides a method for treating a disease in a subject, comprising administering to the subject an effective amount of an antibody or antigen binding fragment thereof disclosed herein, a pharmaceutical composition disclosed herein, a conjugate disclosed herein, or a CAR disclosed herein.

[0095] In some embodiments, the disease is cancer, for example a cancer associated with CDH17 expression.

[0096] In some embodiments, the cancer is selected from 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, hepatoma, cholangiocarcinoma, 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.

[0097] More preferably, the disease is selected from gastric cancer, liver cancer (e.g. hepatocellular carcinoma), colorectal cancer, pancreatic cancer, esophageal cancer, neuroendocrine tumor, and breast cancer.

[0098] In some embodiments, the method further comprises administering to the subject a second therapeutic agent.

[0099] In some embodiments, the second therapeutic agent is selected from an antibody, a chemotherapeutic agent, and a small molecule drug. BRIEF DESCRIPTION OF DRAWINGS

[0100] Figure 1: Western blot detection of eukaryotic recombinant expressed human and cynomolgus CDH17 protein.

[0101] Figure 2: FACS detection of stable cell line HEK293-hCDH17 and HEK293-cyCDH17 cells.

[0102] Figure 3: FACS detection of stable cell line CHO-hCDH17 cells.

[0103] Figure 4: BLI profile of anti-CDH17 chimeric antibody binding to hCDH17.

[0104] Figure 5: Binding of anti-CDH17 chimeric antibody to tumor cells.

[0105] Figure 6: Endocytosis of anti-CDH17 chimeric antibody.

[0106] Figure 7: BLI profile of anti-CDH17 humanized antibody binding to hCDH17.

[0107] Figure 8A-8B: Binding of anti-CDH17 humanized antibody to tumor cells.

[0108] Figure 9A-9B: Endocytosis of anti-CDH17 humanized antibody.

[0109] Figure 10: Killing of tumor cells SNU-16 by anti-CDH17 chimeric antibody ADC.

[0110] Figures 11A-11C: Killing of tumor cells SNU-16 by anti-CDH17 humanized antibody ADC.

[0111] Figure 11D: Killing of tumor cells ASPC-1 by anti-CDH17 humanized antibody ADC.

[0112] Figure 12: Bystander effect of anti-CDH17 humanized antibody ADC.

[0113] Figure 13: Inhibition of tumor growth in mice by anti-CDH17 humanized antibody ADC (targeting ASPC-1 cells).

[0114] Figure 14: Inhibition of tumor growth in mice by anti-CDH17 humanized antibody ADC (targeting SNU-16 cells). DETAILED DESCRIPTION

[0115] 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.

[0116] The embodiments described herein with reference to the drawings are explanatory, illustrative, and for general understanding of the present application. The embodiments are not to be construed as limiting 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.

[0117] In the present application, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by a person skilled in the art. Also, the terms and procedures of protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, immunology, and laboratory operations used herein are terms and procedures commonly used in the corresponding fields. At the same time, in order to better understand the present application, the definitions and explanations of the relevant terms are provided below.

[0118] Definitions

[0119] 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" includes a plurality of antibodies, and reference to "the antibody" in some embodiments includes a plurality of antibodies, and so forth.

[0120] Unless otherwise specified or defined, the term "comprising" and its variants such as "comprise" and "comprises" are to be construed as meaning including, but not excluding, any additional element or step.

[0121] As used herein, the term "antibody" refers to an immunoglobulin molecule having the ability to specifically bind a particular antigen. Such molecules typically comprise 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 sites for the 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).

[0122] 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 CH1 domains and, together 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 binding and binding to Fc receptors on effector cells. The hinge region, found in the 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.

[0123] According to crystallographic studies, the immunoglobulin hinge region can be further subdivided into three regions based on structure and function: the upper hinge, the core hinge, and the lower hinge. The upper hinge includes amino acids from the carboxy-terminal end of CH1 to the first residue in the hinge that restricts movement, usually the first cysteine residue that forms an interchain disulfide bond between the two heavy chains. The length of the upper hinge region correlates with 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 flexible allowed conformational changes in the immunoglobulin hinge region polypeptide sequence can influence the effector functions of the Fc portion of the antibody.

[0124] A "light chain variable region" (VL) or "heavy chain variable region" (VH) is comprised of "framework" regions separated by three "complementarity determining regions" or "CDRs." The framework regions serve to align the CDRs that are primarily responsible for specific antigenic epitope binding. The CDRs include the amino acid residues in the antibody that are primarily responsible for antigen binding. The VL domain and the VH domain each comprise, from amino-terminus to carboxyl-terminus, the following framework regions (FRs) and CDRs: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The CDR1, CDR2, and CDR3 of a VL domain are also referred to herein as LCDR1, LCDR2, and LCDR3, respectively; the CDR1, CDR2, and CDR3 of a VH domain are also referred to herein as HCDR1, HCDR2, and HCDR3, respectively.

[0125] 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.

[0126] 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., IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, etc. Based on the amino acid sequences of the light chains, the light chains of antibodies can be assigned to either a lambda (l) chain or a kappa (K) chain.

[0127] 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., bispecific 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 of glycosylation sites. Antibodies also include post-translationally modified antibodies, fusion proteins containing an antigenic determinant of an antibody, and immunoglobulin molecules containing any other modifications to the antigen recognition site, so long as the antibodies exhibit the desired biological activity.

[0128] 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, and should not be construed as requiring production by any particular method.

[0129] 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 construed 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 modifications to the antigen recognition site, so long as the antibodies exhibit the desired biological activity.

[0130] 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 has a reduced likelihood of eliciting an adverse immune response in a human individual as compared to the parental (e.g., mouse-derived) antibody.

[0131] The term "humanized antibody" generally refers to an antibody in which portions of the amino acid sequence outside of the CDR regions of a non-human antibody (e.g., murine antibody) are replaced with corresponding amino acids from a human immunoglobulin. In the CDR regions, small additions, deletions, insertions, substitutions or modifications of amino acids can also be allowed, as long as they still retain the ability of the antibody to bind to a particular antigen. A humanized antibody can optionally comprise at least a portion of a human immunoglobulin constant region. 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 that includes sequences derived from non-human immunoglobulin. In some cases, CDR region residues in a human immunoglobulin (recipient antibody) can be 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 a human immunoglobulin can be replaced by corresponding non-human residues. Furthermore, a humanized antibody can comprise 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.

[0132] 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.

[0133] 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 consisting of the 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); (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, the term also includes "linear antibodies", which comprise a pair of tandem Fd segments (VH-CH1-VH-CH1) with dual variable domain (D3) antibodies, which form a pair of antigen binding regions with a complementary light chain polypeptide, as well as modifications of any of the foregoing structures.

[0134] These antigen-binding fragments can be obtained using conventional techniques known to those skilled in the art, and the utility of the fragments screened in the same manner as for whole antibodies.

[0135] 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.

[0136] Avidity is a measure of the strength of binding between an antibody and the relevant antigen. Avidity involves both the affinity between the antigenic determinant and the antigen binding site of an antibody and the number of relevant 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 have 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 represents 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, as well as different variations thereof known in the art.

[0137] 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 non-contiguous amino acids juxtaposed by tertiary folding of a protein or proteins. 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 comprise at least 3, more usually at least 5 or 8-10 amino acids in a unique spatial conformation. Epitopes define the minimum binding site of an antibody and are thus the specific targets of an antibody or antigen-binding fragment thereof.

[0138] As used herein, the term "sequence identity" refers to the extent to which two sequences (amino acid) have identical residues in the same positions after alignment. 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.

[0139] 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 of an amino acid residue for another amino acid residue having a similar chemical structure that has little or substantially no effect on the function, activity or other biological properties of the polypeptide. Such conservative amino acid substitutions are well known in the art.

[0140] Such conservative substitutions preferably are substitutions of one amino acid for another that is in the same group (a) to (e) : (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.

[0141] 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.

[0142] As used herein, the term "vector" is intended to refer to a nucleic acid molecule that is capable of transporting another nucleic acid to which it has been linked.

[0143] As used herein, the term "host cell" refers to a cell into which an expression vector has been introduced.

[0144] 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 approvable by a regulatory agency of the Federal or a state government of the United States or listed in the U.S. Pharmacopoeia or other generally recognized pharmacopoeia for inclusion in a drug product for administration to humans.

[0145] As used herein, the terms "treatment," "therapy," "treat," 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 the symptoms of the disease to regress. Treatment can refer to any successful indicia in the treatment or amelioration or prevention of cancer, including any objective or subjective parameters, such as a reduction in symptoms; alleviation of disease symptoms or making the disease condition more tolerable to a patient; slowing in rate of disease progression or degeneration; or a lessening of the last nodes of deterioration. Treatment or amelioration of symptoms is based on one or more objective or subjective parameters; including results of a physician examination. 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.

[0146] As used herein, the term "effective amount" refers to an amount of an agent administered to a subject to treat a disease sufficient to effect treatment of the disease.

[0147] 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.

[0148] Anti-CDH17 antibodies

[0149] 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).

[0150] 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.

[0151] In some embodiments, the VH comprises HCDR1, HCDR2, HCDR3, having an amino acid sequence of SEQ ID NO: 15, 16, 17, respectively, and the VL comprises LCDR1, LCDR2, and LCDR3, having an amino acid sequence of SEQ ID NO: 20, 21, 22, respectively.

[0152] 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.

[0153] 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.

[0154] 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.

[0155] 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.

[0156] 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.

[0157] 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.

[0158] 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.

[0159] 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.

[0160] 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.

[0161] 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 the group consisting of SEQ ID NO: 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 selected from the group consisting of SEQ ID NO: 48, 86, 88, 91, 94, 97.

[0162] 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.

[0163] 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.

[0164] 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.

[0165] 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.

[0166] 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.

[0167] 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.

[0168] 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.

[0169] In some embodiments, the substitution of one or more amino acids can be a conservative substitution of one or more amino acids. Such conservative substitutions preferably are substitutions of one amino acid for another within a 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.

[0170] 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.

[0171] 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.

[0172] 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.

[0173] 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.

[0174] 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.

[0175] 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. 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.

[0176] 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.

[0177] 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.

[0178] 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.

[0179] 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.

[0180] 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.

[0181] 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.

[0182] 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.

[0183] 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.

[0184] 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.

[0185] 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.

[0186] 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.

[0187] 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.

[0188] 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.

[0189] 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.

[0190] 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.

[0191] 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.

[0192] 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.

[0193] 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.

[0194] 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.

[0195] 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.

[0196] 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.

[0197] 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.

[0198] 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.

[0199] 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.

[0200] An 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.

[0201] In some embodiments, the antigen-binding fragment is selected from the group consisting of a Fab, a Fab', a F(ab')2, a Fv, a scFv, and a ds-scFv. In a preferred embodiment, the antigen-binding fragment is a Fab. In another preferred embodiment, the antigen-binding fragment is a Fv. In another preferred embodiment, the antigen-binding fragment is a scFv.

[0202] 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.

[0203] A number of tumor-associated antigens associated with particular cancers have been identified in the art. 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.

[0204] 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.

[0205] 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 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 one of the gamma, delta, epsilon, zeta, and eta chains of CD3.

[0206] 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.

[0207] 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.

[0208] 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).

[0209] In one embodiment, the Fc region comprises a mutation that removes the Asn-linked glycosylation receptor 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.

[0210] In further embodiments, the Fc region is glycoengineered to reduce fucose and thus enhance ADCC, e.g., by adding 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 using FUT8 knockout cells, as described in Yamane-Ohnuki et al. (2004) Biotechnol. Bioeng 87:614. Alternatively, the antibody can be produced in a CHO cell line that has been engineered to express human FUT8, as described in Shinkai et al. (2007) Biotechnol. Bioeng. 97: 1-9. The methods described in Shopes et al. (1999) Nature Biotech 17:176 to optimize ADCC. In another embodiment, the Fc region is engineered to enhance complement activation, e.g., as described in Natsume et al. (2009) Cancer Sci. 100:2411.

[0211] 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-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-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).

[0212] Nucleic Acids

[0213] The present disclosure provides nucleic acids comprising a nucleotide sequence encoding an antibody or antigen-binding fragment thereof disclosed herein.

[0214] The term "nucleic acid" includes single- and double-stranded nucleotide polymers. The nucleic acid can be ribonucleotides or deoxyribonucleotides or a modified form of either type of nucleotide. The modifications include base modifications such as bromouridine and inosine derivatives, ribose modifications such as 2',3'-dideoxyribose, internucleotide linkage modifications such as phosphorothioate, phosphorodithioate, phosphonoselenoate, phosphorodiselenoate, phenylphosphonothioate, phenylphosphonodithioate, and phosphoramidate.

[0215] In some embodiments, the present application provides a nucleic acid molecule encoding any of the heavy chain variable region sequences disclosed herein. The present application also provides a nucleic acid molecule that is at least 90%, at least 95%, at least 98%, or at least 99% identical to a nucleic acid encoding any of the heavy chain variable region sequences disclosed herein. In some embodiments, the present application provides a nucleic acid molecule encoding any of the light chain variable region sequences disclosed herein. The present application also provides a nucleic acid molecule that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to a nucleic acid encoding any of the light chain variable region sequences disclosed herein.

[0216] In some embodiments, the nucleic acid disclosed herein comprises a nucleotide 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: 4, and a nucleotide 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: 9.

[0217] In some embodiments, the nucleic acid disclosed herein comprises a nucleotide 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: 14, and a nucleotide 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: 19.

[0218] In some embodiments, the nucleic acid disclosed herein comprises a nucleotide 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 the group consisting of SEQ ID NO: 24, 100, 102, 104, 106, 109, 111, 113, 115, and a nucleotide 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 the group consisting of SEQ ID NO: 29, 117, 119, 122, 124. In preferred embodiments, the nucleic acid disclosed herein comprises a nucleotide 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: 24, and a nucleotide 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: 29. In preferred embodiments, a nucleotide 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: 102, and a nucleotide 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: 119. In preferred embodiments, a nucleotide 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: 102, and a nucleotide 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: 124. In preferred embodiments, a nucleotide 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: 104, and a nucleotide 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: 119. In preferred embodiments, a nucleotide 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: 104, and a nucleotide 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: 124.

[0219] In some embodiments, the nucleic acid disclosed herein comprises a nucleotide 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: 34, and a nucleotide 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: 39.

