Antibodies, antigen binding fragments and methods of use

Abstract

The present disclosure relates to antibodies, antigen binding fragments and methods of use. Specifically, the present disclosure relates to an antibody or an antigen binding fragment thereof that specifically recognizes human hepatobiliary cadherin, and a pharmaceutical use thereof. It is an object of the present disclosure to develop a potential cancer therapy. According to the technical scheme, the invention provides a human-derived anti-liver-intestine cadherin antibody containing a specific amino acid sequence or an antigen binding fragment of the human-derived anti-liver-intestine cadherin antibody. The antibody or the antigen binding fragment thereof has high specificity and affinity to the hepatobiliary cadherin, and can mediate killing of cells expressing the hepatobiliary cadherin. Therefore, the invention also relates to a pharmaceutical composition containing the antibody or the antigen binding fragment thereof, and application of the pharmaceutical composition in preparation of drugs for preventing and / or treating tumors.

Description

[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. Technical Field

[0002] The present disclosure relates to antibodies, in particular to antibodies that exhibit specificity for entero-cadherin, and uses thereof, such as in the treatment of cancer. Background Art

[0003] Enteric cadherin (cadherin 17) is a cell surface marker that belongs to the cadherin superfamily and has a unique biological structure. Compared with the classic 5 cadherin repeats, it has 7 extracellular cadherin repeats and a very short intracellular region of 20 amino acid residues, lacking a conserved intracellular domain. (Berndorff et al., J Cell Biol. 1994, 125 (6): 1353-1369). Although the biological function of cadherin 17 has not been fully explored, there are reports that cadherin 17 can be used as Ca 2+ Cadherin 17 regulates water absorption in a glycosylated manner (Ahl et al., Biol. Med. Model. 2011, 8(18)). Cadherin 17 also maintains tissue integrity by interacting with integrins within extracellular tight junctions. It is primarily expressed in the human gastrointestinal (GI) tract and pancreas.

[0004] It is reported that cadherin 17 is highly expressed at the DNA and protein levels in tumors (including colorectal tumors, gastric tumors 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. The tumor activity manipulated by cadherin 17 involves multiple signal transduction pathways. One of the most important mechanisms involves cadherin 17-integrin interaction. It has been shown that the RGD motif of cadherin 17 binds to α2β1 integrin, inducing β1 integrin activation, thereby increasing cancer cell proliferation and adhesion (Bartomome et al., J Biol Chem. 2014, 289 (50): 34801-34914). It has also been shown that cadherin 17 regulates cancer invasion in GI cancer through Wnt / β-catenin signaling (Qiu et al., PloS one 2013, 8(3)) and NFκB signaling pathway (Wang et al., Cancer biology & therapy. 2013, 14(3): 262-270). The limited expression of cadherin 17 in normal tissues and its high expression in various cancers make cadherin 17 a good cancer target.

[0005] Several cadherin-17-targeted antibody drugs have been investigated in the field: two bispecific antibody drugs, BI905711 (Boehringer Ingelheim) and ARB202 (Arbele), an anti-cadherin-17-based CAR therapy, and a 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 present disclosure aims to provide novel antibodies or antigen-binding fragments thereof (hereinafter also referred to as antibodies, etc.) that bind to cadherin 17, as well as molecules comprising the antibodies, and pharmaceutical compositions comprising the antibodies, antigen-binding fragments thereof, or the molecules as active ingredients.

[0007] The present inventors conducted extensive research to achieve the above-mentioned objectives and achieved the present disclosure by developing novel anti-cadherin 17 antibodies and molecules containing the antibodies.

[0008] Specifically, the present disclosure includes at least the following aspects:

[0009] The present disclosure provides an anti-cadherin 17 antibody or an antigen-binding fragment thereof, comprising: a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 regions and a light chain variable region comprising LCDR1, LCDR2, and LCDR3 regions, wherein:

[0010] a) a HCDR1 having the amino acid sequence of 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;

[0011] b) a HCDR2 having the amino acid sequence of 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;

[0012] c) a HCDR3 having the amino acid sequence of 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;

[0013] d) a LCDR1 having the amino acid sequence of 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;

[0014] e) LCDR2 having the amino acid sequence shown in SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46 or SEQ ID NO: 47;

[0015] f) 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.

[0016] In some embodiments, the heavy chain variable region sequence comprises:

[0017] HCDR1 having the amino acid sequence of SEQ ID NO: 01, HCDR2 having the amino acid sequence of SEQ ID NO: 11, and HCDR3 having the amino acid sequence of SEQ ID NO: 22, respectively; or

[0018] HCDR1 having the amino acid sequence of SEQ ID NO: 02, HCDR2 having the amino acid sequence of SEQ ID NO: 12, and HCDR3 having the amino acid sequence of SEQ ID NO: 23, respectively; or

[0019] HCDR1 having the amino acid sequence of SEQ ID NO: 03, HCDR2 having the amino acid sequence of SEQ ID NO: 13, and HCDR3 having the amino acid sequence of SEQ ID NO: 24, respectively; or

[0020] HCDR1 having the amino acid sequence of SEQ ID NO: 04, HCDR2 having the amino acid sequence of SEQ ID NO: 14, and HCDR3 having the amino acid sequence of SEQ ID NO: 25, respectively; or

[0021] HCDR1 having the amino acid sequence of SEQ ID NO: 05, HCDR2 having the amino acid sequence of SEQ ID NO: 15, and HCDR3 having the amino acid sequence of SEQ ID NO: 26, respectively; or

[0022] HCDR1 having the amino acid sequence of SEQ ID NO: 06, HCDR2 having the amino acid sequence of SEQ ID NO: 16, and HCDR3 having the amino acid sequence of SEQ ID NO: 27, respectively; or

[0023] HCDR1 having the amino acid sequence of SEQ ID NO: 07, HCDR2 having the amino acid sequence of SEQ ID NO: 17, and HCDR3 having the amino acid sequence of SEQ ID NO: 28, respectively; or

[0024] HCDR1 having the amino acid sequence of SEQ ID NO: 01, HCDR2 having the amino acid sequence of SEQ ID NO: 18, and HCDR3 having the amino acid sequence of SEQ ID NO: 22, respectively; or

[0025] HCDR1 having the amino acid sequence of SEQ ID NO: 08, HCDR2 having the amino acid sequence of SEQ ID NO: 19, and HCDR3 having the amino acid sequence of SEQ ID NO: 29, respectively; or

[0026] HCDR1 having the amino acid sequence of SEQ ID NO: 09, HCDR2 having the amino acid sequence of SEQ ID NO: 20, and HCDR3 having the amino acid sequence of SEQ ID NO: 30, respectively; or

[0027] HCDR1 having the amino acid sequence shown in SEQ ID NO: 10, HCDR2 having the amino acid sequence shown in SEQ ID NO: 21, and HCDR3 having the amino acid sequence shown in SEQ ID NO: 31, respectively.

[0028] In some embodiments, the light chain variable region sequence comprises:

[0029] LCDR1 having the amino acid sequence of SEQ ID NO: 32, LCDR2 having the amino acid sequence of SEQ ID NO: 43, and LCDR3 having the amino acid sequence of SEQ ID NO: 48, respectively; or

[0030] LCDR1 having the amino acid sequence of SEQ ID NO: 33, LCDR2 having the amino acid sequence of SEQ ID NO: 44, and LCDR3 having the amino acid sequence of SEQ ID NO: 49, respectively; or

[0031] LCDR1 having the amino acid sequence of SEQ ID NO: 34, LCDR2 having the amino acid sequence of SEQ ID NO: 43, and LCDR3 having the amino acid sequence of SEQ ID NO: 50, respectively; or

[0032] LCDR1 having the amino acid sequence of SEQ ID NO: 35, LCDR2 having the amino acid sequence of SEQ ID NO: 43, and LCDR3 having the amino acid sequence of SEQ ID NO: 49, respectively; or

[0033] LCDR1 having the amino acid sequence of SEQ ID NO: 36, LCDR2 having the amino acid sequence of SEQ ID NO: 43, and LCDR3 having the amino acid sequence of SEQ ID NO: 51, respectively; or

[0034] LCDR1 having the amino acid sequence of SEQ ID NO: 37, LCDR2 having the amino acid sequence of SEQ ID NO: 43, and LCDR3 having the amino acid sequence of SEQ ID NO: 52, respectively; or

[0035] LCDR1 having the amino acid sequence of SEQ ID NO: 38, LCDR2 having the amino acid sequence of SEQ ID NO: 45, and LCDR3 having the amino acid sequence of SEQ ID NO: 53, respectively; or

[0036] LCDR1 having the amino acid sequence of SEQ ID NO: 39, LCDR2 having the amino acid sequence of SEQ ID NO: 43, and LCDR3 having the amino acid sequence of SEQ ID NO: 48, respectively; or

[0037] LCDR1 having the amino acid sequence of SEQ ID NO: 40, LCDR2 having the amino acid sequence of SEQ ID NO: 46, and LCDR3 having the amino acid sequence of SEQ ID NO: 51, respectively; or

[0038] LCDR1 having the amino acid sequence of SEQ ID NO: 41, LCDR2 having the amino acid sequence of SEQ ID NO: 43, and LCDR3 having the amino acid sequence of SEQ ID NO: 52, respectively; or

[0039] LCDR1 has the amino acid sequence shown in SEQ ID NO: 42, LCDR2 has the amino acid sequence shown in SEQ ID NO: 47, and LCDR3 has the amino acid sequence shown in SEQ ID NO: 54, respectively.

