Anti-CDH17 antibodies and their uses

Abstract

Antibodies with high selectivity and specificity against human CDH17 protein were identified, and these antibodies could be used to treat cancer. These antibodies can be used to prepare antibody-drug conjugates that exhibit effective tumor cell killing in vitro and in vivo.

Description

[0001] This application is a divisional application of the invention patent application filed on September 20, 2024, with application number 202480060753.3 and invention title "Anti-CDH17 Antibody and Its Use". Background Technology

[0002] Gastrointestinal (GI) cancers, including colorectal cancer (CRC), stomach cancer (GC), pancreatic cancer (PC), esophageal cancer (EC), liver cancer, biliary tract cancer, and neuroendocrine tumors (NETs), are a leading cause of cancer death worldwide, with a significant associated incidence. Colorectal cancer alone accounts for approximately 10% of all cancer diagnoses and is the second leading cause of cancer death globally. In China, liver and stomach cancers are among the world's deadliest malignancies, accounting for more than half of all confirmed cases and causing more than 1.42 million deaths globally each year, yet there are currently no effective treatments. Early detection and radical treatments can lead to better outcomes, whereas less than 15% of cases are advanced-stage. Unfortunately, many GI cancers are asymptomatic and are diagnosed at an advanced stage by the time patients seek medical attention. Therefore, in addition to early detection, research is needed on different approaches to treating GI cancers at both early and late stages to improve outcomes.

[0003] Cadmin 17 (CDH17), also known as hepato-intestinal cadmin (LI-cadmin), is a unique member of the cadmin superfamily. It is very similar to cadmin 16 (CDH16), and they form a subgroup called 7D-domain cadmin. Unlike classic cadmins with five cadmin domains and a cytoplasmic tail of over 100 amino acids, CDH17 has a long extracellular domain (7 cadmin domains) and a short cytoplasmic tail (20 amino acid residues). Under physiological conditions, CDH17 expression in humans and mice is primarily confined to epithelial cells in intestinal tissue and pancreatic ducts, and not in vital organs such as the liver, stomach, heart, lungs, and brain. Functionally, CDH17 participates in intercellular adhesion, via Ca2+... 2+ CDH17 regulates intercellular spaces in a dependent manner to maintain tissue integrity and water absorption. Pathophysiologically, CDH17 expression has been extensively studied in various digestive system cancers. Its expression is upregulated in gastric cancer (GC), colorectal cancer (CRC), esophageal cancer (EC), pancreatic cancer (PC), and neuroendocrine tumors (NET). Therefore, CDH17 is considered a prognostic biomarker and an oncogene for cancer intervention.

[0004] The unique expression pattern of CDH17 in GI cancer enables the development of therapeutics that specifically target GI cancer cells using CDH17. Currently, several active clinical studies in GI cancer are evaluating these CDH17-specific drugs. Several modalities targeting CDH17, including bispecific antibodies (BsAbs) and chimeric antigen receptor T-cell therapy (CAR-T), are currently in clinical or preclinical studies. BI905711 is a tetravalent bispecific antibody that crosslinks TRAILR2 with CDH17. These crosslinks drive CDH17-dependent TRAILR2 oligomerization, leading to caspase activation and eventual apoptosis, inhibiting GI tumor growth. In a phase Ia / b study (NCT04137289), BI905711 demonstrated manageable safety in patients (pt) with advanced GI cancer. Disease stability (SD) was achieved in 27% of patients (13 patients achieved SD), and progression-free survival (PFS4) was achieved in 8 patients for ≥4 months. In CRC patients, 6 patients achieved SD, and 3 patients achieved PFS4. In non-CRC patients, 7 achieved SD (PDAC: n = 6) and 5 achieved PFS4 (PDAC: n = 4). Other CDH17-based T-cell conjugates, including ARB202 from Arbele, have also shown promising anti-tumor efficacy in preclinical studies, and a Phase I clinical trial is currently recruiting patients in Australia and Hong Kong (NCT05411133). CHM-2101 is an optimized third-generation CAR-T cell therapy that targets CDH17 for the first time. Preclinical evidence for CHM-2101 shows unique efficacy against solid tumors, completely eradicating tumor cells without recurrence. Preclinical studies have also shown no toxicity to normal tissues. Overall, exploring different types of drugs targeting CDH17 holds promise for providing more options for the treatment of GI cancers. Summary of the Invention

[0005] This disclosure provides anti-CDH17 antibodies or antigen-binding fragments thereof that exhibit efficient binding activity against human CDH17 protein with different epitopes or against CDH17-expressing tumor cells. Antibodies with CDH17 distal membrane conjugates also exhibit efficient internalization rates on CDH17-expressing cells, subsequently demonstrating effective in vitro and in vivo tumor cell killing, particularly when constructed as antibody-drug conjugates (ADCs).

[0006] This disclosure provides an antibody or antigen-binding fragment thereof specific to human cadherin 17 (CDH17) protein, wherein the antibody or antigen-binding fragment comprises a heavy chain variable region (VH) and a light chain variable region (VL), the heavy chain variable region comprising heavy chain complementarity-determining regions HCDR1, HCDR2, and HCDR3, and the light chain variable region comprising light chain complementarity-determining regions LCDR1, LCDR2, and LCDR3, wherein HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 respectively comprise the following amino acid sequences: (a) SEQ ID NO:1-6; (b) SEQ ID NO:9-14; (c) any one of SEQ ID NO:17-21 and SEQ ID NO:22 and 217-221; (d) any one of SEQ ID NO:25, SEQ ID NO:26 or 245, SEQ ID NO:27, SEQ ID NO:28, 246, and 247, SEQ ID NO:29, and SEQ ID (e) SEQ ID NO:30 and 222-230; (f) SEQ ID NO:33-38; (g) SEQ ID NO:41-46; (g) SEQ ID NO:49, 50, 51, SEQ ID NO:52 and 239-241, SEQ ID NO:53 and 242-244, and SEQ ID NO:54; (h) SEQ ID NO:57-62; (i) SEQ ID NO:65-70; (j) SEQ ID NO:73-78; (k) SEQ ID NO:81-86; (l) SEQ ID NO:89-94; (m) SEQ ID NO:97-102; or (n) SEQ ID NO:105-110.

[0007] In some embodiments, HCDR1 contains the amino acid sequence of SEQ ID NO:17, HCDR2 contains the amino acid sequence of SEQ ID NO:18, HCDR3 contains the amino acid sequence of SEQ ID NO:19, LCDR1 contains the amino acid sequence of SEQ ID NO:20, LCDR2 contains the amino acid sequence of SEQ ID NO:21, and LCDR3 contains the amino acid sequence selected from the group consisting of SEQ ID NO:217-221.

[0008] In some embodiments, HCDR1 contains the amino acid sequence of SEQ ID NO:25, HCDR2 contains the amino acid sequence of SEQ ID NO:245, HCDR3 contains the amino acid sequence of SEQ ID NO:27, LCDR1 contains the amino acid sequence of SEQ ID NO:246 or 247, LCDR2 contains the amino acid sequence of SEQ ID NO:29, and LCDR3 contains the amino acid sequence selected from the group consisting of SEQ ID NO:222-230.

[0009] In some embodiments, HCDR1 comprises the amino acid sequence of SEQ ID NO:25, HCDR2 comprises the amino acid sequence of SEQ ID NO:26, HCDR3 comprises the amino acid sequence of SEQ ID NO:27, LCDR1 comprises the amino acid sequence of SEQ ID NO:28, LCDR2 comprises the amino acid sequence of SEQ ID NO:29, and LCDR3 comprises the amino acid sequence selected from the group consisting of SEQ ID NO:30.

[0010] In some embodiments, HCDR1 contains the amino acid sequence of SEQ ID NO:49, HCDR2 contains the amino acid sequence of SEQ ID NO:50, HCDR3 contains the amino acid sequence of SEQ ID NO:51, LCDR1 contains the amino acid sequence selected from the group consisting of SEQ ID NO:239-241, LCDR2 contains the amino acid sequence selected from the group consisting of SEQ ID NO:53 and 242-244, and LCDR3 contains the amino acid sequence of SEQ ID NO:54.

[0011] In some embodiments, HCDR1 contains the amino acid sequence of SEQ ID NO:49, HCDR2 contains the amino acid sequence of SEQ ID NO:50, HCDR3 contains the amino acid sequence of SEQ ID NO:51, LCDR1 contains the amino acid sequence selected from the group consisting of SEQ ID NO:52 and 239-241, LCDR2 contains the amino acid sequence selected from the group consisting of SEQ ID NO:242-244, and LCDR3 contains the amino acid sequence of SEQ ID NO:54.

[0012] In some embodiments, the antibody is a chimeric antibody or a humanized antibody.

[0013] In some embodiments, (a) VH comprises the amino acid sequence of SEQ ID NO: 7, and VL comprises the amino acid sequence of SEQ ID NO: 8; (b) VH contains the amino acid sequence of SEQ ID NO: 15, and VL contains the amino acid sequence of SEQ ID NO: 16; (c) VH contains an amino acid sequence selected from the group consisting of SEQ ID NO: 23 and 125-132, and VL contains an amino acid sequence selected from the group consisting of SEQ ID NO: 24, 134-137 and 203-207; (d) VH contains an amino acid sequence selected from the group consisting of SEQ ID NO: 31, 113-119 and 248, and VL contains an amino acid sequence selected from the group consisting of SEQ ID NO: 32, 121-123, 201-202, 208-216 and 249-250; (e) VH contains an amino acid sequence selected from the group consisting of SEQ ID NO: 39 and 139-140, and VL contains an amino acid sequence selected from the group consisting of SEQ ID NO: 40, 142-145 and 147; (f) VH contains the amino acid sequence of SEQ ID NO: 47, and VL contains the amino acid sequence of SEQ ID NO: 48; (g) VH contains an amino acid sequence selected from the group consisting of SEQ ID NO: 55, 149-152 and 154, and VL contains an amino acid sequence selected from the group consisting of SEQ ID NO: 56, 156-158, 160 and 232-238; (h) VH contains the amino acid sequence of SEQ ID NO: 63, and VL contains the amino acid sequence of SEQ ID NO: 64; (i) VH contains the amino acid sequence of SEQ ID NO: 71, and VL contains the amino acid sequence of SEQ ID NO: 72; (j) VH contains the amino acid sequence of SEQ ID NO: 79, and VL contains the amino acid sequence of SEQ ID NO: 80; (k) VH contains the amino acid sequence of SEQ ID NO: 87, and VL contains the amino acid sequence of SEQ ID NO: 88; (l) VH contains an amino acid sequence selected from the group consisting of SEQ ID NO: 95 and 162-165, and VL contains an amino acid sequence selected from the group consisting of SEQ ID NO: 96, 167-169 and 171; (m) VH contains an amino acid sequence selected from the group consisting of SEQ ID NO: 103 and 173-176, and VL contains an amino acid sequence selected from the group consisting of SEQ ID NO: 104, 178-181, and 183-184; or (n) VH contains an amino acid sequence selected from the group consisting of SEQ ID NO: 111, 186-189 and 191, and VL contains an amino acid sequence selected from the group consisting of SEQ ID NO: 112, 193-196 and 198-199.

[0014] In some embodiments, the antibodies or antigen-binding fragments thereof provided herein comprise VH and VL, which respectively comprise amino acid sequences selected from the group consisting of: (a) SEQ ID NO: 113 and 121; (b) SEQ ID NO: 113 and 122; (c) SEQ ID NO: 113 and 123; (d) SEQ ID NO: 114 and 121; (e) SEQ ID NO: 114 and 122; (f) SEQ ID NO: 114 and 123; (g) SEQ ID NO: 115 and 121; (h) SEQ ID NO: 115 and 122; (i) SEQ ID NO: 115 and 123; (j) SEQ ID NO: 116 and 121; (k) SEQ ID NO: 116 and 122; (l) SEQ ID NO: 116 and 123; (m) SEQ ID NO: 117 and 121; (n) SEQ ID NO: 117 and 122; (o) SEQ ID NO: 118 and 121; (p) SEQ ID NO: 119 and 122; (q) SEQ ID NO: 113 and 201; (r) SEQ ID NO: 114 and 201; (s) SEQ ID NO: 115 and 201; (t) SEQ ID NO: 116 and 201; (u) SEQ ID NO: 113 and 202; (v) SEQ ID NO: 114 and 202; (w) SEQ ID NO: 115 and 202; (x) SEQ ID NO: 116 and 208; (y) SEQ ID NO: 116 and 209; (z) SEQ ID NO: 116 and 210; (aa) SEQ ID SEQ ID NO:116 and 211; (ab) SEQ ID NO:116 and 212; (ac) SEQ ID NO:116 and 213; (ad) SEQ ID NO:116 and 214; (ae) SEQ ID NO:116 and 215; (af) SEQ ID NO:116 and 216; (ag) SEQ ID NO:248 and 123; (ah) SEQ ID NO:116 and 249; (ai) SEQ ID NO:248 and 249; or (aj) SEQ ID NO:249 and 250.

[0015] In some embodiments, the antibodies or antigen-binding fragments thereof provided herein comprise VH and VL, which respectively comprise amino acid sequences selected from the group consisting of: (a) SEQ ID NO: 125 and 134; (b) SEQ ID NO: 125 and 135; (c) SEQ ID NO: 125 and 136; (d) SEQ ID NO: 126 and 134; (e) SEQ ID NO: 126 and 135; (f) SEQ ID NO: 126 and 136; (g) SEQ ID NO: 127 and 134; (h) SEQ ID NO: 127 and 135; (i) SEQ ID NO: 127 and 136; (j) SEQ ID NO: 128 and 134; (k) SEQ ID NO: 128 and 135; (l) SEQ ID NO: 128 and 136; (m) SEQ ID NO: 129 and 134; (n) SEQ ID NO: 129 and 135; (o) SEQ ID NO: 129 and 136; (p) SEQ ID NO: 130 and 134; (q) SEQ ID NO: 130 and 135; (r) SEQ ID NO: 130 and 136; (s) SEQ ID NO: 131 and 134; (t) SEQ ID NO: 125 and 137; (u) SEQ ID NO: 126 and 137; (v) SEQ ID NO: 127 and 137; (w) SEQ ID NO: 128 and 137; (x) SEQ ID NO: 129 and 137; (y) SEQ ID NO: 132 and 137; (z) SEQ ID NO: 131 and 203; (aa) SEQ ID SEQ ID NO:131 and 204; (ab) SEQ ID NO:131 and 205; (ac) SEQ ID NO:131 and 206; or (ad) SEQ ID NO:131 and 207.

[0016] In some embodiments, the antibodies or antigen-binding fragments thereof provided herein comprise VH and VL, which respectively comprise amino acid sequences selected from the group consisting of: (a) SEQ ID NO: 139 and 142; (b) SEQ ID NO: 139 and 143; (c) SEQ ID NO: 139 and 144; (d) SEQ ID NO: 139 and 145; (e) SEQ ID NO: 139 and 147; (f) SEQ ID NO: 140 and 142; (g) SEQ ID NO: 140 and 143; (h) SEQ ID NO: 140 and 144; (i) SEQ ID NO: 140 and 145; or (j) SEQ ID NO: 140 and 147.

[0017] In some embodiments, the antibodies or antigen-binding fragments thereof provided herein comprise VH and VL, which respectively comprise amino acid sequences selected from the group consisting of: (a) SEQ ID NO: 149 and 156; (b) SEQ ID NO: 149 and 157; (c) SEQ ID NO: 149 and 158; (d) SEQ ID NO: 150 and 156; (e) SEQ ID NO: 150 and 157; (f) SEQ ID NO: 150 and 158; (g) SEQ ID NO: 151 and 156; (h) SEQ ID NO: 151 and 157; (i) SEQ ID NO: 151 and 158; (j) SEQ ID NO: 152 and 156; (k) SEQ ID NO: 149 and 160; (l) SEQ ID NO: 152 and 160; (m) SEQ ID NO: 154 and 156; (n) SEQ ID NO: 154 and 160; (o) SEQ ID NO: 152 and 232; (p) SEQ ID NO: 152 and 233; (q) SEQ ID NO: 152 and 234; (r) SEQ ID NO: 152 and 235; (s) SEQ ID NO: 152 and 236; (t) SEQ ID NO: 152 and 237 or (u) SEQ ID NO: 152 and 238.