[0220] In some embodiments, the nucleic acid disclosed herein comprises a nucleotide 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 the group consisting of SEQ ID NO: 44, 74, 76, 78, 80, 83, 85, and a nucleotide 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 the group consisting of SEQ ID NO: 49, 87, 89, 92, 95, 98. In preferred embodiments, the nucleic acid disclosed herein comprises a nucleotide 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: 44, and a nucleotide 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: 49. In preferred embodiments, the nucleic acid disclosed herein comprises a nucleotide 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: 78, and a nucleotide 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: 87. In preferred embodiments, the nucleic acid disclosed herein comprises a nucleotide 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: 83, and a nucleotide 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: 89.

[0221] In some embodiments, the nucleic acid disclosed herein comprises a nucleotide 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: 54, and a nucleotide 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: 59.

[0222] In some embodiments, the nucleic acid disclosed herein comprises a nucleotide 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: 64, and a nucleotide 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: 69.

[0223] In some embodiments, the present application provides nucleic acid molecules encoding the heavy chain sequences disclosed herein. The present application also provides nucleic acid molecules that are at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to the nucleic acids encoding the heavy chain sequences disclosed herein. In some embodiments, the present application provides nucleic acid molecules encoding the light chain sequences disclosed herein. The present application also provides nucleic acid molecules that are at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to the nucleic acids encoding the light chain sequences disclosed herein.

[0224] In some embodiments, the nucleic acid disclosed herein comprises a nucleotide 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: 127, and a nucleotide 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: 129.

[0225] In some embodiments, the nucleic acid disclosed herein comprises a nucleotide 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: 131, and a nucleotide 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: 133.

[0226] In some embodiments, the nucleic acid disclosed herein comprises a nucleotide 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: 135, and a nucleotide 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: 137.

[0227] In some embodiments, the nucleic acid disclosed herein comprises a nucleotide 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: 135, and a nucleotide 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: 139.

[0228] In some embodiments, the nucleic acid disclosed herein comprises a nucleotide 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: 141, and a nucleotide 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: 137.

[0229] In some embodiments, the nucleic acid disclosed herein comprises a nucleotide 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: 141, and a nucleotide 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: 139.

[0230] In some embodiments, the nucleic acid is a ribonucleic acid (RNA) or a deoxyribonucleic acid (DNA). In some embodiments, the present application provides a ribonucleic acid (RNA) comprising a nucleotide sequence encoding an antibody disclosed herein. In some embodiments, the present application provides a deoxyribonucleic acid (DNA) comprising a deoxyribonucleotide sequence encoding an antibody disclosed herein.

[0231] In some embodiments, the deoxyribonucleic acid (DNA) can be introduced into a human cell in vivo. In some embodiments, the deoxyribonucleic acid (DNA) of the present application is comprised in a vector or delivery agent. In some embodiments, the deoxyribonucleic acid (DNA) of the present application is integrated into the genome of the cell.

[0232] In some embodiments, a ribonucleic acid (RNA) can be introduced into a human cell in vivo. In some embodiments, a ribonucleic acid (RNA) of the present application is contained in a vector or delivery agent.

[0233] Vectors

[0234] The present disclosure provides vectors comprising the nucleic acids disclosed herein.

[0235] In some embodiments, a vector is an expression vector capable of expressing a polypeptide comprising a heavy chain or light chain variable region of an antibody. For example, the present application provides an expression vector comprising any of the nucleic acid molecules described above.

[0236] Any vector can be suitable for use in the present disclosure. In some embodiments, a vector is a viral vector. In some embodiments, a vector is a retroviral vector, a DNA vector, a murine leukemia virus vector, an SFG vector, a plasmid, an RNA vector, an adenoviral vector, a baculoviral vector, an Epstein Barr virus vector, a papovaviral vector, a vaccinia virus vector, a herpes simplex virus vector, an adeno-associated vector (AAV), a lentiviral vector, or any combination thereof. Suitable exemplary vectors include, for example, pGAR, pBABE-puro, pBABE-neo large T cDNA, pBABE-hygro-hTERT, pMKO.lGFP, MSCV-IRES-GFP, pMSCV PIG (Puro IRES GFP empty plasmid), pMSCV-loxp-dsRed-loxp-eGFP-Puro-WPRE, MSCV IRES luciferase, pMIG, MDH1-PGK-GFP_2.0, TtRMPVIR, pMSCV-IRES-mCherry FP, pRetroX GFP T2A Cre, pRXTN, pLncEXP, and pLXIN-Luc.

[0237] The expression vector can be any suitable recombinant expression vector. Suitable vectors include those designed for propagation and amplification or for expression or both, such as plasmids and viruses. For example, the vector can be selected from the pUC series (Fermentas Life Sciences, Glen Burnie, Md.), the pBluescript series (Stratagene, La Jolla, Calif.), the pET series (Novagen, Madison, Wis.), the pGEX series (Pharmacia Biotech, Uppsala, Sweden), and the pEX series (Clontech, Palo Alto, Calif.). Bacteriophage vectors such as lambda GT10, lambda GT11, lambda Zap II (Stratagene), lambda EMBL4, and lambda NM1149 can also be used. Examples of plant expression vectors that can be used in the present disclosure include pBI01, pBI101.2, pBI101.3, pBI121, and pBIN19 (Clontech). Examples of animal expression vectors that can be used in the present disclosure include pcDNA, pEUK-Cl, pMAM, and pMAMneo (Clontech).

[0238] Recombinant expression vectors can be prepared using standard recombinant DNA techniques described, for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Press, Cold Spring Harbor, N.Y. 2001; and Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates and John Wiley & Sons, NY, 1994. Circular or linear expression vector constructs can be prepared to contain the functions of a replication system in prokaryotic or eukaryotic host cells. The replication system can be derived from, for example, COLEL, 2μ plasmid, lambda, SV40, bovine papilloma virus, and the like.

[0239] For example, the vector can be an adenoviral vector comprising a nucleotide sequence encoding an antibody disclosed herein. The vector can be administered into a subject, then in vivo into a cell of the subject, thereby integrating the nucleotide sequence encoding the antibody disclosed herein into the genome of the cell, and subsequently the cell expresses the antibody disclosed herein.

[0240] Host cells

[0241] The present disclosure provides a host cell comprising a nucleic acid disclosed herein or a vector disclosed herein.

[0242] Any cell can be used as a host cell for the nucleic acids or vectors of the present disclosure. In some embodiments, the cell can be a prokaryotic cell, a fungal cell, a yeast cell, or a higher eukaryotic cell such as a mammalian cell. Suitable prokaryotic cells include, but are not limited to, eubacteria, such as gram-negative or gram-positive organisms, for example, Enterobactehaceae, such as Escherichia, for example, E. coli; Enterobacter; Erwinia; Klebsiella; Proteus; Salmonella, for example, Salmonella typhimurium; Serratia, for example, Serratia marcescans and Shigella; Bacilli, such as B. subtilis and B. licheniformis; Pseudomonas, such as P. aeruginosa; and Streptomyces. In some embodiments, the cell is a human cell. In some embodiments, the cell is an immune cell. In some embodiments, the host cell includes, for example, CHO cells, such as CHOS cells and CHO-K1 cells, or HEK293 cells, such as HEK293A, HEK293T, and HEK293FS.

[0243] The host cells of the present application are prepared by introducing the vectors disclosed herein or the nucleic acids disclosed herein in vitro or ex vivo. The host cells of the present application can be administered to a subject in vivo, and the host cells express the antibodies disclosed herein in vivo.

[0244] The present application provides host cells into which any of the above-described vectors have been introduced. The present application also provides methods of making the antibodies of the present application, comprising a) culturing the host cells disclosed herein under conditions suitable for production of the antibodies; and b) obtaining the antibodies from the culture.

[0245] Pharmaceutical compositions

[0246] The present disclosure provides pharmaceutical compositions comprising the antibodies or antigen-binding fragments thereof disclosed herein, and a pharmaceutically acceptable carrier or excipient.

[0247] The antibodies or antigen-binding fragments thereof of the present application (also referred to herein as“active compounds”) can be incorporated into pharmaceutical compositions suitable for administration. Such compositions typically comprise the antibody or antigen-binding fragment thereof, together with 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 vehicles and agents for use in formulations of pharmaceutically active substances are well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, its use in the compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions.

[0248] 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.

[0249] 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 ampoules, disposable syringes or multiple dose vials made of glass or plastic.

[0250] Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where 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 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 (for example, 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.

[0251] Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered 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.

[0252] Oral composition 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 use as a mouthwash in which the compound 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.

[0253] For administration by inhalation, the compounds are delivered in the form of an aerosol spray from a pressurized container or a dispenser that contains a suitable propellant, e.g., a gas such as carbon dioxide.

[0254] Systemic administration can also be by 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 compounds are formulated into

[0255] The active compounds can also be prepared in the form of suppositories (e.g., using conventional suppository

[0256] In one embodiment, the active compounds are prepared with carriers that protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for

[0257] The present application provides therapeutic compositions comprising the antibodies or antigen binding fragments thereof of the present application. The therapeutic compositions according to the present application 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 known to all pharmaceutical chemists: Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, PA. These formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, lipid-containing TM conjugates, anhydrous absorption pastes, oil-in-water and water-in-oil emulsions, emulsions with

[0258] Conjugates

[0259] The present disclosure provides conjugates comprising the antibodies or antigen binding fragments thereof disclosed herein, and a chemical moiety conjugated thereto.

[0260] In the context of the present disclosure, a "conjugate" is an antibody or antibody fragment (such as an antigen-binding fragment) covalently linked to a chemical moiety. The chemical moiety can be, for example, a drug, a toxin, a therapeutic agent, a detectable label, a protein, a nucleic acid, a lipid, a nanoparticle, a carbohydrate, or a recombinant virus. Antibody conjugates are often referred to as "immunoconjugates." When the conjugate comprises an antibody linked to a drug (e.g., a cytotoxic agent), the conjugate is often referred to as an "antibody-drug conjugate" or "ADC."

[0261] The term "conjugate" or "linkage" can refer to joining two polypeptides into one continuous polypeptide molecule. In one embodiment, the antibody is linked to a chemical moiety. In another embodiment, the antibody linked to a chemical moiety is further linked to a lipid or other molecule to the protein or peptide to increase its half-life in vivo. The linkage can be by chemical or recombinant means. In one embodiment, the linkage is chemical, in which a reaction between the antibody moiety and the chemical moiety results in a covalent bond formed between the two molecules to form one molecule. A peptide linker (a short peptide sequence) can optionally be included between the antibody and the chemical moiety.

[0262] The chemical moiety can be linked to the antibody of the present application using any number of means known to those of skill in the art. Both covalent and non-covalent means of attachment can be used. The procedure for attaching the chemical moiety to the antibody varies depending on the chemical structure of the chemical moiety. Polypeptides typically contain a variety of functional groups; such as carboxylic acid (COOH), free amine (-NH2), or thiol (-SH) moieties, which can be used to react with a suitable functional group 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. The derivatization can involve the attachment of any of a number of known linker molecules. The linker can be any molecule that serves to link the antibody to the chemical moiety. The linker is capable of forming a covalent bond to both the antibody and the chemical moiety. Suitable linkers are well known to those of skill in the art and include, but are not limited to, straight chain or branched carbon linkers, heterocyclic carbon linkers, or peptide linkers. In the case where the antibody and the chemical moiety are polypeptides, the linker can be attached to a constituent amino acid through a side group (such as through a disulfide bond to a cysteine) or to the alpha carbon amino and carboxyl groups of terminal amino acids.

[0263] In certain cases, it is desirable to release the chemical moiety from the antibody when the immunoconjugate reaches its target site. Thus, in these cases, the immunoconjugate will comprise a linkage that is cleavable in the vicinity of the target site.

[0264] The conditions experienced by the enzymatic activity or immunoconjugate within the target cell or in the vicinity of the target site can prompt cleavage of the linker to release the chemical moiety from the antibody.

[0265] In view of the large number of methods reported for attaching various radiodiagnostic compounds, radiotherapeutic compounds, labels (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.

[0266] Antibodies disclosed herein can be derivatized or linked to another molecule, such as another peptide or protein. Typically, the antibody or portion thereof is derivatized such that binding to the target antigen is not adversely affected by the derivatization or labeling. For example, an antibody can be functionally linked (by chemical coupling, genetic fusion, noncovalent association or otherwise) to one or more other molecular entities, such as another antibody (e.g., a bispecific antibody or a diabody), a detector agent, a pharmaceutical agent, and / or a protein or peptide that can mediate association with another molecule, such as a streptavidin core region or a polyhistidine tag.

[0267] One type of derivatized antibody is produced by cross-linking two or more antibodies (of the same or different type). Suitable cross-linking agents include heterobifunctional or homobifunctional cross-linking agents having two distinctly reactive moieties separated by a suitable spacer (such as m-maleimidobenzoyl-N-hydroxysuccinimide ester) or a sulfosuccinimidyl propionate. Such linkers are commercially available.

[0268] In some embodiments of the conjugates disclosed herein, the chemical moiety is selected from a therapeutic agent, a detectable moiety, and an immunostimulatory molecule.

[0269] In some embodiments, the therapeutic agent includes, but is not limited to, an immunomodulatory agent, a radioactive compound, an enzyme (such as a perforin), a chemotherapeutic agent (such as cisplatin), or a toxin. In some embodiments, the therapeutic agent can be, for example, a maytansinoid, a geldanamycin, a tubulin inhibitor such as a tubulin binding agent (e.g., an auristatin) or a minor groove binder such as a calicheamicin. In some embodiments, the therapeutic agent can be, for example, MMAE, Dxd, or a derivative thereof.

[0270] In some embodiments, the detectable moiety can be selected from biotin, streptavidin, an enzyme or catalytically active fragment thereof, a radionuclide, a nanoparticle, a paramagnetic metal ion, or a fluorescent, phosphorescent, or chemiluminescent molecule. Detectable moieties for diagnostic purposes include, for example, fluorescent labels, radiolabels, enzymes, nucleic acid probes, and contrast agents.

