Antibodies, antigen-binding fragments and methods of use

Abstract

The present disclosure relates to antibodies, antigen-binding fragments, and methods of use. Specifically, the disclosure relates to an antibody or antigen-binding fragment thereof that specifically recognizes human hepatic enteric cadherin and its pharmaceutical use. One objective of the disclosure is to develop potential cancer treatments. As a technical solution, a human anti-hepatic enteric cadherin antibody or antigen-binding fragment thereof containing a specific amino acid sequence is provided. The antibody or antigen-binding fragment thereof has high specificity and affinity for hepatic enteric cadherin and can mediate the killing of hepatic enteric cadherin-expressing cells. Therefore, the disclosure further relates to pharmaceutical compositions containing the antibody or antigen-binding fragment thereof and its use in the manufacture of drugs for preventing and / or treating tumors.

Description

[Technical Field]

[0001] This disclosure claims priority to U.S. Patent Application No. 63 / 486,522, filed February 23, 2023, the entire contents of which are incorporated herein by reference.

[0002] The present disclosure relates to antibodies, particularly antibodies that exhibit specificity for hepatic enteric cadherin, and their uses, for example, in the treatment of cancer. [Background technology]

[0003] Hepatointestinal cadherin (cadherin 17) is a cell surface marker that belongs to the cadherin superfamily and has a unique biological structure. Compared to the five classical cadherins, it has seven extracellular cadherin repeats and a very short intracellular region of 20 amino acid residues that lacks the conserved intracellular domain (Berndorff et al., J Cell Biol. 1994, 125(6):1353-1369). Although the biological function of cadherin 17 remains largely unknown, it is believed to mediate water absorption via Ca 2+ It has been reported that cadherin-17 can be regulated in a cell-dependent manner (Ahl et al., Biol. Med. Model. 2011, 8(18)). Cadherin-17 also maintains tissue integrity by interacting with integrins, which tightly link the extracellular matrix. It is primarily expressed in the human gastrointestinal (GI) tract and pancreas.

[0004] Cadherin-17 has been reported to be highly expressed at the DNA and protein levels in tumors, including colorectal, gastric, and pancreatic tumors (Takamura et al., Med Mol Morphol. 2013, 46:1-7). It plays an important role in regulating cancer metastasis and tumor growth. Cadherin-17-mediated tumor activity involves multiple signaling pathways. One of the most important mechanisms involves cadherin-17-integrin (intergin) interaction. The RGD motif of cadherin-17 has been shown to bind to α2β1 integrin and induce β1 integrin activation, thereby increasing cancer cell proliferation and adhesion (Bartomome et al., J Biol Chem. 2014, 289(50):34801-34914). Cadherin-17 also regulates cancer invasion through Wnt / β-catenin signaling in GI cancer (Qiu et al., PloS one 2013, 8(3)) and NFκB signaling pathway (Wang et al., Cancer biology & therapy. 2013, 14(3): 262-270). Cadherin-17 is a good cancer target due to its limited expression in normal tissues and high expression in various cancers.

[0005] Several targeted antibody drugs based on cadherin-17 have been studied in the field: two bispecific antibody drugs—BI905711 (Boehringer Ingelheim) and ARB202 (Arbele), anti-cadherin-17-based CAR therapy, and one monoclonal antibody drug: PA-0661 (ProAlt). PA-00661 is the only reported monoclonal antibody therapy that specifically detects the cadherin-17 RGD motif and is currently in preclinical trials. Therefore, there is still a need to develop new anti-cadherin-17 antibodies to better treat cancers with high cadherin-17 expression. Summary of the Invention

[0006] The object of the present disclosure is to provide a new antibody or antigen-binding fragment of the antibody (hereinafter also referred to as "antibody") that binds to cadherin-17, and further to provide a molecule comprising the antibody, and a pharmaceutical composition comprising the antibody, its antigen-binding fragment, or the molecule as an active ingredient.

[0007] To achieve the above-mentioned objectives, the present inventors have conducted extensive research and have achieved the present disclosure by developing new anti-cadherin-17 antibodies and molecules containing said antibodies.

[0008] Specifically, the present disclosure includes at least the following aspects: The present disclosure provides an anti-cadherin-17 antibody, or antigen-binding fragment thereof, comprising a heavy chain variable region comprising an HCDR1, HCDR2, and HCDR3 region, and a light chain variable region comprising an LCDR1, LCDR2, and LCDR3 region, wherein: a) HCDR1 having the amino acid sequence shown in SEQ ID NO: 01, SEQ ID NO: 02, SEQ ID NO: 03, SEQ ID NO: 04, SEQ ID NO: 05, SEQ ID NO: 06, SEQ ID NO: 07, SEQ ID NO: 08, SEQ ID NO: 09 or SEQ ID NO: 10; b) an HCDR2 having the amino acid sequence shown in SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20 or SEQ ID NO:21; c) an HCDR3 having the amino acid sequence shown in SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30 or SEQ ID NO:31; d) LCDR1 having the amino acid sequence shown in SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41 or SEQ ID NO:42; e) LCDR2 having the amino acid sequence set forth in SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46 or SEQ ID NO:47; f) An LCDR3 having the amino acid sequence shown in SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53 or SEQ ID NO:54.

[0009] In some embodiments, the heavy chain variable region sequence is HCDR1 having the amino acid sequence set forth in SEQ ID NO: 01, HCDR2 having the amino acid sequence set forth in SEQ ID NO: 11, and HCDR3 having the amino acid sequence set forth in SEQ ID NO: 22, respectively; or HCDR1 having the amino acid sequence shown in SEQ ID NO: 02, HCDR2 having the amino acid sequence shown in SEQ ID NO: 12, and HCDR3 having the amino acid sequence shown in SEQ ID NO: 23, respectively; or HCDR1 having the amino acid sequence set forth in SEQ ID NO: 03, HCDR2 having the amino acid sequence set forth in SEQ ID NO: 13, and HCDR3 having the amino acid sequence set forth in SEQ ID NO: 24, respectively; or HCDR1 having the amino acid sequence set forth in SEQ ID NO: 04, HCDR2 having the amino acid sequence set forth in SEQ ID NO: 14, and HCDR3 having the amino acid sequence set forth in SEQ ID NO: 25, respectively; or HCDR1 having the amino acid sequence set forth in SEQ ID NO: 05, HCDR2 having the amino acid sequence set forth in SEQ ID NO: 15, and HCDR3 having the amino acid sequence set forth in SEQ ID NO: 26, respectively; or HCDR1 having the amino acid sequence set forth in SEQ ID NO: 06, HCDR2 having the amino acid sequence set forth in SEQ ID NO: 16, and HCDR3 having the amino acid sequence set forth in SEQ ID NO: 27, respectively; or HCDR1 having the amino acid sequence set forth in SEQ ID NO: 07, HCDR2 having the amino acid sequence set forth in SEQ ID NO: 17, and HCDR3 having the amino acid sequence set forth in SEQ ID NO: 28, respectively; or HCDR1 having the amino acid sequence set forth in SEQ ID NO: 01, HCDR2 having the amino acid sequence set forth in SEQ ID NO: 18, and HCDR3 having the amino acid sequence set forth in SEQ ID NO: 22, respectively; or HCDR1 having the amino acid sequence set forth in SEQ ID NO: 08, HCDR2 having the amino acid sequence set forth in SEQ ID NO: 19, and HCDR3 having the amino acid sequence set forth in SEQ ID NO: 29, respectively; or HCDR1 having the amino acid sequence set forth in SEQ ID NO: 09, HCDR2 having the amino acid sequence set forth in SEQ ID NO: 20, and HCDR3 having the amino acid sequence set forth in SEQ ID NO: 30, or They each contain an HCDR1 having the amino acid sequence shown in SEQ ID NO:10, an HCDR2 having the amino acid sequence shown in SEQ ID NO:21, and an HCDR3 having the amino acid sequence shown in SEQ ID NO:31.

[0010] In some embodiments, the light chain variable region sequence is LCDR1 having the amino acid sequence set forth in SEQ ID NO:32, LCDR2 having the amino acid sequence set forth in SEQ ID NO:43, and LCDR3 having the amino acid sequence set forth in SEQ ID NO:48, respectively; or LCDR1 having the amino acid sequence set forth in SEQ ID NO:33, LCDR2 having the amino acid sequence set forth in SEQ ID NO:44, and LCDR3 having the amino acid sequence set forth in SEQ ID NO:49, respectively; or LCDR1 having the amino acid sequence set forth in SEQ ID NO:34, LCDR2 having the amino acid sequence set forth in SEQ ID NO:43, and LCDR3 having the amino acid sequence set forth in SEQ ID NO:50, respectively; or LCDR1 having the amino acid sequence set forth in SEQ ID NO:35, LCDR2 having the amino acid sequence set forth in SEQ ID NO:43, and LCDR3 having the amino acid sequence set forth in SEQ ID NO:49, respectively; or LCDR1 having the amino acid sequence set forth in SEQ ID NO:36, LCDR2 having the amino acid sequence set forth in SEQ ID NO:43, and LCDR3 having the amino acid sequence set forth in SEQ ID NO:51, respectively; or LCDR1 having the amino acid sequence set forth in SEQ ID NO:37, LCDR2 having the amino acid sequence set forth in SEQ ID NO:43, and LCDR3 having the amino acid sequence set forth in SEQ ID NO:52, respectively; or LCDR1 having the amino acid sequence set forth in SEQ ID NO:38, LCDR2 having the amino acid sequence set forth in SEQ ID NO:45, and LCDR3 having the amino acid sequence set forth in SEQ ID NO:53, respectively; or LCDR1 having the amino acid sequence set forth in SEQ ID NO: 39, LCDR2 having the amino acid sequence set forth in SEQ ID NO: 43, and LCDR3 having the amino acid sequence set forth in SEQ ID NO: 48, respectively; or LCDR1 having the amino acid sequence set forth in SEQ ID NO:40, LCDR2 having the amino acid sequence set forth in SEQ ID NO:46, and LCDR3 having the amino acid sequence set forth in SEQ ID NO:51, respectively; or LCDR1 having the amino acid sequence set forth in SEQ ID NO: 41, LCDR2 having the amino acid sequence set forth in SEQ ID NO: 43, and LCDR3 having the amino acid sequence set forth in SEQ ID NO: 52, respectively; or They comprise LCDR1 having the amino acid sequence shown in SEQ ID NO:42, LCDR2 having the amino acid sequence shown in SEQ ID NO:47, and LCDR3 having the amino acid sequence shown in SEQ ID NO:54, respectively.