[0018] In some embodiments, the antibodies or antigen-binding fragments thereof provided herein comprise VH and VL, which respectively comprise amino acid sequences selected from the group consisting of: (a) SEQ ID NO: 162 and 167; (b) SEQ ID NO: 162 and 168; (c) SEQ ID NO: 162 and 169; (d) SEQ ID NO: 163 and 167; (e) SEQ ID NO: 163 and 168; (f) SEQ ID NO: 163 and 169; (g) SEQ ID NO: 164 and 167; (h) SEQ ID NO: 164 and 168; (i) SEQ ID NO: 164 and 169; (j) SEQ ID NO: 165 and 167; (k) SEQ ID NO: 162 and 171; or (l) SEQ ID NO: 165 and 171.

[0019] In some embodiments, the antibodies or antigen-binding fragments thereof provided herein comprise VH and VL, which respectively comprise amino acid sequences selected from the group consisting of: (a) SEQ ID NO: 173 and 178; (b) SEQ ID NO: 173 and 179; (c) SEQ ID NO: 173 and 180; (d) SEQ ID NO: 173 and 181; (e) SEQ ID NO: 174 and 178; (f) SEQ ID NO: 174 and 179; (g) SEQ ID NO: 174 and 180; (h) SEQ ID NO: 174 and 181; (i) SEQ ID NO: 175 and 178; (j) SEQ ID NO: 175 and 179; (k) SEQ ID NO: 175 and 180; (l) SEQ ID NO: 175 and 181; (m) SEQ ID NO: 176 and 178; (n) SEQ ID NO: 176 and 179; (o) SEQ ID NO: 176 and 180; (p) SEQ ID NO: 173 and 183; (q) SEQ ID NO: 176 and 183; or (r) SEQ ID NO: 176 and 184.

[0020] In some embodiments, the antibodies or antigen-binding fragments thereof provided herein comprise VH and VL, which respectively comprise amino acid sequences selected from the group consisting of: (a) SEQ ID NO: 186 and 193; (b) SEQ ID NO: 186 and 194; (c) SEQ ID NO: 186 and 195; (d) SEQ ID NO: 186 and 196; (e) SEQ ID NO: 187 and 193; (f) SEQ ID NO: 187 and 194; (g) SEQ ID NO: 187 and 195; (h) SEQ ID NO: 188 and 193; (i) SEQ ID NO: 188 and 194; (j) SEQ ID NO: 189 and 193; (k) SEQ ID NO: 191 and 198; or (l) SEQ ID NO: 191 and 199.

[0021] In one aspect, this disclosure provides an antibody or antigen-binding fragment thereof specific to human CDH17 protein, wherein the antibody or antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising heavy chain complementarity-determining regions HCDR1, HCDR2, and HCDR3, and the light chain variable region comprising light chain complementarity-determining regions LCDR1, LCDR2, and LCDR3, wherein: The HCDR1, HCDR2, and HCDR3 groups are selected from Table 2A or derived from the CDR groups in Table 2A, wherein one, two, or three amino acids are added, deleted, and / or substituted in one or more of these CDRs. The LCDR1, LCDR2 and LCDR3 groups are selected from Table 2B or derived from the CDR groups in Table 2B, wherein one, two or three amino acids are added, deleted and / or substituted in one or more of these CDRs.

[0022] In one respect, this disclosure provides an antibody or antigen-binding fragment thereof that is specific to human CDH17 protein, wherein the antibody or antigen-binding fragment thereof competes with the antibody or antigen-binding fragment thereof provided herein.

[0023] In some embodiments, the antibody or its antigen-binding fragment is selected from the group consisting of full-length antibody, Fab, Fab', F(ab')2, Fd, Fv, single-chain Fv (scFv), single-chain antibody, disulfide-linked Fv (sdFv), nanobody, domain antibody, isolated CDR, and divalent domain antibody.

[0024] In some embodiments, the antibody or its antigen-binding fragment further comprises a heavy chain constant region, a light chain constant region, an Fc region, or a combination thereof.

[0025] In some embodiments, the light chain constant region is the κ or λ chain constant region. In some embodiments, the isotype is IgG1, IgG2, IgG3, or IgG4.

[0026] In one aspect, this disclosure provides an antibody-drug conjugate comprising an antibody or an antigen-binding fragment thereof provided herein, and optionally a drug attached to the antibody or the antigen-binding fragment thereof via a linker.

[0027] In some embodiments, the adapter is linked to the antibody or its antigen-binding fragment provided herein via chemical or enzymatic conjugation.

[0028] In some embodiments, the connector is a cuttable connector or a non-cuttable connector.

[0029] In some embodiments, the cuttable connector is a chemically sensitive connector or an enzyme-cuttable connector; wherein the uncuttable connector contains a thioether or maleimide hexanoyl group (MC).

[0030] In some embodiments, the chemically sensitive adapter is a pH-sensitive adapter or a glutathione-sensitive disulfide adapter; wherein the enzyme-cleavable adapter is a peptide-based adapter or a β-glucuronide adapter.

[0031] In some embodiments, the connector comprises 4-(N-maleimidemethyl)cyclohexane-1-carboxylic acid succinimide ester (SMCC), sulfonyl-SMCC, p-carboxycyclohexylmethylmaleimide, maleimide hexanoyl (MC)-valine-citrulline (VC)-p-aminobenzyloxycarbamoyl (PABC), CL2A, maleimide hexanoyl (MC)-glycine-glycine-phenylalanine-glycine (GGFG), MC, or maleimide propionyl (MP)-PEG8-valine-alanine (VA)-PABC.

[0032] In some embodiments, the drug is selected from the group consisting of cytotoxins, therapeutic peptides, and polypeptides.

[0033] In some embodiments, the drug is selected from the group consisting of: olritamines, maytansines, benzodiazepines, tubulolysins, pyroxine, camptothecin, chalcogenide, eczemab, irinotecan (SN38), doxorubicin, anthracycline, pyrrolobenzodiazepines (PBD), TLR agonists, STING agonists, Pseudomonas aeruginosa exotoxin PE38, diphtheria toxin, Staphylococcus aureus enterotoxin A / E-120, antibacterial antibiotics, shiga toxin, ricin, and urease.

[0034] In some embodiments, the drug is monomethylolpropionate E (MMAE), monomethylolpropionate F (MMAF), maytansine, mertansine (DM1), ravtansine (DM4), tubulolysin A, DX-8951f, DXd, 7-ethyl-10-hydroxycamptothecin (SN-38), DGN462, amberstatin 269, anthramycin, SG3199 / SCX, IRDye®700DX, TLR7 / 8 agonist, diABZI STING agonist-2, or any derivative thereof.

[0035] In some embodiments, the drug and the connector are collectively referred to as ozomicin, virdotin, malfotin, entancin, soravtancin, rivancin, metancin, delutecan, gavitan, or tecillin.

[0036] In some embodiments, the drug and the connector together comprise (a) virdoting having Formula I: (b) Drotecon with Formula II: (c) Ozomi stars with Formula III: ; (d) Mafaltin with Formula IV: ; (e) Entansine with formula V: ; (f) Govitan with Formula VI: ;or (g) Ticillin with Formula VII: .

[0037] In one respect, this disclosure provides a composition comprising an antibody or antigen-binding fragment thereof or an antibody-drug conjugate provided herein, and a pharmaceutically acceptable carrier.

[0038] In one aspect, this disclosure provides an isolated cell containing one or more polynucleotides encoding the antibody or its antigen-binding fragment.

[0039] In one respect, this disclosure provides a polynucleotide that encodes one or more chains of an antibody or an antigen-binding fragment thereof provided herein.

[0040] In one aspect, this disclosure provides a method for treating cancer in patients in need, the method comprising administering to the patient an antibody or antigen-binding fragment thereof, antibody-drug conjugate, or composition provided herein.

[0041] In one respect, this disclosure provides for the use of the antibodies or antigen-binding fragments thereof, antibody-drug conjugates or compositions provided herein in the preparation of medicaments for the treatment of cancer in patients in need.

[0042] In one respect, this disclosure provides for the use of antibodies or antigen-binding fragments thereof, antibody-drug conjugates or compositions provided herein in the treatment of cancer in patients in need.

[0043] In some embodiments, the cancer is selected from the group consisting of bladder cancer, breast cancer, colorectal cancer, endometrial cancer, esophageal cancer, head and neck cancer, kidney cancer, leukemia, liver cancer, lung cancer, lymphoma, melanoma, pancreatic cancer, prostate cancer, and thyroid cancer.

[0044] In some embodiments, the method further includes administering additional therapies to the patient for treating the cancer. In some embodiments, the additional therapies are immunotherapy, chemotherapy, or radiation therapy.

[0045] In one aspect, this disclosure provides a method for detecting CDH17 expression in a sample, the method comprising contacting the sample with the antibody or antigen-binding fragment provided herein under conditions in which an antibody or antigen-binding fragment thereof is bound to CDH17, and detecting the binding indicating CDH17 expression in the sample. Attached Figure Description

[0046] Figures 1A-1B show the binding activity of the CDH17 chimeric antibody to human CDH17 protein.

[0047] Figures 2A-2B show the binding activity of the CDH17 chimeric antibody to human CDH16 protein.

[0048] Figures 3A-3D show the binding activity of the CDH17 chimeric antibody on human CDH17-overexpressing HEK293 cells (3A-3B) and CDH17-negative HEK293 cells (3C-3D).

[0049] Figures 4A-4F show the binding activity of the CDH17 chimeric antibody on CDH17-positive AsPC-1 cells (4A-4B), HCT-8 cells (4C-4D), and AGS cells (4E-4F), respectively.

[0050] Figures 5A-5B show the binding activity of the CDH17 chimeric antibody to HEK293 cells of cynomolgus monkeys overexpressing CDH17.

[0051] Figures 6A-6B show the binding activity of the humanized 29H8D3 antibody against human AsPC-1 cells expressing CDH17. Figures 6C-6G show the binding activity of humanized 29H8D3-z12 against recombinant human (Figure 6C), rhesus macaque (Figure 6D), cynomolgus monkey (Figure 6E), rat (Figure 6F), and mouse (Figure 6G) CDH17 proteins. Figures 6H-6L show the binding activity of humanized 29H8D3-z12 against recombinant human CDH16 (Figure 6H), CDH9 (Figure 6I), CDH10 (Figure 6J), CDH3 (Figure 6K), and CDH6 (Figure 6L) proteins. Figures 6M-6N show the binding activity of humanized 29H8D3-z12 against CDH17 expressed in rhesus macaques (Figure 6M) and cynomolgus monkeys (Figure 6N) on HEK-293 cells.

[0052] Figures 7A-7B show the binding activity of the 29D2D7 humanized antibody to human HEK293 cells expressing CDH17.

[0053] Figure 8 The 61C7F12 humanized antibody was shown to bind to human HEK293 cells expressing CDH17.

[0054] Figure 9 The 69E3H11 humanized antibody was shown to bind to human HEK293 cells expressing CDH17.

[0055] Figures 10A-10B show the binding activity of the 143H10E4 humanized antibody to human HCT-8 cells expressing CDH17.

[0056] Figures 11A-11B show the binding activity of the 152A1D12 humanized antibody to human CDH17-expressing AGS cells.

[0057] Figures 12A-12B show the binding activity of the 155B11C6 humanized antibody to AGS cells expressing CDH17.

[0058] Figures 13A-13D show the binding activity of PTM-removed 29D2D7 humanized antibody (13A-13B) and 29H8D3 humanized antibody (13C-13E) on human CDH17-expressing HCT-8 cells. Figures 13F-13I show the binding activity of affinity-matured 69E3H11 humanized antibody on human CDH17-expressing LoVo (13F), HCT-8 (13G and 13I), and AsPC1 (13H) cells.

[0059] Figures 14A-14B show the internalization of CDH17 chimeric antibodies on human CDH17-positive AsPC-1 cells (14A) and LoVo cells (14B).

[0060] Figures 15A-15E show the internalization of CDH17 humanized antibody and PTM-removed CDH17 humanized antibody on human CDH17-positive HCT-8 cells.

[0061] Figures 16A-16G show the chemical structures of the connector-load compound veldotin (16A), drutecan (16B), ozomicin (16C), malfotin (16D), entansin (16E), gavitan (16F), and tecillin (16G) used in the ADC.

[0062] Figures 17A-17C show the indirect tumor cell killing of AGS cells mediated by CDH17 antibody mediated by Mc-vc-PABC-MMAE-labeled anti-human IgG secondary antibody.

[0063] Figures 18A-18C show the in vitro tumor cell killing effect of the Mc-vc-PABC-MMAE-labeled CDH17 ADC on CDH17-positive AsPC1 (18A), LoVo (18B), and AGS (18C) cells.

[0064] Figures 19A-19D show the binding activity of Mc-GGFG-DXd-labeled CDH17 ADC and parental CDH17 mAb to CDH17-expressing tumor cells, including AsPC1 (19A), AGS (19B), KATO III (19C), and HT29 (19D) cells.

[0065] Figures 20A-20F show the internalization rate (20A, 20C-20F) and percentage (20B) of Mc-GGFG-DXd-labeled ADCs and mAbs in CDH17-expressing HCT-8 (20A), AGS (20C), AsPC1 (20D), SW480-hCDH17 (20E) cells and CDH17-negative SW480 (20F) cells.

[0066] Figures 21A-21D show the in vitro tumor cell killing effect of Mc-GGFG-DXd-labeled CDH17 ADC on CDH17-positive HCT-8 (21A), SW620-hCDH17 (21B), MDA-MB-468-hCDH17 (21C), and SW480-hCDH17 (21D) cells.

[0067] Figures 22A-22I show the in vitro tumor cell killing effect of Mc-GGFG-DXd-labeled CDH17 ADCs on CDH17-positive HCT-8 (22A), AGS (22B), MDA-MB-468-hCDH17 (22C), SW480-hCDH17 (22D), SK-CO-1 (22E), SW620-hCDH17 (22F), LS1034 (22G), Caco2 (22H), and CDH17-negative RKO (22I) cells.

[0068] Figures 23A-23C show the in vitro bystander killing of Mc-GGFG-DXd-labeled CDH17 ADCs in a co-culture system consisting of GFP-labeled CDH17-positive SW480-hCDH17 cells and RFP-labeled CDH17-negative RKO cells (23A), or SW480 cells (23B), or MDA-MB-468 cells (23C).

[0069] Figures 24A-24B This demonstrates the in vivo tumor growth inhibition of the Mc-vc-PABC-MMAE-labeled CDH17 ADC in the LoVo CDX mouse model.

[0070] Figure 25A shows the in vivo tumor growth inhibition of the Mc-GGFG-DXd-labeled CDH17 ADC in the LS1034 CDX mouse model. Figure 25B shows the body weight changes of mice after treatment in the LS1034 CDX mouse model. Figure 25C shows that no body weight changes were observed in any treatment group before the end of the study.

[0071] Figures 26A-26B The in vivo tumor growth inhibition (26A) and body weight change (26B) of GGFG-DXd-labeled CDH17 ADC in four CRC PDX mouse models are shown.

[0072] Figure 27 This demonstrates the in vivo dose-dependent tumor growth inhibition of GGFG-DXd-labeled CDH17 ADC in three CRC PDX mouse models. Detailed Implementation

[0073] definition It should be noted that the term "a / an" refers to one or more of the same entity; for example, "an antibody" is understood to represent one or more antibodies. Therefore, the terms "a / an," "one or more," and "at least one" are used interchangeably in this document.

[0074] As used herein, "antibody" or "antigen-binding moiety" refers to a polypeptide or polypeptide complex that specifically recognizes and binds to an antigen. An antibody can be a complete antibody and any of its antigen-binding fragments or single chains. Therefore, the term "antibody" includes any protein or peptide containing at least a portion of an immunoglobulin molecule that has the biological activity of binding an antigen. Such examples include, but are not limited to, the complementarity-determining region (CDR) of the heavy or light chain or its ligand-binding moiety, the variable region of the heavy or light chain, the constant region of the heavy or light chain, the frame (FR) region or any portion thereof, or at least a portion of the binding protein.