[0271] Antibodies can be conjugated to detectable labels; for example, detectable labels that can be detected by ELISA, spectrophotometry, flow cytometry, microscopy, or diagnostic imaging techniques such as computed tomography (CT), computed axial tomography (CAT) scan, magnetic resonance imaging (MRI), nuclear magnetic resonance imaging NMRI), magnetic resonance tomography (MTR), ultrasound, fiberoptic examination, and laparoscopy. Specific, non-limiting examples of detectable labels include fluorophores, chemiluminescent agents, enzymatic linkages, radioisotopes, and heavy metals or compounds (e.g., superparamagnetic iron oxide nanocrystals for detection by MRI). For example, useful detectable labels include fluorescent compounds, including fluorescein, fluorescein isothiocyanate, rhodamine, 5-dimethylamine-l-napthalenesulfonyl chloride, phycoerythrin, lanthanide phosphors, and the like. Bioluminescent labels such as luciferase, green fluorescent protein (GFP), and yellow fluorescent protein (YFP) can also be used.

[0272] Antibodies or antigen-binding fragments can also be conjugated to enzymes that are useful for detection, such as horseradish peroxidase, beta-galactosidase, luciferase, alkaline phosphatase, glucose oxidase, and the like. When antibodies or antigen-binding fragments are conjugated to detectable enzymes, detection can be achieved by the addition of additional reagents that the enzyme uses to produce a recognizable reaction product. For example, when horseradish peroxidase reagents are present, the addition of hydrogen peroxide and diaminobenzidine results in a colored reaction product that can be detected visually. Antibodies or antigen-binding fragments can also be conjugated to biotin and detected by indirect measurement of avidin or streptavidin binding. It should be noted that avidin itself can be conjugated to an enzyme or fluorescent label.

[0273] Antibodies can be fused to self-labeling protein tags, such as HaloTag. For example, the protein tag can be cloned to the end of the constant region. HaloTag is a self-labeling protein tag derived from a bacterial enzyme (haloalkane dehalogenase) designed to covalently bind to synthetic ligands. In some cases, the synthetic ligand comprises a chloroalkane linker attached to a fluorophore, such as a near-infrared fluorophore (Los et al. (2008) ACS Chem Biol. 3(6):373-82).

[0274] Antibodies can be labeled with magnetic agents such as gadolinium. Antibodies can also be labeled with lanthanides (such as europium and dysprosium) and manganese.

[0275] Paramagnetic particles such as superparamagnetic iron oxide can also be used as labels. Antibodies can also be labeled with predetermined polypeptide epitopes recognized by a second reporter group (such as a leucine zipper pair sequence, a binding site for biotin, a metal binding domain, an epitope tag) that are recognized by a second reporter group. In some embodiments, the tag is attached by a spacer arm of various lengths to reduce potential steric hindrance.

[0276] Antibodies can also be labeled with radiolabeled amino acids. Radiolabels can be used for diagnostic and therapeutic purposes. For example, radiolabels can be used to detect expression of a target antigen by x-ray, emission spectroscopy, or other diagnostic techniques. Examples of polypeptide labels include, but are not limited to, the following radioisotopes or radionucleotides: 3 H、 14 C、 15 N、 35 S、 90 Y、 99 Tc、 111 In、 125 I、 131 I.

[0277] In some embodiments, the immunostimulatory molecule is an immune effector molecule that stimulates an immune response. For example, the immunostimulatory molecule can be a cytokine such as IL-2 and IFN-gamma, a chemokine such as IL-8, platelet factor 4, melanoma growth stimulatory protein, a complement activator; a viral / bacterial protein domain, or a viral / bacterial peptide.

[0278] Chimeric antigen receptor

[0279] The present disclosure provides chimeric antigen receptors (CARs) comprising the antibodies or antigen-binding fragments thereof disclosed herein. The present disclosure also provides genetically modified cells comprising the chimeric antigen receptors disclosed herein.

[0280] The term "chimeric antigen receptor" or "CAR" as used herein refers to a molecule engineered to contain an antigen-binding domain that targets a specific antigen and, when bound to that antigen, activates an immune cell (e.g., a T cell or NK cell, such as a naive T cell, central memory T cell, effector memory T cell, or a combination thereof) to attack and destroy cells bearing that antigen. When these antigens are present on tumor cells, the CAR-expressing immune cells can target and kill the tumor cells.

[0281] A classic chimeric antigen receptor (CAR) is a chimeric type I transmembrane protein that links an extracellular antigen-binding domain to an intracellular signaling domain. The antigen-binding domain is typically an antigen-binding fragment derived from a monoclonal antibody (mAb) (e.g., scFv), but it can be based on other formats containing antibody-like antigen-binding sites or antigen-binding domains derived from natural ligands of the antigen. A hinge domain is often required to separate the antigen-binding domain from the membrane and allow for its proper orientation. A common hinge domain used is the Fc of IgGl. Depending on the antigen, more compact spacers can suffice, such as the stalk from CD8a, and even just the IgGl hinge alone. The transmembrane domain anchors the protein in the cell membrane and links the hinge domain to the intracellular domain (endodomain).

[0282] According to at least one non-limiting view, there have been at least three “generations” of CAR molecules. In the first generation CARs, they were designed to have an intracellular domain derived from the intracellular portion of the gamma chain of Fc epsilon Rl or CD3 zeta. Thus, these first generation CARs transmit immune signal 1, which is sufficient to trigger T cell killing of cognate target cells, but is not able to fully activate T cell proliferation and survival. To overcome this limitation, second generation CARs have been constructed, which have a composite intracellular domain generated by fusing the intracellular portion of a T cell costimulatory molecule with the intracellular portion of CD3 zeta, so that both an activation signal and a costimulatory signal can be transmitted upon antigen recognition. The most commonly used costimulatory domain is that of CD28. This provides the most potent costimulatory signal, immune signal 2, which triggers T cell proliferation. Some CARs have also been described that include TNF receptor family intracellular domains, such as the closely related OX40 and 41BB, which transmit survival signals. Even more potent third generation CARs have now been described, which have intracellular domains capable of transmitting activation, proliferation, and survival signals.

[0283] Thus, a CAR typically comprises: (i) an antigen binding domain; (ii) a hinge domain; (iii) a transmembrane domain; and (iv) an intracellular domain comprising a signaling domain and one or more costimulatory domains.

[0284] “Antigen binding domain” refers to the portion of a chimeric antigen receptor that recognizes an antigen. In classic CARs, the antigen binding domain comprises a single chain variable fragment (scFv) derived from a monoclonal antibody. CARs have also been created with domain antibodies (dAbs), VHH antigen binding domains, or antigen binding domains derived from natural ligands of the antigen. In the present application, the antigen binding domain can be an antibody or antigen binding fragment thereof as described herein.

[0285] A “hinge domain” positions the antigen binding domain away from the surface of the effector cell to enable proper cell / cell contact, antigen binding, and activation to function. CARs optionally include one or more hinge domains between the binding domain and the transmembrane domain (TM). The hinge domain can be derived from natural, synthetic, semi-synthetic, or recombinant sources. The hinge domain can comprise an amino acid sequence of a naturally occurring immunoglobulin hinge region or an altered immunoglobulin hinge region. Illustrative hinge domains suitable for use in the CARs described herein include hinge regions derived from the extracellular region of type 1 membrane proteins such as CD8 (e.g., CD8a), CD4, CD28, 4-1BB, and CD7, which can be from the wild-type hinge region of these molecules or can be altered.

[0286] As used herein, a "transmembrane domain" (TM domain) refers to a portion of a CAR that, optionally via a hinge domain, fuses an extracellular binding moiety with intracellular moieties (e.g., a costimulatory domain and an intracellular signaling domain) and anchors the CAR to the plasma membrane of an immune effector cell. A transmembrane domain is typically a hydrophobic region of a CAR that spans the plasma membrane. The TM domain can be a transmembrane region of a transmembrane protein (e.g., a type I transmembrane protein or other transmembrane protein) or a fragment thereof, an artificial hydrophobic sequence, or a combination thereof.

[0287] An intracellular domain (endodomain) is the signaling portion of a chimeric antigen receptor. It contains a signaling domain and one or more costimulatory domains. Upon antigen recognition, the receptor clusters naturally CD45 and CD148 away from the synapse and signals to the cell, thereby activating one or more immune cell effector functions (e.g., a native immune cell effector function). The most commonly used endodomain component is the endodomain component of CD3 zeta, which contains 3 ITAMs. Upon antigen binding, it transmits an activation signal to the T cell. CD3 zeta can not provide a fully sufficient activation signal and can require additional costimulatory signaling. Costimulatory signals promote T cell proliferation and survival. There are two main types of costimulatory signals: those belonging to the Ig family (CD28, ICOS) and the TNF family (OX40, 41BB, CD27, GITR, etc.).

[0288] An "intracellular signaling domain" refers to a portion of a CAR polypeptide that participates in transducing the information that the CAR is bound to a target antigen, inside the immune effector cell, to elicit effector cell functions such as activation, cytokine production, proliferation, and cytotoxic activity, including release of cytotoxic factors to CAR-bound target cells, or other cellular responses elicited upon antigen binding to the extracellular CAR domain. Non-limiting examples of intracellular signaling domains containing immunoreceptor tyrosine-based activation motifs (ITAMs) include those derived from TCR zeta, FcR gamma, FcR beta, CD3 gamma, CD3 theta, CD3 delta, CD3 eta, CD3 epsilon, CD3 zeta, CD22, CD79a, CD79b, and CD66d.

[0289] A "costimulatory domain" refers to an intracellular signaling domain of a costimulatory molecule. Costimulatory molecules are cell surface molecules other than antigen receptors or Fc receptors that provide a second signal required for efficient activation and function of T lymphocytes upon binding antigen. The costimulatory domain can be a costimulatory domain of, e.g., 4-1BB, CD27, CD28, or OX40.

[0290] Therapeutic methods and uses

[0291] The present disclosure provides methods for treating a disease in a subject comprising administering to the subject an effective amount of an antibody or antigen-binding fragment thereof disclosed herein, a pharmaceutical composition disclosed herein, a conjugate disclosed herein, or a chimeric antigen receptor disclosed herein.

[0292] The present disclosure also provides the use of an antibody or antigen-binding fragment thereof disclosed herein, a pharmaceutical composition disclosed herein, a conjugate disclosed herein, or a chimeric antigen receptor disclosed herein in the manufacture of a medicament for treating a disease in a subject.

[0293] The present disclosure also provides an antibody or antigen-binding fragment thereof disclosed herein, a pharmaceutical composition disclosed herein, a conjugate disclosed herein, or a chimeric antigen receptor disclosed herein for use in treating a disease in a subject.

[0294] In some embodiments, the disease is a cancer, for example a cancer associated with CDH17 expression or a CDH17-positive cancer. In preferred embodiments, the cancer is selected from 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;

[0295] More preferably, the disease is selected from a gastric cancer, a liver cancer (e.g. hepatocellular carcinoma), a colorectal cancer, a pancreatic cancer, an esophageal cancer, a neuroendocrine tumor, and a breast cancer.

[0296] 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 administration 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 antibody and the route of administration.

[0297] A dose of an antibody, composition, conjugate, or chimeric antigen receptor described herein can be administered to a mammal in a suitable period of time either as a single dose or in a series of sub-doses, e.g., as often as necessary, daily, semi-weekly, weekly, bi-weekly, semi-monthly, bi-monthly, semi-annually, or annually. Dose units comprising an effective amount of an antibody, composition, conjugate, or chimeric antigen receptor 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 necessary.

[0298] Suitable modes of administration can be selected by a medical practitioner. 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 by intravenous injection, intramuscular injection, intraperitoneal injection, subcutaneous injection. In some embodiments, the antibody, composition, conjugate or chimeric antigen receptor 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.

[0299] In some embodiments, the methods further comprise administering a second therapeutic agent to the subject. In some embodiments, the antibodies or antigen-binding fragments thereof, pharmaceutical compositions, conjugates or chimeric antigen receptors disclosed herein are 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 antibodies, compositions, conjugates or chimeric antigen receptors disclosed herein are administered prior to, substantially simultaneously with or following administration of the second therapeutic agent.

[0300] Kits / administration devices

[0301] The present disclosure provides kits or administration devices comprising the antibodies or antigen-binding fragments thereof disclosed herein, the nucleic acid molecules disclosed herein, the vectors disclosed herein, the host cells disclosed herein, the conjugates disclosed herein, the pharmaceutical compositions disclosed herein, or the chimeric antigen receptors disclosed herein.

[0302] In some embodiments, the kits or administration devices comprise one or more containers filled with one or more of the ingredients of the pharmaceutical compositions as set out herein, e.g. the antibodies or antigen-binding fragments disclosed herein.

[0303] In specific embodiments, the kits comprise a first container that contains an antibody disclosed herein. In specific embodiments, the kits comprise a first container that is a vial containing the antibody as a lyophilized sterile powder under vacuum and the kit further comprises a second container that contains a pharmaceutically acceptable fluid.

[0304] In specific embodiments, injection devices containing an antibody are provided herein. In specific embodiments, the injection devices comprise an antibody in a sterile solution. In specific embodiments, the injection devices are syringes.

[0305] In one embodiment, the kit includes instructional materials disclosing ways of using the antibodies of the disclosure. The instructional materials can be in written, electronic (such as computer- readable medium or optical disc), or visual (such as video files) form. The kit can also include additional components to facilitate the application for which the kit is designed. Thus, for example, the kit can additionally contain tools for detection of labels (e.g., enzyme substrates for enzymatic labels, filter sets for detection of fluorescent labels, appropriate secondary labels such as secondary antibodies, etc.). The kit can also include buffers and other reagents commonly used in the practice of a particular method. Such kits and appropriate contents are well known to those of skill in the art.

[0306] Examples

[0307] For the purpose of illustrating the present embodiments, the examples that follow are presented in the following order: Preparation of antigen and stable cell line; Preparation of antibodies; Detection of CDH17 in tumor tissues; Detection of CDH17 in serum; and Therapeutic effect of antibodies. These examples are intended to be illustrative only and are not intended to limit the scope of the application in any way. Those of skill in the art will readily recognize a variety of noncritical parameters that can be changed or modified to yield essentially the same results.

