Ligand-cytotoxic drug conjugates and their pharmaceutical uses

Abstract

The present invention provides an antitumor drug with excellent therapeutic effects, which is superior in both antitumor efficacy and safety. It offers an anti-cadherin 17 antibody comprising a heavy chain variable region and a light chain variable region, its drug conjugate, a composition containing the anti-cadherin 17 antibody or its drug conjugate, and its use as a pharmaceutical.

Description

[Technical Field]

[0001] This invention relates to a novel cadherin 17 antibody or a functional fragment thereof comprising modified heavy and light chains. The invention further relates to a conjugate of an improved cadherin 17 antibody with a small molecule drug. The invention further relates to the use of the antibody and its conjugate in the manufacture of drugs for the treatment of cancer. [Background technology]

[0002] Cadherin 17 (CDH17) is a cell surface marker belonging to the cadherin superfamily and possesses a unique biological structure. Compared to the classical five-repeated cadherin, it has seven extracellular cadherin repeats and an extremely short intracellular region of 20 amino acid residues that lacks a conserved intracellular domain (Berndorff et al., J Cell Biol. 1994, 125(6):1353-1369). Although much of the biological function of CDH17 remains unclear, it is thought to be related to water absorption by Ca 2+ It has been reported that CDH17 can be regulated in a dependent manner (Ahl et al., Biol. Med. Model. 2011, 8(18)), and it also maintains tissue integrity through integrin interactions via extracellular tight junctions. It is primarily expressed in the human gastrointestinal (GI) duct and pancreas.

[0003] CDH17 has been reported to be 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 a crucial role in regulating cancer metastasis and tumor growth. The tumor activity involving CDH17 is related to multiple signaling pathways. One of the most important mechanisms is the CDH17-integrin interaction. It has already been demonstrated that the RDG motif of CDH17 increases cancer cell proliferation and adhesion by binding to α2β1 integrin and inducing the activation of β1 integrin (Bartomome et al., J Biol Chem. 2014, 289(50):34801-34914). CDH17 modulates cancer invasion in GI cancers through Wnt / β-catenin signaling (Qiu et al., PloS one 2013, 8(3)) and the NFκB signaling pathway (Wang et al., Cancer biology & therapy. 2013, 14(3): 262-270). Due to its finite expression in normal tissues and high expression in various cancers, CDH17 is a good cancer target.

[0004] In this field, several CDH17-based targeted antibody drugs have been studied. Two bispecific antibody-drugs, BI905711 (Boehringer Ingelheim) and ARB202 (Arbele), are in Phase I trials, and other anti-CDH17 CAR (chimeric antigen receptor) and monoclonal antibody drugs are in preclinical trials. However, there are currently no reports on anti-CDH17 antibody-drug conjugates (ADCs). This invention provides the first anti-CDH17 ADC in this field.

[0005] Antibody-drug conjugates (ADCs) represent a novel category of therapies that include antibodies conjugated with cytotoxic drugs via a chemical linker. The therapeutic concept of ADCs is to combine the binding ability of an antibody with that of a drug, allowing the antibody to bind to an antigen on the target surface and deliver the drug to tumor cells.

[0006] Therefore, there remains a demand in this field for anti-CDH17 antibodies and ADCs that can be used for therapeutic purposes to treat cancers expressing CDH17. These conjugates possess excellent biological activity, stability, and uniformity, as well as low toxicity and side effects. [Overview of the project]

[0007] This invention provides an anti-CDH17 antibody, an antibody-drug conjugate, and a method of using the same. The anticancer effect of antibody-drug conjugates is thought to depend on their uptake by cancer cells expressing surface antigens; therefore, insufficient internalization of monoclonal antibodies targeting CDH17 is an urgent issue that needs to be addressed. Generally, the development of ADCs requires consideration of all these important components, including the selection of the target antigen, antibody, toxin drug, and linker.

[0008] The technical problem that this invention aims to solve is the development of an advanced anti-CDH17 antibody-drug conjugate that exhibits extremely strong antitumor activity in cancer. This antitumor activity may be due to higher DAR and potent endocytosis.

[0009] Specifically, the present invention includes the following embodiments: This disclosure relates to antibody-drug conjugates or pharmaceutically acceptable salts or solvates thereof and their pharmaceutically acceptable uses, wherein the antibody-drug conjugate or pharmaceutically acceptable salt or solvate thereof comprises an anti-CDH17 antibody or its antigen-binding fragment conjugated with a toxic drug (optionally via a linker).

[0010] This disclosure relates to antibody-drug conjugates or pharmaceutically acceptable salts or solvates thereof and their pharmaceutical uses, wherein the antibody-drug conjugate or pharmaceutically acceptable salt or solvate thereof comprises an anti-CDH17 antibody or its antigen-binding fragment conjugated with a toxic drug (optionally via a linker), wherein the anti-CDH17 antibody or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region of the antibody, wherein a) a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 having amino acid sequences shown in SEQ ID NO: 01, SEQ ID NO: 11, and SEQ ID NO: 22, respectively, and a light chain variable region comprising LCDR1, LCDR2, and LCDR3 having amino acid sequences shown in SEQ ID NO: 32, SEQ ID NO: 43, and SEQ ID NO: 48, respectively, or b) the heavy chain variable region comprising SEQ ID NO: 02, SEQ ID NO: 12, and SEQ ID c) The light chain variable region includes HCDR1, HCDR2, and HCDR3 having the amino acid sequence shown in NO:23, and 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 d) The heavy chain variable region includes 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 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 The light chain variable region containing LCDR1, LCDR2, and LCDR3 having the amino acid sequence shown in NO:49, or e) the heavy chain variable region, respectively, is SEQ ID NO:05, SEQ ID NO:15, and SEQ IDThe light chain variable region includes HCDR1, HCDR2, and HCDR3 having the amino acid sequence shown in NO:26, and the light chain variable region includes 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 f) The heavy chain variable region includes 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 includes 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 g) The heavy chain variable region includes HCDR1, HCDR2, and HCDR3 having the amino acid sequences shown in SEQ ID NO:07, SEQ ID NO:17, and SEQ ID NO:28, respectively, and the light chain variable region includes SEQ ID NO:38, SEQ ID NO:45, and SEQ ID h) The heavy chain variable region includes LCDR1, LCDR2, and LCDR3 having the amino acid sequence shown in NO:53, respectively, and the light chain variable region includes 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 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 i) The heavy chain variable region includes 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 includes 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 j) The heavy chain variable region includes SEQ ID NO:09, SEQ ID NO:20, and SEQ ID It contains HCDR1, HCDR2, and HCDR3 having the amino acid sequence shown in NO:30, and respectively SEQ ID NO:41, SEQ ID NO:43 and SEQ IDThe light chain variable region comprising LCDR1, LCDR2 and LCDR3 having the amino acid sequences shown in NO:52, or 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 comprises a light chain variable region comprising 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.

[0011] In some embodiments of the present disclosure, in the antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof, the anti-CDH17 antibody or an antigen-binding fragment thereof according to any one of the foregoing embodiments is a monoclonal antibody or an antigen-binding fragment thereof, a polyclonal antibody or an antigen-binding fragment thereof, a multispecific antibody or an antigen-binding fragment thereof, a mouse antibody or an antigen-binding fragment thereof, a chimeric antibody or an antigen-binding fragment thereof, a humanized antibody or an antigen-binding fragment thereof, a recombinant antibody or an antigen-binding fragment thereof, a human antibody or an antigen-binding fragment thereof, and preferably, the multispecific antibody is a bispecific antibody, a trispecific antibody or a tetravalent antibody.

[0012] In some embodiments of the present disclosure, in the antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof, the anti-CDH17 antibody or an antigen-binding fragment thereof according to any one of the foregoing embodiments comprises a heavy chain variable region containing an amino acid sequence selected from SEQ ID NO:55-65 or a sequence having at least 80%, 85%, 90%, 95% or 99% sequence identity thereto, and / or a light chain variable region containing an amino acid sequence selected from SEQ ID NO:66-76 or a sequence having at least 80%, 85%, 90%, 95% or 99% sequence identity thereto.

[0013] In some embodiments of the present disclosure, in an antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof, the anti-CDH17 antibody or an antigen-binding fragment thereof according to any one of the foregoing embodiments contains a heavy-chain variable region having an amino acid sequence selected from SEQ ID NO: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 having an amino acid sequence selected from SEQ ID NO:76, 72, 70 or a sequence having at least 80%, 85%, 90%, 95% or 99% sequence identity thereto.

[0014] In some embodiments of this disclosure, in an antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof, the anti-CDH17 antibody or antigen-binding fragment according to any one of the embodiments described above comprises: a) a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 55, or an amino acid sequence having at least 80%, 85%, 90%, 95%, or 99% sequence identity thereto; and / or a light chain variable region having the amino acid sequence shown in SEQ ID NO: 66, or an amino acid sequence having at least 80%, 85%, 90%, 95%, or 99% sequence identity thereto; or b) a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 56, or an amino acid sequence having at least 80%, 85%, 90%, 95%, or 99% sequence identity thereto; and / or SEQ ID A light chain variable region having an amino acid sequence shown in NO:67, or an amino acid sequence having at least 80%, 85%, 90%, 95%, or 99% sequence identity thereto, or c) a heavy chain variable region having an amino acid sequence shown in SEQ ID NO:57, or an amino acid sequence having at least 80%, 85%, 90%, 95%, or 99% sequence identity thereto, and / or a light chain variable region having an amino acid sequence shown in SEQ ID NO:68, or an amino acid sequence having at least 80%, 85%, 90%, 95%, or 99% sequence identity thereto, or d) a heavy chain variable region having an amino acid sequence shown in SEQ ID NO:58, or an amino acid sequence having at least 80%, 85%, 90%, 95%, or 99% sequence identity thereto, and / or SEQ ID A light chain variable region having an amino acid sequence shown in NO:69, or an amino acid sequence having at least 80%, 85%, 90%, 95%, or 99% sequence identity thereto, or e) A heavy chain variable region having an amino acid sequence shown in SEQ ID NO:59, or an amino acid sequence having at least 80%, 85%, 90%, 95%, or 99% sequence identity thereto, and / or a light chain variable region having an amino acid sequence shown in SEQ ID NO:70, or an amino acid sequence having at least 80%, 85%, 90%, 95%, or 99% sequence identity thereto, or f) SEQ IDA heavy chain variable region having the amino acid sequence shown in 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 shown in SEQ ID NO:71, or an amino acid sequence having at least 80%, 85%, 90%, 95%, or 99% sequence identity thereto, or g) A heavy chain variable region having the amino acid sequence shown in SEQ ID NO:61, or an amino acid sequence having at least 80%, 85%, 90%, 95%, or 99% sequence identity thereto, and / or a light chain variable region having the amino acid sequence shown in SEQ ID NO:72, or an amino acid sequence having at least 80%, 85%, 90%, 95%, or 99% sequence identity thereto, or h) SEQ ID i) A heavy chain variable region having an amino acid sequence shown in NO:62, or an amino acid sequence having at least 80%, 85%, 90%, 95%, or 99% sequence identity thereto, and / or a light chain variable region having an amino acid sequence shown in SEQ ID NO:73, or an amino acid sequence having at least 80%, 85%, 90%, 95%, or 99% sequence identity thereto, or j) SEQ ID A heavy chain variable region having the amino acid sequence shown in NO:64, or an amino acid sequence having at least 80%, 85%, 90%, 95%, or 99% sequence identity thereto, and / or a light chain variable region having the amino acid sequence shown in SEQ ID NO:75, or an amino acid sequence having at least 80%, 85%, 90%, 95%, or 99% sequence identity thereto, or k) a heavy chain variable region having the amino acid sequence shown in SEQ ID NO:65, or an amino acid sequence having at least 80%, 85%, 90%, 95%, or 99% sequence identity thereto, and / or a light chain variable region having the amino acid sequence shown in SEQ ID NO:76, or an amino acid sequence having at least 80%, 85%, 90%, 95%, or 99% sequence identity thereto.

[0015] In some embodiments of this disclosure, in an antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof, the anti-CDH17 antibody or antigen-binding fragment according to any one of the embodiments described above comprises: a) a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 55 and / or a light chain variable region having the amino acid sequence shown in SEQ ID NO: 66; b) a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 56 and / or a light chain variable region having the amino acid sequence shown in SEQ ID NO: 67; c) a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 57 and / or a light chain variable region having the amino acid sequence shown in SEQ ID NO: 68; d) a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 58 and / or a light chain variable region having the amino acid sequence shown in SEQ ID NO: 69; e) 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; or f) SEQ ID The configuration includes a heavy chain variable region having the amino acid sequence shown in NO:60 and / or a light chain variable region having the amino acid sequence shown in SEQ ID NO:71, or g) a heavy chain variable region having the amino acid sequence shown in SEQ ID NO:61 and / or a light chain variable region having the amino acid sequence shown in SEQ ID NO:72, or h) a heavy chain variable region having the amino acid sequence shown in SEQ ID NO:62 and / or a light chain variable region having the amino acid sequence shown in SEQ ID NO:73, or i) a heavy chain variable region having the amino acid sequence shown in SEQ ID NO:63 and / or a light chain variable region having the amino acid sequence shown in SEQ ID NO:74, or j) a heavy chain variable region having the amino acid sequence shown in SEQ ID NO:64 and / or a light chain variable region having the amino acid sequence shown in SEQ ID NO:75, or k) a heavy chain variable region having the amino acid sequence shown in SEQ ID NO:65 and / or a light chain variable region having the amino acid sequence shown in SEQ ID NO:76.