[0011] In one specific embodiment, the antibody or antigen-binding fragment thereof is a) a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 having the amino acid sequences set forth in SEQ ID NO:01, SEQ ID NO:11 and SEQ ID NO:22, respectively, and a light chain variable region comprising LCDR1, LCDR2 and LCDR3 having the amino acid sequences set forth in SEQ ID NO:32, SEQ ID NO:43 and SEQ ID NO:48, respectively; or b) a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 having the amino acid sequences set forth in SEQ ID NO:02, SEQ ID NO:12 and SEQ ID NO:23, respectively, and a light chain variable region comprising LCDR1, LCDR2 and LCDR3 having the amino acid sequences set forth in SEQ ID NO:33, SEQ ID NO:44 and SEQ ID NO:49, respectively; or c) a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 having the amino acid sequences set forth in SEQ ID NO:03, SEQ ID NO:13 and SEQ ID NO:24, respectively, and a light chain variable region comprising LCDR1, LCDR2 and LCDR3 having the amino acid sequences set forth in SEQ ID NO:34, SEQ ID NO:43 and SEQ ID NO:50, respectively; or d) a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 having the amino acid sequences set forth in SEQ ID NO:04, SEQ ID NO:14 and SEQ ID NO:25, respectively, and a light chain variable region comprising LCDR1, LCDR2 and LCDR3 having the amino acid sequences set forth in SEQ ID NO:35, SEQ ID NO:43 and SEQ ID NO:49, respectively; or e) a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 having the amino acid sequences set forth in SEQ ID NO:05, SEQ ID NO:15 and SEQ ID NO:26, respectively, and a light chain variable region comprising LCDR1, LCDR2 and LCDR3 having the amino acid sequences set forth in SEQ ID NO:36, SEQ ID NO:43 and SEQ ID NO:51, respectively; or f) a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 having the amino acid sequences set forth in SEQ ID NO:06, SEQ ID NO:16 and SEQ ID NO:27, respectively, and a light chain variable region comprising LCDR1, LCDR2 and LCDR3 having the amino acid sequences set forth in SEQ ID NO:37, SEQ ID NO:43 and SEQ ID NO:52, respectively; or g) a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 having the amino acid sequences set forth in SEQ ID NO:07, SEQ ID NO:17 and SEQ ID NO:28, respectively, and a light chain variable region comprising LCDR1, LCDR2 and LCDR3 having the amino acid sequences set forth in SEQ ID NO:38, SEQ ID NO:45 and SEQ ID NO:53, respectively; or h) a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 having the amino acid sequences set forth in SEQ ID NO:01, SEQ ID NO:18 and SEQ ID NO:22, respectively, and a light chain variable region comprising LCDR1, LCDR2 and LCDR3 having the amino acid sequences set forth in SEQ ID NO:39, SEQ ID NO:43 and SEQ ID NO:48, respectively; or i) a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 having the amino acid sequences set forth in SEQ ID NO:08, SEQ ID NO:19 and SEQ ID NO:29, respectively, and a light chain variable region comprising LCDR1, LCDR2 and LCDR3 having the amino acid sequences set forth in SEQ ID NO:40, SEQ ID NO:46 and SEQ ID NO:51, respectively; or j) a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 having the amino acid sequences set forth in SEQ ID NO:09, SEQ ID NO:20 and SEQ ID NO:30, respectively, and a light chain variable region comprising LCDR1, LCDR2 and LCDR3 having the amino acid sequences set forth in SEQ ID NO:41, SEQ ID NO:43 and SEQ ID NO:52, respectively; or k) a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 having the amino acid sequences set forth in SEQ ID NO:10, SEQ ID NO:21 and SEQ ID NO:31, respectively, and a light chain variable region comprising LCDR1, LCDR2 and LCDR3 having the amino acid sequences set forth in SEQ ID NO:42, SEQ ID NO:47 and SEQ ID NO:54, respectively.

[0012] In one specific embodiment, the antibody or antigen-binding fragment thereof is a monoclonal antibody or antigen-binding fragment thereof, a polyclonal antibody or antigen-binding fragment thereof, a bispecific antibody or antigen-binding fragment thereof, a murine antibody or antigen-binding fragment thereof, a chimeric antibody or antigen-binding fragment thereof, a humanized antibody or antigen-binding fragment thereof, a recombinant antibody or antigen-binding fragment thereof, or a human antibody or antigen-binding fragment thereof.

[0013] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising an amino acid sequence selected from SEQ ID NOs: 55-65, or a sequence having at least 80%, 85%, 90%, 95%, or 99% sequence identity thereto, and / or a light chain variable region comprising an amino acid sequence selected from SEQ ID NOs: 66-76, or a sequence having at least 80%, 85%, 90%, 95%, or 99% sequence identity thereto.

[0014] In some embodiments, the antibody or antigen-binding fragment thereof comprises: a) a heavy chain variable region having an amino acid sequence set forth in SEQ ID NO:55, or an amino acid sequence having at least 80%, 85%, 90%, 95%, or 99% sequence identity thereto, and / or a light chain variable region having an amino acid sequence set forth in SEQ ID NO:66, or an amino acid sequence having at least 80%, 85%, 90%, 95%, or 99% sequence identity thereto; or b) a heavy chain variable region having an amino acid sequence set forth in SEQ ID NO:56, or an amino acid sequence having at least 80%, 85%, 90%, 95%, or 99% sequence identity thereto, and / or a light chain variable region having an amino acid sequence set forth in SEQ ID NO:67, or an amino acid sequence having at least 80%, 85%, 90%, 95%, or 99% sequence identity thereto; or c) a heavy chain variable region having an amino acid sequence set forth in SEQ ID NO:57, or an amino acid sequence having at least 80%, 85%, 90%, 95%, or 99% sequence identity thereto, and / or a light chain variable region having an amino acid sequence set forth in SEQ ID NO:68, or an amino acid sequence having at least 80%, 85%, 90%, 95%, or 99% sequence identity thereto; or d) a heavy chain variable region having an amino acid sequence set forth in SEQ ID NO:58, or an amino acid sequence having at least 80%, 85%, 90%, 95%, or 99% sequence identity thereto, and / or a light chain variable region having an amino acid sequence set forth in SEQ ID NO:69, or an amino acid sequence having at least 80%, 85%, 90%, 95%, or 99% sequence identity thereto; or e) a heavy chain variable region having an amino acid sequence set forth in SEQ ID NO:59, or an amino acid sequence having at least 80%, 85%, 90%, 95%, or 99% sequence identity thereto, and / or a light chain variable region having an amino acid sequence set forth in SEQ ID NO:70, or an amino acid sequence having at least 80%, 85%, 90%, 95%, or 99% sequence identity thereto; or f) a heavy chain variable region having an amino acid sequence set forth in SEQ ID NO:60, or an amino acid sequence having at least 80%, 85%, 90%, 95%, or 99% sequence identity thereto, and / or a light chain variable region having an amino acid sequence set forth in SEQ ID NO:71, or an amino acid sequence having at least 80%, 85%, 90%, 95%, or 99% sequence identity thereto; or g) a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO:61, or an amino acid sequence having at least 80%, 85%, 90%, 95%, or 99% sequence identity thereto, and / or a light chain variable region having the amino acid sequence set forth in SEQ ID NO:72, or an amino acid sequence having at least 80%, 85%, 90%, 95%, or 99% sequence identity thereto; or h) a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO:62, or an amino acid sequence having at least 80%, 85%, 90%, 95%, or 99% sequence identity thereto, and / or a light chain variable region having the amino acid sequence set forth in SEQ ID NO:73, or an amino acid sequence having at least 80%, 85%, 90%, 95%, or 99% sequence identity thereto; or i) a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO:63, or an amino acid sequence having at least 80%, 85%, 90%, 95% or 99% sequence identity thereto, and / or a light chain variable region having the amino acid sequence set forth in SEQ ID NO:74, or an amino acid sequence having at least 80%, 85%, 90%, 95% or 99% sequence identity thereto; or j) a heavy chain variable region having an amino acid sequence set forth in SEQ ID NO:64, or an amino acid sequence having at least 80%, 85%, 90%, 95%, or 99% sequence identity thereto, and / or a light chain variable region having an amino acid sequence set forth in SEQ ID NO:75, or an amino acid sequence having at least 80%, 85%, 90%, 95%, or 99% sequence identity thereto; or k) comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO:65, or an amino acid sequence having at least 80%, 85%, 90%, 95%, or 99% sequence identity thereto, and / or a light chain variable region having the amino acid sequence set forth in SEQ ID NO:76, or an amino acid sequence having at least 80%, 85%, 90%, 95%, or 99% sequence identity thereto.

[0015] In one specific embodiment, the antibody or antigen-binding fragment thereof comprises: a) a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO:55 and / or a light chain variable region having the amino acid sequence set forth in SEQ ID NO:66; or b) a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO:56 and / or a light chain variable region having the amino acid sequence set forth in SEQ ID NO:67; or c) a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO:57 and / or a light chain variable region having the amino acid sequence set forth in SEQ ID NO:68; or d) a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO:58 and / or a light chain variable region having the amino acid sequence set forth in SEQ ID NO:69; or e) a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO:59 and / or a light chain variable region having the amino acid sequence set forth in SEQ ID NO:70; or f) a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO:60 and / or a light chain variable region having the amino acid sequence set forth in SEQ ID NO: or g) a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO:61 and / or a light chain variable region having the amino acid sequence set forth in SEQ ID NO:72; or h) a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO:62 and / or a light chain variable region having the amino acid sequence set forth in SEQ ID NO:73; or i) a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO:63 and / or a light chain variable region having the amino acid sequence set forth in SEQ ID NO:74; or j) a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO:64 and / or a light chain variable region having the amino acid sequence set forth in SEQ ID NO:75; or k) a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO:65 and / or a light chain variable region having the amino acid sequence set forth in SEQ ID NO:76.

[0016] In some embodiments, the antibody or antigen-binding fragment thereof comprises: a) a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO:55 and a light chain variable region having the amino acid sequence set forth in SEQ ID NO:66; or b) a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO:56 and a light chain variable region having the amino acid sequence set forth in SEQ ID NO:67; or c) a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO:57 and a light chain variable region having the amino acid sequence set forth in SEQ ID NO:68; or d) a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO:58 and a light chain variable region having the amino acid sequence set forth in SEQ ID NO:69; or e) a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO:59 and a light chain variable region having the amino acid sequence set forth in SEQ ID NO:70; or f) a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO:60 and a light chain variable region having the amino acid sequence set forth in SEQ ID NO:71; or g) a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO:61; or h) a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO:62 and a light chain variable region having the amino acid sequence set forth in SEQ ID NO:73; or i) a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO:63 and a light chain variable region having the amino acid sequence set forth in SEQ ID NO:74; or j) a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO:64 and a light chain variable region having the amino acid sequence set forth in SEQ ID NO:75; or k) a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO:65 and a light chain variable region having the amino acid sequence set forth in SEQ ID NO:76.

[0017] In some embodiments, the antibody or antigen-binding fragment thereof further comprises a human antibody constant region, and preferably the heavy chain constant region of the human antibody constant region is selected from the constant regions of human IgG1, IgG2, IgG3, and IgG4 and common variants thereof, and the light chain constant region of the human antibody constant region is selected from the kappa and lambda chain constant regions of human antibodies and common variants thereof. More preferably, the full-length antibody comprises the human antibody heavy chain constant region of SEQ ID NO:99 or an amino acid sequence having at least 80%, 85%, 90%, 95%, or 99% sequence identity thereto, and the human light chain constant region of SEQ ID NO:100 or an amino acid sequence having at least 80%, 85%, 90%, 95%, or 99% sequence identity thereto. Even more preferably, the full-length antibody comprises the human antibody heavy chain constant region of SEQ ID NO:99 and the human light chain constant region of SEQ ID NO:100.