[0308] Example 1, Preparation of antigen and stable cell line

[0309] 1.1 Preparation of CDH17 recombinant protein

[0310] The nucleic acid sequence encoding the antigen protein with his, Fc tag 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 an appropriate number, and a complex of PEI and plasmid was added dropwise to 293 cells subcultured at a cell density of 1.5 x 10 6 The 293 cells were placed in a cell incubator shaker at 37°C, 120 rpm, 5% CO2, and cultured. The day of transfection was day 0. On days 1 and 3, 5% OPM-293Profeed feed was added to the volume of cells on the day of transfection. 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, in which human CDH17 (hCDH17) and cynomolgus monkey CDH17 (cyCDH17) recombinant proteins were obtained. The amino acid sequences of human and cynomolgus monkey CDH17 proteins are as follows:

[0311] Human CDH17 (SEQ ID NO. 1)

[0312] Cynomolgus CDH17 (SEQ ID NO. 2)

[0313] 1.2 Construction of HEK293-hCDH17 and HEK293-cyCDH17 stable cell lines

[0314] The full length of human or cynomolgus CDH17 gene was cloned into the expression vector containing puromycin resistance gene by molecular cloning technology to obtain plenti-hCDH17-IRES-p or plenti-cyCDH17-IRES-p plasmid. The expression vector plasmid containing human or cynomolgus CDH17 gene was transfected into HEK293 cells. After the target gene entered the host cells, it was integrated into the genome through reverse transcription, thereby expressing human or cynomolgus CDH17 gene. The cell population containing HEK293-hCDH17 or HEK293-cyCDH17 positive cell line (HEK293-hCDH17 mix pool or HEK293-cyCDH17 mix pool) was obtained by adding 2 μg / mL puromycin to the screening medium for continuous screening for 30 days. Next, the HEK293-hCDH17 mix pool or HEK293-cyCDH17 mix pool was subcloned, plated at 1 cell / well into a 96-well cell culture plate, and cultured in a 37°C, 5% CO2 incubator for 2 weeks. The monoclonal cells were expanded to a 24-well plate, and after the cells grew, the monoclonal cells were screened and verified using anti-human or cynomolgus CDH17 antibody. As shown in Figure 2, the expression of human or cynomolgus CDH17 of HEK293 was significantly improved, and the stable cell construction was successful.

[0315] 1.3 Construction of CHO-hCDH17 stable cell line

[0316] The full length of hCDH17 gene was cloned into the expression vector containing puromycin resistance gene by molecular cloning technology to obtain Plasmid. The plasmid was transfected into CHO cells by electroporation method. After the target gene entered the host cell, it was integrated into the genome after reverse transcription, thereby expressing the hCDH17 gene. The cell strain was screened by adding puromycin, and the CHO stable cell strain expressing the hCDH17 gene was obtained. The cell population containing the CHO-hCDH17 positive cell strain (CHO-hCDH17 mix pool) was obtained by continuously screening the medium containing 8 μg / mL puromycin for 2 weeks. Next, the CHO-hCDH17 mix pool was subcloned and plated at 1 cell / well in a 96-well cell culture plate. After 10-15 days of culture at 37°C in a 5% CO2 incubator, the monoclonal cells were expanded to a 24-well plate, and the monoclonal cells were screened and verified using anti-hCDH17 antibodies. As shown in FIG. 3, the monoclonal cell strain of hCDH17 was successfully constructed.

[0317] Example 2. Generation of anti-CDH17 chimeric antibodies

[0318] 2.1 Mouse immunization and antibody screening

[0319] Mouse immunization

[0320] Four to six-week-old female Balb / C mice (Beijing VitoLihua) were selected and immunized using the following four groups of immunization strategies.

[0321] Group 1: Combined immunization using a gene gun and antigen. 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, 3 μg of plasmid per mouse per gene gun immunization, once a week, a total of 4 times. One week later, immunization was performed using hCDH17 protein, 15 μg per mouse, a total of 1 time.

[0322] Group 2: Cell immunization. The first immunization was performed using CHO-hCDH17 cells for subcutaneous and footpad immunization, with Sigma adjuvant system as the adjuvant, 1e 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, once every two weeks, 1e 6 10 cells per mouse, a total of 3 times.

[0323] The third group: adopt antigen immunization. The first immunization of each mouse is subcutaneous and footpad immunization with hCDH17 protein, adjuvant is TiterMax, each mouse immunized with 50 μg, a total of one time. Two weeks later, footpad immunization with hCDH17 protein, adjuvant is Sigma adjuvant system, immunized once every two weeks, each mouse immunized with 15 μg, a total of 3 times.

[0324] The fourth group: adopt antigen immunization. The first immunization of each mouse is subcutaneous and footpad immunization with hCDH17 protein, adjuvant is CFA, each mouse immunized with 50 μg, a total of one time. Two weeks later, footpad immunization with hCDH17 protein, adjuvant is Sigma adjuvant system, immunized once every two weeks, each mouse immunized with 15 μg, a total of 3 times.

[0325] The antibody titers of each group were detected by ELISA and FACS, ELISA detected the binding of antibodies at the antigen level, and FACS detected the binding of antibodies at the cell level, and the antibody titers met the requirements for fusion and screening.

[0326] Antibody screening

[0327] The first group performed two fusions. A total of 845 positive clones were screened, and 739 positive clones were retested. A total of 53 384-well plates were screened in the first fusion, and 1210 clones that bound to hCDH17 were screened by ELISA. Then, 257 clones that bound to both HEK293-hCDH17 and HEK293-cyCDH17 were screened by FACS. Finally, 217 positive clones were obtained by cell level binding, and the supernatant of 217 clones of hybridoma was used to detect endocytosis and killing. A total of 63 384-well plates were screened in the second fusion, and 1749 clones that bound to hCDH17 were screened by ELISA. Then, 588 clones that bound to both HEK293-hCDH17 and HEK293-cyCDH17 were screened by FACS. Finally, 522 positive clones were obtained by cell level binding.

[0328] The second group screened a total of 7 96-well plates selected by clonepix, and 10 clones that bound to hCDH17 were screened by ELISA. Then, 7 clones that bound to both HEK293-hCDH17 and HEK293-cyCDH17 were screened by FACS. Finally, 7 positive clones were obtained by cell level binding.

[0329] The third group screened 62 384-well plates, and a total of 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. Finally, 33 positive clones were obtained by cell level combination.

[0330] The fourth group screened 29 384-well plates, and a total of 394 clones that combined with hCDH17 were screened by ELISA, and 59 clones that combined with HEK293-hCM518 and HEK293-cyCM518 were screened by FACS. The hybridoma supernatant of the 59 clones was detected for endocytosis and killing.

[0331] Based on the cell combination, endocytosis and killing activity of the clones in the four groups, finally 7 clones with better endocytosis and killing activity were selected. The ELISA combination activity and FACS combination activity of the 7 clones are shown in Table 1 and Table 2, respectively.

[0332] Table 1. ELISA combination activity of supernatant culture of different hybridoma clones to human or cynomolgus CDH17 protein

[0333] Table 2. Combination activity of supernatant culture of different hybridoma clones to HEK293 surface human or cynomolgus CDH17 protein

[0334] 2.2 Determination of antibody sequence

[0335] According to the results of hybridoma screening, the positive monoclonal cells were centrifuged at 1000 rpm, the cells were collected, and the total RNA was extracted by Trizol. The first strand cDNA was synthesized, and the variable region DNA sequence of the hybridoma cells was amplified using the first strand cDNA as the subsequent template. In a 50 μL reaction system, cDNA 1 μL, 10×PCR buffer 5 μL, forward and reverse primers each 1 μL, dNTP 1 μL, 25 mmol MgCl2 1 μL, H2O 39 μL, Taq enzyme 1 μL, 95°C pre-denaturation for 10 minutes, temperature cycling, and PCR amplification. The reaction conditions are as follows: 94°C denaturation for 1 minute, 58°C annealing for 1 minute, 72°C extension for 15 seconds, a total of 30 cycles, and then 72°C incubation for 10 minutes. According to the results of phage screening, the variable region sequence of the positive clone was amplified. After sequencing, the heavy chain and light chain variable region sequences of the candidate positive clone were obtained.

[0336] Table 3. Heavy chain variable region (VH) and light chain variable region (VL) sequences of anti-CDH17 murine antibodies

[0337] The heavy chain / light chain variable region sequences of the anti-CDH17 murine antibodies are as follows (wherein the CDR sequences are defined according to the Kabat definition rules):

[0338] mAb1

[0339] The amino acid sequence of the heavy chain VH of mAb1 is shown as SEQ ID NO. 3, the encoding nucleic acid is shown as SEQ ID NO. 4, and the CDR1, CDR2 and CDR3 thereof are shown as SEQ ID NO. 5, 6, 7, respectively.

[0340] Nucleotide sequence

[0341] The amino acid sequence of the light chain VL of mAb1 is shown as SEQ ID NO. 8, the encoding nucleic acid is shown as SEQ ID NO. 9, and the CDR1, CDR2 and CDR3 thereof are shown as SEQ ID NO. 10, 11, 12, respectively.

[0342] Nucleotide sequence

[0343] mAb2

[0344] The amino acid sequence of the heavy chain VH of mAb2 is shown as SEQ ID NO. 13, the encoding nucleic acid is shown as SEQ ID NO. 14, and the CDR1, CDR2 and CDR3 thereof are shown as SEQ ID NO. 15, 16, 17, respectively.

[0345] Nucleotide sequence

[0346] The amino acid sequence of the light chain VL of mAb2 is shown as SEQ ID NO. 18, the encoding nucleic acid is shown as SEQ ID NO. 19, and the CDR1, CDR2 and CDR3 thereof are shown as SEQ ID NO. 20, 21, 22, respectively.

[0347] Nucleotide sequence

[0348] mAb3

[0349] The amino acid sequence of the heavy chain VH of mAb3 is shown as SEQ ID NO. 23, the encoding nucleic acid is shown as SEQ ID NO. 24, and the CDR1, CDR2 and CDR3 thereof are shown as SEQ ID NO. 25, 26, 27, respectively.

[0350] Nucleotide sequence

[0351] The amino acid sequence of the VL of the light chain of mAb 3 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.

[0352] Nucleotide sequence

[0353] mAb 4

[0354] The amino acid sequence of the VH of the heavy chain of mAb 4 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.

[0355] Nucleotide sequence

[0356] The amino acid sequence of the VL of the light chain of mAb 4 is shown in SEQ ID NO. 38, the encoding nucleic acid is shown in SEQ ID NO. 39, and the CDR1, CDR2 and CDR3 are shown in SEQ ID NO. 40, 41, 42 respectively.

[0357] Nucleotide sequence

[0358] mAb 5

[0359] The amino acid sequence of the VH of the heavy chain of mAb 5 is shown in SEQ ID NO. 43, the encoding nucleic acid is shown in SEQ ID NO. 44, and the CDR1, CDR2 and CDR3 are shown in SEQ ID NO. 45, 46, 47 respectively.

[0360] Nucleotide sequence

[0361] The amino acid sequence of the VL of the light chain of mAb 5 is shown in SEQ ID NO. 48, the encoding nucleic acid is shown in SEQ ID NO. 49, and the CDR1, CDR2 and CDR3 are shown in SEQ ID NO. 50, 51, 52 respectively.

[0362] Nucleotide sequence

[0363] mAb 6

[0364] The amino acid sequence of the heavy chain VH of mAb6 is shown in SEQ ID NO. 53, the encoding nucleic acid is shown in SEQ ID NO. 54, and the CDR1, CDR2 and CDR3 are shown in SEQ ID NO. 55, 56, 57, respectively.

[0365] Nucleotide sequence

[0366] The amino acid sequence of the light chain VL of mAb6 is shown in SEQ ID NO. 58, the encoding nucleic acid is shown in SEQ ID NO. 59, and the CDR1, CDR2 and CDR3 are shown in SEQ ID NO. 60, 61, 62, respectively.

[0367] Nucleotide sequence

[0368] mAb7

[0369] The amino acid sequence of the heavy chain VH of mAb7 is shown in SEQ ID NO. 63, the encoding nucleic acid is shown in SEQ ID NO. 64, and the CDR1, CDR2 and CDR3 are shown in SEQ ID NO. 65, 66, 67, respectively.

[0370] Nucleotide sequence

[0371] The amino acid sequence of the light chain VL of mAb7 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.

[0372] Nucleotide sequence

[0373] Example 3, Characterization of Anti-CDH17 Chimeric Antibodies

[0374] 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 IgG1 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, IgG was expressed and secreted into the culture medium, the supernatant was collected and purified after filtration. 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.

[0375] 3.1 Affinity detection of anti-CDH17 chimeric antibody and hCDH17

[0376] Using the biofilm interference technology (BLI / gator prime), the candidate chimeric antibody was fixed with Protein A probe, and then the probe was transferred to the gradient diluted hCDH17 avi cHis antigen, and the affinity of the antibody and the antigen was obtained by detecting the signal change on the probe and by kinetic fitting, as shown in Figure 4 and Table 4. The results showed that each chimeric antibody could bind to hCDH17.

[0377] Table 4 Affinity of anti-CDH17 chimeric antibody

[0378] 3.2 Binding of anti-CDH17 chimeric antibody to tumor cells

[0379] SNU-16 was used as the target cell, and the cell density was adjusted to 1×10 7 cells per 1 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 a 5-fold gradient dilution was prepared. The cell density was diluted to 5×10 5 cells per 1 ml with flow buffer, and 100 μl was inoculated into a U-shaped 96-well plate; centrifugation was performed 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.

[0380] After incubation, the cells were washed 3 times with 200 μl of flow buffer, 50 μl of AF647-Goat Anti-human IgG, Fc (1:300 dilution) was added to each well to label the cell surface of the test sample, 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 average fluorescence intensity of AF647 of SNU-16 single living cells was detected by flow cytometry. The determination results were recorded, with the antibody concentration as the abscissa and the average fluorescence intensity as the ordinate. Four-parameter regression calculation was performed to obtain the EC50 As shown in Figure 5 and Table 5. The irrelevant antibody anti-KLH antibody was used as a control. The results showed that all antibody clones were able to bind SNU-16 cells with high activity, with EC50 in the nM level or below 1 nM.

[0381] Table 5 Binding activity of anti-CDH17 chimeric antibodies to tumor cells

[0382] 3.3 Endocytosis activity of anti-CDH17 chimeric antibodies

[0383] SNU-16 cells were used as target cells, and the cell density was adjusted to 1 ml containing 1 x 10 5 ~ 5 x 10 5 cells per 100 μl in a 96-well flat-bottom plate.