[0016] In some embodiments of this disclosure, in an antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof, the anti-CDH17 antibody or antigen-binding fragment according to any one of the embodiments described above comprises: a) a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 55 and a light chain variable region having the amino acid sequence shown in SEQ ID NO: 66; b) a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 56 and a light chain variable region having the amino acid sequence shown in SEQ ID NO: 67; c) a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 57 and a light chain variable region having the amino acid sequence shown in SEQ ID NO: 68; d) a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 58 and a light chain variable region having the amino acid sequence shown in SEQ ID NO: 69; e) a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 59 and a light chain variable region having the amino acid sequence shown in SEQ ID NO: 70; or f) a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 60 and SEQ The light chain variable region includes the amino acid sequence shown in ID NO:71, or g) a heavy chain variable region having the amino acid sequence shown in SEQ ID NO:61 and a light chain variable region having the amino acid sequence shown in SEQ ID NO:72, or h) a heavy chain variable region having the amino acid sequence shown in SEQ ID NO:62 and a light chain variable region having the amino acid sequence shown in SEQ ID NO:73, or i) a heavy chain variable region having the amino acid sequence shown in SEQ ID NO:63 and a light chain variable region having the amino acid sequence shown in SEQ ID NO:74, or j) a heavy chain variable region having the amino acid sequence shown in SEQ ID NO:64 and a light chain variable region having the amino acid sequence shown in SEQ ID NO:75, or k) a heavy chain variable region having the amino acid sequence shown in SEQ ID NO:65 and a light chain variable region having the amino acid sequence shown in SEQ ID NO:76.

[0017] In some embodiments of the present disclosure, in an antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof, the anti-CDH17 antibody or its antigen-binding fragment according to any one of the embodiments described above 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 their common variants, and the light chain constant region of the human antibody constant region is selected from the constant regions of the κ and λ chains of the human antibody and their common variants, 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, and even more preferably the full-length antibody comprises the human antibody heavy chain constant region of SEQ ID NO:99 and SEQ ID Contains the constant region of the human light chain NO:100.

[0018] In some embodiments of this disclosure, in an antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof, the anti-CDH17 antibody or antigen-binding fragment according to any one of the embodiments described above comprises a heavy chain having the amino acid sequence shown 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 shown in SEQ ID NO:98 or an amino acid sequence having at least 80%, 85%, 90%, 95%, or 99% sequence identity thereto, or A heavy chain having the amino acid sequence shown in SEQ ID NO:89 or an amino acid sequence having at least 80%, 85%, 90%, 95%, or 99% sequence identity thereto, and a light chain having the amino acid sequence shown in SEQ ID NO:90 or an amino acid sequence having at least 80%, 85%, 90%, 95%, or 99% sequence identity thereto, or It includes a heavy chain having the amino acid sequence shown in SEQ ID NO:85 or an amino acid sequence having at least 80%, 85%, 90%, 95%, or 99% sequence identity thereto, and a light chain having the amino acid sequence shown in SEQ ID NO:86 or an amino acid sequence having at least 80%, 85%, 90%, 95%, or 99% sequence identity thereto.

[0019] In some embodiments of this disclosure, in an antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof, the anti-CDH17 antibody or antigen-binding fragment according to any one of the embodiments described above comprises: a) a heavy chain having the amino acid sequence shown in SEQ ID NO: 77 and a light chain having the amino acid sequence shown in SEQ ID NO: 78; b) a heavy chain having the amino acid sequence shown in SEQ ID NO: 79 and a light chain having the amino acid sequence shown in SEQ ID NO: 80; c) a heavy chain having the amino acid sequence shown in SEQ ID NO: 81 and a light chain having the amino acid sequence shown in SEQ ID NO: 82; d) a heavy chain having the amino acid sequence shown in SEQ ID NO: 83 and a light chain having the amino acid sequence shown in SEQ ID NO: 84; e) a heavy chain having the amino acid sequence shown in SEQ ID NO: 85 and a light chain having the amino acid sequence shown in SEQ ID NO: 86; f) a heavy chain having the amino acid sequence shown in SEQ ID NO: 87 and a light chain having the amino acid sequence shown in SEQ ID NO: 88; or g) SEQ ID The heavy chain has the amino acid sequence shown in NO:89 and the light chain has the amino acid sequence shown in SEQ ID NO:90, or h) a heavy chain has the amino acid sequence shown in SEQ ID NO:91 and the light chain has the amino acid sequence shown in SEQ ID NO:92, or i) a heavy chain has the amino acid sequence shown in SEQ ID NO:93 and the light chain has the amino acid sequence shown in SEQ ID NO:94, or j) a heavy chain has the amino acid sequence shown in SEQ ID NO:95 and the light chain has the amino acid sequence shown in SEQ ID NO:96, or k) a heavy chain has the amino acid sequence shown in SEQ ID NO:97 and the light chain has the amino acid sequence shown in SEQ ID NO:98.

[0020] In some embodiments of the present disclosure, the anti-CDH17 antibody or antigen-binding fragment in an antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof, wherein the anti-CDH17 antigen-binding fragment is selected from Fab, Fab', F(ab')2, variable fragment (Fv), single-stranded variable fragment (scFv), dimerization domain V (diabody), disulfide-stabilized Fv (dsFv), and CDR-containing peptide.

[0021] In some embodiments of this disclosure, the toxic drug in the antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof according to any one of the embodiments described above is selected from tubulin inhibitors, topoisomerase inhibitors, DNA intercalators and RNA polymerase inhibitors or pharmaceutically acceptable salts, esters or analogs thereof, preferably the toxic drug is selected from auristatin analogs, camptothecin derivatives and mytansin analogs, and more preferably Alternatively, the toxic drug may be selected from MMAE, MMAF, Exatecan, MMAD, DM1, DM4, eribulin, pyrrolobenzodiazepine (PBD), DGN-549-C, SN-38, irinotecan, topotecan, berotecan, rubitecan, doxorubicin, PNU-159682, duocalmycin, daunorubicin, mitoxantrone, podophyllotoxin, etoposide, α-amanitin, or a pharmaceutically acceptable salt, ester, or analog thereof.

[0022] In some embodiments of this disclosure, the antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof according to any one of the embodiments described above is a drug conjugate of general formula (A), [ka] Here, L1 and L2 are linking units, y is a number selected from 1 to 10, preferably a number selected from 2 to 8, most preferably 2, 4, 5, 6, 7, or 8, and Ab is the anti-CDH17 antibody or its antigen-binding fragment.

[0023] In some embodiments of the present disclosure, the antibody-drug conjugate according to any one of the foregoing embodiments, or a pharmaceutically acceptable salt or solvate thereof, is an antibody-drug conjugate of general formula (I),

Chemical formula

[0024] In some embodiments of the present disclosure, in the antibody-drug conjugate according to any one of the foregoing embodiments, or a pharmaceutically acceptable salt or solvate thereof, L1 is

Chemical formula

[0025] In some embodiments of the present disclosure, in the antibody-drug conjugate according to any one of the foregoing embodiments, or a pharmaceutically acceptable salt or solvate thereof, L2 is -L a -L b -L c -L d -, where said L a is as shown in general formula (II),

Chemical formula

[0026] In some embodiments of this disclosure, the antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof according to any one of the embodiments described above has the following structure: [ka] [ka] [ka] [ka] y is a number selected from 1 to 10, preferably a number selected from 2 to 8, most preferably 2, 4, 5, 6, 7, or 8, and Ab is the anti-CDH17 antibody or its antigen-binding fragment.

[0027] In some embodiments of this disclosure, the antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof according to any one of the embodiments described above is selected from the following compounds: [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] Here, y is a number selected from 1 to 10, preferably a number selected from 2 to 8, more preferably a number selected from 4 to 8, even more preferably a number selected from 4 to 6 or 6 to 8, and most preferably 4, 5, 6, 7 or 8.

[0028] In another embodiment, the present disclosure provides a pharmaceutical composition comprising an antibody-drug conjugate according to any one of the embodiments described above and one or more pharmaceutically acceptable excipients, diluents, or carriers.

[0029] In another aspect, the present disclosure provides drug uses of antibody-drug conjugates or pharmaceutical compositions containing them according to any one of the embodiments described above.

[0030] In another aspect, the present disclosure provides uses of antibody-drug conjugates or pharmaceutical compositions containing them according to any one of the embodiments described above in the manufacture of drugs for treating and / or preventing CDH17-mediated diseases or disorders. CDH17-mediated diseases or disorders are cancers with high CDH17 expression, or cancers with moderate CDH17 expression.

[0031] In another aspect, the present disclosure discloses the use of an antibody-drug conjugate or a pharmaceutical composition containing the same, according to any one of the embodiments described above, in the manufacture of a drug for treating or preventing a tumor or cancer, preferably the tumor or cancer being gastrointestinal cancer, pancreatic cancer, gallbladder cancer, bile duct cancer, stomach cancer, intestinal cancer, ovarian cancer, colorectal cancer, lung cancer, breast cancer, anal cancer, prostate cancer, kidney cancer, bladder cancer, pharyngeal cancer, nasal cavity cancer, skin cancer, oral cancer, tongue cancer, esophageal cancer, vaginal cancer, cervical cancer, spleen cancer, testicular cancer, or glioblastoma.

[0032] In another aspect, the disclosure further relates to a method for treating and / or preventing a tumor or cancer, wherein the method comprises administering to a patient in need a therapeutically effective amount of an antibody-drug conjugate or a pharmaceutical composition comprising the same according to any one of the foregoing embodiments, preferably the tumor or cancer being associated with high CDH17 expression, or the tumor or cancer being associated with moderate CDH17 expression.

[0033] In another embodiment, the Disclosure further relates to a method for treating or preventing a tumor or cancer, wherein the method comprises administering to a patient in need a therapeutically effective amount of an antibody-drug conjugate or a pharmaceutical composition comprising the same according to any one of the foregoing embodiments, preferably the tumor or cancer being gastrointestinal cancer, pancreatic cancer, gallbladder cancer, bile duct cancer, stomach cancer, intestinal cancer, ovarian cancer, colorectal cancer, lung cancer, breast cancer, anal cancer, prostate cancer, kidney cancer, bladder cancer, pharyngeal cancer, nasal cavity cancer, skin cancer, oral cancer, tongue cancer, esophageal cancer, vaginal cancer, cervical cancer, spleen cancer, testicular cancer, or glioblastoma.

[0034] The active compound (e.g., the ligand-drug conjugate according to the Disclosure, or a pharmaceutically acceptable salt or solvate thereof) can be prepared in a form suitable for administration by any appropriate route, preferably in the form of a unit dose or a single dose that can be self-administered by the subject. The unit dose according to the Disclosure may be a tablet, capsule, cachet, vial, powder, granule, lozenge, suppository, regenerated powder, or liquid formulation.

[0035] The dosage of the active compound or composition used in the therapeutic methods disclosed herein usually varies depending on the severity of the disease, the subject's body weight, and the efficacy of the active compound. However, as a general guideline, an appropriate unit dose is 0.01 to 1000 mg.

[0036] In addition to the active compound, the pharmaceutical composition of this disclosure may contain one or more excipients selected from fillers, diluents, adhesives, wetting agents, disintegrants, and other similar agents. Depending on the administration method, the composition may contain 0.01 to 99.9% by weight of the active compound.