[0075] Full-length antibodies consist of two heavy chains and two light chains. The variable regions of the light and heavy chains are responsible for antigen binding. The variable domains of the heavy and light chains can be referred to as “VH” and “VL”, respectively. The variable regions in both chains typically contain three highly variable loops called complementarity-determining regions (CDRs) (including the light chain (LC) CDRs of LC-CDR1, LC-CDR2, and LC-CDR3, and the heavy chain (HC) CDRs of HC-CDR1, HC-CDR2, and HC-CDR3). The CDR boundaries of the antibody-antigen binding fragments disclosed herein can be defined or identified using the following conventions: Kabat, Chothia, or Al-Lazikani (Al-Lazikani 1997; Chothia 1985; Chothia 1987; Chothia 1989; Kabat 1987; Kabat 1991). The three cored red reticulum (CDRs) of either the heavy or light chain are inserted between flanking segments called framework regions (FRs), which are more conserved than the CDRs and form a scaffold supporting the hypervariable loop. The constant regions of the heavy and light chains do not participate in antigen binding but exhibit various effector functions. Antibodies are classified based on the amino acid sequence of the antibody heavy chain constant region. The five major classes or isotypes of antibodies are IgA, IgD, IgE, IgG, and IgM, characterized by the presence of α, δ, ε, γ, and μ heavy chains, respectively. Several major antibody classes are further subdivided into subclasses, such as lgG1 (γ1 heavy chain), lgG2 (γ2 heavy chain), lgG3 (γ3 heavy chain), lgG4 (γ4 heavy chain), lgA1 (α1 heavy chain), or lgA2 (α2 heavy chain).

[0076] As used herein, the term "half-antibody" refers to an immunoglobulin heavy chain associated with an immunoglobulin light chain. Those skilled in the art will readily recognize that a half-antibody may contain fragments of it and may also have an antigen-binding domain consisting of a single variable domain, for example, derived from camelids.

[0077] As used herein, the term "single-chain half antibody" refers to a single-chain polypeptide comprising a VL domain, optionally a CL domain, a linker, a VH domain, optionally a CH1 domain, a hinge domain, a CH2 domain, and a CH3 domain, wherein the domains are positioned relative to each other in the N-terminal to C-terminal direction as follows: VL-linker-VH-hinge-CH2-CH3, VL-linker-VH-partial hinge-CH2-CH3, VL-linker-VH-hinge variant-CH2-CH3, or VL-CL-linker-VH-CH1-hinge-CH2-CH3.

[0078] The expression of "single-domain antibody" (sdAb) or "single-variable-domain (SVD) antibody" generally refers to an antibody in which a single variable domain (VH or VL) can confer antigen binding. In other words, the single variable domain does not need to interact with another variable domain to recognize the target antigen. Examples of single-domain antibodies include those derived from camelids (alpacas and camels) and cartilaginous fish (such as nurse sharks), as well as antibodies derived from human and mouse antibodies from recombinant methods (Nature (1989) 341:544-546; Dev Comp Immunol (2006) 30:43-56; Trend Biochem Sci (2001) 26:230-235; Trends Biotechnol (2003) 21:484-490; WO 2005 / 035572; WO 03 / 035694; Febs Lett (1994) 339:285-290; WO00 / 29004; WO 02 / 051870). When sdAb contains only heavy chains, it can be used interchangeably with "VHH", "single heavy chain variable domain antibody", or "nanobody".

[0079] As used herein, the term "antibody fragment" or "antigen-binding fragment" refers to a part of an antibody, such as F(ab')2, F(ab)2, Fab', Fab, Fv, scFv, etc. Regardless of structure, an antibody fragment binds to the same antigen recognized by the intact antibody. The term "antibody fragment" includes aptamers, spikelers, and dimeric antibodies. The term "antibody fragment" also includes any synthetic or genetically engineered protein that exerts its antibody function by binding to a specific antigen to form a complex.

[0080] The term "Fab" in antibody refers to the monovalent antigen-binding fragment of the antibody, which consists of a single light chain (including both the variable and constant regions) bound by disulfide bonds to the variable region and the first constant region of a single heavy chain. Fab can be obtained by digesting the antibody with papain at residues proximal to the N-terminus of the disulfide bonds between the heavy chains in the hinge region.

[0081] “Fab” refers to a Fab fragment that contains a portion of the hinge region. It can be obtained by digesting an antibody with pepsin at the C-terminus of the disulfide bond between the heavy chains in the hinge region. Therefore, a small number of residues in the hinge region (including one or more cysteine ​​residues) are different from Fab.

[0082] "F(ab)2" refers to the dimer of Fab', which contains two light chains and a portion of two heavy chains.

[0083] The term "Fv" in antibody refers to the smallest fragment of the antibody containing a complete antigen-binding site. An Fv fragment consists of a variable region of a single light chain that binds to the variable region of a single heavy chain. "dsFv" refers to a disulfide-bonded Fv fragment where the connection between the variable regions of the single light chain and the variable regions of the single heavy chain is a disulfide bond.

[0084] "Single-chain variable fragment" or "scFv" refers to the heavy chain (V) of immunoglobulins. H ) and light chains (V L This is a fusion protein containing variable regions. In some respects, these regions are linked by short linker peptides of approximately 10 to 25 amino acids in length. The linkers can be enriched with glycine for flexibility, and serine or threonine for solubility, and can also carry V... H N-terminus and V L The C-terminus is linked, or vice versa. Despite the removal of the constant region and the introduction of a linker, this protein retains the specificity of the original immunoglobulin. scFv molecules are known in the art and are described, for example, in U.S. Patent 5,892,019.

[0085] As used herein, the term "biantibody molecule" refers to a complex of two or more polypeptide chains or proteins, each containing at least one VL and one VH domain or fragments thereof, wherein both domains are contained within a single polypeptide chain. In some embodiments, a "biantibody molecule" includes a molecule containing an Fc or hinge-Fc domain. The polypeptide chains in the complex may be the same or different, i.e., the biantibody molecule may be a homopolymer or a heteropolymer. In certain aspects, a "biantibody molecule" includes a dimer or tetramer or the polypeptide chain containing both the VL and VH domains. The single polypeptide chain containing the multimeric protein may be covalently linked to at least one other peptide of the multimer via interchain disulfide bonds.

[0086] "Domain-specific antibodies" refer to antibody fragments containing only heavy chain variable regions or light chain variable regions. In some embodiments, two or more VH domains are covalently linked to peptide linkers to form bivalent or multivalent domain antibodies. The two VH domains of a bivalent domain antibody can target the same or different antigens.

[0087] The term antibody encompasses a wide range of polypeptide classes that can be distinguished by biochemical methods. Those skilled in the art will recognize that heavy chains are classified as γ, μ, α, δ, or ε, with several subclasses (e.g., γ1-γ4). It is the properties of this chain that determine the "class" of the antibody, such as IgG, IgM, IgA, IgG, or IgE. Immunoglobulin subclasses (isotypes) For example IgG1, IgG2, IgG3, IgG4, IgG5, etc., are well characterized and known to confer functional specificity. In view of this disclosure, those skilled in the art can readily identify these categories and modified versions of isotypes, which are accordingly covered within the scope of this disclosure. All immunoglobulin classes are obviously within the scope of this disclosure, and the following discussion will generally refer to the IgG class of immunoglobulin molecules. Regarding IgG, a standard immunoglobulin molecule comprises two identical light chain polypeptides with a molecular weight of approximately 23,000 Daltons and two identical heavy chain polypeptides with a molecular weight of 53,000-70,000. These four chains are typically linked by disulfide bonds in a “Y” configuration, wherein the light chain begins at the opening of the “Y” and continues to extend into the variable region to surround the heavy chain.

[0088] The antibodies, antigen-binding moieties, variants, or derivatives disclosed herein include, but are not limited to, polyclonal, monoclonal, multispecific, human, humanized, primate-derived, or chimeric antibodies, single-chain antibodies, and epitope-binding fragments. For example Fab, Fab' and F(ab')2, Fd, Fvs, single-chain Fvs (scFv), single-chain antibodies, disulfide-linked Fvs (sdFv), fragments containing VK or VH domains, fragments generated from Fab expression libraries, and anti-idiotypic (anti-Id) antibodies (including...) For example Anti-Id antibodies against the LIGHT antibodies disclosed herein). The immunoglobulin or antibody molecules disclosed herein can be any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass of immunoglobulin molecules.

[0089] As used herein, the term "derivative" in the context of a polypeptide or protein refers to a polypeptide or protein whose amino acid sequence has been altered by the introduction of amino acid residues through substitution, deletion, or addition. As used herein, the term "derivative" also refers to a modified polypeptide or protein, i.e., one that is covalently linked to a polypeptide or protein by any type of molecule. For example, but not limited to, antibodies can be modified, such as by glycosylation, acetylation, polyethylene glycolation, phosphorylation, amidation, derivatization by known protecting / blocking groups, proteolytic cleavage, linking to cellular antigens or other proteins, etc. Derivative polypeptides or proteins can be produced by chemical modification using techniques known to those skilled in the art, including but not limited to specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, etc. Furthermore, derivative polypeptides or proteins have similar or identical functions to the polypeptides or proteins from which they are derived.

[0090] As used herein, the term "derivative" in the context of non-protein derivatives refers to a second organic or inorganic molecule formed based on the structure of a first organic or inorganic molecule. Derivatives of organic molecules include, but are not limited to, molecules modified, for example, by the addition or deletion of hydroxyl, methyl, ethyl, carboxyl, or amino groups. Organic molecules may also be esterified, alkylated, and / or phosphorylated.

[0091] Light chains are classified as κ or λ. Each heavy chain class can bind to either a κ or λ light chain. Generally, light and heavy chains are covalently bonded to each other, and when immunoglobulins are produced by hybridomas, B cells, or genetically engineered host cells, the "tail" portions of the two heavy chains are linked together by covalent disulfide bonds or non-covalent bonds. In this heavy chain, the amino acid sequence extends from the N-terminus at the Y-configuration bifurcation to the C-terminus at the bottom of each chain.

[0092] The light and heavy chains are divided into regions that are structurally and functionally homologous. The terms "constant" and "variable" are used functionally. In this regard, it should be understood that the variable domains (VK and VH) of both the light and heavy chains determine antigen recognition and specificity. Conversely, the constant domains (CK and CH1, CH2, or CH3) of the light and heavy chains confer important biological properties, such as secretion, transplacental activity, Fc receptor binding, and complement binding. By convention, the farther the constant domain is from the antibody's antigen-binding site or N-terminus, the higher its number. The N-terminal portion is the variable region, and the C-terminal portion is the constant region; the CH3 and CK domains actually contain the carboxyl terms of the heavy and light chains, respectively.

[0093] As described above, the variable region enables antibodies to selectively recognize and specifically bind to epitopes on antigens. That is, a subset of the antibody's VK and VH domains, or complementarity-determining regions (CDRs), binds to form a variable region that defines the three-dimensional antigen-binding site. This quaternary antibody structure forms an antigen-binding site located at the end of each arm of this Y-configuration. More specifically, this antigen-binding site is defined by three CDRs (i.e., CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3) on each VH and VK chain. In some cases, such as certain immunoglobulin molecules derived from camel species or engineered based on camel immunoglobulins, the immunoglobulin molecules are complete and may consist only of heavy chains, lacking light chains. See, for example, Hamers-Casterman et al., Nature 363:446-448 (1993).

[0094] In naturally occurring antibodies, each antigen-binding domain contains six "complementarity-determining regions" or "CDRs," which are short, discontinuous sequences of amino acids precisely positioned to form the antigen-binding domain when the antibody assumes its three-dimensional conformation in an aqueous environment. The remaining amino acids within these domains, called "framework" regions, exhibit low intermolecular variability. Framework regions primarily adopt a β-sheet conformation, and CDRs form loops that connect β-sheet structures and, in some cases, form part of the β-sheet structure. Thus, the framework regions act as a scaffold, positioning the CDRs in the correct orientation through interchain, non-covalent interactions. The antigen-binding domain formed by these positioned CDRs defines a surface complementary to an epitope on an immunoreactive antigen. This complementary surface facilitates the non-covalent binding of the antibody to its homologous epitope. For any given heavy or light chain variable region, those skilled in the art can readily identify the amino acids that contain the CDR and framework regions, respectively, as they have been precisely defined (see “Sequences of Proteins of Immunological Interest,” Kabat, E., et al., US Department of Health and Human Services, (1983); and Chothia and Lesk, J. Mol. Biol., 196:901-917 (1987)).

[0095] Where a term used and / or accepted in the art has two or more definitions, the definition of the term as used herein is intended to include all such meanings unless explicitly stated otherwise. A specific example is the use of the term “complementarity-determining region” (“CDR”) to describe discontinuous antigen-binding sites found in the variable regions of both heavy-chain and light-chain polypeptides. This particular region is described in Kabat et al., US Dept. of Health and Human Services, “Sequences of Proteins of Immunological Interest” (1983) and Chothia et al., J. Mol. Biol. 196:901-917 (1987), the entire contents of which are incorporated herein by reference. When comparing the two definitions of CDR by Kabat and Chothia, overlaps or subsets of amino acid residues are included. However, the application of any definition of CDR used to refer to antibodies or their variants is intended to be within the scope of the terminology defined and used herein. For comparison, the corresponding amino acid residues covering these CDRs as defined in the above-cited references are shown in the table below. The exact number of residues covering a particular CDR varies depending on the sequence and size of the CDR. Those skilled in the art can typically determine which residues contain a specific CDR based on the amino acid sequence of the antibody's variable region.

[0096] Kabat et al. also defined a numbering system applicable to the variable domain sequences of any antibody. Those skilled in the art can explicitly assign this “Kabat numbering” system to any variable domain sequence without relying on any experimental data other than the sequence itself. As used herein, “Kabat numbering” refers to the numbering system described in the following reference: Kabat et al., US Dept. of Health and Human Services, “Sequence of Proteins of Immunological Interest” (1983).

[0097] In addition to the table above, the Kabat numbering system describes these CDR regions as follows: CDR-H1 begins at approximately amino acid 31 (approximately 9 residues after the first cysteine ​​residue), consists of approximately 5-7 amino acids, and ends at the next tryptophan residue. CDR-H2 begins at the fifteenth residue after the end of CDR-H1, consists of approximately 16-19 amino acids, and ends at the next arginine or lysine residue. CDR-H3 begins at approximately the thirty-third amino acid residue after the end of CDR-H2; consists of 3-25 amino acids; and ends at the sequence WGXG, where X is any amino acid. CDR-L1 begins at approximately residue 24 (after the cysteine ​​residue); consists of approximately 10-17 residues; and ends at the next tryptophan residue. CDR-L2 begins at approximately the sixteenth residue after the end of CDR-L1, consists of approximately 7 residues. CDR-L3 begins at approximately the thirtieth residue after CDR-L2 (i.e., after the cysteine ​​residue); it consists of approximately 7-11 residues and ends at sequence F or WGXG, where X is any amino acid.

[0098] The antibodies disclosed herein can be derived from any animal source, including birds and mammals. Preferably, the antibodies are human, mouse, donkey, rabbit, goat, guinea pig, camel, llama, horse, or chicken antibodies. In another embodiment, the variable region may be derived from condricthoids (e.g., from sharks).

[0099] As used herein, the term "heavy chain constant region" includes an amino acid sequence derived from the immunoglobulin heavy chain. A polypeptide containing a heavy chain constant region comprises at least one of the following: a CH1 domain, a hinge (e.g., upper, middle, and / or lower hinge region) domain, a CH2 domain, a CH3 domain, or a variant or fragment thereof. For example, an antigen-binding polypeptide for use in this disclosure may comprise a polypeptide chain containing a CH1 domain; a polypeptide chain containing at least a portion of a CH1 domain, a hinge domain, and a CH2 domain; a polypeptide chain containing both a CH1 domain and a CH3 domain; a polypeptide chain containing at least a portion of a CH1 domain, a hinge domain, and a CH3 domain; or a polypeptide chain containing at least a portion of a CH1 domain, a hinge domain, a CH2 domain, and a CH3 domain. In another embodiment, the polypeptide disclosed herein comprises a polypeptide chain containing a CH3 domain. Furthermore, antibodies for use in this disclosure may lack at least a portion of the CH2 domain (e.g., all or part of the CH2 domain). As described above, those skilled in the art will understand that the heavy chain constant regions can be modified so that their amino acid sequences differ from those of naturally occurring immunoglobulin molecules.

[0100] The heavy chain constant region of antibodies disclosed in this article can be derived from different immunoglobulin molecules. For example, the heavy chain constant region of a polypeptide can contain components derived from IgG. l The molecule contains the CH1 domain and a hinge region derived from the IgG3 molecule. In another example, the heavy chain constant region may contain a portion derived from IgG. l The molecule is partially derived from the hinge region of the IgG3 molecule. In another example, the heavy chain portion may contain components partially derived from IgG. l The molecule is a chimeric hinge that is partially derived from the IgG4 molecule.

[0101] As used herein, the term "light chain constant region" includes an amino acid sequence derived from the antibody light chain. Preferably, the light chain constant region comprises at least one of a constant κ domain or a constant λ domain.

[0102] A "light chain-heavy chain pair" refers to a combination of light and heavy chains that can form a dimer through disulfide bonds between the CL domain of the light chain and the CH1 domain of the heavy chain.