[0384] The purified chimeric antibodies and DT3C were diluted with RPMI-1640 base 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 4-fold. 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 molar ratio of 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 horizontal coordinate and the relative viability as the vertical coordinate. Four-parameter regression analysis was performed to obtain the IC 50 As shown in Figure 6 and Table 6. The irrelevant antibody anti-KLH antibody was used as a control. The results showed that all antibody clones had high endocytosis activity, with IC 50 all below 1 nM.

[0385] Table 6 Endocytosis activity of anti-CDH17 chimeric antibodies

[0386] Example 4, Humanization of anti-CDH17 chimeric antibodies

[0387] With mAb3 and mAb5 as candidate molecules, the mouse antibody mAb3 sequence was aligned with human antibody germline sequence, and the key amino acid sequences with good homology, maintaining the antibody structure core (Upper hydrophobic core), which were completely identical and frequently appeared in human body, were found. The human germline light chain gene IMGT_Hvk1-39 or IMGT_Hvk3-11, IGKJ2*01 and human germline heavy chain gene IMGT_hVH1-46 or IMGT_hVH3-23, IGHJ4*01 were used for mouse antibody CDR grafting. Homologous modeling was performed by computer, and the CDR region and its surrounding framework amino acid sequence were analyzed to avoid the concentration distribution 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.

[0388] The mouse antibody mAb5 sequence was aligned with human antibody germline sequence, and the human germline light chain gene IMGT_hVK1-39, IGKJ2*01 and human germline heavy chain gene IMGT_Hvh1-46, IGHJ4*01 were determined for mouse antibody CDR grafting. A total of 6 heavy chain variants mAb5-H1~H6 and 5 light chain variants mAb5-K1~K5 were designed.

[0389] After the light chain variable region and the heavy chain variable region were synthesized, 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, and expressed at 37°C for 2 days. The culture supernatant was collected, and the affinity and biological activity were detected.

[0390] 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.

[0391] Table 7, the sequence of the heavy chain variable region of the anti-CDH17 humanized antibody

[0392] Table 8, the sequence of the light chain variable region of the anti-CDH17 humanized antibody

[0393] 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.

[0394] The amino acid sequence of mAb5 H1 is shown in SEQ ID NO. 73, the encoding nucleic acid is shown in SEQ ID NO. 74, and the CDR1, CDR2 and CDR3 are shown in SEQ ID NO. 45, 46 and 47, respectively.

[0395] Nucleotide sequence

[0396] 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, 47 respectively.

[0397] Nucleotide sequence

[0398] 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, 47 respectively.

[0399] Nucleotide sequence

[0400] 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.

[0401] Nucleotide sequence

[0402] 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.

[0403] Nucleotide sequence

[0404] 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.

[0405] Nucleotide sequence

[0406] 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.

[0407] Nucleotide sequence

[0408] 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.

[0409] Nucleotide sequence

[0410] The amino acid sequence of mAb5 K3 is shown as SEQ ID NO. 91, the encoding nucleic acid is shown as SEQ ID NO. 92, and the CDR1, CDR2 and CDR3 are shown as SEQ ID NO. 90, 93 (WASTRHS), 52, respectively.

[0411] Nucleotide sequence

[0412] The amino acid sequence of mAb5 K4 is shown as SEQ ID NO. 94, the encoding nucleic acid is shown as SEQ ID NO. 95, and the CDR1, CDR2 and CDR3 are shown as SEQ ID NO. 90, 96 (YASTRHS), 52, respectively.

[0413] Nucleotide sequence

[0414] The amino acid sequence of mAb5 K5 is shown as SEQ ID NO. 97, the encoding nucleic acid is shown as SEQ ID NO. 98, and the CDR1, CDR2 and CDR3 are shown as SEQ ID NO. 90, 96, 52, respectively.

[0415] Nucleotide sequence

[0416] The amino acid sequence of mAb3 H1 is shown as SEQ ID NO. 99, the encoding nucleic acid is shown as SEQ ID NO. 100, and the CDR1, CDR2 and CDR3 are shown as SEQ ID NO. 25, 26, 27, respectively.

[0417] Nucleotide sequence

[0418] The amino acid sequence of mAb3 H2 is shown as SEQ ID NO. 101, the encoding nucleic acid is shown as SEQ ID NO. 102, and the CDR1, CDR2 and CDR3 are shown as SEQ ID NO. 25, SEQ ID NO. 125 (AIYPGDGVTRYSQKFKD), SEQ ID NO. 27, respectively.

[0419] Nucleotide sequence

[0420] 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.

[0421] Nucleotide sequence

[0422] 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), SEQ ID NO. 125, SEQ ID NO. 27, respectively.

[0423] Nucleotide sequence

[0424] 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.

[0425] Nucleotide sequence

[0426] 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.

[0427] 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.

[0428] Nucleotide sequence

[0429] 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.

[0430] Nucleotide sequence

[0431] 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.

[0432] Nucleotide sequence

[0433] 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.

[0434] Nucleotide sequence

[0435] 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.

[0436] Nucleotide sequence

[0437] 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.

[0438] Nucleotide sequence

[0439] 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.

[0440] Heavy chain amino acid sequence (SEQ ID NO. 126)

[0441] Heavy chain nucleotide sequence (SEQ ID NO. 127)

[0442] Light chain amino acid sequence (SEQ ID NO. 128)

[0443] Light chain nucleotide sequence (SEQ ID NO. 129)

[0444] The heavy chain amino acid sequence of anti-CDH17 humanized antibody MAB5 H5K2 is shown as SEQ ID NO. 130, the encoding nucleic acid is shown as SEQ ID NO. 131, and the light chain amino acid sequence is shown as SEQ ID NO. 132, the encoding nucleic acid is shown as SEQ ID NO. 133.

[0445] Heavy chain amino acid sequence (SEQ ID NO. 130)

[0446] Heavy chain nucleotide sequence (SEQ ID NO. 131)

[0447] Light chain amino acid sequence (SEQ ID NO. 132)

[0448] Heavy chain nucleotide sequence (SEQ ID NO. 135)

[0449] The heavy chain amino acid sequence of anti-CDH17 humanized antibody MAB3 H2K2 is set forth in SEQ ID NO. 134, the encoding nucleic acid of which is set forth in SEQ ID NO. 135; the light chain amino acid sequence is set forth in SEQ ID NO. 136, the encoding nucleic acid of which is set forth in SEQ ID NO. 137.

[0450] Heavy chain amino acid sequence (SEQ ID NO. 134)

[0451] Heavy chain nucleotide sequence (SEQ ID NO. 135)

[0452] Light chain amino acid sequence (SEQ ID NO. 136)

[0453] Light chain nucleotide sequence (SEQ ID NO. 137)

[0454] The heavy chain amino acid sequence of anti-CDH17 humanized antibody MAB3 H2K2 is set forth in SEQ ID NO. 134, the encoding nucleic acid of which is set forth in SEQ ID NO. 135; the light chain amino acid sequence is set forth in SEQ ID NO. 136, the encoding nucleic acid of which is set forth in SEQ ID NO. 137.

[0455] Heavy chain amino acid sequence (SEQ ID NO. 134)

[0456] Heavy chain nucleotide sequence (SEQ ID NO. 135)

[0457] Light chain amino acid sequence (SEQ ID NO. 138)

[0458] Light chain nucleotide sequence (SEQ ID NO. 139)

[0459] The heavy chain amino acid sequence of anti-CDH17 humanized antibody MAB3 H2K2 is set forth in SEQ ID NO. 134, the encoding nucleic acid of which is set forth in SEQ ID NO. 135; the light chain amino acid sequence is set forth in SEQ ID NO. 136, the encoding nucleic acid of which is set forth in SEQ ID NO. 137.

[0460] Heavy chain amino acid sequence (SEQ ID NO. 140)

[0461] Heavy chain nucleotide sequence (SEQ ID NO. 141)

[0462] Light chain amino acid sequence (SEQ ID NO. 136)

[0463] Light chain nucleotide sequence (SEQ ID NO. 137)

[0464] The heavy chain amino acid sequence of the anti-CDH17 humanized antibody MAB3 H3K4 is shown as SEQ ID NO. 140, and the encoding nucleic acid is shown as SEQ ID NO. 141; the light chain amino acid sequence is shown as SEQ ID NO. 138, and the encoding nucleic acid is shown as SEQ ID NO. 139.

[0465] Heavy chain amino acid sequence (SEQ ID NO. 140)

[0466] Heavy chain nucleotide sequence (SEQ ID NO. 141)

[0467] Light chain amino acid sequence (SEQ ID NO. 138)

[0468] Light chain nucleotide sequence (SEQ ID NO. 139)

[0469] The antibody TORL-3-600 mAb targeting CDH17 is used as a control antibody, which is prepared with reference to the patent WO2023107558A1, the heavy chain amino acid sequence of which is shown as SEQ ID NO. 142, and the encoding nucleic acid 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 is shown as SEQ ID NO. 145.

[0470] Heavy chain amino acid sequence (SEQ ID NO. 142)

[0471] Heavy chain nucleotide sequence (SEQ ID NO. 143)

[0472] Light chain amino acid sequence (SEQ ID NO. 144)

[0473] Light chain nucleotide sequence (SEQ ID NO. 145)

[0474] Example 5, Affinity characterization of anti-CDH17 humanized antibodies

[0475] 5.1 Affinity of anti-CDH17 humanized antibodies to hCDH17

[0476] Using Bio-Layer Interferometry technology (BLI / gator prime), anti-CDH17 humanized antibody molecules in transient supernatant were immobilized with Protein A probe, and then the probe was transferred to gradient-diluted hCDH17 avi cHis antigen. 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.

[0477] Table 9 Affinity of anti-CDH17 humanized antibodies

[0478] 5.2 Binding of anti-CDH17 humanized antibodies to tumor cells

[0479] Using SNU-16 as target cells, the cell density was adjusted to 1x10 7 cells per 1 ml with blocking solution, and the sample was prepared during ice bath for 30 minutes. The antibody was diluted to 30 μg / ml with flow buffer, and a 5-fold gradient dilution was prepared. The cell density was diluted to 5x10 5 cells per 1 ml with flow buffer, and 100 μl was inoculated into a U-shaped 96-well plate. After centrifugation, 100 μl of diluted antibody was added to each well, and the cells were incubated on ice for 60 minutes.

[0480] 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. The mean fluorescence intensity of AF647 of SNU-16 single living cells was detected by flow cytometry. The results are shown in Figure 8A and Table 10, respectively. Using the irrelevant antibody anti-KLH antibody as a control, the binding activity of mAb5 H3K1 and mAb5 H5K2 did not change significantly compared with the parent; the binding activity of mAb3 H2K4 and other four humanized antibodies was slightly higher than that of the parent.

[0481] The binding activity of mAb 5 H3K1, control antibody TORL-3-600, Anti-KLH was detected by the same method as before, with SNU-16 and ASPC-1 as target cells, respectively. The results are shown in Figure 8B and Table 11. The binding plateau of mAb 5 H3K1 was higher than that of control antibody TORL-3-600, indicating that the cell binding activity of mAb 5 H3K1 was superior to TORL-3-600.

[0482] Table 10 Binding activity of anti-CDH17 humanized antibodies to tumor cells Note: " / " represents that no curve is fitted.

[0483] Table 11 Binding activity of anti-CDH17 humanized antibodies to different tumor cells Note: " / " represents that no curve is fitted.

[0484] 5.3 Endocytosis activity of anti-CDH17 humanized antibodies to tumor cells

[0485] SNU-16 was used as target cells, and the cell density was adjusted to 1 ml containing 1x10 5 cells / ml with 20% FBS (Low IgG) RPMI-1640 complete medium, and 100 μl was inoculated into a 96-well flat-bottom plate.

[0486] The purified hybridoma antibody and DT3C were diluted with RPMI-1640 basal 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 times gradient, and 100 μl 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%). The culture was continued for 72 h, and 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 four-parameter regression calculation method was used for analysis, and the IC 50 of the sample was obtained. The results are shown in Figure 9A and Table 12, respectively. The endocytosis activity of the humanized antibodies did not change significantly compared with the parent.

[0487] SNU-16 was used as target cells, and the endocytosis activity of mAb 5 H3K1, control antibody TORL-3-600, Anti-KLH was detected. SNU-16 cells were cultured in RPMI 1640 complete medium at 37°C, 5% CO2, centrifuged at 4°C, 300xg for 10 min to collect the cells, and the cell density was adjusted to 1x10 5 cells / ml with RPMI 1640 reaction buffer, and 100 μl was inoculated into a 96-well cell culture plate at 37°C, 5% CO2 overnight.

[0488] The prepared mAb5 H3K1, TORL-3-600, Anti-KLH and gradient diluted DT3C were mixed in equal volume and incubated, coupled for 30 minutes at 37°C, 100 μl per well was added to the cells, and incubated for 3 days at 37°C, 5% CO2. CCK-8 color developing liquid was added to each well, 20 μl, incubated for 2 hours at 37°C, 5% CO2, and then placed in a microplate reader to measure the light absorption value at a wavelength of 450 nm, and the experimental results were recorded. The target cell wells added with 100 μl of reaction buffer were used as control wells, 200 μl of reaction buffer was used as blank wells, and the same method was used for determination. The results are shown in Figure 9B and Table 13, and the endocytosis activity of mAb5-H3K1 is better than that of TORL-3-600.

[0489] Table 12 Endocytosis activity of anti-CDH17 humanized antibodies Note: " / " represents that the curve is not fitted.

[0490] Table 13 Endocytosis activity of anti-CDH17 humanized antibodies Note: " / " represents that the curve is not fitted.

[0491] Example 6, Preparation of ADC

[0492] The anti-CDH17 antibody of the application is coupled to the linker-drug conjugate mc-vc-PABC-MMAE (also known as VcMMAE) to prepare an ADC, and the specific preparation steps are as follows.

[0493] 6.1 Preparation of ADC-7 to ADC-12

[0494] Using mAb2 and VcMMAE (CAS: 646502-53-6, Lot, manufacturer) as raw materials, the PBS buffer of the exemplary product ADC-7 of the coupling mixture FADC-7A, FADC-7B, FADC-7C was prepared. The specific preparation steps are as follows.