[0037] Beneficial effects of the present invention: The CDH17 antibody and antibody-drug conjugate described herein have good affinity for cell surface antigens, good endocytosis efficiency, high tumor suppression efficiency, and a broader drug application window, making them suitable for clinical drug applications. [Brief explanation of the drawing]

[0038] [Figure 1] This figure illustrates the in vitro binding characteristics of hybridoma clones analyzed by flow cytometry to AsPC1 (A), GP2d (B), and SW480 (C) cells. [Figure 2] This figure shows the characterization of the intracellular translocation activity of selected hybridoma clones using an indirect cytotoxicity assay. [Figure 3]This figure shows flow cytometry analysis and assays characterizing the in vitro binding of anti-CDH17 recombinant antibody to AsPC1 (A) cells and SW480 cells (B), with HBMAB81 used as the positive control and B12 as the isotype control antibody (C). [Figure 4] This is a dose-response curve of internal transfer of the anti-CDH17 recombinant antibody using an indirect cytotoxicity assay. [Figure 5] Competitive binding: (A) 20B4; (B) HBMAB81. [Figure 6] This figure shows the dose-response curve of the in vitro cytotoxic activity of ADC against tumor cells. [Figure 7] This figure shows a comparison of in vitro cytotoxicity in GP2d cell lines of anti-CDH17 ADCs with different DAR values. [Figure 8] This figure shows a comparison of in vitro cytotoxicity in the AsPC1 cell line of anti-CDH17 ADCs with different DAR values. [Figure 9] This figure shows a comparison of in vitro cytotoxicity in the SK-CO-1 cell line of anti-CDH17 ADCs with different DAR values. [Figure 10] This figure shows a comparison of in vitro cytotoxicity in the AsPC1 cell line of anti-CDH17 ADCs with different DAR values. [Figure 11] This figure shows the suppression of tumor growth in AsPC1 tumor-carrying mice by anti-CDH17 ADC. [Figure 12] This figure shows the suppression of tumor growth in GP2d tumor-carrying mice by anti-CDH17 ADC. [Figure 13] This figure shows the suppression of tumor growth in SNU16 tumor-carrying mice by anti-CDH17 ADC. [Figure 14] This figure shows the pharmacokinetic study of anti-CDH17 ADC, with serum concentration levels of total antibody (A) and ADC (B). [Figure 15] This figure shows the pharmacokinetic study of anti-CDH17 ADCs, including the serum concentration levels of total antibody and ADC. [Modes for carrying out the invention]

[0039] Definition of Terms The present invention is based on the development of an antibody that can specifically bind to CDH17. The antibody of the present invention can be optionally conjugated with a growth inhibitor or a cytotoxic agent, the cytotoxic agent being, for example, a topoisomerase inhibitor or auristatin.

[0040] The headings used in this section are for illustrative purposes only and do not limit the invention. Unless otherwise defined herein, scientific and technical terms used herein have the meanings generally understood by those skilled in the art. Also, unless the context specifically requires otherwise, singular forms include plural forms and plural forms include singular forms. Amino acid residue abbreviations are standard three-alphabetic and / or one-alphabetic codes used in the art, and they represent one of the 20 common L-amino acids.

[0041] The terms “CDH17” and “CDH17 antigen” are used synonymously herein and include any variants, isotypes, and species homologs of human CDH17 that are naturally expressed by cells or expressed in cells transfected with the CDH17 gene.

[0042] "CDH17" is broadly interpreted herein as a target and is intended to encompass various forms of CDH17 molecules at different stages in mammals (e.g., humans), including, but not limited to, molecules produced during the amplification, replication, transcription, splicing, translation, and modification processes of the CDH17 gene (e.g., precursor CDH17, mature CDH17, membrane-expressed CDH17, CDH17 splice variants, modified CDH17, or fragments thereof). The term also includes CDH17 that is artificially prepared or expressed in vitro.

[0043] The term "antibody" or "antibodies" is interpreted broadly and includes immunoglobulin molecules, including monoclonal antibodies (including mouse, human, humanized, and chimeric monoclonal antibodies), full-length antibodies, antigen-binding fragments, multispecific antibodies (e.g., bispecific, triplicate, quadruplicate, etc.), dimers, tetramers, or multimers, single-chain antibodies, domain antibodies, and other modified immunoglobulin molecules containing antigen-binding sites with desired specificity.

[0044] "Bispecificity" refers to the specific binding of an antibody to two different antigens or to two different epitopes in the same antigen. Bispecific antibodies may cross-react with other related antigens, such as the same antigen from other species (homologs), e.g., humans or monkeys (e.g., macaques (cynomolgus) or chimpanzees (Pan troglodytes)), or they may bind to epitopes shared by two or more different antigens.

[0045] The term "antibody" refers to a protein comprising at least two heavy (H) chains and two light (L) chains linked to each other via disulfide bonds, or its antigen-binding portion. Each heavy chain consists of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. Each light chain consists of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The VH and VL regions may be further subdivided into hypervariable regions called complementarity-determining regions (CDRs), which contain scattered, more conserved regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs, arranged in the order FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 from the amino acid terminus to the carboxyl group terminus. The variable regions of the heavy and light chains contain binding domains that interact with the antigen. The constant region of an antibody can mediate the binding of immunoglobulins to various cells of the immune system (e.g., effector cells) and to host tissues or factors containing the classical complement system component 1 (C1q).

[0046] As used herein, the antibody term “antigen-binding fragment” refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., CDH17). It has been shown that the antigen-binding function of an antibody can be realized by fragments of a full-length antibody. Examples of binding fragments encompassed by the antibody term “antigen-binding fragment” include (i) a Fab fragment, which is a monovalent fragment consisting of VL, VH, CL, and CHI domains; (ii) a F(ab')2 fragment, which is a bivalent fragment containing two Fab fragments linked via disulfide bonds in the hinge region; (iii) an Fd fragment consisting of VH and CHI domains; (iv) an Fv fragment consisting of VL and VH domains of a single arm of the antibody; (v) a dAb fragment consisting of a VH domain (Ward et al., (1989) Nature 341: 544-546); (vi) an isolated complementarity-determining region (CDR); and (vii) a combination of two or more isolated CDRs that can be optionally linked via a synthetic linker. Although the two domains VL and VH of the Fv fragment are encoded by separate genes, they can be linked via a synthetic linker using recombination methods, thereby creating a single protein chain. In this case, the VL and VH regions pair up to form a monovalent molecule (single-chain Fv (called scFv), e.g., Bird et al. (1988) Science 242: 423-426, and Huston et al. (1988) Proc. Natl. Acad. Sci. USÅ 85: 5879-5883). Such single-chain antibodies are also intended to be covered by the antibody term "antigen-binding moiety".

[0047] As used herein, the term “human antibody” is intended to refer to an antibody having a variable region and a constant region derived from a human germline immunoglobulin sequence. The human antibodies of the present invention may include amino acid residues not encoded by the human germline immunoglobulin sequence (e.g., mutations introduced by in vitro random or site-directed induction or in vivo somatic mutation). However, as used herein, the term “human antibody” does not include antibodies in which a CDR sequence derived from the germline of another mammalian species (e.g., mouse) has been transplanted into a human framework sequence.

[0048] 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) or hybridomas prepared therefrom of human immunoglobulin genes (as further described in Section 1 below), (b) antibodies isolated from transformed host cells expressing antibodies, such as those isolated from transfectomas, (c) antibodies isolated from recombinant combined human antibody libraries, and (d) antibodies prepared, expressed, produced or isolated by any other means relating to splicing human immunoglobulin gene sequences with other DNA sequences. Such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences. However, in some embodiments, in vitro mutagenesis can be performed on such recombinant human antibodies (or, if human Ig sequence transgenic animals are used, in vivo somatic mutagenesis can be performed), and therefore the amino acid sequences of the VH and VL regions of recombinant antibodies may be closely related to human germline VH and VL sequences, but may not naturally exist within the in vivo human antibody germline repertoire.

[0049] The term "CDR" refers to one of the six hypervariable regions within the antibody variable domain, primarily responsible for antigen binding. One of the most commonly used definitions of the six CDRs is provided by Kabat EA et al. (1991) Sequences of proteins of immunological interest. NIH Publication 91-3242. As used herein, the Kabat definition of CDR applies only to the light chain variable domains CDR1, CDR2 and CDR3 (LCDR1, LCDR2, LCDR3 or L1, L2, L3) and the heavy chain variable domains CDR1, CDR2 and CDR3 (HCDR1, HCDR2, HCDR3 or H1, H2, H3).

[0050] Methods for recognizing CDRs within HCVR and LCVR amino acid sequences are well known in the art and can be used to recognize CDRs within HCVR and / or LCVR amino acid sequences as specifically described herein. Exemplary conventions for CDR boundaries include, for example, Chothia (Chothia et al. (1989) Nature 342:877-883) based on the three-dimensional structure of the antibody and the topological structure of the CDR loop; Kabat (Kabat et al., Sequences of Proteins of Immunological Interest, 4th edition, US Department of Health and Human Services, National Institutes of Health (1987)), AbM (University of Bath), Contact (University College London), the international ImMunoGeneTics database (IMGT) (Worldwide Web imgt.cines.fr / ), and the North CDR definition based on affinity propagation clustering utilizing numerous crystal structures. Those skilled in the art can readily identify CDRs defined by each numbering system.

[0051] A useful comparison of CDR numbers is as follows:

[0052] [Table 1]

[0053] Note 1: Some of these definitions (particularly with respect to Chothia loops) vary depending on the individual publication being examined. Note 2: Except for contact definitions that use the Chothia or Martin (extended Chothia) definition, any numbering scheme is applicable to these CDR definitions. Note 3: When numbering using Kabat encryption conventions, the end of a Chothia HCDR1 loop is converted between H32 and H34, which depends on the length of the loop. This is because the Kabat numbering scheme places insertions in H35A and H35B.

[0054] The term "Fab fragment" includes the heavy chain variable domain and the light chain variable domain, and further includes the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. "Fab' fragment" differs from the Fab fragment in that it has several additional residues (including one or more cysteines from the antibody hinge region) at the carboxyl terminus of the heavy chain CH1 domain. "Fab'-SH" refers to a Fab' fragment in which the cysteine ​​residues in its constant domain support a free thiol group. The F(ab')2 antibody fragment was initially produced as a paired Fab' fragment with hinge cysteines between the Fab' fragments. Other chemical couplings of antibody fragments are also known.

[0055] The term “Fc region” is used herein to define the C-terminal region of an immunoglobulin heavy chain, which includes at least a portion of the constant region. The term includes both the native Fc region and mutant Fc regions. In some embodiments, the human IgG heavy chain Fc region extends from Cys226 or Pro230 to the carbonyl end of the heavy chain. However, the C-terminal lysine (Lys447) of the Fc region may or may not be present. Unless otherwise stated, the numbering of amino acid residues in the Fc region or constant region follows the EU numbering system, also known as the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service, National Institutes of Health, Bethesda, Maryland, 1991.

[0056] Those skilled in the art will understand that the precise numbering and location of heavy chain constant region domains may differ between different numbering systems. A useful comparison of heavy chain constant region numbering based on EU and Kabat is provided below, with reference to Edelman et al., 1969, Proc Natl Acad Sci USA 63:78-85 and Kabat et al., 1991, Sequences of Proteins of Immunological Interest, 5th edition, U.S. Public Health Service, U.S. National Institutes of Health, Bethesda, which are incorporated as a whole by reference.

[0057] [Table 2]

[0058] "Conservative modifications," "conservative substitutions," or "conservative variants" refer to substitutions of amino acids in a protein with other amino acids that have similar properties (e.g., charge, side chain size, hydrophobic / hydrophilicity, back chain conformation, and rigidity) that can be frequently substituted without altering the protein's biological activity. Those skilled in the art will generally understand that a single amino acid substitution in a non-essential region of a polypeptide does not alter its biological activity (see, for example, Watson et al. (1987) Molecular Biology of the Gene, The Benjamin / Cummings Pub.Co., p. 224, (4th edition)). Furthermore, substitutions of amino acids with similar structure or function are unlikely to affect biological activity.

[0059] As used herein, the term “nucleic acid molecule” refers to DNA molecules and RNA molecules. Nucleic acid molecules may be single-stranded or double-stranded, but are preferably double-stranded DNA. A nucleic acid is “operably linked” if it is in a functional relationship with another nucleic acid sequence. For example, if a promoter or enhancer affects the transcription of a coding sequence, the promoter or enhancer is effectively linked to the coding sequence.

[0060] The method for producing nucleic acids is a common method in this field. Preferably, it includes the step of obtaining a nucleic acid molecule of the protein by gene cloning technology, or obtaining a nucleic acid molecule encoding the protein by an artificial whole sequence synthesis method.

[0061] As those skilled in the art know, a nucleotide sequence encoding a protein amino acid sequence can be appropriately modified by substitutions, deletions, alterations, insertions, or additions to provide a homolog of a polynucleotide. The homolog of the polynucleotide of the present invention can be produced by substitution, deletion, or addition of one or more nucleotides encoding the gene of the protein sequence, within the range that maintains antibody activity.

[0062] As used herein, the term “linking unit” refers to the portion that links an antibody to a drug in an antibody-drug conjugate (i.e., ADC), which may be cleavable or incleavable. A cleavable linker (i.e., a cleavable linker or a biodegradable linker) can cleave within or on the surface of a target cell to release the drug. In some embodiments, the linking units or linkers of the present invention have good stability and reduce toxicity and side effects by significantly reducing the release of the drug during delivery to the target site (e.g., into the bloodstream). In some specific embodiments, the linking units or linkers of the present invention are selected from cleavable linkers, such as disulfide bond-based linkers (which selectively cleave under high thiol concentrations in tumor cells), peptide linkers (which are cleaved by enzymes in tumor cells), and hydrazone linkers.