[0040] In a specific embodiment, the antibody or antigen-binding fragment thereof comprises:

[0041] a) the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 having the amino acid sequences shown in SEQ ID NO: 01, SEQ ID NO: 11 and SEQ ID NO: 22, respectively; and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 having the amino acid sequences shown in SEQ ID NO: 32, SEQ ID NO: 43 and SEQ ID NO: 48, respectively; or

[0042] b) the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 having the amino acid sequences shown in SEQ ID NO: 02, SEQ ID NO: 12, and SEQ ID NO: 23, respectively; and the light chain variable region comprises LCDR1, LCDR2, and LCDR3 having the amino acid sequences shown in SEQ ID NO: 33, SEQ ID NO: 44, and SEQ ID NO: 49, respectively; or

[0043] c) the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 having the amino acid sequences shown in SEQ ID NO: 03, SEQ ID NO: 13 and SEQ ID NO: 24, respectively; and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 having the amino acid sequences shown in SEQ ID NO: 34, SEQ ID NO: 43 and SEQ ID NO: 50, respectively; or

[0044] d) the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 having the amino acid sequences shown in SEQ ID NO: 04, SEQ ID NO: 14 and SEQ ID NO: 25, respectively; and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 having the amino acid sequences shown in SEQ ID NO: 35, SEQ ID NO: 43 and SEQ ID NO: 49, respectively; or

[0045] e) the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 having the amino acid sequences shown 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 shown in SEQ ID NO: 36, SEQ ID NO: 43, and SEQ ID NO: 51, respectively; or

[0046] f) the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 having the amino acid sequences shown in SEQ ID NO: 06, SEQ ID NO: 16, and SEQ ID NO: 27, respectively; and the light chain variable region comprises LCDR1, LCDR2, and LCDR3 having the amino acid sequences shown in SEQ ID NO: 37, SEQ ID NO: 43, and SEQ ID NO: 52, respectively; or

[0047] g) the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 having the amino acid sequences of 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 of SEQ ID NO: 38, SEQ ID NO: 45, and SEQ ID NO: 53, respectively; or

[0048] h) the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 having the amino acid sequences shown in SEQ ID NO: 01, SEQ ID NO: 18, and SEQ ID NO: 22, respectively; and the light chain variable region comprises LCDR1, LCDR2, and LCDR3 having the amino acid sequences shown in SEQ ID NO: 39, SEQ ID NO: 43, and SEQ ID NO: 48, respectively; or

[0049] i) the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 having the amino acid sequences shown in SEQ ID NO: 08, SEQ ID NO: 19 and SEQ ID NO: 29, respectively; and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 having the amino acid sequences shown in SEQ ID NO: 40, SEQ ID NO: 46 and SEQ ID NO: 51, respectively; or

[0050] j) the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 having the amino acid sequences shown in SEQ ID NO: 09, SEQ ID NO: 20 and SEQ ID NO: 30, respectively; and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 having the amino acid sequences shown in SEQ ID NO: 41, SEQ ID NO: 43 and SEQ ID NO: 52, respectively; or

[0051] k) the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 having the amino acid sequences shown 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 shown in SEQ ID NO: 42, SEQ ID NO: 47 and SEQ ID NO: 54, respectively.

[0052] In a 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.

[0053] 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.

[0054] In some embodiments, the antibody or antigen-binding fragment thereof comprises:

[0055] a) a heavy chain variable region having the amino acid sequence of 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 the amino acid sequence of SEQ ID NO:66, or an amino acid sequence having at least 80%, 85%, 90%, 95% or 99% sequence identity thereto; or

[0056] b) a heavy chain variable region having the amino acid sequence of SEQ ID NO:56, or an amino acid sequence with at least 80%, 85%, 90%, 95% or 99% sequence identity thereto; and / or a light chain variable region having the amino acid sequence of SEQ ID NO:67, or an amino acid sequence with at least 80%, 85%, 90%, 95% or 99% sequence identity thereto; or

[0057] c) a heavy chain variable region having the amino acid sequence of 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 the amino acid sequence of SEQ ID NO:68, or an amino acid sequence having at least 80%, 85%, 90%, 95% or 99% sequence identity thereto; or

[0058] d) a heavy chain variable region having the amino acid sequence of SEQ ID NO:58, or an amino acid sequence with at least 80%, 85%, 90%, 95% or 99% sequence identity thereto; and / or a light chain variable region having the amino acid sequence of SEQ ID NO:69, or an amino acid sequence with at least 80%, 85%, 90%, 95% or 99% sequence identity thereto; or

[0059] e) a heavy chain variable region having the amino acid sequence of 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 of SEQ ID NO:70, or an amino acid sequence having at least 80%, 85%, 90%, 95% or 99% sequence identity thereto; or

[0060] f) a heavy chain variable region having the amino acid sequence of 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 the amino acid sequence of SEQ ID NO:71, or an amino acid sequence having at least 80%, 85%, 90%, 95% or 99% sequence identity thereto; or

[0061] d) a heavy chain variable region having the amino acid sequence of SEQ ID NO:61, or an amino acid sequence with at least 80%, 85%, 90%, 95% or 99% sequence identity thereto; and / or a light chain variable region having the amino acid sequence of SEQ ID NO:72, or an amino acid sequence with at least 80%, 85%, 90%, 95% or 99% sequence identity thereto; or

[0062] h) a heavy chain variable region having the amino acid sequence of SEQ ID NO:62, or an amino acid sequence with at least 80%, 85%, 90%, 95% or 99% sequence identity thereto; and / or a light chain variable region having the amino acid sequence of SEQ ID NO:73, or an amino acid sequence with at least 80%, 85%, 90%, 95% or 99% sequence identity thereto; or

[0063] i) a heavy chain variable region having the amino acid sequence of 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 of SEQ ID NO:74, or an amino acid sequence having at least 80%, 85%, 90%, 95% or 99% sequence identity thereto; or

[0064] j) a heavy chain variable region having the amino acid sequence of SEQ ID NO:64, or an amino acid sequence with at least 80%, 85%, 90%, 95% or 99% sequence identity thereto; and / or a light chain variable region having the amino acid sequence of SEQ ID NO:75, or an amino acid sequence with at least 80%, 85%, 90%, 95% or 99% sequence identity thereto; or

[0065] k) a heavy chain variable region having the amino acid sequence of 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 of SEQ ID NO: 76, or an amino acid sequence having at least 80%, 85%, 90%, 95% or 99% sequence identity thereto.

[0066] In a specific embodiment, the antibody or antigen-binding fragment thereof comprises: a) a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO:55; and / or a light chain variable region having an amino acid sequence as set forth in SEQ ID NO:66; or b) a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO:56; and / or a light chain variable region having an amino acid sequence as set forth in SEQ ID NO:67; or c) a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO:57; and / or a light chain variable region having an amino acid sequence as set forth in SEQ ID NO:68; or d) a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO:58; and / or a light chain variable region having an amino acid sequence as set forth in SEQ ID NO:69; or e) a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO:59; and / or a light chain variable region having an amino acid sequence as set forth in SEQ ID NO:70; or f) a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO:60; and / or a light chain variable region having an amino acid sequence as set forth in SEQ ID NO: NO:71; or g) a heavy chain variable region having the amino acid sequence of SEQ ID NO:61; and / or a light chain variable region having the amino acid sequence of SEQ ID NO:72; or h) a heavy chain variable region having the amino acid sequence of SEQ ID NO:62; and / or a light chain variable region having the amino acid sequence of SEQ ID NO:73; or i) a heavy chain variable region having the amino acid sequence of SEQ ID NO:63; and / or a light chain variable region having the amino acid sequence of SEQ ID NO:74; or j) a heavy chain variable region having the amino acid sequence of SEQ ID NO:64; and / or a light chain variable region having the amino acid sequence of SEQ ID NO:75; or k) a heavy chain variable region having the amino acid sequence of SEQ ID NO:65; and / or a light chain variable region having the amino acid sequence of SEQ ID NO:76.

[0067] In some embodiments, the antibody or antigen-binding fragment thereof comprises: a) a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO:55; and a light chain variable region having an amino acid sequence as set forth in SEQ ID NO:66; or b) a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO:56; and a light chain variable region having an amino acid sequence as set forth in SEQ ID NO:67; or c) a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO:57; and a light chain variable region having an amino acid sequence as set forth in SEQ ID NO:68; or d) a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO:58; and a light chain variable region having an amino acid sequence as set forth in SEQ ID NO:69; or e) a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO:59; and a light chain variable region having an amino acid sequence as set forth in SEQ ID NO:70; or f) a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO:60; and a light chain variable region having an amino acid sequence as set forth in SEQ ID NO:71; or g) a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO: NO:61; and 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 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.