[0495] The antibody is replaced into 50 mM PBS / 1.0 mM EDTA buffer solution (pH 6.5 adjusted by sodium hydroxide solution) using an ultrafiltration tube with a molecular weight cut-off of 50 kD, 2-4 equivalents of 10 mM TCEP aqueous solution are added, and the mixture is shaken at 25°C for 3 hours. The linker-drug conjugate is dissolved in DMSO, 8-20 equivalents of the linker-drug conjugate are taken therefrom, and the mixture is added dropwise to the reduced antibody solution, vortexed to mix uniformly, and shaken at 25°C for 2 hours. After the reaction is completed, 40 equivalents of 100 mM NAC aqueous solution are added, the mixture is shaken at 25°C for 20 minutes to terminate the linker reaction, excess small molecules are removed using an ultrafiltration tube with a molecular weight cut-off of 50 kD or a Sephadex G-25 desalting column, and the antibody-drug conjugate is replaced into 50 mM PBS buffer solution (pH 6.0). After ultrafiltration purification, the sample is filtered with a 0.22 μm filter membrane, and the antibody-drug conjugate is obtained and stored in a refrigerator at 4°C. The DAR value of the conjugate is determined by reverse phase high performance liquid chromatography or mass spectrometry.

[0496] Using mAb3, mAb4, mAb5, mAb6, and mAb7 instead of mAb2, respectively, the ADC-8, ADC-9, ADC-10, ADC-11, and ADC-12 are obtained by using a similar preparation method.

[0497] 6.2 Preparation of ADC-13

[0498] Using the humanized antibody mAb3-H2K2 and VcMMAE (CAS: 646502-53-6) as raw materials, a PBS buffer solution of the exemplary product ADC-13 of the conjugate mixture FADC-13A, FADC-13B, and FADC-13C is prepared by using a method similar to that in Example 6.1. The average drug loading amount calculated by mass spectrometry is y=3.28.

[0499] 6.3 Preparation of ADC-14

[0500] Using the humanized antibody mAb3-H2K4 and VcMMAE (CAS: 646502-53-6) as raw materials, a PBS buffer solution of the exemplary product ADC-14 of the conjugate mixture FADC-14A, FADC-14B, and FADC-14C is prepared by using a method similar to that in Example 6.1. The average drug loading amount calculated by mass spectrometry is y=2.80.

[0501] 6.4 Preparation of ADC-15

[0502] Using humanized antibody mAb 3-H3K2 and VcMMAE (CAS: 646502-53-6) as starting materials, an exemplary product ADC-15 of conjugation mixture FADC-15A, FADC-15B, FADC-15C was prepared in PBS buffer using a similar method as in Example 6.1. The average drug loading was calculated by mass spectrometry: y = 3.19.

[0503] 6.5 Preparation of ADC-16

[0504] Using humanized antibody mAb 3-H3K4 and VcMMAE (CAS: 646502-53-6) as starting materials, an exemplary product ADC-16 of conjugation mixture FADC-16A, FADC-16B, FADC-16C was prepared in PBS buffer using a similar method as in Example 6.1. The average drug loading was calculated by mass spectrometry: y = 5.64.

[0505] 6.6 Preparation of ADC-18

[0506] Using humanized antibody mAb 5-H3K1 and VcMMAE (CAS: 646502-53-6) as starting materials, an exemplary product ADC-18 of conjugation mixture FADC-18A, FADC-18B, FADC-18C was prepared in PBS buffer using a similar method as in Example 6.1. The average drug loading was calculated by mass spectrometry: y = 3.90.

[0507] 6.7 Preparation of ADC-19

[0508] Using humanized antibody mAb 5-H5K2 and VcMMAE (CAS: 646502-53-6) as starting materials, an exemplary product ADC-19 of conjugation mixture FADC-19A, FADC-19B, FADC-19C was prepared in PBS buffer using a similar method as in Example 6.1. The average drug loading was calculated by mass spectrometry: y = 3.83.

[0509] 6.8 Preparation of ADC-Reference 2

[0510] Using anti-KLH antibody and VcMMAE (CAS: 646502-53-6) as starting materials, an exemplary product ADC-Reference 2 of conjugation mixture FADC-Reference 2A, FADC-Reference 2B, FADC-Reference 2C was prepared in PBS buffer using a similar method as in Example 6.1. The average drug loading was calculated by mass spectrometry: y = 5.31.

[0511] 6.9 Preparation of ADC-20

[0512] Using humanized antibody mAb5-H3K1 and LD-114 as raw materials, the PBS buffer of the conjugate mixture FADC-20 was prepared to obtain an exemplary product ADC-20. The specific preparation steps are as follows.

[0513] Preparation of LD-112d

[0514] First step

[0515] LD-13g (500 mg, 1.48 mmol) was dissolved in 5 mL of N,N-dimethylformamide, then 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (564 mg, 1.48 mmol), N,N-diisopropyl ethylamine (574 mg, 4.44 mmol) and 3,6,9-trioxa-1-aminodecane (242 mg, 1.48 mmol) were added in turn, and stirred at 25°C for 2 hours. 15 mL of water was added, extracted with ethyl acetate (30 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 silica gel column chromatography with developing system A to obtain the title product LD-112a (600 mg, yield: 84%) in the form of colorless oil.

[0516] MS m / z (ESI): 483.3 [M+1].

[0517] Second step

[0518] LD-112a (600 mg, 1.24 mmol) was dissolved in 6 mL of methanol, and palladium-carbon (60 mg, 10%, wetted with about 55% water) was added under nitrogen protection. Hydrogen was replaced three times, and stirred at 25°C for 1 hour. Hydrogen was replaced three times, and the reaction solution was stirred at 25°C for 1 hour under hydrogen (15 Psi). The reaction solution was filtered with diatomite, and the filtrate was concentrated by reduced pressure distillation to obtain the title product LD-112b (350 mg, yield: 81%) in the form of colorless oil.

[0519] MS m / z (ESI): 349.3 [M+1].

[0520] Third step

[0521] LD-112b (350 mg, 1.01 mmol) was dissolved in 5 mL of N,N-dimethylformamide, then 2-(7-azobenzotriazol)-N,N,N',N'-tetramethyluronium hexafluorophosphate (384 mg, 1.01 mmol), N,N-diisopropyl ethylamine (390 mg, 3.02 mmol) were added in turn, then LD-13j (271 mg, 1.01 mmol, prepared by the method disclosed in patent application "WO2021228141 A1" page 30, example 2) was added, and stirred at 25°C for 2 hours. 1 mL of water was added, extracted with dichloromethane (3 mL x 2), the organic phase was dried with anhydrous sodium sulfate, filtered, and the filtrate was concentrated by reduced pressure distillation, and the obtained residue was purified by reverse phase high performance liquid chromatography (mobile phase: A-water, B-acetonitrile; isocratic elution: B%: 36%), to obtain the title product LD-112c (301.78 mg, yield: 50%) in the form of a white solid.

[0522] MS m / z (ESI): 599.2 [M+1].

[0523] Fourth step

[0524] LD-112c (50.0 mg, 0.08 mmol) was dissolved in 1 mL of dichloromethane, then 0.5 mL of trifluoroacetic acid was slowly added dropwise, and stirred at 25°C for 0.5 hours. The reaction solution was concentrated by reduced pressure distillation, to obtain the crude title product LD-112d (45 mg) in the form of a colorless oil. The product was used directly in the next step reaction without purification.

[0525] MS m / z (ESI): 543.24 [M+1].

[0526] Preparation of 1e

[0527] First step

[0528] 2-(6-cyano-5-oxo-2,3-dihydro-5H-spiro[indolizine-1,2'-[1,3]dioxolan]-7-yl)-3- cyclopropylpropanoic acid ethyl ester 1b

[0529] Dissolve 1a (1.01 g, 3.31 mmol, prepared using the method disclosed in Example 30, page 28 of the patent application “WO2019238046A1”) 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 distillation under reduced pressure, 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.

[0530] MS m / z (ESI): 359.1 [M+1].

[0531] Second step

[0532] 3-cyclopropyl-2-(6-formyl-5-oxo-2,3-dihydro-5H-spiro[indolizine-1,2'-[1,3]dioxolan]-7- yl)propanoic acid ethyl ester 1c

[0533] Dissolve 1b (1.12 g, 3.05 mmol) in a mixture solvent of 5 mL of water, 5 mL of acetonitrile and 5 mL of 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 over anhydrous sodium sulfate, filter, concentrate the filtrate by distillation under reduced pressure, 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.

[0534] MS m / z (ESI): 362.1 [M+1].

[0535] Third step

[0536] 4-(cyclopropylmethyl)-1,4,7,8-tetrahydro-3H,10H-spiro[pyrano[3,4-f]indolizine-6,2'- [1,3]dioxolan]-3,10-dione 1d

[0537] Dissolve 1c (680 mg, 1.85 mmol) in 10 mL of dichloromethane, protect under nitrogen, and cool to 0 °C in an ice water bath. Add sodium borohydride (108.02 mg, 2.86 mmol) in portions. Stir the reaction at 0 °C for 30 minutes. Add acetic acid (133.15 mg, 2.22 mmol) dropwise at 25 °C. Continue stirring for 2 hours. Add 30 mL of water dropwise at 15 °C. Stir the reaction at 15 °C for 1.5 hours. Wash the reaction with water (50 mL). Add p-toluenesulfonic acid monohydrate (35.15 mg, 184.78 μmol) to the organic phase after washing. Stir at 15 °C for 12 hours. Add 50 mL of water. Extract the reaction with dichloromethane (45 mL x 3). Dry the organic phase over anhydrous sodium sulfate. Filter and concentrate the filtrate by distillation under reduced pressure. Purify the residue obtained by silica gel column chromatography with developing system B to obtain the title product 1d (200 mg, yield: 32%) as a yellow oil.

[0538] MS m / z (ESI): 318.1 [M+1].

[0539] Fourth step

[0540] 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

[0541] 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 for 5 hours under oxygen bubbling (15 psi). Pour the reaction into 10 mL of saturated aqueous ammonium chloride solution. Concentrate by distillation under reduced pressure to remove methanol. 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 obtain the crude title product 1e (140 mg) as a yellow oil. The product was used directly in the next reaction without purification.

[0542] MS m / z (ESI): 334.1 [M+1].

[0543] Preparation of 1i-1

[0544] First step

[0545] (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

[0546] (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

[0547] The 1e (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 1e-1 (301 mg, yield: 22.1 %) as a yellow solid, and the title product 1e-2 (285 mg, yield: 19.4%) as a yellow solid.

[0548] Single configuration compound 1e-1

[0549] 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).

[0550] MS m / z (ESI): 334.0 [M+1].

[0551] Single configuration compound 1e-2

[0552] 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).

[0553] MS m / z (ESI): 333.9 [M+1].

[0554] Second step

[0555] (S)-4-(cyclopropylmethyl)-4-hydroxy-7,8-dihydro-1 H-pyrano[3,4-f]indolizine-3,6,10(4H)- trione 1f-1

[0556] Dissolve 1e-1 (301 mg, 857 μmol) in 2 mL of a mixture of trifluoroacetic acid and 0.5 mL of water, stir at 25 °C for 4 hours. Concentrate the reaction solution under reduced pressure, and the obtained residue is used as crude product of the title product 1f-1 (209 mg) in the form of a yellow solid without purification. The product is directly used in the next step without purification.

[0557] MS m / z (ESI): 290.1 [M+1].

[0558] Third step

[0559] N-((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)acetamide 1h-1

[0560] Dissolve 1f-1 (50.0 mg, 159 μmol) and 1g (48.0 mg, 192 μmol, prepared by the method disclosed in patent application “CN111065621A” page 61, example 5-1) and pyridine 4-methylbenzenesulfonic acid (8.0 mg, 31.8 μmol) in 2 mL of toluene, protect under 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, 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-1 (190 mg, yield: 56%) in the form of a yellow solid.

[0561] MS m / z (ESI): 504.1 [M+1].

[0562] Fourth step

[0563] (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

[0564] Dissolve 1h-1 (60.0 mg, 111 μmol) in 4.5 mL ethyleneglycol dimethyl ether, add 1.5 mL methanesulfonic acid and 1.5 mL water, stir at 85 °C for 16 hours. Add the reaction solution dropwise into 10 mL water with stirring, extract with dichloromethane (10 mL x 5), wash the organic phase with 25 mL 0.05 M aqueous hydrochloric acid, filter the aqueous phase, combine all the aqueous extracts, adjust the pH of the aqueous phase to 7-8 with saturated potassium bicarbonate solution at 0 °C, extract with 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) as a brown solid, use the product directly for the next reaction without purification.

[0565] MS m / z (ESI): 462.1 [M+1].

[0566] Preparation of 1i-1-1

[0567] First step

[0568] (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

[0569] (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

[0570] Separate 1i-1 (550 mg, 1.10 mmol) by SFC (separation conditions: column: DAICEL CHIRALPAK IC 250 mm x 50 mm, 10 μm; mobile phase: A-n-hexane: B-ethanol, isocratic elution: B: 45%), to obtain the title product 1i-1-1 (260 mg, yield: 41%) as a yellow solid, and the title product 1i-1-2 (290 mg, yield: 46%) as a yellow solid.

[0571] Single configuration compound 1i-1-1

[0572] SFC analysis: Retention time 4.951 min. (Chromatography 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).

[0573] MS m / z (ESI): 462.3 [M+1].

[0574] Single configuration compound 1i-1-2

[0575] SFC analysis: Retention time 4.951 min. (Chromatography 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).

[0576] MS m / z (ESI): 462.3 [M+1].

[0577] Preparation of LD-99g

[0578] First step

[0579] (R)-1-(9H-fluoren-9-yl)-10-methyl-3,6-dioxo-2,9-dioxa-4,7-diazoundecanoic acid benzyl ester LD-99b

[0580] LD-99a (3.00 g, 7.87 mmol, prepared using the method disclosed in patent application “WO2022135332 A1” page 226, example 1.15) was dissolved in 50 mL of dichloromethane, 4-methylbenzenesulfonic acid pyridine (1.98 g, 7.87 mmol) and (R)-benzyl 2-hydroxypropanoate (2.84 g, 15.7 mmol) were added in turn, and stirred at 50°C for 10 hours. 40 mL of water was added to the reaction solution, extracted with dichloromethane (50 mL x 3), the organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated by reduced pressure distillation, and the obtained residue was purified by silica gel column chromatography with developing system B to obtain the title product LD-99b (1.55 g, yield: 40%) in the form of yellow gum.