[0063] A “linking unit” may include one or more linker elements. Exemplary linker elements include 6-maleimidocaproyl ("MC"), maleimidopropionyl ("MP"), valine-citrulline ("val-cit" or "vc"), alanine-phenylalanine ("ala-phe"), p-aminobenzyloxycarbonyl ("PAB"), N-succinimidyl-4-(2-pyridylthio)pentanoate ("SPP"), N-succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate ("SMCC", "MCC" as herein) and N-succinimidyl(4-iodoacetyl)aminobenzoate ("SIAB"). TM(Also known as a "linker"). The linker may contain one or more elements or combinations thereof from stretcher units, spacer units, and amino acid units, and can be synthesized by methods known in the art. The linker may be a "cleavable linker" that facilitates the release of a drug into the cell. For example, acid-unstable linkers (e.g., hydrazones), protease-sensitive (e.g., peptidase-sensitive) linkers, photo-unstable linkers, dimethyl linkers, or disulfide-containing linkers can be used (Chari et al., Cancer Research, 52: 127-131 (1992); U.S. Patent No. 5,208,020). The terms "toxic drugs" or "cytotoxic drugs" refer to chemical molecules that can strongly inhibit the normal proliferation of tumor cells. In principle, toxic drugs can kill tumor cells at sufficiently high concentrations, but because they lack specificity, they simultaneously induce apoptosis in normal cells and cause serious side effects. Cytotoxic drugs can be selected from any drugs that are harmful to cells (e.g., kill them). Suitable cytotoxic agents for forming the immune conjugate of the present invention include paclitaxel, tubulin inhibitors, duostatin, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, teniposide, vincristine, vinblastine, 48-haracteri, doxorubicin, daunorubicin, dihydroxyanthraquinone dione, mytansine or its analogs or derivatives, mitoxantrone, mitramycin, actinomycin D, l-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol and puromycin, calicheamicin or its analogs or derivatives, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine), Cytarabine, 48-haracteriz, 5-fluorouracil, 48-haracteriz, hydroxyurea, asparaginase, gemcitabine, cladribine), alkylating agents (e.g., nitrogen mustard, thiophene, chlorambucil, melphalan, carmustine (BSNU), lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, dacarbazine (DTIC), procarbazine, mitomycin C, cisplatin and other platinum derivatives such as carboplatin, as well as duocarbamycin A, duocarbamycin SA, CC-1065 (also called racermycin), or analogs or derivatives of CC-1065), dorastatin, auristatin, pyrrolo[2,1-c][1,4] Benzodiazepine drugs (PDB), indolinobenzodiazepine drugs (IGN) or analogues thereof, antibiotics (e.g., dactinomycin (formerly actinomycin)), bleomycin, daunorubicin (formerly daunomycin), doxorubicin, idarubicin, mitramycin, mitomycin, mitoxantrone, plicamycin, anthramycin (AMC)), antimitotic agents (e.g., tubulin targeting agents), diphtheria toxin and related molecules (e.g., diphtheria A chain) and its active fragments and hybrid molecules), lysine (e.g., lysine A or deglycosylated lysine A chain toxin), cholera toxin, Shiga-like toxin (SLTI, SLT II, ​​SLT IIV) This includes, but is not limited to, LT toxin, C3 toxin, Shiga toxin, pertussis toxin, tetanus toxin, soybean Bowman-Birk protease inhibitors, Pseudomonas exotoxin, alorin, saporin, modeccin, geranin abrin A chain, modeccin A chain, α-salamycin, Aleurites fordii protein, dianthin protein, Phytolacca 48haracter protein (PAPI, PAPII, and PAP S), 48haracter Momordica charantia inhibitor, curcin, crotin, Sapaonaria officinalis inhibitor, geronin, mitomycin, colistin, phenylmycin, and 48haracte toxin. Other suitable coupling molecules include antimicrobial / antilytic peptides such as CLIP, magainin 2, melittin, cecropine, and P18; ribonuclease (RNase), DNase I; Staphylococcus enterotoxin A; pokeweed antiviral protein; diphtheria toxin; and Pseudomonas endotoxin.

[0064] Specifically, the toxic drug may be selected from mitotic inhibitors, DNA alkylating agents, tyrosine kinase inhibitors, topoisomerase inhibitors, and DNA synthesis inhibitors, and is preferably a tubulin inhibitor or a topoisomerase inhibitor.

[0065] The term "alkyl group" refers to a saturated aliphatic hydrocarbon group that is a linear or branched group containing 1 to 20 carbon atoms, preferably an alkyl group containing 1 to 12 carbon atoms, more preferably an alkyl group containing 1 to 10 carbon atoms, and most preferably an alkyl group containing 1 to 6 carbon atoms (1, 2, 3, 4, 5, or 6 carbon atoms). Non-limiting examples include methyl group, ethyl group, n-propyl group, isopropyl group, n-butyl group, isobutyl group, tert-butyl group, sec-butyl group, n-pentyl group, 1,1-dimethylpropyl group, 1,2-dimethylpropyl group, 2,2-dimethylpropyl group, 1-ethylpropyl group, 2-methylbutyl group, 3-methylbutyl group, n-hexyl group, 1-ethyl-2-methylpropyl group, 1,1,2-trimethylpropyl group, 1,1-dimethylbutyl group, 1,2-dimethylbutyl group, 2,2-dimethylbutyl group, 1,3-dimethylbutyl group, 2-ethylbutyl group, 2-methylpentyl group, 3-methylpentyl group, 4-methylpentyl group, 2,3-dimethylbutyl group, n-heptyl group, 2-methylhexyl group, 3-methylhexyl group, 4-methylhexyl group, and 5-methylhexyl group. This includes 2,3-dimethylpentyl group, 2,4-dimethylpentyl group, 2,2-dimethylpentyl group, 3,3-dimethylpentyl group, 2-ethylpentyl group, 3-ethylpentyl group, n-octyl group, 2,3-dimethylhexyl group, 2,4-dimethylhexyl group, 2,5-dimethylhexyl group, 2,2-dimethylhexyl group, 3,3-dimethylhexyl group, 4,4-dimethylhexyl group, 2-ethylhexyl group, 3-ethylhexyl group, 4-ethylhexyl group, 2-methyl-2-ethylpentyl group, 2-methyl-3-ethylpentyl group, n-nonyl group, 2-methyl-2-ethylhexyl group, 2-methyl-3-ethylhexyl group, 2,2-diethylpentyl group, n-decyl group, 3,3-diethylhexyl group, 2,2-diethylhexyl group, and various branched isomers thereof.More preferably, the alkyl group is a lower alkyl group having 1 to 6 carbon atoms. Non-limiting examples include methyl group, ethyl group, n-propyl group, isopropyl group, n-butyl group, isobutyl group, tert-butyl group, sec-butyl group, n-pentyl group, 1,1-dimethylpropyl group, 1,2-dimethylpropyl group, 2,2-dimethylpropyl group, 1-ethylpropyl group, 2-methylbutyl group, 3-methylbutyl group, n-hexyl group, 1-ethyl-2-methylpropyl group, 1,1,2-trimethylpropyl group, 1,1-dimethylbutyl group, 1,2-dimethylbutyl group, 2,2-dimethylbutyl group, 1,3-dimethylbutyl group, 2-ethylbutyl group, 2-methylpentyl group, 3-methylpentyl group, 4-methylpentyl group, 2,3-dimethylbutyl group, and the like. The alkyl base may be substituted or unsubstituted. If substituted, the substituent may be substituted at any available linkage point, where the substituent is preferably one or more groups independently selected from alkyl groups, alkenyl groups, alkynyl groups, alkoxy groups, alkylthio groups, alkylamino groups, halogens, mercapto groups, hydroxyl groups, nitro groups, cyano groups, cycloalkyl groups, heterocycloalkyl groups, aryl groups, heteroaryl groups, cycloalkoxy groups, heterocycloalkoxy groups, cycloalkylthio groups, heterocycloalkylthio groups, carboxyl groups, or oxo groups.

[0066] In some embodiments, anti-CDH17 antibodies can be conjugated with one or more topoisomerase inhibitors to form ADCs for the treatment of cancer. Topoisomerases are enzymes that can alter the DNA topology in eukaryotic cells. They are quite important for cell function and cell proliferation. In some embodiments, topoisomerase inhibitors are camptothecin or camptothecin analogs. Camptothecin is a water-insoluble cytotoxic alkaloid produced from Camptotheca accuminata, native to China, and Nothapodytes foetida, native to India. Camptothecin exhibits tumor cell proliferation inhibitory activity against a variety of tumor cells. Camptothecin analog compounds are typically specific inhibitors of DNA topoisomerase I. The term "topoisomerase inhibitor" refers to any tumor cell proliferation inhibitory compound structurally related to camptothecin.

[0067] In some embodiments, the camptothecin analog is an active metabolite of irinotecan (CPT-11). In some such embodiments, the camptothecin analog is 7-ethyl-10-hydroxycamptothecin (SN-38). As a metabolite, SN-38 is produced by hydrolysis of irinotecan by carboxylesterase. In some embodiments, the camptothecin analog is exatecan mesylate. Exatecan mesylate is a water-soluble camptothecin (CPT) and has more effective topoisomerase I inhibitory activity and antitumor activity compared to other CPT analogs. Exatecan is effective against p-glycoprotein (P-gp)-mediated multidrug-resistant cells. In some embodiments, the camptothecin analog is deruxtecan (Dxd), a highly active derivative of exatecan, which has 10 times higher topoisomerase I inhibitory activity than SN-38.

[0068] The term "ligand-cytotoxic drug conjugate" refers to a ligand being linked to a bioactive drug via a linking unit. In some specific embodiments, the "ligand-cytotoxic drug conjugate" is preferably an antibody-drug conjugate (ADC), which refers to a monoclonal antibody or antibody fragment being linked to a bioactive cytotoxic drug via a linking unit.

[0069] The term "drug-antibody ratio (DAR)" refers to the average amount of cytotoxic drug bound to each ligand, and may also be expressed as the ratio of drug amount to antibody amount. The drug loading for each ligand (Ab) may be 1 to 20 units of cytotoxic drug (D). In embodiments of the present invention, the drug-to-antibody ratio is represented as y. The average amount of drug in the ADC molecule after the coupling reaction can be identified by conventional methods, such as UV / visible spectroscopy, mass spectrometry, ELISA, and HPLC characterization.

[0070] As used herein, the term “transfectoma” includes recombinant eukaryotic host cells of an expressing antibody, such as CHO cells, NS / O cells, HEK293 cells, plant cells, or fungi (including yeast cells).

[0071] The DNA molecule sequence of the antibody or its fragment according to the present invention can be obtained by conventional techniques (e.g., PCR amplification or genome library screening methods). Furthermore, the sequences encoding the light chain and heavy chain can fuse to form a single-chain antibody.

[0072] Once a related sequence is obtained, it can be obtained in large quantities using a recombination method. This is usually done by cloning the sequence into a vector using conventional methods, transforming cells with the vector, and then isolating the related sequence from the proliferated host cells.

[0073] Furthermore, especially when the fragment length is short, related sequences may be artificially synthesized. Typically, multiple small fragments are first synthesized, and then they are concatenated to obtain a longer fragment.

[0074] Currently, the DNA sequence encoding the antibody (or fragment thereof or derivative thereof) of the present invention can be obtained entirely by chemical synthesis. Furthermore, the DNA sequence may be introduced into cells with a conventional DNA molecule known in the art (or, for example, a vector). Additionally, mutations may be introduced into the protein sequence of the present invention by chemical synthesis.

[0075] Generally, the obtained host cells are cultured under conditions suitable for the expression of the antibody according to the present invention. Then, the antibody according to the present invention is purified by conventional immunoglobulin purification steps, such as protein A-agarose chromatography, hydroxyapatite chromatography, gel electrophoresis, dialysis, ion exchange chromatography, hydrophobic chromatography, molecular sieve chromatography, or affinity chromatography, which are conventional separation and purification methods well known to those skilled in the art.

[0076] Monoclonal antibodies obtained can be identified by conventional methods. For example, the binding specificity of monoclonal antibodies can be measured by immunoprecipitation or in vitro binding assays (e.g., radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA)). For example, the binding affinity of monoclonal antibodies can be measured by Scatchard analysis (Munson et al., Anal. Biochem., 107: 220 (1980)).