[0018] In one specific embodiment, the antibody or antigen-binding fragment thereof is a) a heavy chain having an amino acid sequence set forth in SEQ ID NO:77, or an amino acid sequence having at least 80%, 85%, 90%, 95%, or 99% sequence identity thereto, and a light chain having an amino acid sequence set forth in SEQ ID NO:78, or an amino acid sequence having at least 80%, 85%, 90%, 95%, or 99% sequence identity thereto; or b) a heavy chain having an amino acid sequence set forth in SEQ ID NO:79, or an amino acid sequence having at least 80%, 85%, 90%, 95% or 99% sequence identity thereto, and a light chain having an amino acid sequence set forth in SEQ ID NO:80, or an amino acid sequence having at least 80%, 85%, 90%, 95% or 99% sequence identity thereto; or c) a heavy chain having an amino acid sequence set forth in SEQ ID NO:81, or an amino acid sequence having at least 80%, 85%, 90%, 95%, or 99% sequence identity thereto, and a light chain having an amino acid sequence set forth in SEQ ID NO:82, or an amino acid sequence having at least 80%, 85%, 90%, 95%, or 99% sequence identity thereto; or d) a heavy chain having an amino acid sequence set forth in SEQ ID NO:83, or an amino acid sequence having at least 80%, 85%, 90%, 95% or 99% sequence identity thereto, and a light chain having an amino acid sequence set forth in SEQ ID NO:84, or an amino acid sequence having at least 80%, 85%, 90%, 95% or 99% sequence identity thereto; or e) a heavy chain having an amino acid sequence set forth in SEQ ID NO:85, or an amino acid sequence having at least 80%, 85%, 90%, 95% or 99% sequence identity thereto, and a light chain having an amino acid sequence set forth in SEQ ID NO:86, or an amino acid sequence having at least 80%, 85%, 90%, 95% or 99% sequence identity thereto; or f) a heavy chain having an amino acid sequence set forth in SEQ ID NO:87, or an amino acid sequence having at least 80%, 85%, 90%, 95% or 99% sequence identity thereto, and a light chain having an amino acid sequence set forth in SEQ ID NO:88, or an amino acid sequence having at least 80%, 85%, 90%, 95% or 99% sequence identity thereto; or g) a heavy chain having an amino acid sequence set forth in SEQ ID NO:89, or an amino acid sequence having at least 80%, 85%, 90%, 95% or 99% sequence identity thereto, and a light chain having an amino acid sequence set forth in SEQ ID NO:90, or an amino acid sequence having at least 80%, 85%, 90%, 95% or 99% sequence identity thereto; or h) a heavy chain having an amino acid sequence set forth in SEQ ID NO:91, or an amino acid sequence having at least 80%, 85%, 90%, 95% or 99% sequence identity thereto, and a light chain having an amino acid sequence set forth in SEQ ID NO:92, or an amino acid sequence having at least 80%, 85%, 90%, 95% or 99% sequence identity thereto; or i) a heavy chain having an amino acid sequence set forth in SEQ ID NO:93, or an amino acid sequence having at least 80%, 85%, 90%, 95% or 99% sequence identity thereto, and a light chain having an amino acid sequence set forth in SEQ ID NO:94, or an amino acid sequence having at least 80%, 85%, 90%, 95% or 99% sequence identity thereto; or j) a heavy chain having an amino acid sequence set forth in SEQ ID NO:95, or an amino acid sequence having at least 80%, 85%, 90%, 95% or 99% sequence identity thereto, and a light chain having an amino acid sequence set forth in SEQ ID NO:96, or an amino acid sequence having at least 80%, 85%, 90%, 95% or 99% sequence identity thereto; or k) A heavy chain having an amino acid sequence set forth in SEQ ID NO:97, or an amino acid sequence having at least 80%, 85%, 90%, 95% or 99% sequence identity thereto, and a light chain having an amino acid sequence set forth in SEQ ID NO:98, or an amino acid sequence having at least 80%, 85%, 90%, 95% or 99% sequence identity thereto.

[0019] In one specific embodiment, the antibody or antigen-binding fragment thereof comprises: a) a heavy chain having the amino acid sequence set forth in SEQ ID NO:77 and a light chain having the amino acid sequence set forth in SEQ ID NO:78; or b) a heavy chain having the amino acid sequence set forth in SEQ ID NO:79 and a light chain having the amino acid sequence set forth in SEQ ID NO:80; or c) a heavy chain having the amino acid sequence set forth in SEQ ID NO:81 and a light chain having the amino acid sequence set forth in SEQ ID NO:82; or d) a heavy chain having the amino acid sequence set forth in SEQ ID NO:83 and a light chain having the amino acid sequence set forth in SEQ ID NO:84; or e) a heavy chain having the amino acid sequence set forth in SEQ ID NO:85 and a light chain having the amino acid sequence set forth in SEQ ID NO:86; or f) a heavy chain having the amino acid sequence set forth in SEQ ID NO:87 and a light chain having the amino acid sequence set forth in SEQ ID NO:88; or g) a heavy chain having the amino acid sequence set forth in SEQ ID NO:89 and a light chain having the amino acid sequence set forth in SEQ ID NO:90; or h) a heavy chain having the amino acid sequence set forth in SEQ ID NO: or i) a heavy chain having the amino acid sequence set forth in SEQ ID NO:93 and a light chain having the amino acid sequence set forth in SEQ ID NO:94; or j) a heavy chain having the amino acid sequence set forth in SEQ ID NO:95 and a light chain having the amino acid sequence set forth in SEQ ID NO:96; or k) a heavy chain having the amino acid sequence set forth in SEQ ID NO:97 and a light chain having the amino acid sequence set forth in SEQ ID NO:98.

[0020] In one specific embodiment, the antigen-binding fragment is selected from a Fab, a Fab', a F(ab')2, a variable fragment (Fv), a single-chain variable fragment (scFv), a dimerization domain V (diabody), a disulfide-stabilized Fv (dsFv), and a CDR-containing peptide.

[0021] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising an HCDR1, HCDR2, and HCDR3 region, and a light chain variable region comprising an LCDR1, LCDR2, and LCDR3 region, wherein: the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 having the amino acid sequences set forth in SEQ ID NO:05, SEQ ID NO:15, and SEQ ID NO:26, respectively; and the light chain variable region comprises LCDR1, LCDR2, and LCDR3 having the amino acid sequences set forth in SEQ ID NO:36, SEQ ID NO:43, and SEQ ID NO:51, respectively; or the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 having the amino acid sequences set forth in SEQ ID NO: 07, SEQ ID NO: 17, and SEQ ID NO: 28, respectively; the light chain variable region comprises LCDR1, LCDR2, and LCDR3 having the amino acid sequences set forth in SEQ ID NO: 38, SEQ ID NO: 45, and SEQ ID NO: 53, respectively; or the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 having the amino acid sequences set forth in SEQ ID NO: 10, SEQ ID NO: 21, and SEQ ID NO: 31, respectively; and the light chain variable region comprises LCDR1, LCDR2, and LCDR3 having the amino acid sequences set forth in SEQ ID NO: 42, SEQ ID NO: 47, and SEQ ID NO: 54, respectively; wherein the antibody or antigen-binding fragment thereof specifically binds to human and cynomolgus monkey CDH17, and preferably the antibody or antigen-binding fragment thereof specifically binds to K D and more preferably, the antibody or antigen-binding fragment thereof binds to both human and cynomolgus monkey CDH17 with a K of ≦1000 nM. Dand more preferably, the antibody or antigen-binding fragment thereof has the following properties: a) a K of 15 nM or less; D and b) binds to human CDH17 with a K of 15 nM or less. D It binds to cynomolgus monkey CDH17.

[0022] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising an HCDR1, HCDR2, and HCDR3 region, and a light chain variable region comprising an LCDR1, LCDR2, and LCDR3 region, wherein: the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 having the amino acid sequences set forth in SEQ ID NO: 05, SEQ ID NO: 15, and SEQ ID NO: 26, respectively; the light chain variable region comprises LCDR1, LCDR2, and LCDR3 having the amino acid sequences set forth in SEQ ID NO: 36, SEQ ID NO: 43, and SEQ ID NO: 51, respectively; or the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 having the amino acid sequences set forth in SEQ ID NO: 07, SEQ ID NO: 17, and SEQ ID NO: 28, respectively; and the light chain variable region comprises LCDR1, LCDR2, and LCDR3 having the amino acid sequences set forth in SEQ ID NO: 38, SEQ ID NO: 45, and SEQ ID NO: 53, respectively; or the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 having the amino acid sequences set forth in SEQ ID NO:10, SEQ ID NO:21, and SEQ ID NO:31, respectively; and the light chain variable region comprises LCDR1, LCDR2, and LCDR3 having the amino acid sequences set forth in SEQ ID NO:42, SEQ ID NO:47, and SEQ ID NO:54, respectively; wherein the antibody or antigen-binding fragment thereof has the following properties: a) a K D and b) binds to human CDH17 with a K of 3 nM or less. D and binds to cynomolgus monkey CDH17, preferably wherein the antibody or antigen-binding fragment thereof has the following properties: a) a K of 2 nM or less D and b) binds to human CDH17 with a K of 3 nM or less. D It binds to cynomolgus monkey CDH17.

[0023] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising an HCDR1, HCDR2, and HCDR3 region, and a light chain variable region comprising an LCDR1, LCDR2, and LCDR3 region, wherein: the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 having the amino acid sequences set forth in SEQ ID NO:10, SEQ ID NO:21, and SEQ ID NO:31, respectively; and the light chain variable region comprises LCDR1, LCDR2, and LCDR3 having the amino acid sequences set forth in SEQ ID NO:42, SEQ ID NO:47, and SEQ ID NO:54, respectively; wherein the antibody or antigen-binding fragment thereof specifically binds to human and cynomolgus monkey CDH17, and preferably the antibody or antigen-binding fragment thereof specifically binds to K D and more preferably, the antibody or antigen-binding fragment thereof binds to both human and cynomolgus monkey CDH17 with a K of ≦1000 nM. Dand more preferably, the antibody or antigen-binding fragment thereof has the following properties: a) a K of 15 nM or less; D and b) binds to human CDH17 with a K of 15 nM or less. D It binds to cynomolgus monkey CDH17.

[0024] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising an HCDR1, HCDR2, and HCDR3 region, and a light chain variable region comprising an LCDR1, LCDR2, and LCDR3 region, wherein: the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 having the amino acid sequences set forth in SEQ ID NO:10, SEQ ID NO:21, and SEQ ID NO:31, respectively; and the light chain variable region comprises LCDR1, LCDR2, and LCDR3 having the amino acid sequences set forth in SEQ ID NO:42, SEQ ID NO:47, and SEQ ID NO:54, respectively; wherein the antibody or antigen-binding fragment thereof has the following properties: a) a K D and b) binds to human CDH17 with a K of 3 nM or less. D and binds to cynomolgus monkey CDH17, preferably wherein the antibody or antigen-binding fragment thereof has the following properties: a) a K of 2 nM or less D and b) binds to human CDH17 with a K of 3 nM or less. D It binds to cynomolgus monkey CDH17.