[0581] Second step

[0582] (5S, 13R)-5-benzyl-1-(9H-fluoren-9-yl)-13-methyl-3,6,9-trioxo-2,12-dioxa-4,7,10- triazatetradecan-14-oic acid benzyl ester LD-99c

[0583] LD-99b (2.63 g, 5.38 mmol) was dissolved in 30 mL of dichloromethane, and 1,8- diazabicyclo[5.4.0]undec-7-ene (491 mg, 3.23 mmol) was added, and stirred at 25°C for 1 hour. Then (( (9H-fluoren-9-yl)methoxy)carbonyl)-L-phenylalanine (2.29 g, 5.91 mmol), 1- hydroxybenzotriazole (1.09 g, 8.06 mmol), and 1-(3-dimethylaminopropyl)-3- ethylcarbodiimide hydrochloride (1.54 g, 8.06 mmol) were sequentially added, and stirred at 25°C for 2 hours. 10 mL of water was added, extracted with dichloromethane (10 mL x 3), and the organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated by distillation under reduced pressure, and the obtained residue was purified by silica gel column chromatography with the developing system A to obtain the title product LD-99c (1.60 g, yield: 37%) as a yellow solid.

[0584] MS m / z (ESI): 658.4 [M+23].

[0585] Third step

[0586] (11S, 19R)-11-benzyl-1-(9H-fluoren-9-yl)-19-methyl-3,6,9,12,15-pentaoxo-2,18-dioxa-4,7,10,13,16-pentaazaeicosan-20-oic acid benzyl ester LD-99d

[0587] LD-99c (1.60 g, 1.97 mmol) was dissolved in 15 mL of dichloromethane, and 1,8- diazabicyclo[5.4.0]undec-7-ene (179 mg, 1.18 mmol) was added, and stirred at 25°C for 1 hour. Then (( (9H-fluoren-9-yl)methoxy)carbonyl)glycylglycine (836 mg, 2.36 mmol) and 2-(7- azobenzotriazolyl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (1.12 g, 2.95 mmol) were sequentially added, and stirred at 25°C for 1 hour. 10 mL of water was added, extracted with dichloromethane (10 mL x 3), and the organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated by distillation under reduced pressure, and the obtained residue was purified by silica gel column chromatography with the developing system A to obtain the title product LD-99d (1.15 g, yield: 70%) as a yellow solid.

[0588] MS m / z (ESI): 772.4 [M+23].

[0589] Fourth Step

[0590] (9H-Fluoren-9-yl)methyl ((2R,10S)-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)-2-methyl-1,6,9,12,15- pentoxy-3-oxa-5,8,11,14-tetraazahexadec-16-yl)carbamate LD-99f

[0591] LD-99d (500 mg, 602 μmol) and palladium on carbon (448 mg, 10%, wetted with about 55% water) were dissolved in 2 mL of ethyl acetate and 4 mL of ethanol under nitrogen protection, replaced with hydrogen gas for three times, the reaction solution was stirred at 25 °C for 2 hours under hydrogen gas (15 Psi). The reaction solution was filtered with celite, the filtrate was concentrated by distillation under reduced pressure, the crude product of the title compound LD-99e was obtained as a white solid (330 mg, yield: 76%). The product was used directly in the next step without purification.

[0592] MS m / z (ESI): 682.4 [M+23].

[0593] Fifth Step

[0594] (9H-Fluoren-9-yl)methyl ((2R,10S)-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)-2-methyl-1,6,9,12,15- pentoxy-3-oxa-5,8,11,14-tetraazahexadec-16-yl)carbamate LD-99f

[0595] LD-99e (30.2 mg, 41.7 μmol) and 1i-1-1 (15.0 mg, 32.1 μmol) were dissolved in 2 mL of N,N-dimethylformamide, 2-(7-azobenzotriazol)-N,N,N',N'-tetramethyluronium hexafluorophosphate (18.3 mg, 48.1 μmol) and N,N-diisopropyl ethylamine (12.4 mg, 96.2 μmol) were added in turn, stirred at 25 °C for 1 hour. 1 mL of water was added, extracted with dichloromethane (3 mL x 2), the organic phase was dried with anhydrous sodium sulfate, filtered, the filtrate was concentrated by distillation under reduced pressure, the obtained residue was purified by silica gel column chromatography with developing system A, the title product LD-99f was obtained as a yellow solid (25.0 mg, yield: 59%).

[0596] MS m / z (ESI): 1103.6 [M+1].

[0597] Sixth step

[0598] (S)-2-(2-(2-Aminoacetylamino)acetylamino)-N-(2-(((((R)-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-oxopropan-2-yl)oxy)methyl)amino)-2-oxoethyl)-3-phenylpropanamide LD-99g

[0599] LD-99f (44.0 mg, 39.9 μmol) was dissolved in 1 mL of acetonitrile, diethylamine (142 mg, 1.94 mmol) was added, and stirring was performed at 25°C for 0.5 hours. The reaction solution was concentrated by distillation under reduced pressure to obtain the crude title product LD-99g (30.0 mg) as a yellow solid. The product was used directly in the next reaction without purification.

[0600] MS m / z (ESI): 881.5 [M+1].

[0601] Preparation of LD-114

[0602] LD-112d (10.2 mg, 18.7 μmol) and LD-99g (15.0 mg, 17.0 μmol) were dissolved in 1.0 mL of N,N-dimethylformamide, and then 1-hydroxybenzotriazole (3.5 mg, 25.5 μmol), N,N-diisopropylethylamine (6.6 mg, 51 μmol), and 1-(3- dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (4.9 mg, 25.5 μmol) were added in this order, and stirring was performed at 25°C for 2 hours. To the reaction solution was added 3 mL of water, extraction was performed with dichloromethane (5 mL x 2), 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 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%: 28% - 58%), to obtain the title product LD-114 (10.1 mg, yield: 42%) as a white solid.

[0603] MS m / z (ESI): 1405.7 [M+1].

[0604] 1H NMR (400 MHz, CD3OD) δ 8.95-8.88 (m, 2H), 7.68-7.61 (m, 2H), 7.29-7.21 (m, 2H), 7.20-7.12 (m, 3H), 5.68-5.64 (m, 1H), 5.60-5.54 (m, 1H), 5.42-5.33 (m, 2H), 5.16-5.10 (m, 1H), 4.82-4.79 (m, 1H), 4.70-4.66 (m, 1H), 4.43-4.35 (m, 2H), 4.26-4.20 (m, 1H), 3.90-3.80 (m, 4H), 3.78-3.73 (m, 1H), 3.71-3.66 (m, 1H), 3.63-3.54 (m, 6H), 3.54-3.46 (m, 5H), 3.43-3.35 (m, 4H), 3.35-3.33 (m, 3H), 3.27-3.22 (m, 1H), 3.21-3.16 (m, 1H), 3.15-3.12 (m, 1H), 3.07-3.02 (m, 1H), 2.92-2.86 (m, 1H), 2.59-2.52 (m, 2H), 2.47-2.38 (m, 5H), 2.36-2.29 (m, 3H), 2.11-2.04 (m, 1H), 1.99-1.87 (m, 4H), 1.85-1.79 (m, 1H), 1.60-1.51 (m, 3H), 0.92-0.85 (m, 1H), 0.49-0.39 (m, 2H), 0.15-0.08 (m, 1H), 0.06-0.00 (m, 1H).

[0605] Preparation of ADCs

[0606] The antibody was replaced into 50 mM PBS / 1.0 mM EDTA buffer (pH 7.4 adjusted by sodium hydroxide solution) using an ultrafiltration tube with a molecular weight cut-off of 50 kD, 5-10 equivalents of 10 mM TCEP aqueous solution were added, and the mixture was shaken at 25 °C for 3 hours. The linker-drug conjugate LD-114 was dissolved in DMSO, 10-20 equivalents of the linker-drug conjugate were taken therefrom and added to the reduced antibody solution, which was vortexed to mix uniformly, and the mixture was shaken at 25 °C for 2 hours. After the reaction was completed, 40 equivalents of 100 mM NAC aqueous solution were added, and the mixture was shaken at 25 °C for 20 minutes to terminate the linker reaction. Excess small molecules were removed using an ultrafiltration centrifuge tube with a molecular weight cut-off of 50 kD or a Sephadex G-25 desalting column, and the antibody-drug conjugate was replaced into 50 mM PBS buffer (pH 6.0). The sample was filtered using a 0.22 μm filter membrane, and the antibody-drug conjugate was obtained and stored in a refrigerator at 4 °C. The DAR value of the conjugate was determined using reverse phase high performance liquid chromatography or mass spectrometry. An exemplary product ADC-20 of the conjugate mixture FADC-20 was obtained. The average drug loading value calculated by mass spectrometry was y = 8.00.

[0607] 6.10 Preparation of ADC-23

[0608] Using the humanized antibody Anti-KLH and LD-114 as raw materials, an exemplary product ADC-23 of the conjugate mixture FADC-23 in PBS buffer was prepared by a method similar to that of Example 6.9. The average drug loading value calculated by mass spectrometry was y = 7.99.

[0609] 6.11 Preparation of ADC-25

[0610] Using the antibody TORL-3-600 and LD-114 as raw materials, an exemplary product ADC-25 of the conjugate mixture FADC-25 in PBS buffer was prepared by a method similar to that of Example 6.9. The average drug loading value calculated by mass spectrometry was y = 7.46.

[0611] The structures of the ADCs prepared in Examples 6.1-6.8 are summarized as follows:

[0612] Example 7, in vitro killing activity of ADCs

[0613] 7.1 Killing activity of anti-CDH17 chimeric antibody ADC on SUN-16 cells

[0614] Using CDH17-overexpressing SNU-16 cells as target cells, the cell density was adjusted to 5 × 10⁶ cells per ml using RPMI-1640 complete medium containing 20% ​​FBS. 4 100 μl of each cell was seeded into a 96-well plate. The MMAE-conjugated antibody was diluted to 2 μg / ml with RPMI-1640 basal medium, and serially diluted 5-fold. 100 μl of the antibody was added to each well (at this point, the actual working concentration of the ADC was 1 μg / ml, 5-fold dilution, and the FBS concentration was 10%). The cells were cultured for 3 days, stained with 10% CCK-8 reagent, and the absorbance was read at 450 nm. The relative viability was calculated. The IC50 of the sample was determined using a four-parameter regression analysis with ADC concentration on the x-axis and relative viability on the y-axis. 50 The results are shown in Figure 10 and Table 12, respectively. With ADC reference 2 as the control, ADC-7, ADC-8, ADC-9, ADC-10, ADC-11 and ADC-12 can all effectively kill SNU-16 cells, among which ADC-8 has the best killing effect.

[0615] Table 12. Killing activity of anti-CDH17 chimeric antibody ADC Note: " / " indicates that no curve was fitted.

[0616] 7.2 Killing activity of anti-CDH17 humanized antibody ADC against SUN-16 cells

[0617] Using SNU-16 cells, which highly express CDH17, as target cells, the cell density was adjusted to 1×10⁶ cells per ml using RPMI-1640 complete medium containing 20% ​​FBS. 5 100 μl of each cell was seeded into a 96-well plate. The anti-CDH17 humanized antibody ADC was diluted to a concentration of 2 μg / ml with RPMI-1640 basal medium and serially diluted 4-fold. 100 μl of each ADC was added to the cells in each well (at this point, the actual working concentration of ADC was 1 μg / ml, 4-fold dilution, and the FBS concentration was 10%). The cells were cultured for 3 days, stained with 10% CCK-8 reagent, and the absorbance was read at 450 nm to calculate the relative viability.

[0618] The results are shown in Table 13 and Figures 11A and 11B, respectively. ADC-13, ADC-14, ADC-15, ADC-16, ADC-18, and ADC-19 can all effectively kill SNU-16 cells.

[0619] Table 13 Killing activity of anti-CDH17 humanized antibody ADC Note: " / " indicates that no curve was fitted.

[0620] Using CDH17-overexpressing SNU-16 cells as target cells, the cell density was adjusted to 3 × 10⁶ cells per mL of RPMI-1640 complete medium containing 20% ​​FBS. 4 100 μL of each cell was seeded into 96-well plates. ADC-20 and ADC-25 were diluted to 29 μg / mL using RPMI-1640 basal medium, and serially diluted 4-fold. 100 μL of each medium was added to each well (at this point, the actual working concentration of ADC was 14.5 μg / mL, 4-fold dilution, and FBS concentration was 10%). Cells were cultured for 7 days, stained with 10% CCK-8 reagent, and absorbance was read at 450 nm. Relative viability was calculated. A four-parameter regression analysis was performed with ADC concentration on the x-axis and relative viability on the y-axis to obtain the IC50 of the sample. 50 As shown in Table 14, the IC of ADC-20 50 The IC for ADC-25 is 0.26nM. 50 The concentration was 0.31 nM. As shown in Figure 11C, ADC-20 can effectively kill SNU-16 tumor cells, and the killing is more thorough, with better results than ADC-25.

[0621] Table 14. Killing activity of anti-CDH17 humanized antibody ADC Note: " / " indicates that no curve was fitted.

[0622] 7.3 Killing activity of anti-CDH17 humanized antibody ADC against ASPC-1 cells

[0623] Using ASPC-1 cells expressed in CDH17 as target cells, the cell density was adjusted to 1×10⁶ cells per mL of RPMI-1640 complete medium containing 20% ​​FBS. 5 100 μL of each cell was seeded into a 96-well plate. ADC-20 and ADC-25 were diluted to a concentration of 20 μg / mL using RPMI-1640 basal medium, and serially diluted 4-fold. 100 μL of each cell was added to each well (at this point, the actual working concentration of ADC was 10 μg / mL, 4-fold dilution, and FBS concentration was 10%). The cells were cultured for 3 days, stained with 10% CCK-8 reagent, and the absorbance was read at 450 nm to calculate relative viability. The IC50 of the samples was calculated using a four-parameter regression analysis with ADC concentration on the x-axis and relative viability on the y-axis, as shown in Table 15. The IC50 of ADC-20 was 0.26 nM, and the IC50 of ADC-25 was 0.52 nM. As shown in Figure 11D, ADC-20 effectively killed ASPC-1 tumor cells, and the killing was more thorough, showing better results than ADC-25.

[0624] Table 15. Killing activity of anti-CDH17 humanized antibody ADC Note: " / " represents no curve fitting.