[0077] The antibodies according to the present invention can be expressed in cells or on the cell membrane, or secreted extracellularly. If necessary, recombinant proteins can be isolated and purified by various methods depending on the physical, chemical, and other properties of the recombinant protein. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to, conventional regeneration processes, treatment with protein precipitants (salting-out methods), centrifugation, cell lysis by osmosis, sonication, ultracentrifugation, molecular sieve chromatography (gel chromatography), adsorption chromatography, ion exchange chromatography, high-performance liquid chromatography (HPLC), and various other liquid chromatography methods and combinations thereof.

[0078] The term "sequence identity" refers to the relationship between sequences of two or more polypeptide molecules or two or more nucleic acid molecules, as measured, for example, by sequence alignment. Generally, identity refers to the number or percentage of identical positions shared by two amino acid or nucleic acid sequences, taking into account the number of gaps and the length of each gap, and introducing these gaps to achieve optimal alignment of the two sequences. Typically, sequence alignment is performed and gaps (if any) are introduced before calculating the identity percentage between two amino acid or nucleotide sequences. If amino acid residues or bases in two sequences are the same at a certain alignment position, the two sequences are considered identical or matched at that position. If amino acid residues or bases in two sequences are different, they are considered not to match or mismatched at that position. In some algorithms, sequence identity is obtained by dividing the number of matching positions by the total number of positions in the alignment window. In other algorithms, the number and / or length of gaps are also taken into consideration. For the purposes of this disclosure, the well-known alignment software BLAST (available on the website ncbi.nlm.nih.gov) can be used with default settings to obtain optimal sequence alignment and calculate sequence identity between two amino acid or nucleotide sequences. When one amino acid sequence is described as having at least 85%, at least 90%, or at least 95% identity with another amino acid sequence, the difference between the amino acid sequences may be due to conservative substitutions (where all substitutions contained therein are conservative substitutions).

[0079] The term "variant" in the polypeptide context refers to a polypeptide, e.g., an antigen-binding fragment, protein, or antibody, which 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. Protein variants include those modified by protease cleavage, phosphorylation, or other post-translational modifications, but which retain the biological activity of the antibodies disclosed herein, e.g., specificity to bind to CDH17. Variants may be approximately 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, or 80% identical to the sequences of the antibodies or their antigen-binding fragments disclosed herein. The percentage of identity or homology (%) can be calculated by referring to the following explanation.

[0080] In one embodiment, the homology or identity percentage can be calculated as 100 × [(same position) / min (TGA, TGB)], where TGA and TGB are the sum of the number of residues and the number of internal gap positions in the compared sequences A and B (Russell et al., J. Mol Biol., 244: 332-350 (1994)).

[0081] In the present invention, the antibody further comprises its conserved variants, meaning that, compared to the amino acid sequence of the antibody of the present invention, at least 10, preferably at least 8, more preferably at least 5, and most preferably at least 3 amino acids are substituted with amino acids that are similar or have similar properties to form a polypeptide. These conserved variant polypeptides are preferably produced by amino acid substitutions according to Table A.

[0082] [Table 3]

[0083] As used herein, "K DThe term (M) is intended to refer to the equilibrium dissociation constant of a particular antigen interaction. D " refers to the dissociation constant, which is K d and K a The ratio to (i.e., K d / K a ) is obtained from and expressed as molar concentration (M). In view of this disclosure, the K of the antibody is obtained by the methods of the art. D The value can be measured. For example, the K of an antibody D This can be measured using surface plasmon resonance, for example, using a biosensor system (e.g., the RED96 system) or biolayer interference technology (e.g., the Octet RED96 system).

[0084] The term "affinity" refers to the strength of the interaction between an antibody or its antigen-binding fragment and an antigen, which is determined by the characteristics of the antigen (e.g., size, shape, and / or charge) and the CDR sequence of the antibody or antigen-binding fragment. Methods for measuring affinity known in this art can be found below.

[0085] Dissociation constant (K D ) <l0 -6 When M is the case, the antibody or its antigen-binding fragment is said to "specifically bind" to its target (e.g., antigen). D but <l0 -9 When it is M, the antibody specifically binds to its target with "high affinity".

[0086] As used herein, the term “pharmaceutical composition” is intended to mean a mixture containing one or more of the compounds described herein or their physiologically / pharmaceutically acceptable salts or prodrugs with other chemical components (e.g., physiologically / pharmaceutically acceptable carriers and excipients). The purpose of a pharmaceutical composition is to facilitate administration to a living organism, to facilitate the absorption of the active ingredient, and to exert biological activity.

[0087] Applicable to animals, humans, experimental subjects, cells, tissues, organs, or biological fluids, “administration” and “processing” refer to contacting said animals, humans, experimental subjects, cells, tissues, organs, or biological fluids with exogenous drug reagents, therapeutic reagents, diagnostic reagents, or compositions. “Administration” and “processing” may also refer to, for example, therapeutic methods, pharmacokinetic methods, diagnostic methods, research methods, and experimental methods. Cell processing includes contacting cells with a reagent and contacting a liquid with a reagent, where the liquid is in contact with the cells. “Administration” and “processing” also refer to in vitro and ex vivo processing, for example, in vitro and ex vivo processing of cells with reagents, diagnostic agents, conjugate compositions, or other cells. Applicable to humans, veterinary medicine, or research subjects, “treatment” refers to therapeutic treatment, preventive or precautionary measures, research, and diagnostic applications.

[0088] The "therapeutic dose" refers to the amount that effectively achieves the desired therapeutic effect within the required dosage and duration. The therapeutic dose may vary depending on factors such as the individual's disease state, age, sex, weight, and the ability of the treatment or combination of treatments to elicit the desired response in the individual. Exemplary indicators of an effective treatment or combination of treatments include, for example, improvement in the patient's health status.

[0089] The present disclosure further includes various deuterated forms of the compounds of formulas (I) and (A). Each available hydrogen atom bonded to a carbon atom can be independently substituted with a deuterium atom. Those skilled in the art can synthesize the deuterated forms of the compounds of formulas (I) and (A) by referring to relevant literature. The deuterated forms of the compounds of formulas (I) and (A) can be produced using commercially available deuterating initiators or prior art deuterating reagents, including but not limited to deuterated borane, trihydrogenated borane in tetrahydrofuran, deuterated lithium aluminum, deuterated iodoethane, and deuterated iodomethane.

[0090] Furthermore, this disclosure includes a drug for treating CDH17-related disease, which comprises the antibody, its antigen-binding fragment, or antibody-drug conjugate as an active ingredient.

[0091] The diseases associated with CDH17 are not limited to any diseases related to CDH17, and for example, the molecules disclosed herein can reduce the therapeutic response induced by binding to human CDH17. Therefore, in formulations and prescriptions suitable for therapeutic application, the molecules disclosed herein are very useful for people with tumors, cancer, or infections.

[0092] This disclosure relates to a method for immunological detection or measurement of CDH17, a reagent for immunological detection or measurement of CDH17, a method for immunological detection or measurement of cells expressing CDH17, and a diagnostic reagent for diagnosing diseases associated with CDH17-positive cells, which includes as an active ingredient a target antibody or antigen-binding fragment that specifically recognizes human CDH17 of this disclosure.

[0093] In this disclosure, the method for detecting or measuring the amount of CDH17 may be any known method, for example, including immunological detection or measurement.

[0094] Immunological detection or measurement is a method for detecting or measuring the amount of an antibody or antigen using a labeled antigen or antibody. Examples of immunological detection or measurement include radioactive antibody (RIA), enzyme immunoassay (EIA or ELISA), fluorescence immunoassay (FIA), luminescence immunoassay, Western blotting, and physicochemical methods.

[0095] By using the antibody or antibody fragment of the present invention to detect or measure cells expressing CDH17, the above-mentioned diseases associated with CDH17-positive cells can be diagnosed.

[0096] To detect polypeptide-expressing cells, known immunodetectors may be used, preferably immunoprecipitation, fluorescent cell staining, or chemical staining of immunohistochemistry. Alternatively, a fluorescent antibody staining method may be used, specifically the FMAT8100HTS system (Applied Bio system).

[0097] Examples The present invention will be 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. In the following examples, experimental methods without detailed conditions generally follow the conditions described in conventional conditions, e.g., Sambrook, J. et al., "Guidelines for Molecular Cloning Experiments" (translated by Huang Peitang et al., Beijing: Science Press, 2002), or the conditions recommended by the manufacturer (e.g., instruction manual). Unless otherwise specified, percentages and quantities are calculated on a weight basis. Unless otherwise specified, all experimental materials and reagents used in the following examples are commercially available.

[0098] The room temperature described in the examples is a normal room temperature in the art, and is typically between 10 and 30°C.

[0099] Example 1: Mouse immunization and hybridoma fusion Immunology scheme: Anti-CDH17 antibodies were obtained by immunizing genetically modified mice encoding the variable regions of human immunoglobulin heavy chains and κ-light chains using a recombinant human CDH17 His tag (Sino Biological, catalog number 11360-H08H) and then reinforcing them twice with the same antigen. The immune response of the antibodies was monitored by a CDH17 specificity immunoassay. After the desired immune response was achieved, spleen cells were harvested from each mouse, fused with mouse myeloma cells to maintain their viability, and hybridoma cells were formed to screen for CDH17 specificity.

[0100] Spleen cell fusion Hybridoma cells were obtained by fusing splenic lymphocytes and myeloma cells Sp2 / 0 (ATCC® CRL-158) via electrofusion or PEG fusion. Clonacell was prepared according to the manufacturer's instructions. TM PEG fusion was performed using HY technology (STEMCELL technologies). In the case of electrofusion, the ratio of primary cells to mouse myeloma cell lineage is 1:1, while in the case of PEG fusion it is 10:1.

[0101] Example 2: Hybridoma Screening ELISA screening of hybridoma clones that specifically bind to human CDH17 protein. ELISA was performed using the DuoSet ELISA Auxiliary Kit (R&D System, DY008). ELISA plates were coated overnight with 1 μg / ml human CDH17 His tag (Sino Biological, catalog no. 11360-H08H) or BSA. The plates were washed three times with wash buffer to remove excess unbound protein, and then blocked at room temperature for 1 hour. 100 μl of CDH17 hybridoma supernatant was added to each well and incubated at room temperature for 1 hour. Excess unbound antibody was washed away, and 100 μl of goat anti-mouse IgG Fc-HRP (ab5870) secondary antibody, diluted 1:30000, was added to each well and incubated for 1 hour. The plates were washed according to the manufacturer's instructions, and 50 μL of chemiluminescence reagent (color A and color B) was added. The reaction was stopped with 25 μL of stop solution. The light density of the sample at 450 nm was measured using a microplate reader (PerkinElmer). All clones tested selectively bound to human CDH17 but did not bind to BSA.

[0102] [Table 4]

[0103] Flow cytometry screening of hybridoma clones that specifically bind to CDH17-expressing cancer cells Binding tests were performed on hybridoma supernatant using flow cytometry with CDH17-positive cell lines AsPC1 (ATCC, CRL-1682) and GP2d (Creative bioarray, CSC-J9456), and CDH17-negative cell line SW480 (ATCC, CCL-228). A simple 50 μL of cells (2 × 10⁶) in cell staining buffer was used. 6Cells (1 / mL) were mixed with 50 μL of undiluted supernatant. The mixture was incubated on ice for 1 hour and washed twice with ice-cooled staining buffer. The cells were then stained with 50 μL of PE-labeled secondary antibody (diluted 1:250, Biolegend, catalog no. 405307) for 20 minutes. After washing with staining buffer and fixing with 4% PFA, the cells were analyzed by flow cytometry.

[0104] A purified anti-human CDH17 antibody was used as a positive control (Sino Biological, catalog number 11360-MM02). A purified mouse IgG1 antibody was used as an isotype control (Biolegend, catalog number 400102). Figure 1 shows examples of selected cell binding signals measured by flow cytometry. Compared to SW480 cells, hybridoma clones 1H10, 2F9, 4F9, 9E2, 10E11, 13C7, 14B12, 15A4, 17F3, 19C7, and 20B4 were identified as having enhanced binding profiles to AsPC1 and GP2d cells.

[0105] Screening of hybridoma clones with intracellular migration activity using an indirect cytotoxicity assay. In AsPC1 cells, the intracellular translocation activity of hybridoma supernatant was measured using an indirect cytotoxicity assay. AsPC1 cells were inoculated into 96-well plates at a density of 8,000 cells / well, and 500 ng / mL of propidium iodide (abcam, catalog no. ab14083) and SPY650-DNA (Cytoskeleton, Inc., catalog no. CYSC501) diluted 1:2000 were added. The cells were incubated overnight at 37°C in 5% CO2. Hybridoma supernatant from each hybridoma clone was diluted in hybridoma medium containing 500 ng / mL of propidium iodide and SPY650-DNA diluted 2000-fold, mixed with Fab anti-mouse IgG Fc-MMAF conjugate with a cleavable linker (Moradec, AM-202AF), and added to each well. The final concentrations of mouse IgG were approximately 10 nM, 3.33 nM, and 1.11 nM. The final concentration of Fab anti-mouse IgG Fc-MMAF conjugate in each well was 20 nM. Due to the presence of secondary Fab-vc-MMAF, the internally distributed antibody / Fab-vc-MMAF conjugate complex released a cytotoxic payload, killing cells. Cell viability in each well was detected by imaging the plates every 8 hours using Cytation 5 (Agilent).