[0103] As previously mentioned, the subunit structures and three-dimensional conformations of constant regions in various immunoglobulin classes are well known. As used herein, the term "VH domain" includes the N-terminal variable domain of the immunoglobulin heavy chain, and the term "CH1 domain" includes the first (closest to the N-terminus) constant region domain of the immunoglobulin heavy chain. The CH1 domain is adjacent to the VH domain and is located at the N-terminus of the hinge region of the immunoglobulin heavy chain molecule.

[0104] The “CH1 domain” (also known as the “C1” of the “H1” domain) typically extends from about amino acid 118 to about amino acid 215 (EU numbering system).

[0105] As used herein, the term “hinge region” refers to the portion of a heavy chain molecule that links the CH1 domain to the CH2 domain. In IgG, this region corresponds to Glu216 to Pro230 of human IgG1, EU numbering system (Burton, Molec. Immunol. [Molecular Immunology] 22:161-206 (1985)). 。 Other IgG isotypes' hinge regions can be aligned with the IgG1 sequence by placing the first and last cysteine ​​residues forming the inter-heavy chain SS bond in the same position. This hinge region is flexible, thus allowing the two N-terminal antigen-binding regions to move independently. The hinge region can be subdivided into three distinct domains: upper, middle, and lower hinge domains (Roux et al., J. Immuno). l [Journal of Immunology] 161:4083 (1998)).

[0106] As used herein, the term "CH2 domain" encompasses a portion of the heavy chain molecule that extends using conventional numbering schemes, such as from approximately residue 244 to residue 360 ​​of the antibody (residues 244 to 360, Kabat numbering system; and residues 231–340, EU numbering system; see Kabat et al., US Dept. of Health and Human Services, "Sequences of Proteins of Immunological Interest" (1983)). The CH2 domain is unique because it does not pair tightly with another domain. Instead, two N-linked branched carbohydrate chains are inserted between the two CH2 domains of the intact native IgG molecule. It is also well documented that the CH3 domain extends from the CH2 domain to the C-terminus of the IgG molecule and contains approximately 108 residues.

[0107] The “CH3 domain” (also known as the “C3 domain”) contains a string of residues in the Fc region from the C-terminus to the CH2 domain (i.e. from about amino acid residue 341 to the C-terminus of the antibody sequence, usually at amino acid residues 446 or 447 of IgG, EU numbering system).

[0108] The terms “Fc region,” “Fc domain,” or “crystallizable region fragment” used herein are used to define the C-terminal region of the immunoglobulin heavy chain, including native sequence Fc regions and variant Fc regions. Although the boundaries of the Fc region of the immunoglobulin heavy chain can vary, the Fc region of the human IgG heavy chain is generally defined as an amino acid residue extending from the Cys226 position or from Pro230 to its carboxyl terminus. The C-terminal lysine (residue 447 according to the EU numbering system) of the Fc region can be removed, for example, during antibody production or purification or by recombinant engineering of the nucleic acid encoding the antibody heavy chain. Thus, compositions of complete antibodies may include antibody populations with all K447 residues removed, antibody populations without K447 residues removed, and antibody populations having a mixture of antibodies with and without K447 residues. Suitable native sequence Fc regions for the antibodies described herein include human IgG1, IgG2 (IgG2A, IgG2B), IgG3, and IgG4.

[0109] As used herein, the term "disulfide bond" refers to a covalent bond formed between two sulfur atoms. The amino acid cysteine ​​contains a thiol group, which can form a disulfide bond or bridge with a second thiol group. In most naturally occurring IgG molecules, the CH1 and CK regions are linked by disulfide bonds, with the two heavy chains linked by two disulfide bonds located at positions 239 and 242 (positions 226 or 229, EU numbering system) using the Kabat numbering system.

[0110] As used herein, the term "chimeric antibody" should be understood to mean any antibody in which the immune-reactive region or site is derived from or obtained from a first species, and the constant region (which, according to this disclosure, may be whole, partial, or modified) is derived from a second species. In some embodiments, the target binding region or site will be derived from a non-human source (e.g., mouse or primate), and the constant region will be of human origin.

[0111] In this document, "humanized antibody" is used to describe an antibody that contains heavy and light chain variable region sequences derived from a non-human species (e.g., mouse), but at least a portion of the VH and / or VL sequences have been modified to be more "human-like," i.e., more similar to human germline variable sequences. A "humanized antibody" is an antibody or a variant, derivative, analog, or fragment thereof that specifically binds to a target antigen and contains a frame (FR) region and a complementarity-determining region (CDR), wherein the frame region has a substantially identical amino acid sequence to that of a human antibody, and the complementarity-determining region has a substantially identical amino acid sequence to that of a non-human antibody. As used herein, the term "substantially" in the context of a CDR means a CDR having an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to the amino acid sequence of a non-human antibody CDR. Humanized antibodies substantially comprise all of at least one, typically two, variable domains (Fab, Fab', F(ab')2, Fv), where all or substantially all CDR regions within the variable domains correspond to the CDR regions of non-human immunoglobulins (i.e., donor antibodies), and all or substantially all frame regions are frame regions of human immunoglobulin common sequences. In one embodiment, the humanized antibody further comprises at least a portion of an immunoglobulin constant region (Fc), typically the constant region of a human immunoglobulin. In some embodiments, the humanized antibody contains a light chain and at least a variable domain of the heavy chain. The antibody may also include CH1, hinge, CH2, CH3, and CH4 regions of the heavy chain. In some embodiments, the humanized antibody contains only the humanized light chain. In some embodiments, the humanized antibody contains only the humanized heavy chain. In specific embodiments, the humanized antibody contains only the humanized variable domain of the light chain and / or the humanized heavy chain.

[0112] As used herein, the term "epitope" refers to a specific atom or amino acid group on an antigen to which an antibody or antibody moiety binds. If two antibodies or antibody moiety competitively bind to an antigen, they can bind to the same epitope within the antigen.

[0113] Antibody-drug conjugates (ADCs) typically consist of monoclonal antibodies (mAbs) covalently linked to a cytotoxic drug (payload) via a linker. They combine the advantages of highly specific targeting and efficient killing, achieving accurate and efficient clearance of cancer cells, and have become a hot topic in anticancer drug development. The cytotoxic drug and the linker form the structure of the drug linker compound.

[0114] "Specific binding" or "specific to" generally refers to an antibody binding to an epitope via its antigen-binding domain, and this binding requires a certain complementarity between the antigen-binding domain and the epitope. According to this definition, an antibody is said to "specifically bind" to an epitope when it binds more easily to a particular epitope via its antigen-binding domain than to a random, unrelated epitope. In this paper, the term "specificity" is used to confirm the relative affinity of a particular antibody for a particular epitope. For example, antibody "A" can be considered more specific to a given epitope than antibody "B," or antibody "A" can be said to have a higher specificity for binding to epitope "C" than to its specificity for binding to related epitope "D."

[0115] The "sequence identity percentage (%)" for an amino acid sequence (or nucleic acid sequence) is defined as the percentage of amino acid (or nucleotide) residues in the candidate sequence that are identical to amino acid (or nucleic acid) residues in the reference sequence after sequence alignment and, where necessary, the introduction of vacancies to achieve maximum correspondence. Conservative substitutions of amino acid residues may or may not be considered identical residues. Alignment can be used to determine the percentage of identity of amino acid (or nucleic acid) sequences, for example, using publicly available tools such as BLASTN, BLASTp (available on the National Center for Biotechnology Information (NCBI) website, see also Altschul S. F. et al., J. Mol. Biol., 215:403-410 (1990); Stephen F. et al., Nucleic Acids Res., 25:3389-3402 (1997)), ClustalW2 (available on the European Institute for Bioinformatics website, see also Higgins DG et al., Methods in Enzymology, 266:383-402 (1996); Larkin MA et al., Bioinformatics (Oxford, UK), 23(21): 2947-8 (2007)), and ALIGN or Megalign (DNASTAR) software. Those skilled in the art can use the default parameters provided by the tool, or they can customize the parameters based on the comparison results, for example, by selecting a suitable algorithm.

[0116] As used herein, the term "treatment" refers to both therapeutic treatment and preventative or preventative measures aimed at preventing or slowing (alleviating) physiological changes or conditions, such as the progression of cancer. Beneficial or desired clinical outcomes include, but are not limited to, relief of symptoms, reduction of disease severity, stabilization (i.e., non-deterioration) of the disease state, delay or slowing of disease progression, improvement or alleviation of the disease state, and mitigation (whether partial or complete), whether detectable or undetectable. "Treatment" can also refer to extended survival compared to expected survival without treatment. Those in need of treatment include those who already have a condition or symptom, those who are susceptible to a condition or symptom, or those whose condition or symptom requires prevention.

[0117] The terms "subject," "individual," "animal," "patient," or "mammal" refer to any subject who requires diagnosis, prognosis, or treatment, particularly mammalian subjects. Mammal subjects include humans, domestic animals, farm animals and zoo animals, racing animals, or pet animals such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, cows, etc.

[0118] As used herein, phrases such as “to a patient in need of treatment” or “subject in need of treatment” include subjects, such as mammalian subjects, who will benefit from the administration of the antibodies or compositions disclosed herein for purposes such as detection, diagnostic procedures, and / or treatment.

[0119] Anti-CDH17 antibody Through experimentation and trial and error, the inventors were able to identify novel antibodies that can effectively and specifically bind to the human CDH17 protein. The chimeric antibodies include the obtained 16C18, 20C3E8, 29D2D7, 29H8D3, 61C7F12, 67A11B11, 69E3H11, 95F2C2F12, 103G6G1, 120B10C5, 134C7B1, 143H10E4, 152A1D12, and 155B11C6 (Table 1).

[0120] According to one embodiment of this disclosure, an antibody or antigen-binding fragment comprising variable domains of heavy and light chains having CDR regions is provided. CDRs are summarized in Tables 2A-2B (Kabat numbers).

[0121] In some embodiments, VH CDR1, CDR2, and CDR3 are selected from any group of VH CDR1, CDR2, and CDR3 shown in Table 2A, and VL CDR1, CDR2, and CDR3 are selected from any group of VL CDR1, CDR2, and CDR3 shown in Tables 2B, 15B, and 15D. In some embodiments, VH CDR1, CDR2, and CDR3, as well as VL CDR1, CDR2, and CDR3, are selected from those derived from the same antigen-binding moiety.

[0122] In some embodiments, at least one, two, three, four, five, or six of the above-mentioned VH CDR1, CDR2, and CDR3 and VL CDR1, CDR2, and CDR3 are modified by the addition, deletion, substitution, or combination thereof of one, two, or three amino acids.

[0123] The CDR, heavy chain variable region, light chain variable region, or single heavy chain variable domain disclosed herein may be further modified. In some embodiments, the modified heavy chain variable region, light chain variable region, or single heavy chain variable domain retains at least about 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity and is still able to bind to the target site.

[0124] In some embodiments, the modification is a substitution at no more than one hotspot location per CDR. In some embodiments, the modification is a substitution at one, two, or three such hotspot locations. In one embodiment, the modification is a substitution at one of the hotspot locations. In some embodiments, such substitution is a conservative substitution.

[0125] The antibody or its antigen-binding fragment contains a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 7, 15, 23, 31, 39, 47, 55, 63, 71, 79, 87, 95, 103, 111, or a peptide having at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with SEQ ID NO: 7, 15, 23, 31, 39, 47, 55, 63, 71, 79, 87, 95, 103, 111.

[0126] The antibody or its antigen-binding fragment comprises a light chain variable region containing an amino acid sequence selected from the group consisting of SEQ ID NO: 8, 16, 24, 32, 40, 48, 56, 64, 72, 80, 88, 96, 104, 112, or a peptide having at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with SEQ ID NO: 8, 16, 24, 32, 40, 48, 56, 64, 72, 80, 88, 96, 104, 112.

[0127] Post-translational modification (PTM) removal of antibodies can be performed via amino acid substitution in, for example, CDRs or framework regions to remove potential PTM sites leading to deamidation, isomerization, glycosylation, oxidation, and unpaired cysteine ​​residues, thereby improving developability, including long-term stability, manufacturability, and reducing the heterogeneity of the antibodies provided herein. Alternatively, affinity maturation of antibodies can be performed via amino acid substitution in, for example, CDRs to improve binding affinity, thereby enhancing activity. According to this disclosure, exemplary light chain CDRs of 29D2D7-z19, 29H8D3-z12, and 69E3H11-z12 undergoing PTM site removal or affinity maturation are listed in Tables 15B and 15D of Example 4, and the sequences of the resulting light chain variable regions are listed in Tables 15A and 15C.

[0128] The antibody or its antigen-binding fragment comprises (a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 7, a peptide or humanized form thereof having at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity therewith, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 8, a peptide or humanized form thereof having at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity therewith; (b) A heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 15, a peptide having at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity with it, or a humanized form thereof, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 16, a peptide having at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity with it, or a humanized form thereof; (c) A heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 23, a peptide or humanized form thereof having at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity with it, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 24, a peptide or humanized form thereof having at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity with it; (d) A heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 31, a peptide or humanized form thereof having at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity with it, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 32, a peptide or humanized form thereof having at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity with it; (e) A heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 39, a peptide or humanized form thereof having at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity therewith, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 40, a peptide or humanized form thereof having at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity therewith; (f) A heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 47, a peptide having at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity with it, or a humanized form thereof, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 48, a peptide having at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity with it, or a humanized form thereof; (g) Heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 55, a peptide or humanized form thereof having at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity with it, and light chain variable region comprising the amino acid sequence of SEQ ID NO: 56, a peptide or humanized form thereof having at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity with it; (h) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 63, a peptide or humanized form thereof having at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity therewith, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 64, a peptide or humanized form thereof having at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity therewith; (i) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 71, a peptide or humanized form thereof having at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity therewith, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 72, a peptide or humanized form thereof having at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity therewith; (j) A heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 79, a peptide or humanized form thereof having at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity therewith, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 80, a peptide or humanized form thereof having at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity therewith; (k) A heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 87, a peptide or humanized form thereof having at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity therewith, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 88, a peptide or humanized form thereof having at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity therewith; (l) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 95, a peptide or humanized form thereof having at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity with it, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 96, a peptide or humanized form thereof having at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity with it; (m) Heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 103, a peptide having at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity with it, or a humanized form thereof, and light chain variable region comprising the amino acid sequence of SEQ ID NO: 104, a peptide having at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity with it, or a humanized form thereof; (n) A heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 111, a peptide having at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with it, or a humanized form thereof; and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 112, a peptide having at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with it, or a humanized form thereof; or (o) A heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 131 or a peptide having at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity therewith, and a light chain variable region comprising the amino acid sequence selected from the group consisting of SEQ ID NO: 203-207 or a peptide having at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity therewith; (p) A heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 116 or 248 or a peptide having at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity therewith, and a light chain variable region comprising the amino acid sequence selected from the group consisting of SEQ ID NO: 208-216 and 249-250 or a peptide having at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity therewith; or (q) A heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 152 or a peptide having at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity therewith, and a light chain variable region comprising the amino acid sequence selected from the group consisting of SEQ ID NO: 232-238 or a peptide having at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity therewith.

[0129] The antibodies or antigen-binding fragments provided in this article are selected from the group consisting of full-length antibodies, biantibodies, scFv, scFv dimers, BsFv, dsFv, (dsFv)2, dsFv-dsFv', Fv fragments, Fab, Fab', F(ab')2, ds biantibodies, nanobodies, domain antibodies, isolated CDRs, and bivalent domain antibodies.

[0130] According to specific embodiments, the antibody is humanized. The humanized form of a non-human (e.g., mouse) antibody is a chimeric immunoglobulin molecule, an immunoglobulin chain, or a fragment thereof containing a minimal sequence derived from a non-human immunoglobulin (e.g., Fv, Fab, Fab', F(ab')2, or other antigen-binding sequence of the antibody). Humanized antibodies comprise human immunoglobulins (receptor antibodies) in which residues forming the receptor complementarity-determining region (CDR) are replaced by residues from the CDR of a non-human species (donor antibody) (such as a mouse, rat, or rabbit with the desired specificity, affinity, and capability). In some instances, Fv framework residues of the human immunoglobulin may be replaced by corresponding non-human residues. Humanized antibodies may also contain residues not found in the receptor antibody or in the introduced CDR or framework sequence. Typically, a humanized antibody will contain at least one, typically substantially all, of two variable domains, wherein all or substantially all of the CDR regions correspond to those of the non-human immunoglobulin, and all or substantially all of the FR regions are those of the human immunoglobulin common sequence. Humanized antibodies will preferably also contain at least a portion of the immunoglobulin constant region (Fc), typically the constant region of human immunoglobulins (Jones et al., Nature, 321: 522-525 (1986); Riechmann et al., Nature, 332: 323-329 (1988); and Presta, Curr. Op. Struct. Biol., 2: 593-596 (1992)).