[0068] In some embodiments, the antibody or antigen-binding fragment thereof further comprises 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 conventional variants thereof, and the light chain constant region of the human antibody constant region is selected from the constant regions of κ chain and λ chain of human antibodies and conventional 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, 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; further 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.

[0069] In a specific embodiment, the antibody or antigen-binding fragment thereof comprises:

[0070] a) a heavy chain having the amino acid sequence of SEQ ID NO:77, or an amino acid sequence with at least 80%, 85%, 90%, 95% or 99% sequence identity thereto; and a light chain having the amino acid sequence of SEQ ID NO:78, or an amino acid sequence with at least 80%, 85%, 90%, 95% or 99% sequence identity thereto; or

[0071] b) a heavy chain having the amino acid sequence of SEQ ID NO:79, or an amino acid sequence with at least 80%, 85%, 90%, 95% or 99% sequence identity thereto; and a light chain having the amino acid sequence of SEQ ID NO:80, or an amino acid sequence with at least 80%, 85%, 90%, 95% or 99% sequence identity thereto; or

[0072] c) a heavy chain having the amino acid sequence of SEQ ID NO:81, or an amino acid sequence with at least 80%, 85%, 90%, 95% or 99% sequence identity thereto; and a light chain having the amino acid sequence of SEQ ID NO:82, or an amino acid sequence with at least 80%, 85%, 90%, 95% or 99% sequence identity thereto; or

[0073] d) a heavy chain having the amino acid sequence of SEQ ID NO:83, or an amino acid sequence with at least 80%, 85%, 90%, 95% or 99% sequence identity thereto; and a light chain having the amino acid sequence of SEQ ID NO:84, or an amino acid sequence with at least 80%, 85%, 90%, 95% or 99% sequence identity thereto; or

[0074] e) a heavy chain having the amino acid sequence of SEQ ID NO:85, or an amino acid sequence with at least 80%, 85%, 90%, 95% or 99% sequence identity thereto; and a light chain having the amino acid sequence of SEQ ID NO:86, or an amino acid sequence with at least 80%, 85%, 90%, 95% or 99% sequence identity thereto; or

[0075] f) a heavy chain having the amino acid sequence of SEQ ID NO:87, or an amino acid sequence with at least 80%, 85%, 90%, 95% or 99% sequence identity thereto; and a light chain having the amino acid sequence of SEQ ID NO:88, or an amino acid sequence with at least 80%, 85%, 90%, 95% or 99% sequence identity thereto; or

[0076] g) a heavy chain having the amino acid sequence of SEQ ID NO:89, or an amino acid sequence with at least 80%, 85%, 90%, 95% or 99% sequence identity thereto; and a light chain having the amino acid sequence of SEQ ID NO:90, or an amino acid sequence with at least 80%, 85%, 90%, 95% or 99% sequence identity thereto; or

[0077] h) a heavy chain having the amino acid sequence of SEQ ID NO:91, or an amino acid sequence with at least 80%, 85%, 90%, 95% or 99% sequence identity thereto; and a light chain having the amino acid sequence of SEQ ID NO:92, or an amino acid sequence with at least 80%, 85%, 90%, 95% or 99% sequence identity thereto; or

[0078] i) a heavy chain having the amino acid sequence of SEQ ID NO:93, or an amino acid sequence with at least 80%, 85%, 90%, 95% or 99% sequence identity thereto; and a light chain having the amino acid sequence of SEQ ID NO:94, or an amino acid sequence with at least 80%, 85%, 90%, 95% or 99% sequence identity thereto; or

[0079] j) a heavy chain having the amino acid sequence of SEQ ID NO:95, or an amino acid sequence with at least 80%, 85%, 90%, 95% or 99% sequence identity thereto; and a light chain having the amino acid sequence of SEQ ID NO:96, or an amino acid sequence with at least 80%, 85%, 90%, 95% or 99% sequence identity thereto; or

[0080] k) 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.

[0081] In a specific embodiment, the antibody or antigen-binding fragment thereof comprises: a) a heavy chain having the amino acid sequence of SEQ ID NO:77 and a light chain having the amino acid sequence of SEQ ID NO:78; or b) a heavy chain having the amino acid sequence of SEQ ID NO:79 and a light chain having the amino acid sequence of SEQ ID NO:80; or c) a heavy chain having the amino acid sequence of SEQ ID NO:81 and a light chain having the amino acid sequence of SEQ ID NO:82; or d) a heavy chain having the amino acid sequence of SEQ ID NO:83 and a light chain having the amino acid sequence of SEQ ID NO:84; or e) a heavy chain having the amino acid sequence of SEQ ID NO:85 and a light chain having the amino acid sequence of SEQ ID NO:86; or f) a heavy chain having the amino acid sequence of SEQ ID NO:87 and a light chain having the amino acid sequence of SEQ ID NO:88; or g) a heavy chain having the amino acid sequence of SEQ ID NO:89 and a light chain having the amino acid sequence of SEQ ID NO: NO:90; or h) a heavy chain having the amino acid sequence set forth in SEQ ID NO:91 and a light chain having the amino acid sequence set forth in SEQ ID NO:92; 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.

[0082] In a specific embodiment, 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 CDR-containing peptide.

[0083] 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:

[0084] The heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 having the amino acid sequences shown 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 shown in SEQ ID NO: 36, SEQ ID NO: 43, and SEQ ID NO: 51, respectively; or

[0085] the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 having the amino acid sequences of 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 of SEQ ID NO: 38, SEQ ID NO: 45, and SEQ ID NO: 53, respectively;

[0086] or the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 having the amino acid sequences shown 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 shown in SEQ ID NO: 42, SEQ ID NO: 47 and SEQ ID NO: 54, respectively, and

[0087] wherein the antibody or its antigen-binding fragment specifically binds to human and cynomolgus monkey CDH17; preferably, wherein the antibody or its antigen-binding fragment specifically binds to human and cynomolgus monkey CDH17; D ≤1000 nM binds to both human and cynomolgus monkey CDH17; more preferably, wherein the antibody or antigen-binding fragment thereof binds to both human and cynomolgus monkey CDH17 with a K D ≤500nM, 400nM, 300nM, 200nM, 100nM, 90nM, 80nM, 70nM, 60nM, 50nM, 40nM, 30nM, 20nM, 19nM, 18nM, 17nM, 16nM, 15nM, 14nM, 13nM, 12nM, 11nM, 10nM, 9nM, 8nM, 7nM, 6nM, 5nM, 4nM or 3nM; further preferably, wherein the antibody or antigen-binding fragment thereof has the following properties: a) with a K of 15nM or less D Binds to human CDH17; b) with a K of 15 nM or lessD Binds to cynomolgus monkey CDH17.

[0088] 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:

[0089] the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 having the amino acid sequences of 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 of SEQ ID NO: 36, SEQ ID NO: 43, and SEQ ID NO: 51, respectively;

[0090] or the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 having the amino acid sequences of 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 of SEQ ID NO: 38, SEQ ID NO: 45, and SEQ ID NO: 53, respectively;

[0091] or the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 having the amino acid sequences shown 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 shown in SEQ ID NO: 42, SEQ ID NO: 47 and SEQ ID NO: 54, respectively; and wherein the antibody or antigen-binding fragment thereof has the following properties: a) with a K of 3 nM or less D Binds to human CDH17; b) with a K of 3 nM or less D Binds to cynomolgus monkey CDH17; preferably, wherein the antibody or antigen-binding fragment thereof has the following properties: a) with a K of 2 nM or less D Binds to human CDH17; b) with a K of 3 nM or less D Binds to cynomolgus monkey CDH17.

[0092] 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:

[0093] The heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 having the amino acid sequences shown 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 shown in SEQ ID NO: 42, SEQ ID NO: 47 and SEQ ID NO: 54, respectively; and wherein the antibody or antigen-binding fragment thereof specifically binds to human and cynomolgus monkey CDH17; preferably, ... D ≤1000 nM binds to both human and cynomolgus monkey CDH17; more preferably, wherein the antibody or antigen-binding fragment thereof binds to both human and cynomolgus monkey CDH17 with a K D ≤500nM, 400nM, 300nM, 200nM, 100nM, 90nM, 80nM, 70nM, 60nM, 50nM, 40nM, 30nM, 20nM, 19nM, 18nM, 17nM, 16nM, 15nM, 14nM, 13nM, 12nM, 11nM, 10nM, 9nM, 8nM, 7nM, 6nM, 5nM, 4nM or 3nM; further preferably, wherein the antibody or antigen-binding fragment thereof has the following properties: a) with a K of 15nM or less D Binds to human CDH17; b) with a K of 15 nM or less D Binds to cynomolgus monkey CDH17.

[0094] 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:

[0095] The heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 having the amino acid sequences shown 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 shown in SEQ ID NO: 42, SEQ ID NO: 47 and SEQ ID NO: 54, respectively, and wherein the antibody or antigen-binding fragment thereof has the following properties: a) with a K of 3 nM or less D Binds to human CDH17; b) with a K of 3 nM or less D Binds to cynomolgus monkey CDH17; preferably, wherein the antibody or antigen-binding fragment thereof has the following properties: a) with a K of 2 nM or less D Binds to human CDH17; b) with a K of 3 nM or less D Binds to cynomolgus monkey CDH17.