[0106] Purified anti-human CDH17 antibody was used as a positive control (Invitrogen, catalog number MA5-29135). Purified mouse IgG1 antibody was used as an isotype control (Biolegend, catalog number 400102). Cells treated with selected hybridoma supernatant showed decreased viability and internal transfer of the antibody, as shown in Figure 2. Using an indirect injury assay, clones 1H10, 2F9, 4F9, 9E2, 10E11, 13C7, 14B12, 15A4, 17F3, 19C7, and 20B4 were identified as having the potential for internal transfer to CDH17-positive cell lines.

[0107] Example 3: Sequencing of positive hybridoma clones The sequencing process for positive hybridoma clones was as follows: Hybridoma cells in the logarithmic growth phase were collected, RNA was extracted, reverse transcription was performed, and the VDJ region was amplified. Next-generation sequencing was performed on the cDNA libraries amplified from each clone. The amino acid sequences of the heavy and light chain variable region DNA sequences corresponding to antibodies 1H10, 2F9, 4F9, 9E2, 10E11, 13C7, 14B12, 15A4, 17F3, 19C7, and 20B4 were obtained. The amino acid sequences of the heavy and light chain variable regions and CDR sequences for each antibody are shown in the following tables (Tables 2 to 4). In VH / VL, the amino acid residues of the CDR were numbered and annotated using the Kabat numbering system.

[0108] [Table 5]

[0109] [Table 6]

[0110] [Table 7-1] [Table 7-2]

[0111] Example 4 Expression, purification, and binding characterization of recombinant antibodies Molecular cloning of recombinant antibodies The cDNA sequences encoding the VH and VL regions of the selected clones were directly synthesized as DNA fragments containing the 5' in-frame reader sequence (MGWSCIILFLVATATGVHS). These DNA fragments were cloned into the selected vectors using a cloning kit (New England Biolabs) assembled with NEBuilder DNA. The VH region was cloned into the pFUSE-CHIg_hG1 vector (InvivoGen #pfuse-hchg1) and is in the same framework 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) and is in the same framework as the constant region of the hIg κ light chain in the vector. The amino acid sequences of the hIgG1 heavy chain constant region and the hIg κ light chain constant region are as follows: [ka] The IgG format of the antibodies was disclosed as the full-length heavy and light chains shown in Table 5 below: 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).

[0112] [Table 8-1] [Table 8-2] [Table 8-3] [Table 8-4] [Table 8-5] [Table 8-6]

[0113] Recombinant antibody expression and purification Heavy chain expression plasmids and light chain plasmids were co-transfected into CHO cells (ATCC, catalog number #CCL-61) using the ExpiFectamine 293 transfect kit (ThermoFisher, A14524), or into ExpiCHO-S cells (ThermoFisher, #A29127) using the ExpiFectamine CHO transfect kit (ThermoFisher, A29129). Following the manufacturer's instructions, the plasmid DNA concentration reached 1.0 μg per ml of suspended cells, and the LC:HC vector ratio was 1:1. Transfected cells were cultured for 5-7 days in an orbital shaker at 37°C and 8% CO2. Conditional media were collected, and antibodies were purified using a HiTrap MabSelect SuRe column (Cytiva, #17549112) on an AKTA Pure 25 machine (Cytiva). The eluted antibodies were neutralized with Tris buffer (pH 9.0), and the PBS buffer was replaced. The product concentration was measured by UV absorption spectroscopy, and the mass was determined by SDS-PAGE and HPLC.

[0114] Characterization of the binding of anti-CDH17 recombinant antibodies to CDH17-positive and CDH17-negative cell lines by flow cytometry. Using a cancer cell line containing CDH17-positive AsPC1 cells and CDH17-negative SW480 cells, the binding of recombinant antibody (human IgG1) to cell surface CDH17 was measured by FACS analysis.

[0115] 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 humid atmosphere of 5% CO2.

[0116] To measure the binding of recombinant antibodies to the cell surface CDH17 receptor, cells were first harvested and measured in 1.3 × 10⁶ units. 6 ~1.5×10 6 Cells were suspended in cell staining buffer (BioLegend, catalog number 420201) at a concentration of 10 cells / mL. The cells were then treated on ice for 10 minutes with human Fc receptor blocking reagent (BioLegend, catalog number 422302). The resulting cell suspensions were divided into 50 μL portions. 25 μL of recombinant antibodies from different serf species were mixed with cell aliquots, resulting in a final concentration range of recombinant antibodies in the mixture from 1.1 pM to 200 nM. The cells were incubated on ice for 1 hour and then washed twice with cell staining buffer. Secondary antibody (PE conjugate goat anti-human Fc, eBioscience) was used. TM 50 μL (diluted 1:250) was added to each sample to resuspend the cells. The cells were incubated on ice for a further 20 minutes. The cells were then washed twice with cell staining buffer and fixed by resuspending them in 4% PFA. The samples were analyzed using iQue3, and the median fluorescence intensity was measured in the corresponding channel.

[0117] HBMAB-81 is a re-formed mAb possessing a CDH17-binding arm and a human IgG1 backbone from the bispecific antibody BI 905771 (see WO 2018115231A2; SEQ ID:116, SEQ ID:117). B12 is an internal isotype control antibody disclosed in patent US005652138A. The results are disclosed in Figure 3 and Table 6. As a result, it was demonstrated that the anti-CDH17 antibodies of this disclosure bind specifically and in a concentration-dependent manner to human CDH17 originally expressed in cells, while HBMAB81 binds nonspecifically to the CDH17-negative cell line SW480.

[0118] [Table 9]

[0119] Example 5: Characterization of intracellular location of anti-CDH17 recombinant antibody by indirect cytotoxicity assay in CDH17-expressing cells To evaluate the endocytotic activity of antibodies induced by anti-CDH17 antibody binding, antibody internalization was characterized using an indirect injury assay. Fab anti-human IgG Fc-MMAF conjugates with a cleavable linker (Moradec, AH-202AF) were incubated with recombinant anti-CDH17 antibody. The resulting complex granules were then incubated with CDH17-expressing cells to form a complex. After binding to the cell surface CDH17 receptor, the complex was internalized, and after lysosomal cleavage of the linker, the conjugated MMAF was released. The released MMAF subsequently inhibited cell division by inhibiting tubulin polymerization. Briefly, CDH17-expressing AsPC1 cells were inoculated into 96-well plates at a rate of 5,000 cells / well and incubated overnight. Recombinant anti-CDH17 antibody was mixed with Fab anti-human IgG Fc-MMAF conjugates with a cleavable linker in a 1:6 (mol / mol) ratio and incubated for 10 minutes to form a complex. Then, a series of dilutions of the complex (4.5 pM to 30 nM) were added to each well, and cell viability was measured for 48 hours using the Cytation 5 imaging system. Dose-response curves were created and fitted using GraphPad Prism 9. As shown in Figure 4 and Table 7, all antibody clones showed toxic effects in the AsPC1 cell line compared to the negative control clone (B12), indicating that MMAF was effectively delivered into the cells.

[0120] [Table 10]

[0121] Example 6 Octet-based characterization of the binding activity of anti-CDH17 clones to interspecies CDH17 and homologs. The affinity between the CDH17 antibody and the CDH17 antigen and homolog was measured using an Octet (Octet Red 384) instrument. An anti-hIgG Fc capture (AHC) biosensor was selected and the sensor was equilibrated in buffer solution for 10 minutes. The sensor was then immersed in wells containing 1 μg / ml CDH17 lead antibody to load the antibody onto the probe. Excess unbound antibody was washed away. The antigens bound to wells containing a series of diluted human CDH17-His tags (Sino biological, catalog number 11360-H08H), cynomolgus monkey CDH17-His tags (Acro Biosystems, catalog number CA7-C52H4), homologous human CDH6-His tags (Acro Biosystems, catalog number CA6-H5229), and human CDH16-His tags (Sino biological, catalog number 10915-H08H), with concentrations ranging from 200 nM to 3.1 nM, and lasting for 5 minutes. Well probes containing buffer were immersed to initiate another 15-minute dissociation.

[0122] The results are shown in Table 8. 10E11, 14B12, and 20B4 all bind to human and cynomolgus monkey CDH17 proteins, but HBMAB81 binds only to human CDH17 protein.

[0123] [Table 11] Note: N / A: Not combined.

[0124] Example 7: Competitive coupling Following manufacturer instructions (ThermoFisher #A20186), 20B4 and HBMAB81 mAbs were labeled with Alexa Flour 647. In a competitive binding assay, 20B4-AF647 and HBMAB81-AF647 were immobilized at EC80 concentrations of 1.51 nM and 0.97 nM, respectively. First, 20B4 and HBMAB81 in the range of 0.03 nM to 1800 nM were mixed with 1.51 nM 20B4-AF647 or 0.97 nM HBMAB81-647, and incubated with AsPC1 cells on ice for 1 hour. Cells were washed twice with cell staining buffer. Samples were analyzed using iQue3, and the median fluorescence intensity was measured in the corresponding pathway.

[0125] The results are disclosed in Figure 5. The "20B4-AF647+HBMAB81" group and the "HBMAB-AF647+20B4" group did not show binding inhibition. This indicates that 20B4 and HBMAB81 do not bind to the same or similar epitopes.

[0126] Example 8 Production of anti-CDH17 antibody drug conjugate (ADC) The antibody of the present invention possesses cell affinity activity and endocytosis activity, thereby conjugating with drugs to form antibody-drug conjugates, making it suitable for use in the treatment of CDH17-mediated diseases.

[0127] Purification of monoclonal antibodies For a method of producing the anti-CDH17 antibody, refer to Example 4. Mota, 1H10, 2F9, 4F9, 9E2, 10E11, 13C7, 14B12, 17F3, 19C7, 20B4, and HBMAB-81 are purified and used for conjugation with the drug. Mota is an internal isotype antibody obtained from patent US008568726B2. Other antibodies used for conjugation may include any antibodies described herein (see Example 4).

[0128] Production of drug intermediates The following intermediate compounds are for producing an antibody-drug conjugate (ADC) of an anti-CDH17 antibody and can produce an mc-vc-MMAE drug linker in a manner similar to that described in US 2005 / 0238649. Compound C is produced by the method disclosed in PCT patent application (see WO 2020063673, filed September 25, 2019), and compound D is produced by the method disclosed in PCT patent application (see WO 2022161385, filed January 26, 2022). [ka]

[0129] Coupling of monoclonal antibodies and drug molecules Compound D Anti-CDH17 antibody (5 mg / mL in PBS solution, pH 7.4) was treated with an excess equivalent of 10 mM (tris(2-carboxyethyl)phosphine (TCEP)) at 37°C for 2 hours. A sufficient molar equivalent (6-12 eq) of drug linker (compound D in DMSO) was added to the reducing antibody in PBS. The sample and compound D were then incubated overnight at room temperature. The drug-antibody ratio (DAR) of ADC was measured using a 6230 LC / MS-TOF system (Agilent), and the mean values ​​are summarized in Table 9.

[0130] Compound C Anti-CDH17 antibody (5 mg / mL in PBS solution, pH 7.4) was treated with an excess equivalent of 10 mM (tris(2-carboxyethyl)phosphine (TCEP)) at 37°C for 2 hours. A sufficient molar equivalent (6-12 eq) of drug linker (compound C in DMSO) was added to the reducing antibody in PBS. The sample and compound C were then rotated and incubated overnight at 4°C. The drug-antibody ratio (DAR) of ADCs was measured using a 6230 LC / MS-TOF system (Agilent), and the mean values ​​are summarized in Table 9.

[0131] Vc-MMAE Anti-CDH17 antibody (5 mg / mL, PBS solution, pH 7.4) was treated with an excess equivalent of 10 mM (tris(2-carboxyethyl)phosphine (TCEP)) at 37°C for 1 hour. A sufficient molar equivalent (8 eq) of drug linker (vc-MMAE in DMSO) was added to the reducing antibody in PBS. The sample and vc-MMAE were then incubated at room temperature for 1 hour. The drug-antibody ratio (DAR) of the ADCs was measured using a 6230 LC / MS-TOF system (Agilent), and the mean values ​​are summarized in Table 9. The mean DAR value of anti-CDH17 ADCs coupled with vc-MMAE was approximately 4.0.