[0131] Methods for humanizing nonhuman antibodies are well known in the art. Typically, humanized antibodies have one or more amino acid residues introduced from a nonhuman source. These nonhuman amino acid residues are generally referred to as input residues, and they are typically derived from the input variable domain. Humanization can be performed essentially as described by Winter et al. (Jones et al., Nature, 321: 522-525 (1986); Riechmann et al., Nature, 332: 323-327 (1988); Verhoeyen et al., Science, 239: 1534-1536 (1988)) by replacing the corresponding sequence of a human antibody with a rodent CDR or CDR sequence. Thus, such humanized antibodies are chimeric antibodies (US Patent No. 4,816,567), in which substantially less than the fully human variable domain has been replaced by the corresponding sequence from a nonhuman species. In practice, humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are replaced by residues from similar sites in rodent antibodies.

[0132] "Conservative amino acid substitution" refers to the substitution of amino acid residues with amino acid residues having similar side chains. Families of amino acid residues with similar side chains have been defined in the art, including: basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Therefore, it is preferable to replace non-essential amino acid residues in immunoglobulin peptides with another amino acid residue from the same side chain family. In another embodiment, a string of amino acids can be replaced with a string of structurally similar amino acids that differ in sequence and / or composition of side chain family members.

[0133] The table below provides non-limiting examples of conserved amino acid substitutions, where a similarity score of 0 or higher indicates a conserved substitution between two amino acids.

[0134] Amino acid similarity matrix Conservative amino acid substitution In some embodiments, the humanized antibody or antigen-binding fragments provided herein contain no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residue substitutions in each person's FR sequence, or no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residue substitutions in all FRs of the variable domain of the heavy or light chain. In some embodiments, such changes in amino acid residues may be present only in the heavy chain FR region, only in the light chain FR region, or in both chains.

[0135] In some embodiments, one or more amino acid residues are mutated, for example, reverting to the corresponding residues found in the nonhuman parent antibody (e.g., in the mouse frame region) of the derived CDR sequence. Those skilled in the art can select a suitable mutation site based on principles known in the art. For example, a mutation site can be selected where: 1) the residues in the human germline sequence frame are rare (e.g., less than 20% or less than 10% in the human variable region sequence); 2) the site is adjacent to one or more of the three CDRs in the primary sequence of the human germline chain, as it is likely to interact with residues in the CDRs; or 3) the site is close to the CDRs in a three-dimensional model, and therefore is likely to interact with amino acids in the CDRs. The residues at the selected site can be reverted to the corresponding residues in the parent antibody, or mutated back to residues that are neither the corresponding residues in the human germline sequence nor the corresponding residues in the parent antibody, but rather typical residues of the human sequence, i.e., residues that occur more frequently at that location in known human sequences belonging to the same subgroup as the human germline sequence (see U.S. Patent No. 5,693,762).

[0136] If desired, the reversion mutation can be introduced into the human germline framework sequence. In some embodiments, the humanized antibody 29H8D3 can be incorporated into the human germline sequence IGHV1-18. The heavy chain framework of 01 contains one or more reversion mutations selected from the following groups: A24V, V37M, M48I, R67K, V68A, M70L, T72A, T74K, M81I, R84N, R87T, D89E, and Y95F. The humanized antibody 29H8D3 can be expressed in the human germline sequence IGKV2-28. The light chain frame of 01 contains one or more reversion mutations selected from the group consisting of: Y41F, L51V, D75A, and Q105A. Specific variable region sequences having reversion mutations can be found in Table 7A. In some embodiments, the heavy chain variable region having one or more reversion mutations comprises amino acid sequences selected from the group consisting of SEQ ID NO: 114-119. In some embodiments, the light chain variable region having one or more reversion mutations comprises amino acid sequences selected from the group consisting of SEQ ID NO: 122-123 and 201-202. Specific combinations of heavy chain and light chain variable regions can be found in Table 7B.

[0137] In some embodiments, the humanized antibody 29D2D7 can be derived from the human germline sequence IGHV1-18. The heavy chain framework of 01 contains one or more reversion mutations selected from the following groups: R38K, M48I, R67K, V68A, M70L, T72A, T74K, R87T, and D89E. The humanized antibody 29D2D7 can be derived from the human germline sequence IGKV4-1. The light chain framework of 01 contains one or more reversion mutations selected from the group consisting of: V3G, T5S, and P49S. Specific variable region sequences having reversion mutations can be found in Table 8A. In some embodiments, the heavy chain variable region having one or more reversion mutations comprises amino acid sequences selected from the group consisting of SEQ ID NO: 126-132. In some embodiments, the light chain variable region having one or more reversion mutations comprises amino acid sequences selected from the group consisting of SEQ ID NO: 135-137. Specific combinations of heavy chain and light chain variable regions can be found in Table 8B.

[0138] In some embodiments, the humanized antibody 61C7F12 can be derived from the human germline sequence IGHV3-21. The heavy chain framework of 01 contains an S49A reversion mutation. The humanized antibody 61C7F12 can be used in the human germline sequence IGKV1-9. The light chain framework of 01 contains one or more reversion mutations selected from the group consisting of: D1Q, Q3V, A44S, L48W, E71S, F72Y, and T73S. Specific variable region sequences having reversion mutations can be found in Table 9A. In some embodiments, the heavy chain variable region having one or more reversion mutations comprises the amino acid sequence of SEQ ID NO: 140. In some embodiments, the light chain variable region having one or more reversion mutations comprises amino acid sequences selected from the group consisting of SEQ ID NO: 143-145. Specific combinations of heavy chain and light chain variable regions can be found in Table 9B.

[0139] In some embodiments, the humanized antibody 69E3H11 can be derived from the human germline sequence IGHV1-3. The heavy chain framework of 01 contains one or more reversion mutations selected from the following groups: R38K, M48I, R67K, V68A, I70L, R72V, T74K, and R98L; or in the hominid sequence IGHV1-46. The heavy chain framework of 01 contains one or more reversion mutations selected from the group consisting of M70L, V79A, and R87T. The humanized antibody 69E3H11 can be expressed in the human germline sequence IGKV6-21. The light chain framework of 01 contains one or more reversion mutations selected from the group consisting of K50Y and F72Y. Specific variable region sequences having reversion mutations can be found in Table 10A. In some embodiments, the heavy chain variable region having one or more reversion mutations comprises amino acid sequences selected from the group consisting of SEQ ID NOs: 150-152 and 154. In some embodiments, the light chain variable region having one or more reversion mutations comprises amino acid sequences selected from the group consisting of SEQ ID NOs: 157-158. Specific combinations of heavy chain and light chain variable regions can be found in Table 10B.

[0140] In some embodiments, the humanized antibody 143H10E4 can be derived from the human germline sequence IGHV1-69. The heavy chain framework of 02 contains one or more reversion mutations selected from the following groups: G27S, S30T, M48I, R67K, V68A, I70L, S84N, and Y95F. The humanized antibody 143H10E4 can be expressed in the human germline sequence IGKV6-21. The light chain framework of 01 contains one or more reversion mutations selected from the group consisting of: L47P, L48W, K50Y, F72Y, and T73S. Specific variable region sequences having reversion mutations can be found in Table 11A. In some embodiments, the heavy chain variable region having one or more reversion mutations comprises amino acid sequences selected from the group consisting of SEQ ID NO: 163-165. In some embodiments, the light chain variable region having one or more reversion mutations comprises amino acid sequences selected from the group consisting of SEQ ID NO: 167-169. Specific combinations of heavy chain and light chain variable regions can be found in Table 11B.

[0141] In some embodiments, the humanized antibody 152A1D12 can be used in the human germline sequence IGHV1-3. The heavy chain framework of 01 contains one or more reversion mutations selected from the following groups: R38K, M48I, R67K, V68A, I70L, and R72V. Humanized antibody 152A1D12 can be derived from IGKV1-13. 02 and IGKV3-11 The 01 frame combination produces a heterozygous light chain frame of a pedigree sequence containing one or more reversion mutations selected from the following groups: L46R, L47W, and F71Y; or in the pedigree sequence IGKV3-11. The light chain framework of 01 contains one or more reverse mutations selected from the group consisting of L47W and I58V. Specific variable region sequences with reversion mutations can be found in Table 12A. In some embodiments, the heavy chain variable region with one or more reversion mutations comprises amino acid sequences selected from the group consisting of SEQ ID NO: 174-176. In some embodiments, the light chain variable region with one or more reversion mutations comprises amino acid sequences selected from the group consisting of SEQ ID NO: 179-181 and 184. Specific combinations of heavy chain and light chain variable regions can be found in Table 12B.

[0142] In some embodiments, the humanized antibody 155B11C6 can be derived from the human germline sequence IGHV1-2. The heavy chain framework of 02 contains one or more reversion mutations selected from the following groups: R38K, M48I, R67K, V68A, M70L, R72A, and T74K; and in the human germline sequence IGHV1-46 The heavy chain framework of 01 contains one or more reverse mutations selected from the following groups: M70L, R72A, T74K, S85G, and R87T. The humanized antibody 155B11C6 can be expressed in the human germline sequence IGKV3-20. The light chain framework of 01 contains one or more reversion mutations selected from the following groups: A44S, L47P, L48W, I59V, D71S, and F72Y, as well as in the hominid sequence IGKV6-21. The light chain frame of 01 contains one or more reverse mutations selected from the group consisting of: L47P, L48W, K50Y, and F72Y. Specific variable region sequences with reversion mutations can be found in Table 13A. In some embodiments, the heavy chain variable region with one or more reversion mutations comprises amino acid sequences selected from the group consisting of SEQ ID NOs: 187-189 and 191. In some embodiments, the light chain variable region with one or more reversion mutations comprises amino acid sequences selected from the group consisting of SEQ ID NOs: 194-196 and 199. Specific combinations of heavy chain and light chain variable regions can be found in Table 13B.

[0143] In one embodiment, an antibody or antigen-binding fragment thereof competitively binds to human CDH17 protein against 16C18 is provided. In one embodiment, an antibody or antigen-binding fragment thereof competitively binds to human CDH17 protein against 20C3E8 is provided. In one embodiment, an antibody or antigen-binding fragment thereof competitively binds to human CDH17 protein against 29D2D7 is provided. In one embodiment, an antibody or antigen-binding fragment thereof competitively binds to human CDH17 protein against 29H8D3 is provided. In one embodiment, an antibody or antigen-binding fragment thereof competitively binds to human CDH17 protein against 61C7F12 is provided. In one embodiment, an antibody or antigen-binding fragment thereof competitively binds to human CDH17 protein against 67A11B11 is provided. In one embodiment, an antibody or antigen-binding fragment thereof competitively binds to human CDH17 protein against 69E3H11 is provided. In one embodiment, an antibody or antigen-binding fragment thereof competitively binds to human CDH17 protein against 95F2C2F12 is provided. In one embodiment, an antibody or antigen-binding fragment thereof competitively binds to human CDH17 protein against 103G6G1 is provided. In one embodiment, an antibody or antigen-binding fragment thereof competitively binds to human CDH17 protein with 120B10C5 is provided. In one embodiment, an antibody or antigen-binding fragment thereof competitively binds to human CDH17 protein with 143H10E4 is provided. In one embodiment, an antibody or antigen-binding fragment thereof competitively binds to human CDH17 protein with 152A1D12 is provided. In one embodiment, an antibody or antigen-binding fragment thereof competitively binds to human CDH17 protein with 155B11C6 is provided.

[0144] In one embodiment, an antibody or antigen-binding fragment thereof is provided that binds to the same amino acid residue as 16C18 on the human CDH17 protein. In one embodiment, an antibody or antigen-binding fragment thereof is provided that binds to the same amino acid residue as 20C3E8 on the human CDH17 protein. In one embodiment, an antibody or antigen-binding fragment thereof is provided that binds to the same amino acid residue as 29D2D7 on the human CDH17 protein. In one embodiment, an antibody or antigen-binding fragment thereof is provided that binds to the same amino acid residue as 29H8D3 on the human CDH17 protein. In one embodiment, an antibody or antigen-binding fragment thereof is provided that binds to the same amino acid residue as 61C7F12 on the human CDH17 protein. In one embodiment, an antibody or antigen-binding fragment thereof is provided that binds to the same amino acid residue as 67A11B11 on the human CDH17 protein. In one embodiment, an antibody or antigen-binding fragment thereof is provided that binds to the same amino acid residue as 69E3H11 on the human CDH17 protein. In one embodiment, an antibody or antigen-binding fragment thereof is provided that binds to the same amino acid residue as 95F2C2F12 on the human CDH17 protein. In one embodiment, an antibody or antigen-binding fragment thereof is provided that binds to the same amino acid residue as 103G6G1 on the human CDH17 protein. In one embodiment, an antibody or antigen-binding fragment thereof is provided that binds to the same amino acid residue as 120B10C5 on the human CDH17 protein. In one embodiment, an antibody or antigen-binding fragment thereof is provided that binds to the same amino acid residue as 143H10E4 on the human CDH17 protein. In one embodiment, an antibody or antigen-binding fragment thereof is provided that binds to the same amino acid residue as 152A1D12 on the human CDH17 protein. In one embodiment, an antibody or antigen-binding fragment thereof is provided that binds to the same amino acid residue as 155B11C6 on the human CDH17 protein.

[0145] In one embodiment, an antibody or antigen-binding fragment thereof binding to the EC1 domain of the human CDH17 protein is provided (domain regions are shown in Table A). In one embodiment, an antibody or antigen-binding fragment thereof binding to the EC2 domain of the human CDH17 protein is provided. In one embodiment, an antibody or antigen-binding fragment thereof binding to the EC3 domain of the human CDH17 protein is provided. In one embodiment, an antibody or antigen-binding fragment thereof binding to the EC4 domain of the human CDH17 protein is provided. In one embodiment, an antibody or antigen-binding fragment thereof binding to the EC5 domain of the human CDH17 protein is provided. In one embodiment, an antibody or antigen-binding fragment thereof binding to the EC6 domain of the human CDH17 protein is provided. In one embodiment, an antibody or antigen-binding fragment thereof binding to the EC7 domain of the human CDH17 protein is provided.

[0146] In one embodiment, an antibody or antigen-binding fragment thereof binding to the EC1-EC2 domains of the human CDH17 protein is provided. In one embodiment, an antibody or antigen-binding fragment thereof binding to the EC2-EC3 domains of the human CDH17 protein is provided. In one embodiment, an antibody or antigen-binding fragment thereof binding to the EC3-EC4 domains of the human CDH17 protein is provided. In one embodiment, an antibody or antigen-binding fragment thereof binding to the EC4-EC5 domains of the human CDH17 protein is provided. In one embodiment, an antibody or antigen-binding fragment thereof binding to the EC5-EC6 domains of the human CDH17 protein is provided. In one embodiment, an antibody or antigen-binding fragment thereof binding to the EC6-EC7 domains of the human CDH17 protein is provided.

[0147] Table A. CDH17 domains In some embodiments, the humanized light and heavy chains disclosed herein are substantially non-immunogenic in the human body and retain substantially the same or even higher affinity for CDH17 as the parent antibody.

[0148] Those skilled in the art will also understand that the antibodies disclosed herein can be modified to differ in amino acid sequence from the naturally occurring binding polypeptides from which they are derived. For example, the polypeptide or amino acid sequence derived from a specified protein may be similar to the starting sequence, for example, having a certain percentage of identity, such as 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% identity with the starting sequence.

[0149] Antibodies can be produced through an affinity maturation process in which modified antibodies exhibit increased affinity for antigens compared to unmodified parental antibodies. Affinity-maturing antibodies can be produced by methods known in the art, such as Marks et al., Rio / Technology 10:779-783 (1992); Barbas et al., Proc Nat. Acad. Sci. USA 91:3809-3813 (1994); Schier et al., Gene 169:147-155 (1995); Yelton et al., J. Immunol. 155:1994-2004 (1995); Jackson et al., J. Immunol. 154(7):3310-159 (1995); and Hawkins et al., J. Mol. Biol. 226:889-896 (1992).

[0150] In some embodiments, one or more amino acid modifications may be introduced into the Fc domain to create Fc domain variants. The Fc domain variants may contain a human Fc domain sequence (e.g., derived from the Fc region of human IgG1, IgG2, IgG3, or IgG4) with amino acid modifications (e.g., substitutions) at one or more amino acid positions. In some embodiments, the Fc domain variants alter one or more functional and / or pharmacokinetic properties of the antibody.