[0096] In some embodiments, the present disclosure provides an isolated anti-CDH17 antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof competes with the above-mentioned antibodies or antigen-binding fragments thereof for binding to CDH17.

[0097] 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 amino acid sequences as shown 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 amino acid sequences as shown 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 amino acid sequences as shown 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 amino acid sequences as shown 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 amino acid sequences as shown in SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15 NO: 21 and SEQ ID NO: 31; and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 having the amino acid sequences shown 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, 14B12 or 10E11 for binding to human CDH17.

[0098] 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 shown 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 shown 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.

[0099] In some embodiments, the present disclosure provides an isolated nucleic acid molecule encoding any of the above-mentioned antibodies or antigen-binding fragments thereof.

[0100] In one aspect, the present disclosure also provides a recombinant vector comprising the isolated nucleic acid molecule described above.

[0101] In another aspect, the present disclosure further provides a host cell comprising the above-mentioned recombinant vector, wherein the host cell is selected from prokaryotic cells and eukaryotic cells, preferably eukaryotic cells, and more preferably mammalian cells.

[0102] In one aspect, the present disclosure further provides a therapeutic conjugate comprising the above-mentioned 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, the therapeutic agent is a cytotoxic agent.

[0103] On the one hand, the present disclosure also provides a diagnostic conjugate comprising the above-mentioned anti-CDH17 antibody or its antigen-binding fragment, wherein the antibody or its antigen-binding fragment is conjugated to one or more diagnostic agents; preferably, the diagnostic agent can be, but is not limited to, a detectable label, an enzyme, a metal-containing substance, a fluorescent substance, a luminescent substance, a colored substance, or a quantum dot.

[0104] In one aspect, the present disclosure also 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 the above-mentioned anti-CDH17 antibody or an antigen-binding fragment thereof.

[0105] In one aspect, the present disclosure also provides a method for producing the above-mentioned anti-CDH17 antibody or its antigen-binding fragment, wherein the method comprises culturing the host cell in a culture medium to produce and accumulate the antibody or its antigen-binding fragment, and harvesting the antibody or its antigen-binding fragment from the culture.

[0106] In one aspect, the present disclosure also provides a method for immunologically detecting or measuring CDH17, wherein the method comprises detecting or measuring CDH17 by contacting a sample with the above-mentioned anti-CDH17 antibody or antigen-binding fragment thereof or diagnostic conjugate.

[0107] In one aspect, the present disclosure also 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-mentioned anti-CDH17 antibody or antigen-binding fragment thereof or diagnostic conjugate.

[0108] In one aspect, the present disclosure also provides a pharmaceutical composition comprising a therapeutically effective amount or a prophylactically effective amount of the above-mentioned anti-CDH17 antibody or antigen-binding fragment thereof, or bispecific antibody or therapeutic conjugate, and one or more pharmaceutically acceptable carriers, diluents or excipients.

[0109] In one aspect, the present disclosure also provides a method for treating or preventing a disease or condition mediated by CDH17, comprising administering a therapeutically effective amount or a prophylactically effective amount of the above-mentioned anti-CDH17 antibody or antigen-binding fragment thereof, or bispecific antibody, or therapeutic conjugate, or pharmaceutical composition to a subject in need of treatment or prevention of the disease or condition.

[0110] 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.

[0111] In another aspect, the present disclosure provides a kit comprising a device for implementing the methods of the present disclosure. In a specific embodiment, the present disclosure provides a kit comprising at least one container comprising the above-mentioned anti-CDH17 antibody or antigen-binding fragment thereof, therapeutic conjugate or pharmaceutical composition. The kit may also include instructions for preparing, administering and / or delivering the above-mentioned anti-CDH17 antibody or antigen-binding fragment thereof, therapeutic conjugate or pharmaceutical composition.

[0112] 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. In addition, the antibody or antigen-binding fragment thereof is sufficiently internalized in human cells expressing CDH17, can effectively bind to CDH17 and mediate the killing of CDH17-expressing cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0113] Figure 1 . In vitro binding characterization of hybridoma clones to AsPC1(A), GP2d(B), and SW480(C) cells.

[0114] Figure 2 The cell internalization activity of selected hybridoma clones was characterized using an indirect killing assay.

[0115] Figure 3 In vitro binding characterization of anti-CDH17 recombinant antibodies to AsPC1 (A) and SW480 cells (B) was determined by flow cytometry analysis, using HBMAB81 as a positive control and B12 as an isotype control antibody (C).

[0116] Figure 4 Dose-response curve of internalization of anti-CDH17 recombinant antibody as determined by indirect killing.

[0117] Figure 5 Octet characterization of the binding activity of HBMAB81 to cross-species CDH17, its homolog CDH6, or CDH16.

[0118] Figure 6 Octet characterization of the binding activity of anti-CDH17 clone 10E11 to cross-species CDH17, homolog CDH6, or CDH16.

[0119] Figure 7 Octet characterization of the binding activity of anti-CDH17 clone 14B12 to cross-species CDH17, homolog CDH6, or CDH16.

[0120] Figure 8 Octet characterization of the binding activity of anti-CDH17 clone 20B4 to cross-species CDH17, homolog CDH6, or CDH16.

[0121] Figure 9 Competitive binding: (A) 20B4; (B) HBMAB81. DETAILED DESCRIPTION

[0122] Definition of terms

[0123] Before describing the present disclosure in detail below, it will be understood that the present disclosure is not limited to the specific methods, protocols and reagents described herein, as they may vary. It will also be understood that the terms used herein are only used for the purpose of describing specific embodiments and are not intended to limit the scope of the present invention, which will be limited only by the appended claims. Unless otherwise defined, all technical terms and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention belongs.

[0124] For interpretation of the specification, the following definitions will apply and, wherever appropriate, terms used in the singular may also include the plural, and vice versa. It will be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0125] The terms used herein are for the purpose of describing specific embodiments only and are not intended to limit the present disclosure. Unless the context clearly indicates otherwise, the open-ended expressions "include" and "including" should be interpreted as including unmentioned structural components or method steps, but it should be noted that the open-ended expressions also cover the case where only mentioned components and method steps are included (i.e., it covers the case where the closed expression "consists of...").

[0126] The terms "CDH17" and "CDH17 antigen" are used interchangeably herein and include any variants, isoforms, and species homologs of human CDH17 that are naturally expressed by cells or expressed on cells transfected with a CDH17 gene.

[0127] "CDH17" as a target should be interpreted broadly herein and is intended to encompass various forms of CDH17 molecules at different stages in mammals (such as humans), such as, but not limited to, molecules produced during 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 artificially prepared or in vitro expressed CDH17.

[0128] The term "antibody" as referred to herein includes whole antibodies and any antigen-binding fragment (ie, "antigen-binding portion") or single chains thereof.

[0129] The term "antibody" refers to an immunoglobulin comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, or an antigen-binding portion thereof. Each heavy chain is composed of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region (CH). Each light chain is composed of a light chain variable region (abbreviated herein as VL) and a light chain constant region (CL). The VH and VL regions can be further subdivided into hypervariable regions called complementarity determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). Both the heavy and light chains have three complementarity determining regions, referred to as HCDR1, HCDR2, HCDR3, and LCDR1, LCDR2, LCDR3, respectively. Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxyl-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 region of an antibody can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (such as effector cells) and the first component (C1q) of the classical complement system. Antibodies are classified based on the amino acid sequence of their heavy chain constant region. 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 chains. The same type of Ig can also be divided into different subclasses based on the amino acid composition of its hinge region and the number and position of heavy chain disulfide bonds. For example, IgG can be divided into IgG1, IgG2, IgG3, and IgG4. Generally speaking, the heavy chain constant region of an IgG antibody includes three constant domains, namely CH1, CH2, and CH3, and the light chain constant region includes one constant domain, namely CL. Light chains can also be divided into kappa chains or lambda chains based on the constant region. Each of the five types of Ig can have either a kappa chain or a lambda chain.

[0130] 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 its epitope (e.g., CDH17). It has been shown that the antigen-binding function of an antibody can be achieved by fragments of a full-length antibody. Examples of binding fragments encompassed within the term "antigen-binding fragment" of an antibody include: (i) Fab fragment: a monovalent fragment consisting of VL, VH, CL, and CHI domains; (ii) F(ab')2 fragment: a bivalent fragment comprising two Fab fragments linked by a disulfide bond at the hinge region; (iii) an Fd fragment consisting of VH and CHI domains; (iv) an Fv fragment consisting of the VL and VH domains of a single arm of an antibody; (v) a dAb fragment consisting of a VH domain (Ward et al., (1989) Nature 341: 544-546); (vi) isolated complementary determining regions (CDRs), and (vii) a combination of two or more isolated CDRs, which may optionally be connected by a synthetic linker. In addition, although the two domains VL and VH of the Fv fragment are encoded by separate genes, they can be connected by synthetic linkers using recombinant methods, making it possible to make them into a single protein chain, wherein the VL and VH regions are paired to form a monovalent molecule (referred to as single-chain Fv (scFv); see, for example, Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Sci. USA 85:5879-5883). Such single-chain antibodies are also intended to be included in the term "antigen-binding portion" of an antibody. In some examples, the term "antigen-binding fragment" also includes dimerization domain V (diabodies) and disulfide-stabilized Fv (dsFv).