[0025] In some embodiments, the disclosure provides an isolated anti-CDH17 antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof competes with the above-described antibody or antigen-binding fragment thereof for binding to CDH17.

[0026] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 regions, and a light chain variable region comprising LCDR1, LCDR2, and LCDR3 regions, wherein the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 having the amino acid sequences set forth in SEQ ID NO:05, SEQ ID NO:15, and SEQ ID NO:26, respectively, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3 having the amino acid sequences set forth in SEQ ID NO:36, SEQ ID NO:43, and SEQ ID NO:51, respectively; or the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 having the amino acid sequences set forth in SEQ ID NO:07, SEQ ID NO:17, and SEQ ID NO:28, respectively, and the light chain variable region comprises LCDR1, HCDR2, and HCDR3 having the amino acid sequences set forth in SEQ ID NO:38, SEQ ID NO:45, and SEQ ID NO:51, respectively. or the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 having the amino acid sequences set forth in SEQ ID NO:10, SEQ ID NO:21, and SEQ ID NO:31, respectively, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3 having the amino acid sequences set forth in SEQ ID NO:42, SEQ ID NO:47, and SEQ ID NO:54, respectively, wherein the antibody or antigen-binding fragment thereof competes with antibody 20B4, 14B12, or 10E11 for binding to human CDH17.

[0027] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 regions, and a light chain variable region comprising LCDR1, LCDR2, and LCDR3 regions, wherein the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 having the amino acid sequences set forth in SEQ ID NO:10, SEQ ID NO:21, and SEQ ID NO:31, respectively, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3 having the amino acid sequences set forth in SEQ ID NO:42, SEQ ID NO:47, and SEQ ID NO:54, respectively, and wherein the antibody or antigen-binding fragment thereof competes with antibody 20B4 for binding to human CDH17.

[0028] In some embodiments, the present disclosure further provides an isolated nucleic acid molecule encoding any of the above-mentioned antibodies or antigen-binding fragments thereof.

[0029] In one aspect, the present disclosure provides a recombinant vector comprising the isolated nucleic acid molecule described above.

[0030] In another aspect, the present disclosure further provides a host cell comprising the above-described recombinant vector, wherein said host cell is selected from a prokaryotic cell and a eukaryotic cell, preferably a eukaryotic cell, and more preferably a mammalian cell.

[0031] In one aspect, the present disclosure further provides a therapeutic conjugate comprising the above-described anti-CDH17 antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof is conjugated to one or more therapeutic agents, preferably a cytotoxic agent.

[0032] In one aspect, the present disclosure further provides a diagnostic conjugate comprising the above-described anti-CDH17 antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof is conjugated to one or more diagnostic agents, preferably, the diagnostic agent may be, but is not limited to, a detectable label, an enzyme, a metal-containing material, a fluorescent material, a luminescent material, a colored material, or a quantum dot.

[0033] In one aspect, the present disclosure further provides a bispecific antibody comprising a first binding domain that specifically binds to CDH17 and a second binding domain that specifically binds to a second target protein, wherein the first binding domain is an anti-CDH17 antibody or an antigen-binding fragment thereof as described above.

[0034] In one aspect, the present disclosure further provides a method for producing the anti-CDH17 antibody or antigen-binding fragment thereof described above, wherein the method comprises culturing the host cell in a culture medium to produce and accumulate the antibody or antigen-binding fragment thereof, and recovering the antibody or antigen-binding fragment thereof from the culture.

[0035] In one aspect, the present disclosure further provides a method for immunologically detecting or measuring CDH17, wherein the method comprises detecting or measuring CDH17 by contacting a sample with the above-described anti-CDH17 antibody or antigen-binding fragment thereof, or diagnostic conjugate.

[0036] In one aspect, the present disclosure further provides a method for diagnosing a disease associated with human CDH17-positive cells, wherein the method comprises detecting or measuring CDH17-positive cells by contacting a sample with the above-described anti-CDH17 antibody or antigen-binding fragment thereof, or diagnostic conjugate.

[0037] In one aspect, the disclosure further provides a pharmaceutical composition comprising a therapeutically or prophylactically effective amount of the above-described anti-CDH17 antibody or antigen-binding fragment thereof, or bispecific antibody or therapeutic conjugate, and one or more pharmaceutically acceptable carriers, diluents, or excipients.

[0038] In one aspect, the present disclosure further provides a method for treating or preventing a CDH17-mediated disease or condition, comprising administering to a subject in need of treatment or prevention of the disease or condition a therapeutically or prophylactically effective amount of the above-described anti-CDH17 antibody or antigen-binding fragment thereof, or bispecific antibody, or therapeutic conjugate, or pharmaceutical composition.

[0039] In some embodiments, the disease is cancer, preferably, the cancer is colorectal cancer, gastric cancer, pancreatic cancer, liver cancer, gallbladder cancer, ovarian cancer, or bile duct cancer.

[0040] In another aspect, the present disclosure provides a kit comprising an apparatus for carrying out a method of the present disclosure. In one specific embodiment, the present disclosure provides a kit comprising at least one container, the container comprising the anti-CDH17 antibody or antigen-binding fragment thereof, therapeutic conjugate, or pharmaceutical composition. The kit may further comprise instructions for manufacturing, administering, and / or delivering the anti-CDH17 antibody or antigen-binding fragment thereof, therapeutic conjugate, or pharmaceutical composition.

[0041] Advantages of the present disclosure: The antibody or antigen-binding fragment thereof can specifically recognize / bind to human and cynomolgus monkey CDH17 with desired affinity, and the antibody or antigen-binding fragment thereof exhibits sufficient internalization in human cells expressing CDH17, effectively binds to CDH17, and mediates killing of CDH17-expressing cells. [Brief explanation of the drawings]

[0042] [Figure 1]Figure 1 shows the characterization of in vitro binding of hybridoma clones to AsPC1 (A), GP2d (B), and SW480 (C) cells. [Figure 2] FIG. 1 shows characterization of the cell internalization activity of selected hybridoma clones by indirect cytotoxicity assay. [Figure 3] Flow cytometric analysis and confirmation of in vitro binding characterization of anti-CDH17 recombinant antibodies to AsPC1 (A) and SW480 cells (B), with HBMAB81 as a positive control and B12 as an isotype control antibody (C). [Figure 4] 1 shows a dose-response curve of anti-CDH17 recombinant antibody internalization by indirect cytotoxicity assay. [Figure 5] FIG. 1 shows Octet characterization of the binding activity of HBMAB81 to cross-species CDH17, homologue CDH6, or CDH16. [Figure 6] FIG. 1 shows Octet characterization of the binding activity of anti-CDH17 clone 10E11 to cross-species CDH17, homologue CDH6, or CDH16. [Figure 7] FIG. 1 shows Octet characterization of the binding activity of anti-CDH17 clone 14B12 to cross-species CDH17, homologue CDH6, or CDH16. [Figure 8] FIG. 1 shows Octet characterization of the binding activity of anti-CDH17 clone 20B4 to cross-species CDH17, homologue CDH6, or CDH16. [Figure 9] Competitive binding: (A) 20B4; (B) HBMAB81. DETAILED DESCRIPTION OF THE INVENTION

[0043] Definition of Terms Before describing the present disclosure in detail below, it is to be understood that the present disclosure is not limited to the particular methods, formats, and reagents described herein, as these may vary. It is further understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention, which will be limited only by the appended claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0044] For the purposes of interpretation of the specification, the following definitions will apply, and where appropriate, terms used in the singular will also include the plural and vice versa. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0045] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the disclosure. Unless the context clearly dictates otherwise, the open-ended phrases "include" and "including" should be interpreted to include unrecited structural elements or method steps, but it should be noted that the open-ended phrase also encompasses only the recited elements and method steps (i.e., it also encompasses the open-ended phrase "consisting of").

[0046] The terms "CDH17" and "CDH17 antigen" are used interchangeably herein and include any variant, isotype, and species homolog of human CDH17 that is naturally expressed by a cell or expressed on a cell transfected with the CDH17 gene.

[0047] "CDH17" is herein broadly construed as a target and is intended to encompass various forms of CDH17 molecules at different stages in mammals (e.g., humans), including, but not limited to, molecules produced during the amplification, replication, transcription, splicing, translation, and modification of the CDH17 gene (e.g., precursor CDH17, mature CDH17, membrane-expressed CDH17, CDH17 splice variants, modified CDH17, or fragments thereof). The term also encompasses CDH17 artificially prepared or expressed in vitro.

[0048] The term "antibody" referred to herein includes whole antibodies and any antigen-binding fragment (ie, "antigen-binding portion") or single chains thereof.

[0049] The term "antibody" refers to an immunoglobulin, or antigen-binding portion thereof, comprising at least two heavy (H) chains and two light (L) chains linked together via disulfide bonds. Each heavy chain consists of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region (CH). Each light chain consists of a light chain variable region (abbreviated herein as VL) and a light chain constant region (CL). The VH and VL regions may be further subdivided into hypervariable regions called complementarity-determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). Each heavy and light chain has three complementarity-determining regions, designated HCDR1, HCDR2, HCDR3, and LCDR1, LCDR2, LCDR3, respectively. Each VH and VL consists of three CDRs and four FRs, arranged from the amino acid terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens or their epitopes. The constant regions of antibodies mediate the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component of the classical complement system (C1q). Antibodies are classified based on the amino acid sequence of their heavy chain constant regions. The five major classes or isotypes of antibodies are IgA, IgD, IgE, IgG, and IgM, and the corresponding heavy chains are μ, δ, γ, α, and ε. Igs of the same class may be further divided into various subclasses based on differences in the amino acid composition of their hinge regions and the number and location of heavy chain disulfide bonds; for example, IgG may be divided into IgG1, IgG2, IgG3, and IgG4. Generally, the heavy chain constant region of an IgG antibody contains three constant domains, namely, CH1, CH2, and CH3, and the light chain constant region contains one constant domain, namely, CL. Light chains can be further divided into kappa chains and lambda chains depending on the constant region. Each of the five classes of Ig can have either kappa chains or lambda chains.

[0050] 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 or an epitope thereof (e.g., CDH17). It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed by the term "antigen-binding fragment" of an antibody include: (i) a Fab fragment, which is a monovalent fragment consisting of the VL, VH, CL, and CH1 domains; (ii) an F(ab')2 fragment, which is a bivalent fragment comprising two Fab fragments linked via a disulfide bond at the hinge region; (iii) an Fd fragment consisting of the VH and CH1 domains; (iv) an Fv fragment consisting of the VL and VH domains of a single arm of an antibody; (v) a dAb fragment (Ward et al., (1989) Nature 341: 544-546) consisting of the VH domain; (vi) an isolated complementarity-determining region (CDR); and (vii) a combination of two or more isolated CDRs, which may optionally be linked via a synthetic linker. Although the two domains, VL and VH, of an Fv fragment are encoded by separate genes, they can be linked via a synthetic linker using recombinant methods, thereby producing a single protein chain, in which the VL and VH regions pair to form a monovalent molecule (referred to as a single-chain Fv (scFv)); see, for example, Bird et al. (1988) Science 242: 423-426 and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85: 5879-5883). Such single-chain antibodies are also intended to be covered by the term "antigen-binding portion" of an antibody. In some examples, the term "antigen-binding fragment" further includes the dimerization domain V (diabodies) and disulfide-stabilized Fvs (dsFv).