[0151] The Fc region can also be engineered to enhance or eliminate effector function. IgG antibodies can induce direct antitumor effects in an indirect manner through Fc-mediated effector functions that are involved in other immune cell or killing mechanisms. As used herein, “effector function” or “antibody effector function” refers to the biological activity attributable to the binding of the antibody’s Fc region to its effectors, such as the C1 complex and Fc receptors (FcγRIIa or FcγRIIIa)). Exemplary effector functions include: complement-dependent cytotoxicity (CDC) induced by the interaction of antibody and C1q on the C1 complex; antibody-dependent cell-mediated cytotoxicity (ADCC) induced by the binding of the antibody’s Fc region to Fc receptors on effector cells; and antibody-dependent cell-mediated phagocytosis (ADCP), in which nonspecific cytotoxic cells expressing the Fcγ receptor (FcγR) recognize antibodies bound to target cells and subsequently induce phagocytosis of the target cells.

[0152] In some embodiments, the Fc region provided herein preserves or improves effector functionality, such as ADCC and / or CDC.

[0153] Of the four IgG subclasses, IgG1 and IgG3 induce the strongest Fc effector functions. However, due to the longest half-life and greater stability of IgG1 than IgG3, most therapeutic antibodies with Fc-mediated functions are IgG1 isotypes.

[0154] Both IgG2 and IgG4 isoforms exhibit significantly lower binding affinity for FcγR. Recent evidence suggests that the IgG2 isoform is not entirely devoid of effector function, while the IgG4 isoform can undergo in vivo Fab arm exchange, producing bispecific antibodies and off-target effects.

[0155] Antibody-drug conjugates In some embodiments, the antibody comprises an amino acid sequence or one or more portions that do not normally associate with the antibody. Exemplary modifications are described in more detail below. For example, the antibody disclosed herein may comprise a flexible linker sequence or may be modified to add a functional portion (e.g., PEG, a drug, a toxin, or a label).

[0156] The antibodies, variants, or derivatives disclosed herein include modified derivatives, i.e., those obtained by covalently attaching any type of molecule to the antibody such that the covalent attachment does not impede the antibody's binding to the epitope. For example, but not limited to, antibodies can be modified, such as by glycosylation, acetylation, polyethylene glycolation, phosphorylation, amidation, derivatization by known protecting / blocking groups, proteolytic cleavage, or linkage to cellular antigens or other proteins. Any of many of these chemical modifications can be performed using known techniques, including but not limited to specific chemical cleavage, acetylation, formylation, and the metabolic synthesis of tunicamycin. Additionally, antibodies may contain one or more non-classical amino acids.

[0157] In some embodiments, antibodies may be conjugated with therapeutic agents, prodrugs, peptides, proteins, enzymes, viruses, lipids, biological response modifiers, pharmaceuticals, or PEG.

[0158] Antibodies can be conjugated or fused with therapeutic agents, which may include detectable markers (such as radioactive markers), immunomodulators, hormones, enzymes, oligonucleotides, photoactive therapeutics or diagnostics, cytotoxic agents (which may be drugs or toxins), ultrasound enhancers, non-radioactive markers, combinations thereof, and other such agents known in the art.

[0159] The antibodies or antigen-binding fragments thereof described herein can be conjugated or linked to one or more drug linker compounds to form antibody-drug conjugates (ADCs). Drug linker compounds include a linker and a payload (drug).

[0160] The ADCs derived from the novel antibodies disclosed herein have been tested in the accompanying experimental examples and have demonstrated excellent properties. In Example 6, the in vitro tumor cell killing activity of the ADC with vc-MMAE was measured. All ADCs with antibodies that bind to distal domains (e.g., EC1, EC2, EC3, and EC4) exhibited excellent tumor cell killing activity consistent with the internalization efficiency of these antibodies.

[0161] Surprisingly, ADCs can even exhibit higher internalization efficiencies than their naked antibody counterparts. For example, when 29H8D3-z12 is conjugated to GGFG-DXd, the resulting ADC (29H8D3-z12-DXd) shows a higher internalization rate in CDH17-expressing tumor cells than the naked antibody (Example 8).

[0162] Due to such excellent internalization efficiency and cytotoxic activity, the tested ADCs exhibited effective bystander killing (Example 8) and in vivo antitumor efficacy (Examples 9 and 10), including against tumors with driver gene mutations or resistance to chemotherapy drugs (Example 11).

[0163] Drug linker compounds can be linked to antibodies via cysteine ​​or lysine residues on the antibody. ADCs can be constructed chemically, for example via appropriate coupling reactions, such as amide coupling (lysine) and thiol coupling (cysteine), to randomly couple pre-existing lysine or cysteine ​​residues. Active carboxylic acid esters (when available in the linker) are used to link the payload to lysine residues on the antibody. The primary amine in Lys readily reacts with N-hydroxysuccinimide (NHS) esters introduced into the drug-linker to form stable amides. A typical IgG1 antibody molecule has approximately 90 Lys residues, of which about 30 can be modified for conjugation, meaning that 1 to 30 payloads can be covalently coupled to the antibody. For cysteine, upon reduction, the disulfide bond can be transformed to expose free cysteine ​​residues, which can be used for coupling reactions such as Michael addition, disulfide bond formation, and α-halocarbonyl alkylation.

[0164] In some embodiments, the drug linker compound is linked to the antibody provided herein via a free thiol group of a cysteine ​​residue on the antibody.

[0165] ADCs can also be constructed via enzymatic methods, such as site-specific conjugation. Site-specific conjugation includes the introduction of engineered reactive cysteine ​​residues, disulfide rebridging, non-natural amino acids, enzyme-assisted linking or glycan remodeling, sugar conjugation, or click chemistry. Details of conjugation methods can be found in the art, for example, Fu et al., Signal Transduction and Targeted Therapy 7:93 (2022).

[0166] In some embodiments, the drug-antibody ratio (DAR) can be greater than 2, greater than 4, greater than 6, greater than 8, greater than 10, greater than 16, greater than 20, or greater than 30. In some embodiments, the drug-antibody ratio (DAR) can be 1-30, 1-20, 1-10, or 1-8, for example, 4-8 or 2-4. In some embodiments, the drug-antibody ratio (DAR) can be 2, 4, 6, 8, 10, 16, 20, or 30. In some embodiments, the DAR is homogeneous.

[0167] The adapters contained in drug-proximity compounds can be either uncuttable or cuttable. Cuttable adapters mainly include enzyme-cuttable adapters and chemically sensitive adapters.

[0168] Uncleavable linkers consist of stabilizing bonds that resist protein hydrolysis and ensure greater plasma stability. The mechanism of action of uncleavable linkers is based on the internalization of the ADC complex, followed by the degradation of the mAb component in the lysosome, leading to the release of cytotoxic drugs that kill tumor cells. They do not release cytotoxic agents at off-target sites and therefore do not harm healthy cells. Uncleavable linkers are classified into two groups: thioethers or maleimide hexanoyl (MC). Examples of uncleavable linkers include, but are not limited to, 4-maleimide methylcyclohexane-1-carboxylate (MCC), 4-(N-maleimide methyl)cyclohexane-1-carboxylate succinimide (SMCC), maleimide hexanoyl (MC), and p-carboxycyclohexylmethylmaleimide.

[0169] Cleavable adapters mainly include chemically sensitive adapters and enzyme-cleavable adapters. Chemically sensitive adapters are usually cleaved by environmental differences (such as redox potential and pH), while enzyme-cleavable adapters are cleaved by specific enzymes in response to extracellular and intracellular environments.

[0170] Chemically sensitive connectors include, but are not limited to, pH-sensitive connectors and glutathione-sensitive disulfide connectors (Khongorzul et al., Mol Cancer Res; 18(1) (2020)).

[0171] pH-sensitive connectors are a group of connectors that are sensitive to acidic environments but stable in alkaline environments (e.g., systemic circulation), such as hydrazone-based connectors. A successful example of an ADC design using pH-sensitive connectors is IMMU-110, which consists of a humanized anti-CD74 mAb conjugated with doxorubicin via an acid-labile hydrazone.

[0172] Compared to plasma, glutathione-sensitive disulfide linkers utilize differences in reduction potential in the cytoplasm. Higher concentrations of glutathione can be found in cancer cells than in normal cells. Glutathione-sensitive linkers are stable in the bloodstream and are particularly cleaved by elevated intracellular glutathione concentrations in tumor cells, releasing the active drug from a non-toxic prodrug at the tumor site (see above).

[0173] Enzyme-cleavable adapters include, but are not limited to, peptide-based adapters, β-glucuronide-based adapters, and phosphate-based adapters.

[0174] Peptide-based linkers, also known as protease-sensitive linkers, are the most commonly used ADC linkers. These linkers can be cleaved by specific extracellular and / or intracellular proteases. For intracellular cleavage, tumor cells exhibit high expression of lysosomal proteases (such as cathepsin B) compared to normal cells; therefore, protease-sensitive peptide linker ADCs selectively bind and transform into cancer cells via receptor-mediated endocytosis. Peptide linkers are stable in systemic circulation and release drugs only in target cells (see above). Examples are valine-citrulline (VC or Val-Cit), valine-alanine (VA), phenylalanine-lysine (PL), and glycine-glycine-phenylalanine-glycine (GGFG).

[0175] The β-glucuronide-based linker is recognized and hydrolyzed by β-glucuronidase or β-galactosidase to release the drug, and both β-glucuronidase and β-galactosidase are enriched in lysosomes and tumor necrosis regions. β-glucuronidase is inactive at physiological pH (circulatory) but active at lysosomal pH. This selective site of action allows for the cleavage of the glycosidic bond of the β-glucuronide-sensitive linker, thereby enabling the selective release of cytotoxic payloads (see above).

[0176] Phosphate-based linkers are a class of enzyme-cleavable linkers that are specifically expressed to target enzymes in lysosomal compartments. These linkers target pyrophosphatases and acid phosphatases, which hydrolyze pyrophosphates and terminal monophosphates into their respective alcohols.

[0177] Parts that can be introduced as part of a linker to facilitate drug-linker or antibody-linker linkage include maleimide hexanoyl, maleimide-hexanoyl (MC) moiety, maleimide-methylene-cyclohexyl carbonyl moiety spacer, maleimide propionyl (MP), p-aminobenzylcarbamate (PABC) spacer, p-aminobenzyloxycarbamate (PABC) spacer, aminomethoxymethylene carbonyl (-NH-CH2-217 O-CH2-CO-) spacer, 4-(4'-acetylphenoxy)butyric acid moiety, acylhydrazide (-CO-NH-NH2) and mercapto (-SH) moiety, Lys-PABC, PEG chain, and PEG8.

[0178] Examples of connectors include, but are not limited to, 4-(N-maleimidemethyl)cyclohexane-1-carboxylic acid succinimide ester (SMCC), sulfon-SMCC, MC-VC-PABC, CL2A, MC-GGFG, MC, or MP-PEG8-VA-PABC.

[0179] The drug / payload in the drug-connector compound can be a cytotoxin, a therapeutic peptide, or a polypeptide.

[0180] Peptides or polypeptides can be any peptide or polypeptide with therapeutic properties, such as analgesic, antidiabetic, antitumor, or antiviral activity. Alternatively or concurrently, the drug preferably contains an amino, thiol, or carboxylic acid group, as these types of groups provide ideal sites for the conjugation of the drug with the linkers disclosed herein. Examples of biologic drugs are Pseudomonas aeruginosa exotoxin PE38, diphtheria toxin, Staphylococcus aureus enterotoxin A / E-120, Shiga toxin, ricin, and urease.

[0181] Cytotoxins are activated upon release from ADCs within the cytoplasm of tumor cells and can destroy these cells. The main types of cytotoxins that can be used in ADC design fall into two categories: microtubule disruptors (such as iorestatins and maytansine) and DNA damage agents (such as chalcogenides, benzodiazepines, and doxorubicin). Examples include, but are not limited to, iorestatins, maytansines, benzodiazepines, tubulolysins, benzodiazepines, cimetidine, irinotecan (SN38), doxorubicin, anthracyclines, pyrrolobenzodiazepines (PBDs), TLR agonists, and STING agonists.

[0182] Orestatins are synthetic antitumor agents derived from the natural product salicylindrica 10. They block the microtubule polymerization process, leading to cell cycle arrest and apoptosis. Examples include monomethyl olrestatin E (MMAE) and monomethyl olrestatin F (MMAF).

[0183] Maytansine derivatives are isolated from maytansine (a benzo[a]-bridged macrolide). These drugs inhibit microtubule polymerization. Examples include DM1 and DM4.

[0184] Kachizomycin is a class of enediyne antitumor antibiotics derived from Micromonospora echinococcosis. Kachizomycin recognizes the minor groove of DNA and stops DNA replication, leading to mitotic arrest and cell death. One example is N-acetyl-kachizomycin, a derivative of kachizomycin.

[0185] Betainecin is a natural product derivative extracted from Streptomyces bacteria. It is another type of DNA minor groove binding alkylating agent. These drugs exert their effects by binding to the minor groove of DNA, subsequently causing irreparable alkylation of the DNA, thereby disrupting nucleic acid structure and structural integrity.

[0186] Doxorubicin exerts its effect by embedding itself into DNA that inhibits DNA synthesis. One example is IMMU-110.

[0187] Iciticon is derived from the Chinese tree *Epipremnum aureum* (…). Camptotheca acuminata Ecinotecan is a synthetic derivative of the natural cytotoxin camptothecin, isolated from [a specific organism]. Like camptothecin, eccinotecan binds to the topoisomerase 1-DNA complex, preventing DNA rejoining, leading to the accumulation of DNA strand breaks and ultimately cell death. Examples of eccinotecan are DX-8951f and DXd. 7-Ethyl-10-hydroxycamptothecin (SN-38) is also a derivative of camptothecin.

[0188] Other examples of cytotoxins include, but are not limited to, tubulolysin A, camptothecin, DGN462, ambroxol 269, anthraquinone, SG3199 / SCX, IRDye® 700DX, TLR7 / 8 agonists, and diABZI STING agonist-2.

[0189] Cytotoxins may be chemotherapeutic agents, and based on their mechanism of action, they can be classified into the following groups: - Antimetabolites / anticancer agents, such as pyrimidine analogues fluorouracil, capecitabine and cytarabine; - Purine analogues, folic acid antagonists and related inhibitors; - Antiproliferative / antimitotic agents, including natural products such as vinca alkaloids (vincrine, vincristine) and microtubules such as taxanes (paclitaxel, docetaxel), vincristine, nocodazole, epothilone, and novibine. ® ) and epipodophyllotoxin (etoposide, teniposide); - DNA damaging agents, such as actinomycin, acridine, busulfan, carboplatin, chlorambucil, cisplatin, cyclophosphamide (CYTOXAN) ®), daunorubicin, doxorubicin, epirubicin, ifosfamide, melphalan, dichloromethyldiethylamine, mitomycin, mitoxantrone, nitrosourea, procarbazine, paclitaxel, tessotericin, teniposide, etoposide, and triethylene thiophosphoramide; - Antibiotics, such as daunorubicin, doxorubicin, idarubicin, anthracyclines, mitoxantrone, bleomycin, procainox (photomycin), and mitomycin; - Enzymes, such as L-asparaginase, metabolize L-asparagine systemically and deprive cells that are unable to synthesize their own asparagine. - Antiplatelet agents; - Antiproliferative / antimitotic alkylating agents, such as nitrogen mustard cyclophosphamide and analogues (melphalan, chlorambucil, hexamethylmelamine and thiotepa), alkylnitrosoureas (carmustine) and analogues, streptozoline and triazine (dacarbazine). - Antiproliferative / antimitotic antimetabolites, such as folic acid analogs (methotrexate). - Platinum coordination complexes (cisplatin, oxaliplatin and carboplatin), procarbazine, hydroxyurea, mitotane and ammonia glutide; - Hormones, hormone analogs (estrogens, tamoxifen, goserelin, bicalutamide and nilumid) and aromatase inhibitors (letrozole and anastrozole); - Anticoagulants, such as heparin, synthetic heparin salts and other inhibitors of thrombin; - Fibrinolytic agents, such as tissue plasminogen activator, streptokinase, urokinase, aspirin, dipyridamole, ticlopidine, and clopidogrel; - Anti-migration agent; - anti-secretion agent (breveldin); - Immunosuppressants tacrolimus, sirolimus, azathioprine, and mycophenolate mofetil; - Compounds (TNP-470, genistein) and growth factor inhibitors (vascular endothelial growth factor inhibitor and fibroblast growth factor inhibitor). - Angiotensin receptor blockers, nitric oxide donors; - Antisense oligonucleotides; - Antibodies, such as trastuzumab and rituximab; - Cell cycle inhibitors and differentiation inducers, such as retinoic acid; - Inhibitors, topoisomerase inhibitors (doxorubicin, daunorubicin, daunorubicin, iniposide, epirubicin, etoposide, idarubicin, irinotecan, mitoxantrone, topotecan and irinotecan) and corticosteroids (cortisone, dexamethasone, hydrocortisone, methylprednisolone, prednisone and prednisolone). - Growth factor signal transduction kinase inhibitors; - Dysfunction inducers; - Toxins, such as cholera toxin, ricin, Pseudomonas exotoxin, Bordetella pertussis adenylate cyclase toxin, diphtheria toxin and caspase activator; - and chromatin.