[0131] As used herein, the term "human antibody" is intended to include antibodies 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 mutagenesis in vitro or by somatic mutation in vivo).

[0132] "Humanized antibodies" include antibodies having variable and constant regions of human immunoglobulin sequences in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.

[0133] "Chimeric antibodies" generally refer to antibody molecules formed from antibody domain fragments of different origins, for example, an antibody comprising a mouse variable region inserted into a human constant region.

[0134] 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 transgenic or transchromosomal animals (e.g., mice) of human immunoglobulin genes or hybridomas prepared therefrom, (b) antibodies isolated from host cells transformed to express the antibody, such as 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 to other DNA sequences. Such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences. However, in certain embodiments, such recombinant human antibodies may be subjected to in vitro mutagenesis (or, when using animals transgenic for human Ig sequences, in vivo somatic mutagenesis), such that the amino acid sequences of the VH and VL regions of the recombinant antibodies are sequences that, although derived from and related to human germline VH and VL sequences, may not naturally exist in the human antibody germline repertoire in vivo.

[0135] The term "CDR" refers to one of the six hypervariable regions within an antibody variable domain that primarily contribute to 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 a light chain variable domain, and CDR1, CDR2, and CDR3 (HCDR1, HCDR2, HCDR3, or H1, H2, H3) of a heavy chain variable domain. In addition, CDRs can be defined according to 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 definition of Martin's related research (Martin ACR, Cheetham JC, Rees AR (1989) Modelling antibody hypervariable loops: A combined algorithm. Proc Natl Acad Sci USA 86:9268-9272). The correspondence between different numbering systems is well known to those skilled in the art (for example, see Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003). Those skilled in the art can readily identify the CDRs defined by each numbering system.

[0136] "Conservative modification" or "conservative replacement or substitution" or "conservative variant" refers to the replacement of an amino acid in a protein with another amino acid having similar properties (e.g., charge, side chain size, hydrophobicity / hydrophilicity, main chain conformation and rigidity, etc.), so that substitutions can be made frequently without changing the biological activity of the protein. It will be understood by those skilled in the art that, in general, single amino acid substitutions in non-essential regions of a polypeptide will not substantially change the biological activity (see, e.g., Watson et al. (1987) Molecular Biology of the Gene, The Benjamin / Cummings Pub. Co., p. 224, (4th ed.)). In addition, substitutions of amino acids with similar structure or function are less likely to affect biological activity.

[0137] In some embodiments, the anti-CDH17 antibodies or fragments thereof of the present disclosure encompass antibodies or fragments thereof having post-translational modifications on the light chain variable region, heavy chain variable region, light chain, or heavy chain.

[0138] In certain embodiments, the antibodies provided herein are modified to increase or decrease the degree of glycosylation. The addition or deletion of antibody glycosylation sites can be conveniently accomplished by changing the amino acid sequence to introduce or remove one or more glycosylation sites. In some applications, it may be useful to remove modifications that do not require glycosylation sites, for example, modifications that remove fucose modules to enhance antibody-dependent cellular cytotoxicity (ADCC) function (see Shield et al., (2002) JBC 277: 26733). In other applications, galactosylation modifications can be performed to modify complement-dependent cytotoxicity (CDC).

[0139] In certain embodiments, one or more amino acid modifications can be introduced into the Fc region of an antibody provided herein to generate an Fc region variant to enhance the efficacy of the antibody, e.g., in the treatment of cancer or cell proliferative disorders. The Fc region variant can comprise a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3, or IgG4 Fc region) comprising an amino acid modification (e.g., substitution) at one or more amino acid positions.

[0140] 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 are preferably double-stranded DNA. A nucleic acid is "operably linked" when it is placed in a functional relationship with another nucleic acid sequence. For example, a promoter or enhancer is considered to be operably linked to a coding sequence when it affects the transcription of the coding sequence.

[0141] The method for preparing the nucleic acid is a conventional method in the art. Preferably, it comprises the following steps: obtaining a nucleic acid molecule encoding the above protein by gene cloning technology, or obtaining a nucleic acid molecule encoding the above protein by artificial full-length sequence synthesis.

[0142] Those skilled in the art will appreciate that the base sequence encoding the protein amino acid sequence may be appropriately replaced, deleted, altered, inserted, or added to provide a polynucleotide homolog. Homologs of the polynucleotides of the present disclosure may be prepared by replacing, deleting, or adding one or more bases of a gene encoding a protein sequence while maintaining antibody activity.

[0143] The term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it is connected. In one embodiment, a vector is a "plasmid," which refers to a circular double-stranded DNA loop to which additional DNA fragments can be connected. In another embodiment, a vector is a viral vector, in which additional DNA fragments can be connected to a viral genome. Vectors disclosed herein can self-replicate in the host cell into which they are introduced (e.g., bacterial vectors and episomal mammalian vectors having bacterial origins of replication), or can be incorporated into the genome of the host cell when introduced into the host cell, thereby replicating together with the host genome (e.g., non-episomal mammalian vectors).

[0144] Recombinant expression vectors can be obtained by conventional methods in the art, that is, by linking the nucleic acid molecules disclosed herein to various expression vectors. The expression vector can be any of a variety of conventional vectors in the art, as long as it can carry the nucleic acid molecules. Preferred vectors include various plasmids, cosmids, phages, or viral vectors.

[0145] As used herein, the term "transfectoma" includes recombinant eukaryotic host cells, such as CHO cells, NS / 0 cells, HEK293 cells, plant cells, or fungi (including yeast cells), that express the antibody.

[0146] The sequence of the DNA molecule of the antibody or its fragment according to the present disclosure can be obtained by conventional techniques (e.g., methods such as PCR amplification or genomic library screening). In addition, the sequences encoding the light chain and heavy chain can be fused together to form a single-chain antibody.

[0147] Once the related sequence is obtained, it can be obtained in bulk using recombinant methods. This is usually done by cloning the sequence into a vector by conventional methods, transforming cells with the vector, and then isolating the related sequence from the proliferating host cells.

[0148] In addition, related sequences can be synthesized artificially, especially when the fragment length is short. Usually, several small fragments are synthesized first and then connected together to obtain fragments of the long sequence.

[0149] Currently, DNA sequences encoding the antibodies (or fragments thereof or derivatives thereof) of the present disclosure can be obtained entirely by chemical synthesis. The DNA sequences can then be introduced into various existing DNA molecules (or, for example, vectors) and cells known in the art. In addition, mutations can also be introduced into the protein sequences of the present disclosure by chemical synthesis.

[0150] Typically, the obtained host cells are cultured under conditions suitable for expressing the antibodies of the present disclosure. The antibodies of the present disclosure are then purified using conventional immunoglobulin purification procedures, such as conventional separation and purification methods well known to those skilled in the art, such as protein A-agarose, hydroxyapatite chromatography, gel electrophoresis, dialysis, ion exchange chromatography, hydrophobic chromatography, molecular sieve chromatography, or affinity chromatography.

[0151] The obtained monoclonal antibodies can be identified by conventional means. For example, the binding specificity of the monoclonal antibodies can be determined by immunoprecipitation or in vitro binding assays such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA). The binding affinity of the monoclonal antibodies can be determined by, for example, Scatchard analysis (Munson et al., Anal. Biochem., 107:220 (1980)).

[0152] Antibodies according to the present disclosure can be expressed in cells or on cell membranes, or can be secreted extracellularly. If necessary, the recombinant protein can be separated and purified by various separation methods based 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, treatment with a protein precipitant (such as salt precipitation), centrifugation, cell lysis by osmosis, ultrasonic treatment, ultracentrifugation, molecular sieve chromatography (gel chromatography), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC) and any other liquid chromatography, and combinations thereof.

[0153] 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, as determined by comparing sequences. Generally speaking, identity refers to the quantity or percentage of the same position shared by two amino acid or nucleic acid sequences. Considering the number of rooms and the length of each room, it is necessary to introduce these rooms to achieve the best comparison of two sequences. Usually, before calculating the identity percentage between two amino acid or nucleotide sequences, a sequence alignment is performed, and a room (if any) is introduced. If the amino acid residues or bases in the two sequences are identical at a certain comparison position, it is considered that the two sequences are identical or match at that position. If the amino acid residues or bases in the two sequences are different, it is considered that they are inconsistent or mismatched at that position. In some algorithms, the number of matching positions is divided by the total number of positions in the comparison window to obtain sequence identity. In other algorithms, the number of notches and / or the length of notches are also considered. For purposes of the present disclosure, well-known comparison software BLAST (which can be found on the webpage ncbi.nlm.nih.gov) can be used, and default settings are used to obtain the best sequence alignment and calculate the sequence identity between two amino acid or nucleotide sequences. When an amino acid sequence is described as being at least 85%, or at least 90%, or at least 95% identical to another amino acid sequence, the amino acid sequences may differ by conservative substitutions (including where all substitutions are conservative substitutions).