[0051] As used herein, the term "human antibody" is intended to refer to an antibody having variable and constant regions derived from human germline immunoglobulin sequences. The human antibodies of the present disclosure may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific induction in vitro or by somatic mutation in vivo).

[0052] "Humanized antibodies" include antibodies having human immunoglobulin sequence variable and constant regions, in which CDR sequences from the germline of another mammalian species (e.g., a mouse) have been grafted onto human framework sequences.

[0053] "Chimeric antibodies" generally refer to antibody molecules composed of antibody domain fragments from different sources, for example, antibodies in which a murine variable region has been inserted into a human constant region.

[0054] As used herein, the term "recombinant human antibody" includes all human antibodies prepared, expressed, produced, or isolated by recombinant means, such as (a) antibodies isolated from human immunoglobulin gene transgenic or transchromosomic animals (e.g., mice) or hybridomas prepared therefrom, (b) antibodies isolated from host cells that have been transformed to express the antibody, e.g., those isolated from transfectomas, (c) antibodies isolated from recombinant combinatorial human antibody libraries, and (d) antibodies prepared, expressed, produced, or isolated by any other means involving splicing human immunoglobulin gene sequences into other DNA sequences. Such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences. However, in some embodiments, such recombinant human antibodies can be subjected to in vitro mutagenesis (or, when human Ig sequence transgenic animals are used, to in vivo somatic mutagenesis) so that the amino acid sequences of the VH and VL regions of the recombinant antibodies are sequences that are derived from and closely related to human germline VH and VL sequences, but which may not naturally occur within the human antibody germline repertoire in vivo.

[0055] The term "CDR" refers to one of the six hypervariable regions within an antibody variable domain, which is primarily responsible for antigen binding. One of the most commonly used definitions of the six CDRs is provided by Kabat EA et al. (1991) Sequences of proteins of immunological interest. NIH Publication 91-3242. As used herein, the Kabat definition of CDR applies only to CDR1, CDR2, and CDR3 (LCDR1, LCDR2, LCDR3 or L1, L2, L3) of the light chain variable domain and CDR1, CDR2, and CDR3 (HCDR1, HCDR2, HCDR3 or H1, H2, H3) of the heavy chain variable domain. CDRs can also be defined based on various other numbering systems known in the art, such as the Chothia numbering system (Chothia & Lesk (1987) J. Mol. Biol. 196:901-917, Chothia et al. (1989) Nature 342:878-883), the IMGT numbering system (Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003), or the Martin related study definition (Martin ACR, Cheetham JC, Rees AR (1989) Modeling antibody hypervariable loops: A combined algorithm. Proc Natl Acad Sci USA 86:9268-9272), and the correspondence between different numbering systems is well known to those skilled in the art (e.g., Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003). One of skill in the art can readily identify the CDRs defined by each numbering system.

[0056] "Conservative modifications" or "conservative substitutions or replacements" or "conservative variants" refer to the substitution of amino acids in a protein with other amino acids having similar properties (e.g., charge, side chain size, hydrophobicity / hydrophilicity, main-chain conformation, and rigidity) that can frequently be substituted without altering the biological activity of the protein. Those skilled in the art will understand that single amino acid substitutions in non-essential regions of a polypeptide generally do not alter biological activity (see, e.g., Watson et al. (1987) Molecular Biology of the Gene, The Benjamin / Cummings Pub. Co., p. 224, (4th ed.)). Furthermore, substitutions of amino acids that are similar in structure or function are unlikely to affect biological activity.

[0057] In some embodiments, the anti-CDH17 antibodies or fragments thereof of the present disclosure encompass antibodies or fragments thereof that have a light chain variable region, a heavy chain variable region, or a post-translational modification on the light chain or heavy chain.

[0058] In some embodiments, antibodies herein are modified to increase or decrease the degree of glycosylation. Addition or deletion of glycosylation sites in an antibody can be easily accomplished by altering the amino acid sequence to introduce or remove one or more glycosylation sites. In some applications, modification of unnecessary glycosylation sites can be useful; for example, removing a fucose module improves modification of antibody-dependent cellular cytotoxicity (ADCC) function (Shield et al., (2002) JBC 277:26733). In other applications, galactosylation modifications can be performed to modify complement-dependent cytotoxicity (CDC).

[0059] In some embodiments, one or more amino acid modifications can be introduced into the Fc region of an antibody according to the present invention to produce an Fc region variant that can improve the efficacy of the antibody, for example, in treating cancer or a proliferative disorder in cells. The Fc region variant may comprise a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3, or IgG4 Fc region) that comprises an amino acid modification (e.g., a substitution) at one or more amino acid positions.

[0060] As used herein, the term "nucleic acid molecule" refers to DNA molecules and RNA molecules. Nucleic acid molecules can be single-stranded or double-stranded, but preferably are double-stranded DNA. A nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, a promoter or enhancer is considered to be operably linked to a coding sequence if the promoter or enhancer affects the transcription of the coding sequence.

[0061] The method for producing a nucleic acid is a common method in the art, and preferably includes the steps of obtaining a nucleic acid molecule of the protein by gene cloning technology or obtaining a nucleic acid molecule encoding the protein by artificial total sequence synthesis.

[0062] As known to those skilled in the art, a polynucleotide homolog can be produced by appropriately introducing substitutions, deletions, modifications, insertions, or additions into a base sequence encoding a protein amino acid sequence. A polynucleotide homolog of the present disclosure can be produced by substituting, deleting, or adding one or more bases encoding the protein sequence gene, as long as antibody activity is maintained.

[0063] The term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. In one embodiment, a vector is a "plasmid," which refers to a circular double-stranded DNA loop into which other DNA fragments can be ligated. In another embodiment, the vector is a viral vector, wherein other DNA fragments can be ligated into the viral genome. The vectors disclosed herein can either autonomously replicate in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors), or can be integrated into the genome of the host cell upon introduction into the host cell and replicate along with the host genome (e.g., non-episomal mammalian vectors).

[0064] The recombinant expression vector can be constructed by ligating the nucleic acid molecule of the present disclosure into various expression vectors, which may be any of a number of common vectors in the art, as long as they are capable of carrying the nucleic acid molecule. The vectors preferably include various plasmids, cosmids, phages, or viral vectors.

[0065] As used herein, the term "transfectoma" includes recombinant eukaryotic host cells for expressing antibodies, such as CHO cells, NS / 0 cells, HEK293 cells, plant cells or fungi (including yeast cells).

[0066] The sequence of a DNA molecule encoding an antibody or fragment thereof according to the present disclosure can be obtained by conventional techniques (e.g., PCR amplification or genomic library screening methods). Note that the sequences encoding the light and heavy chains can be fused to form a single-chain antibody.

[0067] Once the relevant sequence is obtained, recombinant methods can be used to obtain large amounts of the relevant sequence, usually by cloning the sequence into a vector, transforming cells with the vector, and isolating the relevant sequence from grown host cells by conventional methods.

[0068] It should be noted that, particularly when the fragment length is short, related sequences may also be artificially synthesized, usually by first synthesizing several small fragments and then joining them to obtain a long fragment of the sequence.

[0069] Currently, DNA sequences encoding the antibodies (or fragments or derivatives thereof) of the present disclosure can be obtained entirely by chemical synthesis, and the DNA sequences can be introduced into conventional DNA molecules (or vectors, for example) and cells known in the art. Mutations can also be introduced into the protein sequences of the present disclosure by chemical synthesis.

[0070] Typically, the resulting host cells are cultured under conditions suitable for expression of an antibody of the present disclosure, and the antibody of the present disclosure is purified by conventional immunoglobulin purification steps, such as protein A-agarose, hydroxyapatite chromatography, coagulated gel electrophoresis, dialysis, ion exchange chromatography, hydrophobic chromatography, molecular sieve chromatography, or affinity chromatography, or other conventional separation and purification means well known to those skilled in the art.

[0071] The monoclonal antibodies obtained can be identified by conventional means. For example, the binding specificity of the monoclonal antibody can be determined by immunoprecipitation or an in vitro binding assay (e.g., radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA)). For example, the binding affinity of the monoclonal antibody can be determined by Scatchard analysis (Munson et al., Anal. Biochem., 107:220 (1980)).

[0072] Antibodies according to the present disclosure can be expressed intracellularly or on the cell membrane, or can be secreted extracellularly. If necessary, recombinant proteins can be isolated and purified by a variety of methods, depending on the physical, chemical, and other properties of the recombinant protein. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to, conventional renaturation, treatment with protein precipitants (salting-out methods), centrifugation, osmotic lysis, sonication, ultracentrifugation, molecular sieve chromatography (gel chromatography), adsorption chromatography, ion exchange chromatography, high-performance liquid chromatography (HPLC) and various other liquid chromatography methods, and combinations thereof.

[0073] The term "identity" of a sequence refers to the relationship between the sequences of two or more polypeptide molecules or two or more nucleic acid molecules, e.g., as determined by aligning the sequences. Generally, identity refers to the number or percentage of identical positions shared by two amino acid or nucleic acid sequences, taking into account the number of gaps and the length of each gap, which must be introduced to achieve optimal alignment of the two sequences. Typically, before calculating the percent identity between two amino acid or nucleotide sequences, sequence alignment is performed and gaps (if any) are introduced. If the amino acid residue or base in the two sequences is the same at a certain aligned position, the two sequences are considered to be identical or matched at that position. If the amino acid residue or base in the two sequences is different, they are considered to be unmatched or mismatched at that position. In some algorithms, sequence identity is obtained by dividing the number of matching positions by the total number of positions in the alignment window. In other algorithms, the number and / or length of gaps are also taken into account. For the purposes of this disclosure, the well-known alignment software BLAST (available at the website ncbi.nlm.nih.gov) can be used with default settings to obtain optimal sequence alignment and calculate sequence identity between two amino acid or nucleotide sequences. When one amino acid sequence is described as having at least 85%, at least 90%, or at least 95% identity with another amino acid sequence, the differences in the amino acid sequences may be conservative substitutions (all of the substitutions contained therein are conservative substitutions).

[0074] The term "variant" of a polypeptide, e.g., antigen-binding fragment, protein, or antibody, refers to a polypeptide in which one or more amino acid residues are inserted, deleted, added, and / or substituted relative to another polypeptide sequence, and includes fusion polypeptides. Protein variants are modified by enzymatic digestion, phosphorylation, or other post-translational modifications, but retain the biological activity of the antibodies disclosed herein, e.g., CDH17-binding specificity. A variant may be about 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, or 80% identical to the sequence of an antibody or antigen-binding fragment thereof disclosed herein. Percent identity or homology (%) can be calculated with reference to the following description.