[0190] Other examples of chemotherapy agents include: - Alkylating agents, such as thiotepa and cyclophosphamide (CYTOXAN) ® ); - Alkyl sulfonates, such as busulfan, improsulfan, and piposulfan. - Aziridines, such as benzodopa, carboquone, meturedopa, and uredopa; - Ethyleneimine and methylmelamine, including hexamethylmelamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide and tris(hydroxymethylmelamine); - Polyacetogenins, especially bullatacin and bullatacinone; - Camptothecin, including its synthetic analogue topotecan; - Bryophyte toxin; - Callystatin; - CC-1065, including its synthetic analogues adozelesin, carzelesin and bizelesin; - Cryptocycins, especially cryptocycin 1 and cryptocycin 8; - Dolastatin; - Duocarmycin, including synthetic analogues KW-2189 and CBI-TMI; - eleutherobin; - Pancratistatin; - Sarcodictyin; - Spongistatin; - Nitrogen mustards, such as chlorambucil, naphthylambucil, cyclophosphamide, estradiol, ifosfamide, nitrogen mustard, mechlorethamine oxide hydrochloride, melphalan, novombhichin, phenesterine, prednimustine, trofosfamide, and uramustine; - Nitrosoureas, such as carmustine, chlorozotocin, foremustine, lomustine, nimustine, and ranimustine; - Antibiotics, such as enediyne antibiotics (e.g., kazimycin, especially kazimycin γII and kazimycin phiI1), danendomycin (including danendomycin A), bisphosphonates (e.g., clodronate), esporamycin, neocarcinogen chromophores and related chromogenin enediyne antibiotic chromophores, aclarubicin, actinomycin, azaserine, bleomycin, actinomycin C, carbapenem, carminoxetine, chromomycin, daunorubicin, detoxin, 6-hexamethasone Nitrogen-5-oxo-L-leucine, doxorubicin (including morpholino doxorubicin, cyanomorpholino doxorubicin, 2-pyrrolin-doxorubicin and deoxydoxorubicin), epirubicin, idarubicin, idarubicin, mesorubicin, mitomycin (such as mitomycin C), mycophenolic acid, nopramine, olivomycin, pepromycin, pofibromycin, puromycin, quelamycin, rodorubicin, streptomycin, streptozotocin, tuberculin, ubenmexicin, fenestrated statin and levorubicin; - Antimetabolites, such as methotrexate and 5-fluorouracil (5-FU); - Folic acid analogues, such as demopterin, methotrexate, pteropterin, and trimethoprim; - Purine analogues, such as fludarabine, 6-mercaptopurine, thiamiprine, and thioguanine; - Pyrimidine analogues, such as ancitabine, azacitidine, 6-azauridine, carmofur, dodeoxyuridine, doxifluridine, enoxabin, and fluorouridine. - Androgens, such as calusterone, drotaldone propionate, cyclothionol, meandrolone, and testosterone; - Anti-adrenergic drugs, such as aminoglutethimide, mitotane, and triplostane; - Folic acid supplements, such as folinic acid; - Trichothecenes, especially T-2 toxin, verracurin A, roridin A and anguidine. - Taxanes, such as paclitaxel (TAXOL) ® ) and TAXOTERE ® ); - Platinum analogues, such as cisplatin and carboplatin; - Acetaldehyde lactone; Aldehydephosphatidylglycoside; Aminolevulinic acid; Eniluracil; Acridine; Hestrabucil; Bisantrene; Edatraxate; Defofamine; Demecolcine; Diaziquone; Eflumicin; Elliptinium acetate); epsilon; etoglucid; gallium nitrate; hydroxyurea; lentinan; formicoyltetrahydrofolate; chlordamine; maytansine derivatives, such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopiperazine; diammonium nitrate; pentostatin; phenamet; pirarubicin; losoxantrone; fluoropyrimidine; leucovorin; podophyllinic acid; ethylhydrazide; procarbazine; polysaccharide-K (PSK); razoxane; rhizomycin; cizonan; spirogermanium; tenuazonic acid acid); triaminoquinone; 2,2',2''-trichlorotriethylamine; urethane; vindesine; dacarbazine; mannitol mustard; dibromomannitol; dibromoeutherol; piperobromo; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; chlorambucil; gemcitabine ® ); 6-Thioguanine; mercaptopurine; methotrexate; vincristine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; vinorelbine (NAVELBINE) ®); novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeoloda; ibandronate; CPT-11; topoisomerase inhibitor RFS 2000; difluoromethylornithine (DFMO); retinoids such as retinoic acid; capecitabine; FOLFIRI (fluorouracil, leucovorin, and irinotecan); - and any of the above pharmaceutically acceptable salts, acids or derivatives.

[0191] In some embodiments disclosed herein, the drug linker compounds are linker-cytotoxic structures of ozomicin (a derivative of chachiomycin, see U.S. Patent No. 5,773,001), virdotin (MC-VC-PABC-MMAE, see U.S. Patent No. 7,659,241), malfotin (MC-MMAF, see U.S. Patent No. 7,498,298), entancin (SMCC-DM1, U.S. Patent No. 5,208,020), delutecan (MC-GGFG-DXd, see U.S. Patent No. 10,195,288), gavitan (CL2A-SN38, see U.S. Patent No. 8,420,086), or tecillin (MP-PEG8-VA-PABC-SG3199 / SCX, see U.S. Patent No. 9,889,207), the entire contents of which are incorporated herein by reference.

[0192] Other examples of drug linker compounds include pasudotox (PE38), soravtansine (DM4), rivtansine (DM4), and metansine (DM1).

[0193] In some embodiments disclosed herein, the drug connector compound is virdocin or a derivative thereof having Formula I: (Mc-vc-PABC-MMAE).

[0194] In some embodiments disclosed herein, the drug connector compound is a derutin having formula II or a derivative thereof: In some embodiments disclosed herein, the drug connector compound is ozomicin having formula III or a derivative thereof: .

[0195] In some embodiments disclosed herein, the drug connector compound is malfotin having formula IV or a derivative thereof: In some embodiments of this disclosure, the drug connector compound is entansine having formula V or a derivative thereof: In some embodiments disclosed herein, the drug connector compound is gavitan having formula VI or a derivative thereof: In some embodiments disclosed herein, the drug connector compound is tecillin or a derivative thereof having formula VII: In some embodiments, virdoting and delutecan can be linked to the antibody disclosed herein via the free sulfhydryl group of the cysteine ​​residue.

[0196] Treatment and Uses As described herein, the antibodies, variants or derivatives or ADCs disclosed herein may be used in certain therapeutic and diagnostic approaches.

[0197] This disclosure further relates to antibody-based therapies involving the administration of the antibodies disclosed herein to patients, such as animals, mammals, and humans, to treat one or more of the disorders or conditions described herein. The therapeutic compounds disclosed herein include, but are not limited to, the antibodies disclosed herein (including variants and derivatives thereof described herein) and nucleic acids or polynucleotides encoding the antibodies disclosed herein (including variants and derivatives thereof described herein).

[0198] In some embodiments, methods for treating cancer in patients in need are provided. In one embodiment, the method involves administering to the patient an effective amount of the disclosed antibody or its antigen-binding fragment, or ADC.

[0199] In some embodiments, the use of the antibody disclosed herein or its antigen-binding fragment or ADC in the preparation of a medicament for treating cancer in patients in need is provided.

[0200] In some embodiments, antibodies or antigen-binding fragments or ADCs disclosed herein are provided for use in treating cancer in patients in need.

[0201] Unless otherwise stated, the terms “cancer” and “tumor” are used interchangeably in this document. These terms specifically refer to, but are not limited to, cancers and tumors selected from the following groups: basal cell carcinoma; bladder cancer; bone cancer, such as osteosarcoma; central nervous system tumors, such as cerebellar astrocytoma, brain astrocytoma / malignant glioma, craniopharyngioma, ependymoblastoma, ependymoma, medulloblastoma, medullary epithelioma, moderately differentiated pineal parenchymal tumor, primitive neuroectodermal tumor, pineal blastoma, and spinal cord tumors; Burkitt lymphoma; breast cancer; cervical cancer; chronic myeloid leukemia; colon cancer; rectal cancer; colorectal cancer; esophageal cancer; Ewing tumor family; extrahepatic bile duct cancer; gallbladder cancer; gastrointestinal stromal tumor (GIST); glioma; head and neck cancer; islet cell tumors; Kaposi's sarcoma; leukemia; liver cancer; lymphoma. Tumors; Hodgkin's lymphoma; non-Hodgkin's lymphoma; T-cell lymphoma; mesothelioma; multiple myeloma / plasma cell tumor; myeloid leukemia; multiple myeloma; nasopharyngeal carcinoma; neuroblastoma; small cell lung cancer; non-small cell lung cancer; oropharyngeal carcinoma; osteosarcoma; ovarian cancer; pancreatic cancer; parathyroid carcinoma; penile cancer; pharyngeal cancer; pheochromocytoma; pituitary adenoma; prostate cancer; renal cell carcinoma; respiratory tract cancer; retinoblastoma; skin cancer (melanoma); small intestine cancer; soft tissue sarcoma; squamous cell carcinoma; squamous neck carcinoma; stomach / gastric cancer; testicular cancer; laryngeal cancer; thyroid cancer; transitional cell carcinoma of the renal pelvis and ureter; urethral cancer; uterine cancer; vaginal cancer; vulvar cancer and nephroblastoma.

[0202] Other diseases or conditions associated with increased cell survival (which can be treated, prevented, diagnosed, and / or predicted by the antibodies disclosed herein or their variants or derivatives) include, but are not limited to, the progression and / or metastasis of malignancies and related disorders, such as leukemia (including acute leukemia (e.g., acute lymphoblastic leukemia, acute myeloid leukemia (including myeloblastic, promyelocytic, myelomonocytic, monocytic, and erythroleukemia)) and chronic leukemia (e.g., chronic myeloid (granulocytic) leukemia and chronic lymphocytic leukemia)), polycythemia vera, lymphoma (e.g., Hodgkin's disease and non-Hodgkin's disease), multiple myeloma, Waldenström macroglobulinemia, heavy chain disease, and solid tumors, including but not limited to sarcomas and carcinomas such as fibrosarcoma, myxosarcoma, liposarcoma, and chondrosarcoma). Osteosarcoma, chordoma, angiosarcoma, endothelial sarcoma, lymphangiosarcoma, lymphangioendothelial sarcoma, synovoma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary gland carcinoma, cystadenoma, medullary carcinoma, bronchial carcinoma, renal cell carcinoma, liver cancer, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, nephroblastoma, cervical cancer, testicular tumor, lung cancer, small cell lung cancer, bladder cancer, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pineal tumor, hemangioblastoma, acoustic neuroma, oligodendroglioma, hemangioma, melanoma, neuroblastoma, and retinoblastoma.

[0203] The specific dosage and treatment regimen for any given patient will depend on a number of factors, including the specific antibody used, its variant or derivative or ADC, the patient's age, weight, general health condition, sex, and diet, as well as the timing of administration, excretion rate, drug combination, and the severity of the specific disease being treated. The judgment of healthcare professionals regarding such factors is within the scope of those skilled in the art. The dosage will also depend on the individual patient to be treated, the route of administration, the type of formulation, the characteristics of the compound used, the severity of the disease, and the desired effect. The dosage used can be determined using pharmacological and pharmacokinetic principles well known in the art.

[0204] Methods of administering antibodies, variants, or ADCs include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. Antigen-binding peptides or compositions can be administered via any convenient route, such as by infusion or bolus injection, absorption through epithelial or mucosal skin linings (e.g., oral mucosa, rectal and intestinal mucosa), and can be administered together with other bioactive agents. Therefore, pharmaceutical compositions containing the antigen-binding peptides disclosed herein can be administered orally, rectally, parenterally, intracerebrospinal, intravaginally, intraperitoneally, topically (e.g., by powder, ointment, drops, or transdermal patch), buccally, or as oral or nasal sprays.

[0205] As used in this article, the term “parenteral” refers to administration methods including intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous, and intra-articular injections and infusions.

[0206] Administration can be systemic or local. Alternatively, it is preferable to introduce the disclosed antibodies into the central nervous system via any suitable route, including intraventricular and intrathecal injection; intraventricular injection can be facilitated by, for example, an intraventricular catheter attached to a reservoir (such as an Ommaya reservoir). Pulmonary administration can also be used, for example, by using an inhaler or nebulizer, and formulations containing nebulizers.

[0207] Preferably, the antigen-binding peptides or compositions disclosed herein are applied topically to the area requiring treatment; this can be achieved, for example, but not limited to, local infusion during surgery, topical application (e.g., in conjunction with postoperative wound dressings), by injection, via catheter, via suppository, or via implantation of a porous, non-porous, or gelatinous material, including membranes such as sialastic membranes or fibers. Preferably, when administering the proteins (including antibodies) disclosed herein, care must be taken to use materials that do not absorb the proteins.

[0208] In some embodiments, a method for detecting human CDH17 protein expression in a sample is also provided, the method comprising contacting the sample with an antibody or a fragment thereof and detecting binding indicating CDH17 expression in the sample.

[0209] In some embodiments, the use of the antibodies or antigen-binding fragments thereof disclosed herein is provided in the preparation of kits for detecting human CDH17 protein expression in samples.

[0210] Polynucleotides encoding antibodies and methods for preparing antibodies This disclosure also provides isolated polynucleotide or nucleic acid molecules encoding antibodies, variants, or derivatives thereof disclosed herein. The polynucleotides disclosed herein may encode the entire heavy and light chain variable regions of an antigen-binding polypeptide, variant, or derivative thereof, either on the same polynucleotide molecule or on separate polynucleotide molecules. Furthermore, the polynucleotides disclosed herein may encode portions of the heavy and light chain variable regions of an antigen-binding polypeptide, variant, or derivative thereof, either on the same polynucleotide molecule or on separate polynucleotide molecules.

[0211] Methods for preparing antibodies are well known in the art and are described herein. In some embodiments, both the variable and constant regions of the antigen-binding polypeptide disclosed herein are fully human. Fully human antibodies can be prepared using techniques described in the art and herein. For example, fully human antibodies against a specific antigen can be prepared by administering the antigen to a transgenic animal that has been modified to produce such antibodies in response to antigen challenge, but whose endogenous sites have been disabled. Exemplary techniques that can be used to prepare such antibodies are described in U.S. Patents 6,150,584, 6,458,592, and 6,420,140, ​​the entire contents of which are incorporated herein by reference.

[0212] Composition This disclosure also provides pharmaceutical compositions. Such compositions comprise an effective amount of antibody and an acceptable carrier. In some embodiments, the composition further comprises a second anticancer agent (e.g., an immune checkpoint inhibitor).

[0213] In certain embodiments, the term "pharmaceuticalally acceptable" means approved by federal or state regulatory agencies, or listed in the United States Pharmacopeia or other recognized pharmacopoeia, for use in animals, and more particularly in humans. Furthermore, "pharmaceuticalally acceptable carriers" are typically non-toxic solid, semi-solid, or liquid fillers, diluents, encapsulating materials, or any type of formulation aid.