[0154] The term "variant" of a polypeptide, such as an antigen-binding fragment, protein or antibody, is a polypeptide in which one or more amino acid residues are inserted, deleted, added and / or substituted compared to another polypeptide sequence, and includes fusion polypeptides. In addition, protein variants include modifications by enzymatic digestion, phosphorylation or other post-translational modifications, but retain the biological activity of the antibodies disclosed herein, such as binding and specificity to CDH17. The 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 the antibody or antigen-binding fragment thereof disclosed herein. The identity or homology percentage (%) can be calculated with reference to the following description.

[0155] In the present disclosure, the antibodies of the present disclosure also include conservative variants thereof (conventional variants), which means that compared with the amino acid sequence of the antibodies of the present disclosure, up to 10, preferably up to 8, more preferably up to 5, and most preferably up to 3 amino acids are substituted with amino acids having similar or similar properties to form polypeptides. These conservative variant polypeptides are preferably generated by conservative substitutions according to Table A.

[0156] Table A

[0157] original residue Representative replacement Preferred replacement Ala Val; Leu; Ile Val Arg Lys; Gln; Asn Lys Asn Gln; His; Lys; Arg Gln Asp Glu Glu Cys Ser Ser Gln Asn Asn Glu Asp Asp Gly Pro; Ala Ala His Asn; Gln; Lys; Arg Arg Ile Leu; Val; Met; Ala; Phe Leu Leu Ile; Val; Met; Ala; Phe Ile Lys Arg; Gln; Asn Arg Met Leu; Phe; Ile Leu Phe Leu; Val; Ile; Ala; Tyr Leu Pro Ala Ala Ser Thr Thr Thr Ser Ser Trp Tyr; Phe Tyr Tyr Trp; Phe; Thr; Ser Phe Val Ile;Leu;Met;Phe;Ala Leu

[0158] As used herein, the term "K D "(M) is intended to refer to the dissociation equilibrium constant for a specific antibody-antigen interaction. "K D ” refers to the dissociation constant, which is given by K d With K a The ratio of Kd / Ka is obtained and expressed as molar concentration (M). In view of the present disclosure, the K of an antibody can be determined using methods in the art. D For example, the K D It can be determined by using surface plasmon resonance, such as by using a biosensor system (eg, the AUTO(R) system) or using biolayer interferometry (eg, the Octet RED96 system).

[0159] The term "affinity" refers to the strength of the interaction between an antibody or antigen-binding fragment thereof and an antigen, and is determined by properties of the antigen, such as the size, structure, and / or charge of the antigen, and the CDR sequences of the antibody or antigen-binding fragment thereof. Methods for determining affinity are known in the art and can be referenced below.

[0160] When the dissociation constant (K D ) <l0 -6 When K is greater than 1, an antibody or antigen-binding fragment thereof is said to "specifically bind" to its target (e.g., antigen). D yes <l0 -9 When M, the antibody binds specifically to its target with "high affinity".

[0161] 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 (such as physiologically / pharmaceutically acceptable carriers and excipients). The purpose of a pharmaceutical composition is to facilitate administration to an organism, which facilitates the absorption of the active ingredient and the exertion of its biological activity.

[0162] When applied to an animal, a human, a laboratory subject, a cell, a tissue, an organ, or a biological fluid, "administering" and "treating" refer to contacting the animal, human, subject, cell, tissue, organ, or biological fluid with an exogenous pharmaceutical agent, therapeutic agent, diagnostic agent, or composition. "Administering" and "treating" can, for example, refer to therapeutic methods, pharmacokinetic methods, diagnostic methods, research methods, and experimental methods. Treatment of cells encompasses contacting cells with an agent, as well as contacting a fluid with an agent, wherein the fluid is in contact with cells. "Administering" and "treating" also refer to in vitro or ex vivo manipulation, for example, in vitro or ex vivo manipulation of a cell by an agent, a diagnostic, a binding composition, or by another cell. When applied to a human, veterinary, or research subject, "treatment" refers to therapeutic treatment, prophylactic or preventative measures, research and diagnostic applications.

[0163] Furthermore, the present disclosure includes a drug for treating a disease associated with CDH17, comprising the antibody or antigen-binding fragment thereof of the present disclosure as an active ingredient.

[0164] There is no limitation on the disease associated with CDH17, as long as it is a disease associated with CDH17. For example, the therapeutic response induced by the molecules disclosed in this disclosure can be reduced by binding to human CDH17. Therefore, when in a formulation suitable for therapeutic use, the molecules disclosed in this disclosure are very useful for those suffering from tumors, cancers or infectious diseases.

[0165] Furthermore, the present disclosure relates to methods for immunodetection or measurement of CDH17, reagents for immunodetection or measurement of CDH17, methods for immunodetection or measurement of cells expressing CDH17, and diagnostic reagents for diagnosing diseases associated with CDH17-positive cells, comprising the antibody or antigen-binding fragment thereof that specifically recognizes human CDH17 of the present disclosure as an active ingredient.

[0166] In the present disclosure, the method for detecting or determining the amount of CDH17 may be any known method, including, for example, immunoassays or assays.

[0167] Immunoassay or assay is a method for detecting or determining the amount of an antibody or antigen by using a labeled antigen or antibody. Examples of immunoassays or assays include radiolabeled immunoassays (RIA), enzyme immunoassays (EIA or ELISA), fluorescent immunoassays (FIA), luminescent immunoassays, Western blotting, physicochemical methods, and the like.

[0168] The above-mentioned diseases associated with CDH17-positive cells can be diagnosed by detecting or measuring cells expressing CDH17 using the antibodies or antibody fragments or diagnostic conjugates thereof of the present disclosure.

[0169] To detect cells expressing the polypeptide, known immunoassays can be used, preferably immunoprecipitation, fluorescent cell staining, immunohistochemical staining, etc. In addition, fluorescent antibody staining methods using the FMAT8100 HTS system (Applied Biosystem) can be used.

[0170] Example

[0171] The present invention is further illustrated 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. The experimental methods without detailed conditions in the following examples are generally in accordance with the conditions described in conventional conditions, such as Sambrook.J et al. "Molecular Cloning Experimental Guide" (Huang Peitang et al., Beijing: Science Press, 2002), or according to the conditions recommended by the manufacturer (such as product instructions). Unless otherwise stated, percentages and parts are all by weight. Unless otherwise stated, the experimental materials and reagents used in the following examples are all commercially available.

[0172] The room temperature described in the examples is a conventional room temperature in the art and is usually 10-30°C.

[0173] Example 1 Mouse Immunization and Hybridoma Fusion

[0174] Preparation of antigens

[0175] Mice were immunized using a combination of recombinant protein antigen human CDH17 His tag (Sino biological, catalog number 11360-H08H).

[0176] Immunization operation plan:

[0177] Anti-CDH17 antibodies were obtained by immunizing genetically modified mice encoding the variable regions of human immunoglobulin heavy and kappa light chains with a recombinant protein antigen, human CDH17 His-tag (Sino biological, catalog number 11360-H08H), and boosting with the same antigen. Antibody immune responses were monitored using a CDH17-specific immunoassay. When the desired immune response was achieved, splenocytes were harvested from each mouse and fused with mouse myeloma cells to maintain their viability and form hybridomas, which were then screened for CDH17 specificity.

[0178] Spleen cell fusion

[0179] Splenic lymphocytes and myeloma cells Sp2 / 0 ( CRL-158) were fused to obtain hybridoma cells. TMPEG 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.

[0180] Example 2 Hybridoma Screening

[0181] Screening of hybridoma clones specifically binding to CDH17 protein by ELISA

[0182] ELISA was performed using the DuoSet ELISA Accessory Kit (R&D System, DY008). ELISA plates were coated overnight with 1 μg / ml human CDH17 His tag (Sino biological, catalog number 11360-H08H) or BSA. Excess unbound protein was washed away by washing the plates three times with wash buffer 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 washed away and 100 μl of a 1:30,000 diluted secondary antibody goat anti-mouse IgG Fc-HRP (ab5870) was added to each well for 1 hour. The plates were washed according to the manufacturer's protocol and 50 μL of chemiluminescent agent (color A and color B) was added. The reaction was terminated using 25 μL of stop solution. The optical density of the samples at 450 nm was measured by a microplate reader (PerkinElmer). All clones tested selectively bound to human CDH17 but not to BSA.

[0183] Table 1. Characterization of hybridoma clones binding to CDH17 protein by ELISA (OD450)

[0184]

[0185] Screening of hybridoma clones that specifically bind to CDH17-expressing cancer cells by flow cytometry

[0186] Hybridoma supernatants were tested for binding to CDH17-positive cell lines AsPC1 (ATCC, CRL-1682), GP2d (Creative bioarray, CSC-J9456) and CDH17-negative cell line SW480 (ATCC, CCL-228) using flow cytometry analysis. Briefly, 50 μL of cells (2 × 10 6Cells were collected and 50 μL of undiluted supernatant was added. 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 (1:250 dilution, Biolegend, catalog number 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 number 11360-MM02). Purified mouse IgG1 antibody was used as an isotype control (Biolegend, catalog number 400102).