[0075] In the present disclosure, the antibodies of the present disclosure further include conservative variants (conventional variants) thereof, which means that, compared to the amino acid sequence of an antibody of the present disclosure, at most 10, preferably at most 8, more preferably at most 5, and most preferably at most 3 amino acids are substituted with amino acids that are similar or have similar properties to form a polypeptide. These conservative variant polypeptides are preferably produced by conservative substitutions according to Table A.

[0076] [Table 1]

[0077] As used herein, "K D The term "K" (M) is intended to refer to the equilibrium dissociation constant of a particular antibody-antigen interaction. D " refers to the dissociation constant, which is K d and K. a (i.e., K d / K a ) and expressed as molar concentration (M). In light of the present disclosure, the K D For example, the K Dcan be measured using surface plasmon resonance and determined, for example, using a biosensor system (eg, system) or biolayer interference technology (eg, Octet RED96 system).

[0078] The term "affinity" refers to the strength of the interaction between an antibody or antigen-binding fragment thereof and an antigen, which is determined by the characteristics of the antigen, such as its size, structure, and / or charge, and the CDR sequences of the antibody or antigen-binding fragment thereof. Methods for determining affinity known in the art can be found in the following:

[0079] Dissociation constant (K D ) <l0 -6 M, the antibody or antigen-binding fragment thereof is said to "specifically bind" to its target (e.g., antigen). D but <l0 -9 If M, then the antibody specifically binds to its target with "high affinity."

[0080] As used herein, the term "pharmaceutical composition" is intended to refer to a mixture containing one or more antibodies (or antigen-binding fragments thereof) described herein, or physiologically / pharmaceutically acceptable salts thereof, or prodrugs thereof, and other chemical components (e.g., physiologically / pharmaceutically acceptable carriers and excipients). The purpose of a pharmaceutical composition is to facilitate administration to an organism, favor absorption of the active ingredient, and exert biological activity.

[0081] "Administration" and "treatment," as applied to an animal, human, experimental subject, cell, tissue, organ, or biological fluid, refer to contacting the animal, human, experimental subject, cell, tissue, organ, or biological fluid with an exogenous drug reagent, therapeutic reagent, diagnostic reagent, or composition. "Administration" and "treatment" may refer, for example, to therapeutic, pharmacokinetic, diagnostic, research, and experimental methods. Treatment of a cell includes contacting a cell with a reagent and contacting a fluid with a reagent, where the fluid contacts the cell. "Administration" and "treatment" also refer to in vitro and ex vivo treatments, e.g., treatment of a cell with a reagent, vibration, binding composition, or another cell. When applied to a human, veterinary, or research subject, "treatment" refers to therapeutic treatment, prophylactic or preventative measures, research, and diagnostic applications.

[0082] Furthermore, the present disclosure includes drugs for treating diseases associated with CDH17, which contain the antibody or antigen-binding fragment thereof of the present disclosure as an active ingredient.

[0083] The CDH17-associated disease is not limited to, and may be any disease associated with CDH17. For example, binding to human CDH17 can reduce the therapeutic response induced by the molecules disclosed in the present disclosure. Therefore, the molecules of the present disclosure, when used in preparations and formulations suitable for therapeutic application, are very useful for people with tumors, cancers, or infectious diseases.

[0084] The present disclosure relates to a method for immunologically detecting or measuring CDH17, a reagent for immunologically detecting or measuring CDH17, a method for immunologically detecting or measuring cells expressing CDH17, and a diagnostic reagent for diagnosing diseases associated with CDH17-positive cells, which comprises, as an active ingredient, a target antibody or an antigen-binding fragment thereof that specifically recognizes human CDH17 of the present disclosure.

[0085] In the present disclosure, the method for detecting or determining the amount of CDH17 may be any known method, for example, it includes immunological detection or measurement.

[0086] Immunological detection or measurement is a method for detecting or determining the amount of an antibody or antigen using a labeled antigen or antibody. Examples of immunological detection or measurement include radioactively labeled immunoassay (RIA), enzyme immunoassay (EIA or ELISA), fluorescent immunoassay (FIA), luminescence immunoassay, Western blotting, physicochemical methods, etc.

[0087] The above-mentioned diseases associated with CDH17-positive cells can be diagnosed by detecting or measuring cells expressing CDH17 using the antibody or antibody fragment thereof, or diagnostic conjugate of the present disclosure.

[0088] To detect polypeptide-expressing cells, known immunodetection methods may be used, preferably immunoprecipitation, fluorescent cell staining, or immunohistochemical staining. Fluorescent antibody staining may also be used, using the FMAT8100HTS system (Applied Biosystems).

[0089] Example The present invention will be further described by the following specific examples. It will be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. In the following examples, experimental methods without detailed conditions generally follow the conditions described in conventional conditions, such as Sambrook, J. et al., "Molecular Cloning Experimental Guidelines" (translated by Huang Pedang et al., Beijing: Science Press, 2002), or the conditions recommended by the manufacturer (e.g., the instruction manual). Unless otherwise specified, percentages and parts are all calculated by weight. Unless otherwise specified, all experimental materials and reagents used in the following examples are commercially available products.

[0090] The room temperature described in the examples is a normal room temperature in the art, and is usually 10 to 30°C.

[0091] Example 1 Mouse Immunization and Hybridoma Fusion Antigen production Mice were immunized with a combination of recombinant protein antigen human CDH17 His tag (Sino biological, Cat. No. 11360-H08H).

[0092] Immuno-engineering scheme: Anti-CDH17 antibodies were obtained by immunizing genetically modified mice encoding human immunoglobulin heavy and kappa light chain variable regions with the recombinant protein antigen human CDH17 His-tag (Sino biological, catalog no. 11360-H08H) and boosting them with the same antigen. Antibody immune responses were monitored using a CDH17-specific immunoassay. After the required immune response was achieved, spleen cells were harvested from each mouse and fused with mouse myeloma cells to maintain their viability and form hybridoma cells, which were then screened for CDH17 specificity.

[0093] Spleen cell fusion Hybridoma cells were obtained by fusing splenic lymphocytes with myeloma cells Sp2 / 0 (ATCC® CRL-158) by electrofusion or PEG fusion. TM PEG fusion was performed using HY technology (STEMCELL technologies). The ratio of primary cells to mouse myeloma cell lines was 1:1 for electrofusion and 10:1 for PEG fusion.

[0094] Example 2 Hybridoma screening Screening of hybridoma clones that specifically bind to CDH17 protein by ELISA ELISA was performed using the DuoSet ELISA Auxiliary Kit (R&D System, DY008). ELISA plates were coated overnight with 1 μg / ml human CDH17 His tag (Sino biological, catalog no. 11360-H08H) or BSA. The plates were washed three times with wash buffer to remove excess unbound protein and then blocked at room temperature for 1 hour. 100 μl of CDH17 hybridoma supernatant was added to each well and incubated at room temperature for 1 hour. Excess unbound antibody was removed by washing, and 100 μl of goat anti-mouse IgG Fc-HRP (ab5870) secondary antibody diluted 1:30,000 was added to each well and incubated for 1 hour. The plate was washed according to the manufacturer's instructions, and 50 μL of chemiluminescence reagent (color A and color B) was added. The reaction was stopped with 25 μL of stop solution. The optical density of the samples was measured at 450 nm using a microplate reader (PerkinElmer). All clones tested selectively bound to human CDH17 but did not bind to BSA.

[0095] [Table 2]

[0096] Flow cytometric screening of hybridoma clones that specifically bind to CDH17-expressing cancer cells Hybridoma supernatants were tested for binding to the CDH17-positive cell lines AsPC1 (ATCC, CRL-1682), GP2d (Creative bioarray, CSC-J9456), and the CDH17-negative cell line SW480 (ATCC, CCL-228) by flow cytometry. Briefly, 50 μL of cells (2 × 10 cells) in cell staining buffer were cultured. 6Cells (cells / mL) were mixed with 50 μL of undiluted supernatant. The mixture was incubated on ice for 1 hour and then washed twice with ice-cold staining buffer. The cells were then stained with 50 μL of PE-labeled secondary antibody (diluted 1:250, Biolegend, catalog no. 405307) for 20 minutes. After washing with staining buffer and fixing with 4% PFA, the cells were analyzed by flow cytometry. Purified anti-human CDH17 antibody was used as a positive control (Sino Biological, catalog no. 11360-MM02). Purified mouse IgG1 antibody was used as an isotype control (Biolegend, catalog no. 400102).

[0097] Examples of selected cell binding signals measured by flow cytometry are shown in Figure 1. Compared to SW480 cells, hybridoma clones 1H10, 2F9, 4F9, 9E2, 10E11, 13C7, 14B12, 15A4, 17F3, 19C7, and 20B4 were identified as having enhanced binding profiles with AsPC1 and GP2d cells.

[0098] Screening of hybridoma clones with cell internalization activity by indirect cytotoxicity assay The internalization activity of hybridoma supernatants was measured using an indirect cytotoxicity assay in AsPC1 cells. AsPC1 cells were seeded into 96-well plates at a density of 8,000 cells / well and treated with 500 ng / mL propidium iodide (Abcam, catalog no. ab14083) and a 1:2000 dilution of SPY650-DNA (Cytoskeleton, Inc., catalog no. CYSC501). The cells were incubated overnight at 37°C in 5% CO2. Hybridoma supernatant from each hybridoma clone was diluted with hybridoma medium containing 500 ng / mL propidium iodide and a 2000-fold dilution of SPY650-DNA, mixed with a cleavable linker-containing Fab anti-mouse IgG Fc-MMAF conjugate (Moradec, AM-202AF), and added to each well. The final concentrations of mouse IgG were approximately 10 nM, 3.33 nM, and 1.11 nM. The final concentration of Fab anti-mouse IgG Fc-MMAF conjugate in each well was 20 nM. Due to the presence of the secondary Fab-vc-MMAF, the internalizing antibody / Fab-vc-MMAF conjugate complex released its cytotoxic payload and killed the cells. The plate was imaged every 8 hours using a Cytation 5 (Agilent) to detect cell viability in each well. Purified anti-human CDH17 antibody was used as a positive control (Invitrogen, catalog no. MA5-29135). Purified mouse IgG1 antibody was used as an isotype control (Biolegend, catalog no. 400102).

[0099] Cells treated with selected hybridoma supernatants showed reduced viability and antibody internalization, as shown in Figure 2. Using an indirect killing assay, clones 1H10, 2F9, 4F9, 9E2, 10E11, 13C7, 14B12, 15A4, 17F3, 19C7, and 20B4 were identified as capable of internalization into CDH17-positive cell lines.