[0625] 7.4 Bystander effect of anti-CDH17 humanized antibody ADC

[0626] Prepare cell suspension with 1640 complete medium containing 10% FBS, add positive cells SNU-16 and negative cells KATO III into the same well of a 6-well plate, the number is 1 x 10 5 and 5 x 10 4 , the culture volume is 2 ml, and plate overnight. The next day, prepare ADC samples with complete medium, the concentration is 10 μg / ml, 5-fold dilution, a total of 3 concentrations, add 1 ml of prepared ADC sample into the well, and set up untreated control to add culture medium, continue to culture for 3 days of killing. After the corresponding time of killing, collect all cells in the well, count the viable cells using a cell counter, and then perform flow cytometry analysis, calculate the proportion of positive and negative cells, and combine the viable cell count results to calculate the actual number of the two types of cells in the well. The results are shown in Figure 12. Compared with the control, ADC-18 exhibits bystander effect and can effectively kill KATO III cells negative for CDH17.

[0627] Example 8, in vivo efficacy of anti-CDH17 humanized antibody ADC

[0628] Take ASPC-1 cells expressing CDH17 as target cells, passaged with RPMI-1640 complete medium containing 10% FBS, and culture the cells to a total amount of 4.5 x 10 8 cells. Take the tumor cells in the logarithmic growth phase (the growth density is not more than 70%), centrifuge at 300 g for 7 min, wash with PBS for 3 times, count, adjust the cell density to 4 x 10 7 cells / ml, resuspend in an appropriate volume of PBS, inject 100 μL of cell suspension subcutaneously into each mouse, and the day of inoculation is recorded as D0. Measure the tumor size 3 times a week thereafter, make a tumor growth curve, and monitor the change in body weight of the mice. On D10, when the tumor grows to 100-200 mm 3 , start grouping and dosing. The dosing groups include ADC-Reference 2 (6 mg / kg) and ADC-18 (6 mg / kg), and the dosing regimen is 1 dose per week for a total of 3 doses. The dosing method is tail vein injection.

[0629] The in vivo efficacy results are shown in Figure 13. The results show that the tumor growth inhibition rate (TGI) of the ADC-18 6 mg / kg group is 81.1%, indicating that it has excellent tumor inhibition effect.

[0630] Using CDH17-overexpressing SNU-16 cells as target cells, cells were passaged in RPMI-1640 complete medium containing 10% FBS until the total cell count reached 4.5 × 10⁻⁶. 8 After counting the cells, tumor cells in the logarithmic growth phase (growth density not exceeding 70%) were collected, centrifuged at 300g for 7 minutes, washed three times with PBS, and then counted. The cell density was adjusted to 4 × 10⁻⁶ cells / year. 7 Cells were resuspended at a density of 100 μL / ml in an appropriate volume of PBS. Each mouse was injected with 100 μL of the cell suspension subcutaneously for tumor inoculation, with the day of inoculation recorded as D0. Tumor size was measured three times per week to create tumor growth curves; mouse body weight was also monitored. On day 10, when the tumors reached 100-200 mm³, drug administration was initiated in different groups. The treatment groups included ADC-reference 2 and ADC-18 (1, 0.3, and 0.1 mg / kg), administered as a single dose via tail vein injection.

[0631] The in vivo efficacy results are shown in Figure 14. The TGI of ADC-18 at the three dose groups of 1, 0.3 and 0.1 mg / kg were 55.31%, 28.25% and 20.46%, respectively, indicating that it has excellent antitumor effect.

[0632] While preferred embodiments of the invention have been shown and described herein, these embodiments will be apparent to those skilled in the art as provided by way of example only. Many variations, changes, and substitutions will occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments described herein may be employed. The following claims are intended to define the scope of the invention and cover the methods and structures within the scope of these claims and their equivalents.

Claims

1. An antibody or antigen-binding fragment thereof that binds CDH17, 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 of SEQ ID NOs: 5, 6, 7, respectively, and the VL comprises LCDR1, LCDR2, and LCDR3 having the amino acid sequences of SEQ ID NOs: 10, 11, 12, respectively; or (2) 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 (3) the VH comprises HCDR1 having the amino acid sequence of SEQ ID NO: 25 or 107, HCDR2 having the amino acid sequence of SEQ ID NO: 26 or 125, and HCDR3 having the amino acid sequence of SEQ ID NO: 27, and the VL comprises LCDR1 having the amino acid sequence of SEQ ID NO: 30 or 120, LCDR2 having the amino acid sequence of SEQ ID NO: 31, and LCDR3 having the amino acid sequence of SEQ ID NO: 32; or (4) the VH comprises HCDR1, HCDR2, HCDR3 having the amino acid sequences of SEQ ID NOs: 35, 36, 37, respectively, and the VL comprises LCDR1, LCDR2, and LCDR3 having the amino acid sequences of SEQ ID NOs: 40, 41, 42, respectively; or (5) the VH comprises HCDR1 having the amino acid sequence of SEQ ID NO: 45, HCDR2 having the amino acid sequence of SEQ ID NO: 46 or 81, and HCDR3 having the amino acid sequence of SEQ ID NO: 47, and the VL comprises LCDR1 having the amino acid sequence of SEQ ID NO: 50 or 90, LCDR2 having the amino acid sequence of SEQ ID NO: 51, 93, or 96, and LCDR3 having the amino acid sequence of SEQ ID NO: 52; or (6) the VH comprises HCDR1, HCDR2, HCDR3 having the amino acid sequences of SEQ ID NOs: 55, 56, 57, respectively, and the VL comprises LCDR1, LCDR2, and LCDR3 having the amino acid sequences of SEQ ID NOs: 60, 61, 62, respectively; or (7) the VH comprises HCDR1, HCDR2, HCDR3, each having an amino acid sequence as set forth in SEQ ID NO: 65, 66, 67, respectively, and the VL comprises LCDR1, LCDR2, and LCDR3, each having an amino acid sequence as set forth in SEQ ID NO: 70, 71, 72, respectively.

2. The antibody or antigen-binding fragment thereof of claim 1, wherein: (1) the VH comprises HCDR1, HCDR2, HCDR3, each having an amino acid sequence as set forth in SEQ ID NO: 5, 6, 7, respectively, and the VL comprises LCDR1, LCDR2, and LCDR3, each having an amino acid sequence as set forth in SEQ ID NO: 10, 11, 12, respectively; or (2) the VH comprises HCDR1, HCDR2, HCDR3, each having an amino acid sequence as set forth in SEQ ID NO: 15, 16, 17, respectively, and the VL comprises LCDR1, LCDR2, and LCDR3, each having an amino acid sequence as set forth in SEQ ID NO: 20, 21, 22, respectively; or (3) the VH comprises HCDR1, HCDR2, HCDR3, each having an amino acid sequence as set forth in SEQ ID NO: 25, 26, 27, respectively, and the VL comprises LCDR1, LCDR2, and LCDR3, each having an amino acid sequence as set forth in SEQ ID NO: 30, 31, 32, respectively; or (4) the VH comprises HCDR1, HCDR2, HCDR3, each having an amino acid sequence as set forth in SEQ ID NO: 35, 36, 37, respectively, and the VL comprises LCDR1, LCDR2, and LCDR3, each having an amino acid sequence as set forth in SEQ ID NO: 40, 41, 42, respectively; or (5) the VH comprises HCDR1, HCDR2, HCDR3, each having an amino acid sequence as set forth in SEQ ID NO: 45, 46, 47, respectively, and the VL comprises LCDR1, LCDR2, and LCDR3, each having an amino acid sequence as set forth in SEQ ID NO: 50, 51, 52, respectively; or (6) the VH comprises HCDR1, HCDR2, HCDR3, each having an amino acid sequence as set forth in SEQ ID NO: 55, 56, 57, respectively, and the VL comprises LCDR1, LCDR2, and LCDR3, each having an amino acid sequence as set forth in SEQ ID NO: 60, 61, 62, respectively; or (7) the VH comprises HCDR1, HCDR2, HCDR3, each having an amino acid sequence as set forth in SEQ ID NO: 65, 66, 67, respectively, and the VL comprises LCDR1, LCDR2, and LCDR3, each having an amino acid sequence as set forth in SEQ ID NO: 70, 71, 72, respectively. (8) the VH comprises HCDR1, HCDR2, HCDR3, each having an amino acid sequence as set forth in SEQ ID NO: 25, 125, 27, respectively, and the VL comprises LCDR1, LCDR2, and LCDR3, each having an amino acid sequence as set forth in SEQ ID NO: 120, 31, 32, respectively; or (9) the VH comprises HCDR1, HCDR2, HCDR3, each having an amino acid sequence as set forth in SEQ ID NO: 45, 46, 47, respectively, and the VL comprises LCDR1, LCDR2, and LCDR3, each having an amino acid sequence as set forth in SEQ ID NO: 90, 51, 52, respectively.

3. The antibody or antigen-binding fragment thereof of claim 1, 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: 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; 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: 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 (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 any one selected from the group consisting of 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 the group consisting of SEQ ID NO: 28, 116, 118, 121, 123; 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: 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 (5) the VH comprises an amino acid sequence with 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, 73, 75, 77, 79, 82, 84, and the VL comprises an amino acid sequence with 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, 91, 94, 97; or (6) the VH comprises an amino acid sequence with 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 with 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 (7) the VH comprises an amino acid sequence with 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 with 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.

4. The antibody or antigen-binding fragment thereof of claim 3, wherein: (1) the VH comprises an amino acid sequence with 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 with 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; or (2) the VH comprises an amino acid sequence with 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 with 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 (3) the VH comprises an amino acid sequence with 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 with 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 (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: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 (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: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 (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: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 (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: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 (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:118; or (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: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 (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: 118; or (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: 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 (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: 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 (13) 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.

5. The antibody or antigen-binding fragment thereof of claim 4, wherein: (1) 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; or (2) 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 (3) 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 (4) 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 (5) 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 (6) 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 (7) 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 (8) the VH comprises the amino acid sequence set forth in SEQ ID NO: 73 and the VL comprises the amino acid sequence set forth in SEQ ID NO: 78; or (9) the VH comprises the amino acid sequence set forth in SEQ ID NO: 83 and the VL comprises the amino acid sequence set forth in SEQ ID NO: 88; or (10) the VH comprises the amino acid sequence set forth in SEQ ID NO: 93 and the VL comprises the amino acid sequence set forth in SEQ ID NO: 98; or (11) the VH comprises the amino acid sequence set forth in SEQ ID NO: 103 and the VL comprises the amino acid sequence set forth in SEQ ID NO: 108; or (12) the VH comprises the amino acid sequence set forth in SEQ ID NO: 113 and the VL comprises the amino acid sequence set forth in SEQ ID NO: 118; or (13) the VH comprises the amino acid sequence set forth in SEQ ID NO: 123 and the VL comprises the amino acid sequence set forth in SEQ ID NO:

128. (6) 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 (7) 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 (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: 118; or (9) 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 (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: 118; or (11) 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 (12) 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 (13) 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.

6. The antibody or antigen-binding fragment thereof of any one of claims 1-5, wherein the antibody is a murine antibody, a chimeric antibody, or a humanized antibody.

7. The antibody or antigen-binding fragment thereof of any one of claims 1-6, wherein the antibody is of an isotype selected from the group consisting of IgG, IgA, IgM, IgE, and IgD.

8. The antibody or antigen-binding fragment thereof of any one of claims 1-7, wherein the antibody is of a subtype selected from the group consisting of IgGl, IgG2, IgG3, and IgG4.

9. The antibody or antigen-binding sequence thereof of any one of claims 1-8, 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 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; 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 to SEQ ID NO: 134 or 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 or SEQ ID NO:

138.

10. The antibody or antigen-binding fragment thereof of claim 9, 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 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; 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 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; 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 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; 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 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; 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 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; 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.

11. The antibody or antigen-binding fragment thereof of claim 10, wherein: (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.

12. The antibody or antigen-binding fragment thereof according to any one of claims 1-11, wherein the antigen-binding fragment is selected from the group consisting of a Fab, a Fab’, a F(ab’)2, a Fv, a scFv, and a ds-scFv.

13. The antibody or antigen-binding fragment thereof according to any one of claims 1-12, wherein the antibody is a monoclonal antibody, a bispecific antibody, or a multispecific antibody.

14. The antibody or antigen-binding fragment thereof according to claim 13, wherein the antibody is a bispecific antibody further comprising a second antigen-binding region that binds to a second antigen.

15. A nucleic acid comprising a nucleotide sequence encoding the antibody or antigen-binding fragment thereof of any one of claims 1-14.

16. A vector comprising the nucleic acid of claim 15.

17. A host cell comprising the nucleic acid of claim 15 or the vector of claim 16.

18. A pharmaceutical composition comprising (i) the antibody or antigen-binding fragment thereof of any one of claims 1-14; and (ii) a pharmaceutically acceptable carrier or excipient.

19. The pharmaceutical composition of claim 18, further comprising a second therapeutic agent.

20. The pharmaceutical composition of claim 19, wherein the second therapeutic agent is selected from the group consisting of an antibody, a chemotherapeutic agent, and a small molecule drug.

21. A conjugate comprising the antibody or antigen-binding fragment thereof of any one of claims 1-14, and a chemical moiety conjugated thereto.

22. The conjugate of claim 21, wherein the chemical moiety is selected from a therapeutic agent, a detectable moiety, and an immunostimulatory molecule.

23. A chimeric antigen receptor (CAR) comprising the antibody or antigen-binding fragment thereof of any one of claims 1-14.

24. A method for treating a disease in a subject, comprising administering to the subject an effective amount of the antibody or antigen-binding fragment thereof of any one of claims 1-14, the pharmaceutical composition of any one of claims 18-20, the conjugate of claim 21 or 22, or the CAR of claim 23.

25. The method of claim 24, wherein, the disease is a cancer, e.g., a cancer associated with CDH17 expression.

26. The method of claim 25, wherein the cancer is selected from 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 carcinoma, a non-small cell lung carcinoma, a bladder carcinoma, or an epithelial carcinoma; More preferably, the disease is selected from a gastric cancer, a liver cancer (e.g., a hepatocellular carcinoma), a colorectal cancer, a pancreatic cancer, an esophageal cancer, a neuroendocrine tumor, and a breast cancer.

27. The method of any one of claims 24-26, further comprising administering to the subject a second therapeutic agent.

28. The method of claim 27, wherein the second therapeutic agent is selected from an antibody, a chemotherapeutic agent, and a small molecule drug.

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