[0187] Figure 1 Examples of selected cell binding signals measured by flow cytometry are shown. Hybridoma clones 1H10, 2F9, 4F9, 9E2, 10E11, 13C7, 14B12, 15A4, 17F3, 19C7, and 20B4 were identified as having enhanced binding profiles to AsPC1 and GP2d cells compared to SW480 cells.

[0188] Screening of hybridoma clones with cell internalization activity by indirect killing assay

[0189] In AsPC1 cells, the cellular internalization activity of hybridoma supernatants was measured using an indirect killing assay. AsPC1 cells were seeded on 96-well plates at a density of 8,000 cells / well, and 500 ng / mL of propidium iodide (abcam, catalog number ab14083) and a 1:2000 dilution of SPY650-DNA (Cytoskeleton, Inc., catalog number CYSC501) were added. The cells were incubated overnight at 37°C, 5% CO2. The hybridoma supernatant from each hybridoma clone was diluted with hybridoma culture medium containing 500 ng / mL propidium iodide and 2000-fold diluted SPY650-DNA and mixed with a Fab anti-mouse IgG Fc-MMAF conjugate with a cleavable linker (Moradec, AM-202AF) and then added to each well. The final concentration of mouse IgG was approximately 10 nM, 3.33 nM, and 1.11 nM. The final concentration of Fab anti-mouse IgG Fc-MMAF conjugate in each well is 20nM. Due to the presence of secondary Fab-vc-MMAF, internalization antibody / Fab-vc-MMAF conjugate complex will release cytotoxic payload and kill cells. Cytation5 (Agilent) is used to image the plate every 8 hours to detect the viability of cells in each well. Purified anti-human CDH17 antibody is used as a positive control (Invitrogen, catalog number (Cat. No.) MA5-29135). Purified mouse IgG1 antibody is used as an isotype control (Biolegend, catalog number (Cat. No.) 400102).

[0190] Cells treated with selected hybridoma supernatants showed reduced viability, e.g. Figure 2 As shown, internalization of the antibodies was demonstrated. Clones 1H10, 2F9, 4F9, 9E2, 10E11, 13C7, 14B12, 15A4, 17F3, 19C7, and 20B4 were identified as being able to internalize into CDH17-positive cell lines using an indirect killing assay.

[0191] Example 3: Sequencing of positive hybridoma clones

[0192] The process of sequencing from positive hybridoma clones is as follows. Hybridoma cells in the logarithmic growth phase are collected, RNA is extracted, reverse transcribed, and then the VDJ region is amplified. The cDNA library amplified from each clone is subjected to next generation sequencing. The amino acid sequences of the heavy chain and light chain variable region DNA sequences corresponding to antibodies 1H10, 2F9, 4F9, 9E2, 10E11, 13C7, 14B12, 15A4, 17F3, 19C7, and 20B4 are obtained. The amino acid sequences of the heavy chain variable region, light chain variable region, and CDR sequences of each antibody are shown in the following table (Tables 2 to 4). The amino acid residues of the CDRs in VH / VL are numbered and annotated according to the Kabat numbering system.

[0193] Table 2. CDR sequences of the heavy chain variable domain in CDH17 hybridoma clones

[0194]

[0195]

[0196] Table 3. CDR sequences of light chain variable domains in CDH17 hybridoma clones

[0197]

[0198] Table 4. Sequences of the heavy and light chain variable domains of CDH17 hybridoma clones

[0199]

[0200]

[0201]

[0202] Example 4 Expression, Purification and Binding Characterization of Recombinant Antibodies Molecular Cloning of Recombinant Antibodies

[0203] The cDNA sequences encoding the VH and VL regions of the clones selected were directly synthesized into DNA fragments with a 5' end in-frame leader sequence (MGWSCIILFLVATATGVHS). These DNA fragments were cloned into the selected vector using the NEBuilder DNA assembly cloning kit (New England Biolabs). The VH region was cloned into the pFUSE-CHIg_hG1 vector (InvivoGen#pfuse-hchg1) in the same frame as the constant region of the hIgG1 heavy chain in the vector. The VL region was cloned into the pFUSE2-CLIg_hk vector (InvivoGen, #pfuse2-hclk) in the same frame as the constant region of the hIgκ light chain in the vector. The amino acid sequences of the hIgG1 heavy chain constant region and the hIgκ light chain constant region are as follows:

[0204] >Heavy chain constant region (SEQ ID NO: 99):

[0205] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK;

[0206] >Light chain constant region (SEQ ID NO: 100):

[0207] RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSG NSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNR GEC.

[0208] The IgG forms of the antibodies are disclosed as the following heavy and light chain full lengths in Table 5. 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: 95 (heavy chain) and 96 (light chain); 20B4: SEQ ID NO: 97 (heavy chain) and 98 (light chain).

[0209] Table 5. Full-length sequences of heavy and light chains of anti-CDH17 recombinant antibodies

[0210]

[0211]

[0212]

[0213]

[0214]

[0215]

[0216] Expression and purification of recombinant antibodies

[0217] Heavy chain expression plasmids and light chain plasmids were co-transfected into CHO cells (ATCC, catalog #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). Based on the manufacturer's instructions, the plasmid DNA concentration was 1.0 μg per ml of suspended cells, with a 1:1 LC:HC vector ratio. Transfected cells were cultured on an orbital shaker at 37°C, 8% CO₂ for 5 to 7 days. Conditioned medium was collected and antibodies were purified using HiTrap MabSelect SuRe columns (Cytiva, #17549112) on an AKTA Pure 25 machine (Cytiva). The eluted antibodies were neutralized with Tris buffer (pH 9.0) and exchanged into PBS. Product concentration was measured by UV absorption, and mass was determined by SDS-PAGE and HPLC.

[0218] Binding characterization of anti-CDH17 recombinant antibodies to CDH17-positive and CDH17-negative cell lines by flow cytometry

[0219] Using cancer cell lines including CDH17-positive cancer cell AsPC1 cells and CDH17-negative cancer cell SW480 cells, the binding of the recombinant antibody (human IgG1) to cell surface CDH17 was determined by FACS analysis.

[0220] 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.

[0221] To determine the binding of the recombinant antibody to the cell surface CDH17 receptor, cells were first harvested and cultured at 1.3 × 10 6 to 1.5×10 6Cells / mL were resuspended in cell staining buffer (BioLegend, catalog number 420201). The cells were then treated with human Fc receptor blocking reagent (BioLegend, catalog number 422302) on ice for 10 minutes. The resulting cell suspension was aliquoted into 50 μL portions. 25 μL of recombinant antibodies at different concentrations were mixed with the cell aliquots, and the final concentration of the recombinant antibodies in the mixture ranged from 1.1 pM to 200 nM. The cells were incubated on ice for 1 hour and then washed twice with cell staining buffer. 50 μL of secondary antibody (PE-conjugated goat anti-human Fc, eBioscience TM , diluted 1:250) was added to each sample to resuspend the cells. The cells were incubated on ice for an additional 20 min. The cells were then washed twice with cell staining buffer and resuspended in 4% PFA to fix the cells. The samples were analyzed using iQue3 to measure median fluorescence intensity using the corresponding channel.

[0222] The results are published on Figure 3 and Table 6. HBMAB81 is a positive control monoclonal antibody disclosed in patent application WO2018115231A2. B12 is an internal isotype control antibody disclosed in patent US005652138A. As a result, it was confirmed that the anti-CDH17 antibody of the present disclosure specifically binds to human CDH17 originally expressed in cells in a concentration-dependent manner, while HBMAB81 non-specifically binds to the CDH17-negative cell line SW480.

[0223] Table 6. K values ​​of anti-CDH17 recombinant antibodies binding to CDH17-positive AsPC1 cells D value

[0224] Antibody <![CDATA[K D (nM)]]> 1H10 0.31 2F9 0.17 4F9 1.69 9E2 0.31 10E11 0.10 13C7 0.28 14B12 0.07 15A4 2.32 17F3 0.74 19C7 0.13 20B4 0.10

[0225] Example 5 Characterization of cellular internalization of anti-CDH17 recombinant antibodies by indirect killing assay in cells expressing CDH17

[0226] To evaluate the endocytosis of antibodies induced by anti-CDH17 antibody binding, an indirect killing assay was used to characterize antibody internalization. A Fab anti-human IgG Fc-MMAF conjugate (Moradec, AH-202AF) with a cleavable linker was incubated with a recombinant anti-CDH17 antibody to form a complex. The formed complex was then incubated with cells expressing CDH17. After binding to the cell surface CDH17 receptor, the complex was internalized, and the conjugated MMAF was released after lysosomal cleavage of the linker. The released MMAF then inhibited cell division by blocking the polymerization of tubulin. In brief, AsPC1 cells, a cell line expressing CDH17, were seeded into 96-well plates at 5,000 cells / well and incubated overnight. Recombinant anti-CDH17 antibodies were mixed with a 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 a complex. Then, serial dilutions of the complex (from 4.5 pM to 30 nM) were added to each well, and cell viability was measured for 48 h using the Cytation 5 imaging system. Dose-response curves were plotted and fitted using GraphPad Prism 9.