[0132] [Table 12]

[0133] Example 9: Inhibitory effect of ADC on tumor cell proliferation In vitro cytotoxicity of ADCs in diverse cell lines This example analyzed ADC immunoconjugates of different antibodies targeting CDH17 with varying degrees of internalization. The CDH17-mediated cytotoxicity of these conjugates was tested in cell cultures to measure the efficacy of various linker cytotoxic agent combinations. Different cancer cell lines were tested, including colorectal cancer cell lines GP2d, NCI-H716, SK-CO-1, SNU16, ESO26, and SW480, and pancreatic cancer cell line AsPC1.

[0134] The inventors tested the cytotoxicity of the immunoconjugate in the above cell line using two methods (Cell titer glo and Cytation5). In the Cytation5 method, cells were harvested during the exponential growth phase and distributed in a 96-well plate at a density of 5000 cells / well. 500 ng / mL of propidium iodide (abcam, catalog no. ab14083) and SPY650-DNA (Cytoskeleton, Inc., catalog no. CYSC501) diluted 1:2000 were added. The cells were incubated overnight at 37°C in 5% CO2. The immunoconjugate was diluted in cell medium containing 500 ng / mL of propidium iodide and SPY650-DNA diluted 2000-fold, and then added to each well. The final concentration range of the immunoconjugate was 0.003 nM to 200 nM. The plates were imaged every 12 hours for a total of 96 hours using Cytation5 (Agilent) to detect cell viability in each well. For the Cell Titer Glo method, cells were inoculated into 96-well plates at a concentration of 2000 cells / well. After overnight incubation, the immunoconjugate was added to each well. The final concentration range of the immunoconjugate in the wells was 0.0001 nM to 200 nM. After incubation for 5 or 7 days, cell viability in each well was measured by the Cell Titer Glo 2.0 assay (Promega).

[0135] In GraphPad Prism, curves and ICs are obtained by S-type dose-response nonlinear regression fitting. 50 Values ​​were generated. The data from these experiments are summarized in Tables 10-11 and Figure 6. All ADC molecules showed good cytotoxic potential against tumor cells. The ADC molecules exhibited CDH17 expression level-dependent cytotoxicity; the higher the CDH expression level, the greater the cytotoxicity of the ADC to cells. This indicates that the ADC molecules are less toxic to non-CDH17 expressing cells and are safer in vivo. Anti-CDH17 antibodies coupled with mc-vc-MMAE also exhibit excellent tumor cytotoxicity.

[0136] [Table 13]

[0137] Note 1: *: Cytotoxicity was measured using Cytation5 (96 hours). Other measurements were taken using Celltiter Glo (7 days).

[0138] Note 2: NA: Not applicable, or current dose-response curve IC 50 According to this, it is not conclusive (indicating no / low toxicity). Note 3: "-": Not tested.

[0139] [Table 14]

[0140] Note 1: *: Cytotoxicity was measured using Cytation5 (96 hours). Others were measured using Celltiter Glo for 5 days or #: 7 days.

[0141] Note 2: NA: Not applicable, or not conclusive according to the current dose-response curve IC50 (indicating no / low toxicity).

[0142] In vitro cytotoxicity comparison of anti-CDH17 ADCs with different DAR values The cytotoxicity of anti-CDH17 ADCs with different DAR values ​​was tested using the same method (Cell Titer Glo and Cytation 5). Plates were imaged every 12 hours for a total of 96 hours using Cytation 5 (Agilent), and AsPC1 cell viability in each well was detected. The final concentration range of the immunoconjugate was 0.003 nM to 200 nM. After incubation for 7 days, cell viability of GP2d and SKCO1 cell lines was detected by the Cell Titer Glo 2.0 assay (Promega). The final concentration range of the immunoconjugate in the wells was 0.0007 nM to 50 nM.

[0143] In GraphPad Prism, curves and ICs are obtained by S-type dose-response nonlinear regression fitting. 50 Values ​​were generated. Data for anti-CDH17 ADCs containing compound D are summarized in Table 12 and Figures 7-8. All ADCs with different DAR values ​​showed cytotoxicity against tumor cells. Data for anti-CDH17 ADCs containing compound C are summarized in Table 13 and Figures 9-10. The cytotoxicity of all ADCs was within a reasonable range.

[0144] [Table 15]

[0145] *: Cytotoxicity was measured using Cytation5 (96 hours). For other measurements, the Celltiter Glo assay was used (7 days).

[0146] [Table 16]

[0147] *: Cytotoxicity was measured using Cytation5 (96 hours). Other measurements were taken using Celltiter Glo (7 days).

[0148] Example 10 Tumor suppression experiment on a nude mouse subcutaneous tumor model of ADC-positive CDH17 cancer cells Therapeutic efficacy of anti-CDH17 ADCs in a mouse model of human pancreatic cancer cells. AsPC1 cells (pancreatic cancer) were subcutaneously transplanted into female BALB / c nude mice. The tumor volume was approximately 150-200 mm². 3 When this was reached, the transplanted mice were randomly divided into 7 groups (5 mice per group). These groups were carrier control, ADC-02, ADC-05, ADC-06, ADC-07, ADC-10, and ADC-11. The mice were treated with intravenous injection of ADC (6 mg / kg) and Q4D x 3. The mean tumor growth inhibition (TGI) was calculated using the following formula: TGI = ((mean (C) - mean (C0)) - (mean (T) - mean (T0))) / (mean (C) - mean (C0)) * 100%, where T is the value for the current group, C is the value for the control group, and T0 and C0 represent the tumor volume at the start of the study.

[0149] The research results are shown in Table 14 and Figure 11. Compared to the carrier agent / PBS group, tumor growth was suppressed in mice treated with ADC-02, ADC-05, ADC-06, ADC-07, and ADC-10. ADC-10 showed the highest tumor suppression rate (80.76%), followed by ADC-02 (79.25%), ADC-07 (77.77%), ADC-05 (76.27%), and ADC-06 (76.26%), all of which were superior to the positive control ADC-11 (67.62%). The anti-CDH17 antibody coupled with compound C also showed excellent tumor suppression (Table 15 and Figure 11).

[0150] [Table 17]

[0151] [Table 18]

[0152] Therapeutic efficacy of anti-CDH17 ADCs in a mouse model of human colorectal cancer cells. GP2d cells (colon and rectal cancer) were xenotransplanted into female BALB / c nude mice. Tumor volume was approximately 100-150 mm. 3 When the target was reached, the transplanted mice were randomly divided into seven groups (5 mice per group). These groups were carrier control, ADC-02, ADC-05, ADC-06, ADC-07, ADC-10, and ADC-11. The mice were treated with intravenous injection of ADC (6 mg / kg) and Q4D x 3.

[0153] The research results are shown in Tables 16-17 and Figure 12. In terms of in vivo therapeutic effect, all ADC molecules effectively suppressed tumor volume increase compared to the control group (PBS). Compared to the carrier / PBS group, tumor growth was suppressed in mice treated with ADC-02, ADC-05, ADC-06, ADC-07, and ADC-10, with TGI values ​​of 104.20%, 104.22%, 92.03%, 104.22%, and 104.10%, respectively. Compared to the ADC-11 control group, tumor growth was suppressed for ADC-02, ADC-05, ADC-07, and ADC-10. Tumor volume in the ADC-11 group increased approximately 30 days after the first dose. Compared to the positive control ADC-11, our ADCs showed better therapeutic efficacy.

[0154] Anti-CDH17 antibodies coupled with compound C also exhibit excellent tumor suppression (Table 18, Figure 12). In the GP2d CDX model, compound C-coupled ADCs with different DAR values ​​were previously compared. Tumor suppression was observed in all treatment groups. When ADCs had the same equivalent payload, they showed comparable in vivo therapeutic efficacy (Table 19, Figure 12).

[0155] [Table 19]

[0156] [Table 20]

[0157] [Table 21]

[0158] [Table 22]

[0159] Therapeutic efficacy of anti-CDH17 ADCs in a xenograft mouse model of human gastric cancer cells SNU16 cells (gastric cancer) were subcutaneously transplanted into female NU / J mice. The tumor volume was approximately 150-200 mm². 3 When this was reached, the transplanted mice were randomly divided into 10 groups (5 mice per group). Detailed administration policies are described in Table 20. Compound C-coupling ADCs have different DAR values. In the ADC-24 3 mg / kg group, tumors began to regrow after 17 days of treatment (Figure 13). At low doses, there is a significant difference in tumor volume between ADC-24 and ADC-21.

[0160] [Table 23]

[0161] Example 11: Pharmacokinetic study of ADCs To study the pharmacokinetics of ADCs, ADC-02, ADC-05, ADC-06, ADC-07, ADC-10, and ADC-11 were intravenously injected at a concentration of 5 mg / kg into Balb / c mice (3 mice per group). After injection, mouse serum samples were collected at predetermined time points (pre-blood collection (blank), 1 hour, 4 hours, 24 hours, 2 days, 3 days, 5 days, 7 days, 14 days, and 21 days) and stored at -80°C.

[0162] Detection of total antibodies in mouse serum Human CDH17 His Tag protein (Sino Biological, catalog number 11360-H08H) was coated at a concentration of 4 μg / mL onto MSD bare standard plates (Meso Scale Discovery, catalog number L15XA-3) overnight at 4°C. After washing away excess unbound protein with PBST buffer, the plates were blocked at room temperature for 1 hour with 3% BSA in PBS buffer at a shaking rate of 700 rpm. After washing three times with PBST, standard samples, QC samples, SC samples, and mouse serum samples were added to the plates and incubated at room temperature for 1 hour at a shaking rate of 700 rpm. The final concentration range of the standard samples was 0.02 ng / mL to 1000 ng / mL. Quality control (QC) samples and sample control (SC) samples were diluted to four different concentrations (0.06 ng / mL, 0.244 ng / mL, 3.906 ng / mL, and 250 ng / mL). Mouse serum samples were also diluted to the detection range. The plates were then washed three times with PBST, and the anti-Fc antibody coupled to the sulfotag was incubated with shaking at room temperature for 1 hour. Finally, 150 μL of MSD GOLD Read Buffer B was added to each well, and the plates were read using a MESO QuickPlex SQ 120 MM (Meso Scale Discovery). The raw data was analyzed using MSD discovery stage software, and the AUC data for total antibody in mouse serum was calculated using a pK solver.

[0163] Detection of ADCs in mouse serum Human CDH17 His Tag protein (Sino Biological, catalog number 11360-H08H) was coated onto MSD bare standard plates (Meso Scale Discovery, catalog number L15XA-3) at a concentration of 8 μg / mL overnight at 4°C. After washing away excess unbound protein with PBST buffer, the plates were blocked at room temperature for 1 hour with 3% BSA in PBS buffer at a shaking rate of 700 rpm. After washing three times with PBST, standard samples, QC samples, SC samples, and mouse serum samples were added to the plates and incubated at room temperature for 1 hour at a shaking rate of 700 rpm. The final concentration range of the standard samples was 0.02 ng / mL to 1000 ng / mL. Quality control (QC) samples and sample control (SC) samples were diluted to four different concentrations (0.06 ng / mL, 0.244 ng / mL, 3.906 ng / mL, and 250 ng / mL). Mouse serum samples were also diluted to the detection range. The plates were then washed three times with PBST, and the anti-compound D antibody, which couples with the sulfotag, was incubated with shaking at room temperature for 1 hour. Finally, 150 μL of MSD GOLD Read buffer B was added to each well, and the plates were read using a MESO QuickPlex SQ 120 MM (Meso Scale Discovery). The raw data was analyzed using MSD discovery stage software, and the AUC data for mouse serum ADCs was calculated using a pK solver.

[0164] Here, total antibody refers to antibodies from ADCs, and the inventors measured total antibody and ADCs simultaneously during the measurement. The research results are shown in Figure 14 and Tables 21-22. All ADC molecules have good pharmacokinetic properties. The AUCs of ADC-02, ADC-05, ADC-06, ADC-07, and ADC-10 were significantly larger than those of the control group ADC-11. ADC-02, ADC-05, ADC-06, ADC-07, and ADC-10 were detected from 7-day mouse serum samples, but ADC-11 was detected only from 5-day mouse serum samples.

[0165] [Table 24]

[0166] [Table 25]

[0167] Similar administration, detection, and analysis methods were used for ADC-21, ADC-24, and ADC-34. Balb / c mice (3 mice per group) were administered 5 mg / kg intravenously. Serum samples were collected at 13 time points: pre-collection, and at 1 hour, 4 hours, 24 hours, 2 days, 3 days, 5 days, 7 days, 9 days, 12 days, 14 days, 17 days, and 21 days. MSD bare standard plates were coated with a concentration of 2 μg / mL and used for detection of total antibody and ADC PK. Quality control (QC) samples and sample control (SC) samples were diluted to three different concentrations (0.22 ng / ml, 3.52 ng / ml, and 225 ng / mL).