[0100] Example 3: Sequencing of positive hybridoma clones The sequencing process for positive hybridoma clones was as follows: Hybridoma cells in the logarithmic growth phase were harvested, RNA was extracted, reverse transcribed, and VDJ region amplification was performed. Next-generation sequencing was performed on the cDNA library amplified from each clone. The amino acid sequences of the heavy and light chain variable region DNA sequences corresponding to antibodies 1H10, 2F9, 4F9, 9E2, 10E11, 13C7, 14B12, 15A4, 17F3, 19C7, and 20B4 were obtained. The amino acid sequences of the heavy and light chain variable regions and CDR sequences of each antibody are shown in the following tables (Tables 2 to 4). In VH / VL, the amino acid residues of the CDRs were numbered and annotated using the Kabat numbering system.

[0101] [Table 3]

[0102] [Table 4]

[0103] [Table 5-1] [Table 5-2]

[0104] Example 4 Expression, purification and binding characterization of recombinant antibodies Molecular cloning of recombinant antibodies The cDNA sequences encoding the VH and VL regions of the selected clones were directly synthesized as DNA fragments with a 5' in-frame leader sequence (MGWSCIILFLVATATGVHS). Using the NEBuilder DNA cloning kit (New England Biolabs), these DNA fragments were cloned into the selected vectors. The VH region was cloned into the pFUSE-CHIg_hG1 vector (InvivoGen, #pfuse-hchg1) in the same framework as the hIgG1 heavy chain constant region in the vector. The VL region was cloned into the pFUSE2-CLIg_hk vector (InvivoGen, #pfuse2-hclk) in the same framework as the hIg κ light chain constant region in the vector. The amino acid sequences of the hIgG1 heavy chain constant region and the hIg κ light chain constant region are as follows: [ka] The IgG formats of the antibodies are disclosed as the full-length heavy and light chains in Table 5 as follows: 1H10: SEQ ID NO: 77 (heavy chain) and 78 (light chain), 2F9: SEQ ID NO: 79 (heavy chain) and 80 (light chain), 4F9: SEQ ID NO: 81 (heavy chain) and 82 (light chain), 9E2: SEQ ID NO: 83 (heavy chain) and 84 (light chain), 10E11: SEQ ID NO: 85 (heavy chain) and 86 (light chain), 13C7: SEQ ID NO: 87 (heavy chain) and 88 (light chain), 14B12: SEQ ID NO: 89 (heavy chain) and 90 (light chain), 15A4: SEQ ID NO: 91 (heavy chain) and 92 (light chain), 17F3: SEQ ID NO: 93 (heavy chain) and 94 (light chain), 19C7: SEQ ID NO: 88 (heavy chain) and 89 (light chain), 14B12: SEQ ID NO: 89 (heavy chain) and 90 (light chain), 15A4: SEQ ID NO: 91 (heavy chain) and 92 (light chain), 17F3: SEQ ID NO: 93 (heavy chain) and 94 (light chain), 19C7: SEQ ID NO: 89 (heavy chain) and 90 (light chain). NO:95 (heavy chain) and 96 (light chain), 20B4: SEQ ID NO:97 (heavy chain) and 98 (light chain).

[0105] [Table 6-1] [Table 6-2] [Table 6-3]

[0106] Recombinant antibody expression and purification The heavy chain expression plasmid and light chain plasmid were co-transfected into CHO cells (ATCC, catalog number #CCL-61) using the ExpiFectamine 293 Transfection Kit (ThermoFisher, A14524) or into ExpiCHO-S cells (ThermoFisher, #A29127) using the ExpiFectamine CHO Transfection Kit (ThermoFisher, A29129). According to the manufacturer's instructions, the plasmid DNA concentration reached 1.0 μg per ml of suspended cells, with a 1:1 LC:HC vector ratio. Transfected cells were cultured for 5–7 days in an orbital shaker at 37°C and 8% CO2. Conditioned medium was collected, and antibodies were purified using a HiTrap MabSelect SuRe column (Cytiva, #17549112) in an AKTA Pure 25 machine (Cytiva). The eluted antibody was neutralized with Tris buffer (pH 9.0) and exchanged with PBS. The product concentration was determined by UV absorption, and the mass was measured by SDS-PAGE and HPLC.

[0107] Binding characterization of anti-CDH17 recombinant antibodies to CDH17-positive and CDH17-negative cell lines by flow cytometry Using cancer cell lines including CDH17-positive cancer cells AsPC1 cells and CDH17-negative cancer cells SW480 cells, binding of recombinant antibody (human IgG1) to cell surface CDH17 was measured by FACS analysis.

[0108] AsPC1 cells were maintained in RPMI-1640 medium supplemented with 10% FBS and 1% penicillin and streptomycin. SW480 cells were maintained in DMEM medium supplemented with 10% FBS and 1% penicillin and streptomycin. Cells were cultured at 37°C in a humidified atmosphere of 5% CO2.

[0109] To determine the binding of the recombinant antibody to the cell surface CDH17 receptor, cells were first harvested and 1.3 × 10 6 ~1.5×10 6 The cells were suspended in cell staining buffer (BioLegend, Cat. No. 420201) at 10 cells / mL. Then, the cells were treated with human Fc receptor blocking reagent (BioLegend, Cat. No. 422302) on ice for 10 minutes. The resulting cell suspension was divided into 50 μL aliquots. 25 μL of recombinant antibodies from different strains were mixed with the cell aliquots, with the final recombinant antibody concentrations ranging from 1.1 pM to 200 nM. The cells were incubated on ice for 1 hour and then washed twice with cell staining buffer. Secondary antibodies (PE-conjugated goat anti-human Fc, eBioscience) were added. TM 50 μL of 1:250 diluted PBS was added to each sample to resuspend the cells. The cells were then incubated on ice for an additional 20 min. The cells were then washed twice with cell staining buffer and resuspended in 4% PFA for fixation. Samples were analyzed using iQue3 to measure the median fluorescence intensity at the corresponding passage.

[0110] The results are shown in Figure 3 and Table 6. HBMAB81 is a positive control monoclonal antibody disclosed in patent application WO 2018115231A2. B12 is an internal isotype control antibody disclosed in patent US005652138A. The results demonstrate that the anti-CDH17 antibodies of the present disclosure specifically bind to human CDH17 expressed in native cells in a concentration-dependent manner, while HBMAB81 nonspecifically binds to the CDH17-negative cell line SW480.

[0111] [Table 7]

[0112] Example 5 Characterization of Cellular Internalization of Anti-CDH17 Recombinant Antibodies by Indirect Cytotoxicity Assay in CDH17-Expressing Cells To evaluate the endocytic activity of anti-CDH17 antibody conjugates, we characterized antibody internalization using an indirect cytotoxicity assay. A Fab anti-human IgG Fc-MMAF conjugate with a cleavable linker (Moradec, AH-202AF) was incubated with recombinant anti-CDH17 antibody to form a complex. The complex was then incubated with CDH17-expressing cells. After binding to the cell surface CDH17 receptor, the complex was internalized, and after lysosomal cleavage of the linker, the conjugated MMAF was released. The released MMAF subsequently inhibited cell division by inhibiting tubulin polymerization. Briefly, CDH17-expressing cell line AsPC1 cells were seeded at 5,000 cells / well in a 96-well plate and incubated overnight. The recombinant anti-CDH17 antibody was mixed with the Fab anti-human IgG Fc-MMAF conjugate with a cleavable linker at a ratio of 1:6 (mol / mol) and incubated for 10 minutes to form the complex. Serial dilutions of the complex (4.5 pM to 30 nM) were then added to each well, and cell viability was measured for 48 hours using a Cytation 5 imaging system. Dose-response curves were generated and fitted using GraphPad Prism 9.

[0113] As shown in FIG. 4 and Table 7, all antibody clones exhibited cytotoxic activity in the AsPC1 cell line compared with the negative control clone (B12), indicating that MMAF was effectively delivered into cells.

[0114] [Table 8]

[0115] Example 6. Characterization of the binding activity of anti-CDH17 clones to cross-species CDH17 and homologs by Octet. The affinity between the CDH17 antibody and the CDH17 antigen and homologs was measured using an Octet (Octet Red 384) instrument. An anti-hIgG Fc capture (AHC) biosensor was selected and equilibrated with a buffer solution for 10 minutes. The sensor was then immersed in a well containing 1 μg / ml of CDH17 lead antibody to load the antibody onto the probe. Excess unbound antibody was removed by washing. Antigens were bound to serially diluted human CDH17-His tag (Sino biological, catalog no. 11360-H08H), cynomolgus monkey CDH17-His tag (Acro Biosystems, catalog no. CA7-C52H4), homologous human CDH6-His tag (Acro Biosystems, catalog no. CA6-H5229), and human CDH16-His tag (Sino biological, catalog no. 10915-H08H) at concentrations ranging from 200 nM to 3.1 nM for 10 min. Dissociation was initiated by immersion of well probes containing buffer for another 30 min.

[0116] The results showed that 10E11, 14B12, and 20B4 all bind to human and cynomolgus monkey CDH17 proteins, whereas HBMAB81 only binds to human CDH17 protein (Figures 5 to 8, Table 8).

[0117] [Table 9]

[0118] Example 7 Competitive Binding 20B4 and HBMAB81 mAbs were labeled with Alexa Flour 647 according to the manufacturer's instructions (ThermoFisher #A20186). 20B4-AF647 and HBMAB81-AF647 were immobilized in competitive binding assays at EC80 concentrations of 1.51 nM and 0.97 nM, respectively. 20B4 and HBMAB81 concentrations ranging from 0.03 nM to 1800 nM were mixed with 1.51 nM 20B4-AF647 or 0.97 nM HBMAB81-AF647 and incubated with AsPC1 cells on ice for 1 hour. Cells were washed twice with cell staining buffer. Samples were analyzed using iQue3 to measure the median fluorescence intensity at the corresponding passage.

[0119] The results are shown in Figure 9. The "20B4-AF647+HBMAB81" group and the "HBMAB-AF647+20B4" group did not show binding inhibition, indicating that 20B4 and HBMAB81 do not bind to the same or similar epitopes.

[0120] Although specific embodiments of the present invention have been described in detail, it will be understood that those skilled in the art can make various modifications and changes to the details based on all the teachings disclosed, and these still fall within the scope of the present invention. The protection scope of the present invention is limited by the appended claims and any equivalents thereof.

Claims

1. 1. An anti-CDH17 antibody, or antigen-binding fragment thereof, comprising: a heavy chain variable region comprising an HCDR1, HCDR2, and HCDR3 region; and a light chain variable region comprising an LCDR1, LCDR2, and LCDR3 region, wherein: HCDR1, each having the amino acid sequence shown in SEQ ID NO: 10; HCDR2 having the amino acid sequence set forth in SEQ ID NO: 21, and HCDR3 having the amino acid sequence set forth in SEQ ID NO: 31; Or, HCDR1, each having the amino acid sequence shown in SEQ ID NO: 07; HCDR2 having the amino acid sequence set forth in SEQ ID NO: 17, and HCDR3 having the amino acid sequence set forth in SEQ ID NO: 28; Or, HCDR1, each having the amino acid sequence shown in SEQ ID NO: 05; HCDR2 having the amino acid sequence set forth in SEQ ID NO: 15, and HCDR3 having the amino acid sequence set forth in SEQ ID NO: 26; and / or LCDR1, each having the amino acid sequence shown in SEQ ID NO: 42; LCDR2 having the amino acid sequence set forth in SEQ ID NO: 47, and an LCDR3 having the amino acid sequence set forth in SEQ ID NO: 54; Or, LCDR1, each having the amino acid sequence shown in SEQ ID NO: 38; LCDR2 having the amino acid sequence set forth in SEQ ID NO: 45, and an LCDR3 having the amino acid sequence set forth in SEQ ID NO: 53; Or, LCDR1, each having the amino acid sequence shown in SEQ ID NO: 36; LCDR2 having the amino acid sequence set forth in SEQ ID NO: 43, and An anti-CDH17 antibody or antigen-binding fragment thereof, wherein the LCDR3 has the amino acid sequence set forth in SEQ ID NO:

51.