[0227] like Figure 4 As shown in Table 7 , all antibody clones exhibited killing effects in the AsPC1 cell line compared with the negative control clone (B12), indicating that MMAF was effectively delivered into the cells.

[0228] Table 7. IC of indirect killing assay used to evaluate internalization of recombinant anti-CDH17 antibodies in AsPC1 cells 50 .

[0229] Clone ID <![CDATA[IC 50 (nM)]]> 1H10 0.19 2F9 0.14 4F9 0.29 9E2 0.15 10E11 0.08 13C7 0.09 14B12 0.09 15A4 1.99 17F3 0.08 19C7 0.09 20B4 0.07

[0230] Example 6 Characterization of the binding activity of anti-CDH17 clones to cross-species CDH17 and homologs by Octet.

[0231] The affinity between CDH17 antibodies and CDH17 antigens and homologs was determined using an Octet (Octet Red 384) instrument. An anti-hIgG Fc capture (AHC) biosensor was selected and the sensor was equilibrated with buffer solution for 10 minutes. Subsequently, the sensor was immersed in a well containing 1 μg / ml CDH17 lead antibody to load the antibody onto the probe. Excess unbound antibody was washed away. Antigen was bound to a solution containing serially diluted human CDH17-His tag (Sino biological, catalog number 11360-H08H), cynomolgus monkey CDH17-His tag (Acro Biosystems, catalog number CA7-C52H4), homolog human CDH6-His tag (Acro Biosystems, catalog number CA6-H5229) and human CDH16-His tag (Sino biological, catalog number 10915-H08H) at concentrations ranging from 200 nM to 3.1 nM for 10 minutes. The probes were then immersed in wells containing refreshing buffer to initiate dissociation for another 30 min.

[0232] The results showed that 10E11, 14B12 and 20B4 all bound to human and cynomolgus monkey CDH17 proteins, while HBMAB81 only bound to human CDH17 protein ( Figures 5 to 8 , Table 8).

[0233] Table 8. Binding activity of anti-CDH17 clones to CDH17 and its homologs across species.

[0234]

[0235] Example 7 Competitive Binding

[0236] 20B4 and HBMAB81 mAbs were labeled with Alexa Flour 647 according to the manufacturer's instructions (ThermoFisher #A20186). In competitive binding assays, 20B4-AF647 and HBMAB81-AF647 were immobilized at EC80 concentrations of 1.51 nM and 0.97 nM, respectively. 20B4 and HBMAB81, ranging from 0.03 nM to 1800 nM, were first mixed with 1.51 nM 20B4-AF647 or 0.97 nM HBMAB81-647 and then incubated with AsPC1 cells on ice for 1 hour. Cells were washed twice with cell staining buffer. Samples were analyzed using iQue3 to measure median fluorescence intensity using the corresponding channel.

[0237] The results are as follows Figure 9The "20B4-AF647+HBMAB81" group and the "HBMAB-AF647+20B4" group showed no binding inhibition, indicating that 20B4 and HBMAB81 do not bind to the same or similar epitopes.

[0238] Although the specific embodiments of the present invention have been described in detail, those skilled in the art will appreciate that various modifications and changes can be made to the details based on all disclosed teachings, which are also included in the scope of the present invention. The scope of protection of the present invention is defined by the appended claims and any equivalent solutions thereof.

Claims

1. An anti-CDH17 antibody or antigen-binding fragment thereof, comprising: a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 regions and a light chain variable region comprising LCDR1, LCDR2, and LCDR3 regions, wherein: Each of the HCDR1s has the amino acid sequence shown in SEQ ID NO: 10, HCDR2 having the amino acid sequence shown in SEQ ID NO: 21, and a HCDR3 having the amino acid sequence shown in SEQ ID NO: 31; or Each of the HCDR1s has the amino acid sequence shown in SEQ ID NO: 07, HCDR2 having the amino acid sequence shown in SEQ ID NO: 17, and a HCDR3 having the amino acid sequence shown in SEQ ID NO: 28; or Each of the HCDR1s has the amino acid sequence shown in SEQ ID NO: 05, HCDR2 having the amino acid sequence shown in SEQ ID NO: 15, and a HCDR3 having the amino acid sequence shown in SEQ ID NO: 26; and / or 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; or LCDR1 having the amino acid sequence shown in SEQ ID NO: 38, LCDR2 having the amino acid sequence shown in SEQ ID NO: 45, and LCDR3 having the amino acid sequence shown in SEQ ID NO: 53; or LCDR1 having the amino acid sequence shown in SEQ ID NO: 36, LCDR2 having the amino acid sequence shown in SEQ ID NO: 43, and LCDR3 having the amino acid sequence shown in SEQ ID NO:

51.

2. The anti-CDH17 antibody or antigen-binding fragment thereof according to claim 1, wherein: The heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 having the amino acid sequences of 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 of 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 of 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 of 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 shown 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 shown in SEQ ID NO: 36, SEQ ID NO: 43 and SEQ ID NO: 51, respectively.

3. The anti-CDH17 antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein 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; Preferably, the multispecific antibody is 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, 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, 70, or a sequence having at least 80%, 85%, 90%, 95% or 99% sequence identity thereto.

5. The anti-CDH17 antibody or antigen-binding fragment thereof according to claim 4, wherein the antibody or antigen-binding fragment thereof comprises: a heavy chain variable region having the amino acid sequence of 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 of 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 of 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 of SEQ ID NO:72, 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 of 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 of SEQ ID NO: 70, or an amino acid sequence having at least 80%, 85%, 90%, 95% or 99% sequence identity thereto.

6. The anti-CDH17 antibody or antigen-binding fragment thereof according to claim 5, wherein the antibody or antigen-binding fragment thereof comprises: a heavy chain variable region having the amino acid sequence of SEQ ID NO: 65; and / or a light chain variable region having the amino acid sequence of SEQ ID NO: 76; or a heavy chain variable region having the amino acid sequence of SEQ ID NO: 61; and / or a light chain variable region having the amino acid sequence of SEQ ID NO: 72; or 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 anti-CDH17 antibody or antigen-binding fragment thereof according to claim 6, wherein the antibody or antigen-binding fragment thereof comprises: a heavy chain variable region having the amino acid sequence of SEQ ID NO: 65; and a light chain variable region having the amino acid sequence of SEQ ID NO: 76; or a heavy chain variable region having the amino acid sequence of SEQ ID NO: 61; and a light chain variable region having the amino acid sequence of SEQ ID NO: 72; or 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. The anti-CDH17 antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, 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 conventional variants thereof, and the light chain constant region of the human antibody constant region is selected from the constant regions of human antibody kappa chain and lambda chain and conventional 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, 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; 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.

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

10. The anti-CDH17 antibody or antigen-binding fragment thereof according to claim 9, wherein the antibody or antigen-binding fragment thereof comprises: a heavy chain having the amino acid sequence of SEQ ID NO: 97 and a light chain having the amino acid sequence of SEQ ID NO: 98; or a heavy chain having the amino acid sequence of SEQ ID NO: 89 and a light chain having the amino acid sequence of SEQ ID NO: 90; or The heavy chain has the amino acid sequence shown in SEQ ID NO: 85 and the light chain has the amino acid sequence shown 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 the group consisting of: Fab, Fab', F(ab')2, variable fragment (Fv), single-chain variable fragment (scFv), dimerization domain V (diabody), disulfide-stabilized Fv (dsFv) and CDR-containing peptide.

12. The anti-CDH17 antibody or antigen-binding fragment thereof according to any one of claims 1 to 11, wherein the antibody or antigen-binding fragment thereof has a K of ≤ 1000 nM. D Binds both human and cynomolgus CDH17. 13 . The anti-CDH17 antibody or antigen-binding fragment thereof according to 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 . 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 according to any one of claims 1 to 14.

16. A recombinant vector comprising the isolated nucleic acid molecule of claim 15. A host cell comprising the recombinant vector according to claim 16 , wherein the host cell is selected from prokaryotic cells and eukaryotic cells, preferably eukaryotic cells, more preferably mammalian cells.

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

19. A diagnostic conjugate comprising the anti-CDH17 antibody or antigen-binding fragment thereof according to 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 agent is selected from a detectable label, an enzyme, a metal-containing substance, a fluorescent substance, a luminescent substance, a colored substance and quantum dots.

20. 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 according to any one of claims 1 to 14.

21. A method for producing the anti-CDH17 antibody or antigen-binding fragment thereof according to any one of claims 1 to 14, wherein the method comprises: The host cell of claim 17 is cultured in a culture medium to produce and accumulate the anti-CDH17 antibody or antigen-binding fragment thereof according to any one of claims 1 to 14, and the antibody or antigen-binding fragment thereof is harvested from the culture.

22. A method for immunologically detecting or measuring CDH17, wherein the method comprises the step of contacting a sample with the anti-CDH17 antibody or antigen-binding fragment thereof according to any one of claims 1 to 14.

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

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

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

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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