[0168] Anti-Fc antibodies and anti-payload antibodies coupled with sulfotags were used to detect total antibody and ADCs, respectively. Tables 23-24 show that the AUC of ADC-21 and ADC-34 was larger than that of ADC-24 (Figure 15).

[0169] [Table 26]

[0170] [Table 27]

[0171] While specific embodiments of the present invention have been described in detail, those skilled in the art will understand that various modifications and changes can be made to the details based on all disclosed teachings, and these too fall within the scope of the present invention. The scope of protection of the present invention is limited by the appended claims and any equivalents thereof.

Claims

1. An antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof comprising an anti-CDH17 antibody or its antigen-binding fragment, which is optionally conjugated with a toxic drug via a linker, wherein the anti-CDH17 antibody or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region of the antibody, wherein The heavy chain variable region includes HCDR1, HCDR2, and HCDR3 having 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 includes LCDR1, LCDR2, and LCDR3 having amino acid sequences shown in SEQ ID NO: 42, SEQ ID NO: 47, and SEQ ID NO: 54, respectively, or The heavy chain variable region includes HCDR1, HCDR2, and HCDR3 having amino acid sequences shown in SEQ ID NO: 07, SEQ ID NO: 17, and SEQ ID NO: 28, respectively, and the light chain variable region includes LCDR1, LCDR2, and LCDR3 having amino acid sequences 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 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 shown in SEQ ID NO: 36, SEQ ID NO: 43, and SEQ ID NO: 51, respectively, an antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof.

2. The anti-CDH17 antibody or its antigen-binding fragment is a monoclonal antibody or its antigen-binding fragment, a polyclonal antibody or its antigen-binding fragment, a multispecific antibody or its antigen-binding fragment, a mouse antibody or its antigen-binding fragment, a chimeric antibody or its antigen-binding fragment, a humanized antibody or its antigen-binding fragment, a recombinant antibody or its antigen-binding fragment, a human antibody or its antigen-binding fragment, and preferably the multispecific antibody is a bispecific antibody, a triplicate antibody, or a quadruplicate antibody, according to claim 1, an antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof.

3. The antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof according to claim 1 or 2, wherein the anti-CDH17 antibody or its antigen-binding fragment comprises a heavy chain variable region containing an amino acid sequence selected from SEQ ID NO: 65, 61, 59 or a sequence having at least 80%, 85%, 90%, 95%, or 99% sequence identity therewith, and / or a light chain variable region containing an amino acid sequence selected from SEQ ID NO: 76, 72, 70 or a sequence having at least 80%, 85%, 90%, 95%, or 99% sequence identity therewith.

4. The anti-CDH17 antibody or its antigen-binding fragment is A heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 65 or an amino acid sequence having at least 80%, 85%, 90%, 95%, or 99% sequence identity thereto, and / or a light chain variable region having the amino acid sequence shown in SEQ ID NO: 76 or an amino acid sequence having at least 80%, 85%, 90%, 95%, or 99% sequence identity thereto, or A heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 61 or an amino acid sequence having at least 80%, 85%, 90%, 95%, or 99% sequence identity thereto, and / or a light chain variable region having the amino acid sequence shown in SEQ ID NO: 72 or an amino acid sequence having at least 80%, 85%, 90%, 95%, or 99% sequence identity thereto, or An antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof according to any one of claims 1 to 3, comprising a heavy chain variable region having an amino acid sequence shown in SEQ ID NO: 59 or an amino acid sequence having at least 80%, 85%, 90%, 95%, or 99% sequence identity therewith, and / or a light chain variable region having an amino acid sequence shown in SEQ ID NO: 70 or an amino acid sequence having at least 80%, 85%, 90%, 95%, or 99% sequence identity therewith.

5. The anti-CDH17 antibody or its antigen-binding fragment is A heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 65, and / or a light chain variable region having the amino acid sequence shown in SEQ ID NO: 76, or A heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 61, and / or a light chain variable region having the amino acid sequence shown in SEQ ID NO: 72, or An antibody-drug conjugate according to any one of claims 1 to 4, or a pharmaceutically acceptable salt or solvate thereof, comprising a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 59, and / or a light chain variable region having the amino acid sequence shown in SEQ ID NO:

70.

6. The anti-CDH17 antibody or its antigen-binding fragment is A heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 65 and a light chain variable region having the amino acid sequence shown in SEQ ID NO: 76, or A heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 61 and a light chain variable region having the amino acid sequence shown in SEQ ID NO: 72, or An antibody-drug conjugate according to any one of claims 1 to 5, or a pharmaceutically acceptable salt or solvate thereof, comprising a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 59 and a light chain variable region having the amino acid sequence shown in SEQ ID NO:

70.

7. The anti-CDH17 antibody or its antigen-binding fragment 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 their common variants, and the light chain constant region of the human antibody constant region is selected from the κ chain and λ chain constant regions of human antibodies and their common variants. More preferably, the full-length antibody comprises the human antibody heavy chain constant region of SEQ ID NO: 99 or an amino acid sequence having at least 80%, 85%, 90%, 95%, or 99% sequence identity thereto, and the human light chain constant region of SEQ ID NO: 100 or an amino acid sequence having at least 80%, 85%, 90%, 95%, or 99% sequence identity thereto. More preferably, the full-length antibody comprises a human antibody heavy chain constant region of SEQ ID NO: 99 and a human light chain constant region of SEQ ID NO: 100, the antibody-drug conjugate according to any one of claims 1 to 6, or a pharmaceutically acceptable salt or solvate thereof.

8. The anti-CDH17 antibody or its antigen-binding fragment is A heavy chain having the amino acid sequence shown 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 shown in SEQ ID NO: 98 or an amino acid sequence having at least 80%, 85%, 90%, 95%, or 99% sequence identity thereto, or A heavy chain having the amino acid sequence shown in SEQ ID NO: 89 or an amino acid sequence having at least 80%, 85%, 90%, 95%, or 99% sequence identity thereto, and a light chain having the amino acid sequence shown in SEQ ID NO: 90 or an amino acid sequence having at least 80%, 85%, 90%, 95%, or 99% sequence identity thereto, or An antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof according to any one of claims 1 to 7, comprising a heavy chain having an amino acid sequence shown in SEQ ID NO: 85 or an amino acid sequence having at least 80%, 85%, 90%, 95%, or 99% sequence identity therewith, and a light chain having an amino acid sequence shown in SEQ ID NO: 86 or an amino acid sequence having at least 80%, 85%, 90%, 95%, or 99% sequence identity therewith.

9. The anti-CDH17 antibody or its antigen-binding fragment is A heavy chain having the amino acid sequence shown in SEQ ID NO: 97 and a light chain having the amino acid sequence shown in SEQ ID NO: 98, or A heavy chain having the amino acid sequence shown in SEQ ID NO: 89 and a light chain having the amino acid sequence shown in SEQ ID NO: 90, or An antibody-drug conjugate according to any one of claims 1 to 8, or a pharmaceutically acceptable salt or solvate thereof, comprising a heavy chain having the amino acid sequence shown in SEQ ID NO: 85 and a light chain having the amino acid sequence shown in SEQ ID NO:

86.

10. The anti-CDH17 antigen-binding fragment is selected from Fab, Fab', F(ab')2, variable fragment (Fv), single-stranded variable fragment (scFv), dimerization domain V (diabody), disulfide-stabilized Fv (dsFv), and CDR-containing peptide, and is an antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof according to any one of claims 1 to 9.

11. The toxic drug is selected from tubulin inhibitors, topoisomerase inhibitors, DNA intercalators, and RNA polymerase inhibitors or pharmaceutically acceptable salts, esters, or analogs thereof, preferably the toxic drug is selected from auristatin analogs, camptothecin derivatives, and mytansin analogs, and more preferably the toxic drug is MMAE, MMAF, exatecan, MMAD, DM1, DM4, eribulin, pyrrolobenzodiazepine An antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof according to any one of claims 1 to 10, selected from PBD, DGN-549-C, SN-38, irinotecan, topotecan, berotecan, rubitecan, doxorubicin, PNU-159682, duocalmycin, daunorubicin, mitoxantrone, podophyllotoxin, etoposide, α-amanitin, or a pharmaceutically acceptable salt, ester, or analog thereof.

12. The antibody-drug conjugate is as shown in general formula (A), 【Chemistry 1】 Here, L １ and L ２ This is a connecting unit, y is a number selected from 1 to 10, preferably a number selected from 2 to 8, and most preferably 2, 4, 5, 6, 7, or 8. Ab is an anti-CDH17 antibody according to any one of claims 1 to 10 or an antigen-binding fragment thereof, wherein the antibody-drug conjugate according to any one of claims 1 to 11 or a pharmaceutically acceptable salt or solvate thereof.

13. The antibody-drug conjugate is as shown in general formula (I), 【Chemistry 2】 Here, L １ and L ２ This is a connecting unit, y is a number selected from 1 to 10, preferably a number selected from 2 to 8, and most preferably 2, 4, 6, or 8. Ab is an anti-CDH17 antibody according to any one of claims 1 to 10 or an antigen-binding fragment thereof, wherein the antibody-drug conjugate according to any one of claims 1 to 11 or a pharmaceutically acceptable salt or solvate thereof.

14. L １ teeth, 【Transformation 3】 An antibody-drug conjugate according to claim 13, or a pharmaceutically acceptable salt or solvate thereof, selected from the above.

15. L ２ is -L ａ -L ｂ -L ｃ -L ｄ - and where Said L ａ This is as shown in general formula (II), 【Chemistry 4】 Here, s １ and s ２ Each of these is an integer selected independently from 0 to 8, preferably s １ and s ２ It is independently selected from 1, 2, 3, 4, 5 or 6, Or, s １ s is an integer from 1 to 8, ２ is 0, preferably s １ is selected from 4, 5, 6, 7, or 8, s ２ It is 0, Or, s ２ is an integer selected from 2 to 8, and s １ is 2, preferably s ２ is selected from 2, 3, 4, 5 or 6, s １ It is 2, L ｂ It is a chemical bond, L ｃ is a tetrapeptide residue, preferably L ｃ This is a tetrapeptide residue of glycine-glycine-phenylalanine-glycine (GGFG), L ｄ -NR １ (CR ２ R ３ )s ３ - and here, R １ , R ２ and R ３ They are either the same or different, and each is independently a hydrogen atom or an alkyl group, s ３ is 1 or 2, where L ａ The terminal is connected to Ab, L ｄ The end is L １ An antibody-drug conjugate according to any one of claims 13 to 14, or a pharmaceutically acceptable salt or solvate thereof, which is linked to the antibody-drug conjugate according to any one of claims 13 to 14.

16. The antibody-drug conjugate has the following structure: 【Transformation 5】 【Transformation 6】 【Transformation 7】 y is a number selected from 1 to 10, preferably a number selected from 2 to 8, and most preferably 2, 4, 5, 6, 7, or 8. Ab is an anti-CDH17 antibody according to any one of claims 1 to 10 or an antigen-binding fragment thereof, wherein the antibody-drug conjugate according to any one of claims 12 to 15 or a pharmaceutically acceptable salt or solvate thereof.

17. The antibody-drug conjugate is selected from the following compounds: 【Transformation 8】 【Chemistry 9】 【Chemistry 10】 Here, The antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof according to any one of claims 1 to 16, wherein y is a number selected from 1 to 10, preferably a number selected from 2 to 8, more preferably a number selected from 4 to 8, even more preferably a number selected from 4 to 6 or 6 to 8, and most preferably 4, 5, 6, 7 or 8.

18. A pharmaceutical composition comprising an antibody-drug conjugate according to any one of claims 1 to 17 or a pharmaceutically acceptable salt or solvate thereof, and one or more pharmaceutically acceptable carriers.

19. Uses of an antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof according to any one of claims 1 to 17, or a pharmaceutical composition according to claim 18, in the manufacture of a drug for treating and / or preventing a CDH17-mediated disease or disorder.

20. The use according to claim 19, wherein the CDH17-mediated disease or disorder is a cancer having high CDH17 expression.

21. The use according to claim 19, wherein the CDH17-mediated disease or disorder is a cancer having moderate CDH17 expression.

22. Uses of an antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof according to any one of claims 1 to 17, or a pharmaceutical composition according to claim 18, in the manufacture of a drug for treating and / or preventing a tumor or cancer, wherein the tumor or cancer is preferably gastrointestinal cancer, pancreatic cancer, gallbladder cancer, bile duct cancer, stomach cancer, intestinal cancer, ovarian cancer, colorectal cancer, lung cancer, breast cancer, anal cancer, prostate cancer, kidney cancer, bladder cancer, pharyngeal cancer, nasal cavity cancer, skin cancer, oral cancer, tongue cancer, esophageal cancer, vaginal cancer, cervical cancer, spleen cancer, testicular cancer, or glioblastoma.

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