2. the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 having the amino acid sequences set forth in SEQ ID NO: 10, SEQ ID NO: 21, and SEQ ID NO: 31, respectively; and the light chain variable region comprises LCDR1, LCDR2, and LCDR3 having the amino acid sequences set forth in SEQ ID NO: 42, SEQ ID NO: 47, and SEQ ID NO: 54, respectively; Or, the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 having the amino acid sequences set forth in SEQ ID NO: 07, SEQ ID NO: 17, and SEQ ID NO: 28, respectively; and the light chain variable region comprises LCDR1, LCDR2, and LCDR3 having the amino acid sequences set forth in SEQ ID NO: 38, SEQ ID NO: 45, and SEQ ID NO: 53, respectively; Or, 2. The anti-CDH17 antibody or antigen-binding fragment thereof of claim 1, wherein the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 having the amino acid sequences set forth in SEQ ID NO: 05, SEQ ID NO: 15, and SEQ ID NO: 26, respectively; and the light chain variable region comprises LCDR1, LCDR2, and LCDR3 having the amino acid sequences set forth in SEQ ID NO: 36, SEQ ID NO: 43, and SEQ ID NO: 51, respectively.

3. the antibody or antigen-binding fragment thereof is a monoclonal antibody or antigen-binding fragment thereof, a polyclonal antibody or antigen-binding fragment thereof, a multispecific antibody or antigen-binding fragment thereof, a murine antibody or antigen-binding fragment thereof, a chimeric antibody or antigen-binding fragment thereof, a humanized antibody or antigen-binding fragment thereof, a recombinant antibody or antigen-binding fragment thereof, or a human antibody or antigen-binding fragment thereof; The anti-CDH17 antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein the multispecific antibody is preferably a bispecific antibody, a trispecific antibody, or a tetraspecific antibody.

4. The anti-CDH17 antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising an amino acid sequence selected from SEQ ID NOs: 65, 61, and 59, or a sequence having at least 80%, 85%, 90%, 95%, or 99% sequence identity thereto, and / or a light chain variable region comprising an amino acid sequence selected from SEQ ID NOs: 76, 72, and 70, or a sequence having at least 80%, 85%, 90%, 95%, or 99% sequence identity thereto.

5. The antibody or antigen-binding fragment thereof a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 65, or an amino acid sequence having at least 80%, 85%, 90%, 95%, or 99% sequence identity thereto, and / or a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 76, or an amino acid sequence having at least 80%, 85%, 90%, 95%, or 99% sequence identity thereto; Or, a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 61, or an amino acid sequence having at least 80%, 85%, 90%, 95%, or 99% sequence identity thereto, and / or a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 72, or an amino acid sequence having at least 80%, 85%, 90%, 95%, or 99% sequence identity thereto; Or, 5. The anti-CDH17 antibody or antigen-binding fragment thereof of claim 4, comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 59, or an amino acid sequence having at least 80%, 85%, 90%, 95%, or 99% sequence identity thereto, and / or a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 70, or an amino acid sequence having at least 80%, 85%, 90%, 95%, or 99% sequence identity thereto.

6. The antibody or antigen-binding fragment thereof a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 65, and / or a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 76; Or, a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 61, and / or a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 72; Or, The anti-CDH17 antibody or antigen-binding fragment thereof of claim 5, comprising a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 59 and / or a light chain variable region having the amino acid sequence shown in SEQ ID NO:

70.

7. The antibody or antigen-binding fragment thereof a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 65, and a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 76; Or, a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 61, and a light chain variable region having the amino acid sequence shown in SEQ ID NO: 72; Or, 7. The anti-CDH17 antibody or antigen-binding fragment thereof of claim 6, comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 59 and a light chain variable region having the amino acid sequence set forth in SEQ ID NO:

70.

8. further comprising a human antibody constant region; Preferably, the heavy chain constant region of the human antibody constant region is selected from the constant regions of human IgG1, IgG2, IgG3, and IgG4 and common variants thereof, and the light chain constant region of the human antibody constant region is selected from the κ chain and λ chain constant regions of human antibodies and common variants thereof; More preferably, the full-length antibody comprises a human antibody heavy chain constant region of SEQ ID NO: 99 or an amino acid sequence having at least 80%, 85%, 90%, 95% or 99% sequence identity thereto, a human light chain constant region of SEQ ID NO: 100 or an amino acid sequence having at least 80%, 85%, 90%, 95% or 99% sequence identity thereto, More preferably, the full-length antibody comprises a human antibody heavy chain constant region of SEQ ID NO: 99 and a human light chain constant region of SEQ ID NO:

100.

9. The antibody or antigen-binding fragment thereof a heavy chain having the amino acid sequence set forth in SEQ ID NO: 97 or an amino acid sequence having at least 80%, 85%, 90%, 95% or 99% sequence identity thereto, and a light chain having the amino acid sequence set forth in SEQ ID NO: 98 or an amino acid sequence having at least 80%, 85%, 90%, 95% or 99% sequence identity thereto; or a heavy chain having the amino acid sequence set forth in SEQ ID NO: 89 or an amino acid sequence having at least 80%, 85%, 90%, 95% or 99% sequence identity thereto, and a light chain having the amino acid sequence set forth in SEQ ID NO: 90 or an amino acid sequence having at least 80%, 85%, 90%, 95% or 99% sequence identity thereto; or 9. The anti-CDH17 antibody or antigen-binding fragment thereof of any one of claims 1 to 8, comprising a heavy chain having the amino acid sequence set forth in SEQ ID NO: 85 or an amino acid sequence having at least 80%, 85%, 90%, 95%, or 99% sequence identity thereto, and a light chain having the amino acid sequence set forth in SEQ ID NO: 86 or an amino acid sequence having at least 80%, 85%, 90%, 95%, or 99% sequence identity thereto.

10. The antibody or antigen-binding fragment thereof a heavy chain having the amino acid sequence set forth in SEQ ID NO: 97 and a light chain having the amino acid sequence set forth in SEQ ID NO: 98; or a heavy chain having the amino acid sequence set forth in SEQ ID NO: 89 and a light chain having the amino acid sequence set forth in SEQ ID NO: 90; or 10. The anti-CDH17 antibody or antigen-binding fragment thereof of claim 9, comprising a heavy chain having the amino acid sequence set forth in SEQ ID NO: 85 and a light chain having the amino acid sequence set forth in SEQ ID NO:

86.

11. The anti-CDH17 antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, wherein the antigen-binding fragment is selected from Fab, Fab', F(ab')2, variable fragment (Fv), single-chain variable fragment (scFv), dimerization domain V (diabody), disulfide-stabilized Fv (dsFv), and a CDR-containing peptide.

12. The antibody or antigen-binding fragment thereof has a K Ｄ The anti-CDH17 antibody or antigen-binding fragment thereof according to any one of claims 1 to 11, which binds to both human and cynomolgus monkey CDH17 at the

13. The anti-CDH17 antibody or antigen-binding fragment thereof of any one of claims 1 to 11, wherein the antibody or antigen-binding fragment thereof competes with antibody 20B4 for binding to human CDH17.

14. 12. An isolated anti-CDH17 antibody or antigen-binding fragment thereof, wherein the antibody competes with the antibody or antigen-binding fragment thereof of any one of claims 1 to 11 for binding to human CDH17.

15. An isolated nucleic acid molecule encoding the anti-CDH17 antibody or antigen-binding fragment thereof of any one of claims 1 to 14.

16. A recombinant vector comprising the isolated nucleic acid molecule of claim 15.

17. 17. A host cell comprising the recombinant vector of claim 16, wherein the host cell is selected from a prokaryotic cell and a eukaryotic cell, preferably a eukaryotic cell, more preferably a mammalian cell.

18. 15. A therapeutic conjugate comprising the anti-CDH17 antibody or antigen-binding fragment thereof of any one of claims 1 to 14, wherein the antibody or antigen-binding fragment thereof is conjugated to one or more therapeutic agents, preferably wherein the therapeutic agents are cytotoxic agents.

19. 15. A diagnostic conjugate comprising the anti-CDH17 antibody or antigen-binding fragment thereof of any one of claims 1 to 14, wherein the antibody or antigen-binding fragment thereof is conjugated to one or more diagnostic agents, preferably the diagnostic agents are selected from a detectable label, an enzyme, a metal-containing substance, a fluorescent substance, a luminescent substance, a colored substance, and a quantum dot.

20. 15. A bispecific antibody comprising a first binding domain that specifically binds to CDH17 and a second binding domain that specifically binds to a second target protein, wherein the first binding domain is the anti-CDH17 antibody or antigen-binding fragment thereof of any one of claims 1 to 14.

21. 15. A method for producing the anti-CDH17 antibody or antigen-binding fragment thereof of any one of claims 1 to 14, the method comprising culturing the host cell of claim 17 in a culture medium to produce and accumulate the anti-CDH17 antibody or antigen-binding fragment thereof of any one of claims 1 to 14, and recovering the antibody or antigen-binding fragment thereof from the culture.

22. 15. A method for immunologically detecting or measuring CDH17, said method comprising contacting a sample with an anti-CDH17 antibody or antigen-binding fragment thereof of any one of claims 1 to 14.

23. 20. A method for diagnosing a disease associated with human CDH17-positive cells, the method comprising the step of contacting a sample with the anti-CDH17 antibody or antigen-binding fragment thereof of any one of claims 1 to 14, or the diagnostic conjugate of claim 19.

24. 21. A pharmaceutical composition comprising a therapeutically or prophylactically effective amount of the anti-CDH17 antibody or antigen-binding fragment thereof of any one of claims 1 to 14, or the therapeutic conjugate of claim 18, or the bispecific antibody of claim 20, and one or more pharmaceutically acceptable carriers, diluents or excipients.

25. 26. A method for treating or preventing a CDH17-mediated disease or condition, comprising the step of administering to a subject in need thereof a therapeutically or prophylactically effective amount of the anti-CDH17 antibody or antigen-binding fragment thereof of any one of claims 1 to 14, or the therapeutic conjugate of claim 18, or the bispecific antibody of claim 20, or the pharmaceutical composition of claim 24.

26. 26. The method of claim 25, wherein the disease or condition is cancer, preferably the cancer is colorectal cancer, gastric cancer, pancreatic cancer, liver cancer, gallbladder cancer, ovarian cancer, or bile duct cancer.

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