[0214] The term "carrier" refers to a diluent, adjuvant, excipient, or medium that is administered with a therapeutic agent. Such drug carriers can be sterile liquids, such as water and oils, including petroleum, animal, plant, or synthetic oils, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. Water is the preferred carrier when the drug composition is administered intravenously. Saline solutions and aqueous solutions of dextran and glycerol can also be used as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, skim milk powder, glycerol, propylene, ethylene glycol, water, ethanol, etc. If desired, the composition may also contain small amounts of wetting agents or emulsifiers, or pH buffers, such as acetates, citrates, or phosphates. Antibacterial agents, such as benzyl alcohol or methylparaben; antioxidants, such as ascorbic acid or sodium bisulfite; chelating agents, such as ethylenediaminetetraacetic acid; and agents for regulating tension, such as sodium chloride or dextrose. These compositions can be in the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release formulations, etc. Compositions can be formulated as suppositories containing conventional binders and carriers, such as triglycerides. Oral formulations may include standard carriers, such as pharmaceutical-grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, etc. Examples of suitable drug carriers are described in EW Martin's Remington's Pharmaceutical Sciences, which is incorporated herein by reference. Such compositions will contain a therapeutically effective amount of an antigen-binding polypeptide, preferably in a purified form, and an appropriate amount of carrier to provide a suitable form of administration to the patient. The formulation should be suitable for the route of administration. Parenteral preparations can be packaged in ampoules, disposable syringes, or multi-dose vials made of glass or plastic.

[0215] In the embodiments, the composition is formulated into a pharmaceutical composition suitable for intravenous administration to humans according to conventional procedures. Typically, the composition for intravenous administration is a solution in a sterile isotonic buffer solution. If necessary, the composition may also include a solubilizer and a local anesthetic, such as lidocaine, to relieve pain at the injection site. These components are typically supplied individually or mixed together in unit dosage forms, for example, as a dry lyophilized powder or anhydrous concentrate, in a sealed container (e.g., an ampoule or sachet) indicating the amount of active agent. When the composition is administered by infusion, it can be dispensed using an infusion bottle containing sterile pharmaceutical-grade water or saline. When the composition is administered by injection, ampoules of sterile water for injection or saline can be provided to allow mixing of the components prior to administration.

[0216] The compounds disclosed herein can be formulated into neutral or salt forms. Pharmaceutically acceptable salts include salts that form with anions, such as salts derived from hydrochloric acid, phosphoric acid, acetic acid, oxalic acid, tartaric acid, etc., and salts that form with cations, such as salts derived from sodium, potassium, ammonium, calcium, ferric hydroxide, isopropylamine, triethylamine, 2-ethylaminoethanol, histidine, procaine, etc.

[0217] Example Example 1. Production of anti-human CDH17 antibodies in mice This example describes the use of hybridoma technology to generate mouse anti-human CDH17 monoclonal antibodies.

[0218] Immunogen: In mouse immunization, a full-length extracellular domain (ECD) and a truncated human CDH17 protein were used as immunogens. In short, the full-length ECD of the human CDH17 protein (UniProt reference sequence: Q12864, Q23-M787) was fused to its C-terminus with a his tag, and termed hCDH17-his (Acro Bio, catalog number CA7H52H3; or custom-made by Biointron). A truncated human CDH17 protein (hereinafter referred to as hCDH17 EC6-7-hFc, custom-made by Biointron) was generated by fusing the EC6 (S567-L667) and EC7 (A668-M787) regions of human CDH17 to the N-terminus of the C (Fc) region of a fragment of human immunoglobulin G (IgG).

[0219] Mouse immunization regimen: To generate mouse monoclonal antibodies against human CDH17, BALB / c, AJ, and SJL mice were immunized intraperitoneally or subcutaneously every two weeks with hCDH17-his or hCDH17EC6-7-hFc protein. Serum titers of immunized mice were monitored by ELISA against human hCDH17-his protein or hCDH17 EC6-7-hFc and by FACS against human CDH17 overexpressed on the HEK293 cell line (HEK293-hCDH17, custom-made by Genomeditech), with the HEK293 parental cell line serving as a negative control. After 3–6 rounds of immunization, mice with sufficient titers were enhanced with hCDH17-his protein and selected for fusion.

[0220] Cell fusion and hybridoma screening:Spleen cells from selected mice were fused with the mouse myeloma cell line Sp2 / 0 via electrofusion. These hybridoma cells were then plated in 96-well flat-bottom microplates and allowed to secrete mouse antibodies in the supernatant. For initial screening, positive clones were screened in a high-throughput manner using cells that bound the hCDH17-his protein via ELISA and cells that bound HEK293-hCDH17 via FACS. Confirmatory screening was performed by ELISA to identify clones that bound the Fc-tagged cynomolgus CDH17 ECD protein (cynomolgus CDH17-hFc, Sino Biopharmaceutical Co., Ltd., catalog number 90147-C02H) (isotype 1, NCBI reference sequence: XP_005563762.1, M1-M787).

[0221] Subcloning and sequencing: Positive primary clones from each fusion that meet the above criteria were subcloned by restriction dilution to ensure that the hybridoma subclones originated from a single parental cell. The subclone screening criteria were the same as those for the initial cloning screening described above. Subclones with effective binding efficacy to human CDH17 were selected for subsequent sequencing.

[0222] The variable region of the resulting mouse antibody was fused with the constant region of human IgG1 to generate a chimeric CDH17 mAb. The DNA sequence of the chimeric antibody was cloned into the pcDNA3.4 plasmid and expressed in CHO-K1 cells. The antibody was then purified from the culture supernatant using a protein A affinity chromatography column or beads. The purified chimeric antibody underwent a series of in vitro screening processes to determine affinity, binding capacity, epitopes, specificity, and species cross-reactivity.

[0223] Based on the performance in the screening assays, a series of CDH17 chimeric mAbs, including 16C18, 20C3E8, 29D2D7, 29H8D3, 61C7F12, 67A11B11, 69E3H11, 95F2C2F12, 103G6G1, 120B10C5, 134C7B1, 143H10E4, 152A1D12, and 155B11C6, were selected for further analysis. The amino acid sequences of the variable regions of the selected chimeric CDH17 antibodies are shown in Table 1 below, and the CDR sequences are summarized in Tables 2A and 2B.

[0224] Table 1. Sequences of the variable regions of CDH17 chimeric mAbs (underlined CDRs) Table 2A. CDR sequences (Kabat numbers) in the heavy chain of CDH17 chimeric mAbs Table 2B. CDR sequences (Kabat numbers) in the light chains of CDH17 chimeric mAbs Example 2. Binding activity of chimeric monoclonal antibodies targeting CDH17 2.1. Binding to human CDH17 ELISA To determine the binding affinity of the chimeric mAb to human CDH17 protein, an ELISA-based binding assay was performed as follows. Briefly, hCDH17-his protein was diluted 2 μg / mL with 1×ELISA coating buffer (Solarbio, catalog number C1050) and adsorbed into the wells of a 96-well microplate overnight at 4°C. After blocking the wells with 1% bovine serum albumin (BSA) to prevent nonspecific binding, the CDH17 chimeric mAb or isotype control was titrated 4-fold at 100 nM and added to the pre-adsorbed antigen-rich wells. The mixture was incubated at room temperature (RT) for 1 hour. The bound CDH17 mAb was recognized by a detection antibody against human IgG Fc conjugated to horseradish peroxidase (HRP) (Jackson Immuno, catalog number 109-035-008). The HRP substrate tetramethylbenzidine (TMB) was added to the wells to visualize the binding signal. After sufficient color development, stop solution was added to the wells. The absorbance of the signal was detected at 450 nm using an Envision multi-label plate reader (PerkinElemer). Graphs were generated and statistical analysis was performed using a four-parameter nonlinear regression curve fitted in Graphpad Prism 9 software.

[0225] As shown in Figure 1, all CDH17 chimeric mAbs effectively bound to human CDH17 protein. Table 3 summarizes the EC50 and maximum values ​​of the binding curves for each antibody.

[0226] Table 3. Binding activity of CDH17 antibody to antigen 2.2. Binding to human CDH16 ELISA Since CDH16 and CDH17 belong to the same 7D-cadherin subfamily, it is important to identify the specificity of CDH17 chimeric monoclonal antibodies.

[0227] To determine the nonspecific binding of the chimeric mAb to human CDH16 protein (hCDH16-his, Novoprotein, catalog number CJ16), an ELISA binding assay was performed as described previously. hCDH16-his protein was used as the coating antigen at a concentration of 1 μg / mL.

[0228] As shown in Figure 2, all CDH17 chimeric mAbs showed negligible binding to human CDH16 protein, indicating their specificity for human CDH17.

[0229] 2.3. Affinity Measurement Use Biacore TM The binding affinity of the chimeric monoclonal antibody to human CDH17 protein was determined using 8K assays. Briefly, the antibody was captured at a concentration of 2 μg / mL using a Pro-A chip. A single dose (…) was then delivered at a flow rate of 30 μL / min. Human CDH17-his protein was injected onto the capture antibody at doses of 100 nM or 400 nM, or two doses (25 nM and 100 nM). Antigen association was allowed for 120-150 seconds, followed by dissociation for 200-500 seconds. Biacore was used. TM 8K evaluation software is used for data analysis.

[0230] The results showed that all chimeric CDH17 monoclonal antibodies exhibited high affinity for human CDH17, with equilibrium dissociation constants (KD) ranging from 1 × 10⁻⁶. -7 Up to 1 × 10 -9 M (Table 4).

[0231] Table 4. Affinity ranking results of CDH17 chimeric antibodies Single-dose affinity 2.4. Epitope / Domain Mapping To assess the exact binding domains of the chimeric mAb to human CDH17, cell-based and protein-based binding assays were employed as follows. Briefly, HEK293 cells stably expressing a range of human CDH17 ECDs were constructed. The human CDH17 protein possesses a unique extracellular structure composed of seven cadherin domains (ECs). Therefore, cell lines expressing human CDH17 conjugates of the transmembrane domain (TM) and intracellular domain (ICD) of human CDH17 were constructed (in-house manufactured or custom-made by Jimon Biotechnology Co., Ltd.). In addition, a truncated human CDH17EC3 (V245-C340) protein (EC3-mFc, custom-made by Nearshore Protein Technology Co., Ltd.) fused to a mouse IgG1 Fc tag was constructed to help determine the exact binding domains of certain EC3-4 conjugates. For cell binding assays, the designated CDH17 chimeric mAb or isotype control was diluted to 100 nM in staining buffer (DPBS buffer containing 2% FBS). The antibody dilution was then mixed with 5 x 10⁻⁶ antibodies. 4 Cells were incubated in 96-well microplates at 4°C for 30 minutes. The cell-antibody mixture was then washed twice with staining buffer. The solution was then used with Alexa Fluor. TM 488 (Thermo Fisher Scientific, catalog number A11013) conjugated goat anti-human IgG (H+L) cross-adsorption secondary antibody was used to detect antibodies binding to cell surface antigens at a dilution of 1:2000 at 4°C for 30 min, followed by thorough washing. The antibody was then analyzed by flow cytometry using an LSR Tortessa flow cytometer. TM Cells were analyzed using a cell analyzer (BD Biosciences). Data were analyzed using Flowjo 10.0 software. Graphs were generated in Graphpad Prism 9 software. For the ELISA binding assay, the experiment was performed as described in Example 2.1 above.

[0232] As shown in Table 5, these CDH17 chimeric antibodies specifically bind to different ECD domains of human CDH17 or truncated human CDH17 protein expressed on HEK293 cells. More specifically, 29H8D3, 143H10E4, 152A1D12, and 155B11C6 bind to the EC1 domain of human CDH17. 120B10C5 binds to the EC2 domain. 29D2D7 binds to the EC3 domain. 20C3E8 and 103G6G1 bind to the EC4 domain. 16C8 binds to the EC5 domain. 61C7F12 and 67A11B11 specifically bind to the EC6 domain of human CDH17. 69E3H11 and 95F2C2F12 bind to the proximal EC7 domain of human CDH17.

[0233] Table 5. Domain mapping of CDH17 chimeric antibodies 2.5 Binding ability with HEK293 cells overexpressing human CDH17 To evaluate the binding ability of the chimeric mAb to human CDH17 expressed on cells, a cell-based binding assay was performed as follows. Briefly, a stable HEK293 cell line expressing full-length human CDH17 (designated HEK293-hCDH17) was constructed (custom-made by Jimon Biotech). Parental HEK293 cells were used as a negative control. For the cell binding assay, the designated CDH17 chimeric mAb or isotype control was diluted fourfold, starting at a concentration of 100 nM, in staining buffer (DPBS buffer containing 2% FBS). The antibody dilution was mixed with 5 x 10⁻⁶ cells / mL. 4 Cells were incubated in 96-well microplates at 4°C for 30 minutes. The cell-antibody mixture was then washed twice with staining buffer. The solution was then used with Alexa Fluor. TM 488 (Thermo Fisher Scientific, catalog number A11013) conjugated goat anti-human IgG (H+L) cross-adsorption secondary antibody was used to detect antibodies binding to cell surface antigens at a dilution of 1:2000 at 4°C for 30 min, followed by thorough washing. The antibody was then analyzed by flow cytometry using an LSR Tortessa flow cytometer. TM Cell analysis was performed using a cell analyzer (BD Biosciences). Data analysis was conducted using Flowjo 10.0 software. Graphs and statistical analyses were generated using three- or four-parameter nonlinear regression curve fitting in Graphpad Prism 9 software.

[0234] As shown in Figures 3A and 3B, all CDH17 chimeric monoclonal antibodies effectively bound human CDH17 expressed on HEK293 cells in a dose-dependent manner. In contrast, no specific binding was observed on parental HEK293 cells (Figures 3C and 3D).

[0235] Table 6 summarizes the EC50 values ​​of the binding ability of the CDH17 chimeric antibody to human CDH17 expressed on HEK293 cells.

[0236] 2.6 Binding ability with tumor cells expressing human CDH17 To assess the binding ability of the chimeric mAb to human CDH17 expressed on tumor cells, several human tumor cell lines, including pancreatic cancer cell line AsPC1, colorectal adenocarcinoma cell line HCT-8, and gastric adenocarcinoma cell line AGS, were used in a cell-based binding assay, following the protocol described in Example 2.5, to represent high, medium, and low levels of CDH17 expression and different tumor types.

[0237] As shown in Figures 4A-4F, all CDH17 chimeric antibodies effectively bound to human CDH17 expressed on tumor cells in a dose-dependent manner. More importantly, the binding ability of individual CDH17 chimeric antibodies showed a similar trend across these three tumor cell lines. Interestingly, antibodies binding to the distal membrane region of human CDH17 showed better binding efficiency compared to antibodies binding to the proximal membrane region. These phenomena highlight the superior binding potency of distal membrane region conjugates compared to proximal membrane region conjugates.

[0238] Table 6 summarizes the EC50 values ​​of the binding ability of CDH17 chimeric antibodies to endogenously expressed human CDH17 on tumor cells.

[0239] 2.7 Binding ability with cells overexpressing CDH17 in cynomolgus monkeys To assess the species cross-reactivity of the chimeric mAb with cynomolgus CDH17 expressed on cells, a cell-based binding assay was performed following the protocol described in Example 2.5. Briefly, a HEK293 cell line (custom-made by Jimon Biotechnology) stably expressing a spliced ​​isotype of full-length cynomolgus CDH17 (isotype 2, Uniprot reference sequence: A0A2K5X8I8, M1-M840, designated HEK293-cynomolgus CDH17) was constructed. This isotype differs from cynomolgus CDH17 isotype 1 (NCBI reference sequence: XP_005563762.1, M1-S832) and human CDH17 in the N-terminal region. Designated CDH17 chimeric Abs or isotype controls were tested in this assay.

[0240] As shown in Figures 5A and 5B, the CDH17 chimeric antibody, including EC2 to EC6 domain binders, effectively binds to cynomolgus CDH17 isotype 2 expressed on HEK293 cells. However, the EC1 and EC7 binding mAbs hardly bind to cynomolgus CDH17 isotype 2 expressed on cells.

[0241] Table 6 summarizes the EC50 values ​​of the binding ability of the CDH17 chimeric antibody to CDH17 expressed on HEK293 cells in cynomolgus monkeys.

[0242] Table 6. Binding characteristics of chimeric antibodies to hCDH17 on cells and CDH17 in cynomolgus monkeys Note: -- indicates non-specific binding. Example 3. Humanization of CDH17 chimeric antibodies 3.1 Humanization design of CDH17 chimeric antibodies Based on the performance of the chimeric antibodies, several antibodies were selected for further humanization. The variable region of the CDH17 chimeric antibody was chosen for humanization. Briefly, the amino acid sequences of VH and VL were compared with available human Ig gene sequence databases to identify the most generally matching human germline Ig gene sequence. Then, the CDRs of the heavy and light chains of the CDH17 chimeric antibody were transplanted into the candidate germlines. A 3D model of the transplanted antibody was generated using a Molecular Operating Environment (MOE) to determine if any key human amino acids in the frame region must be reverted to their corresponding mouse amino acids to maintain CDR conformation and function.

[0243] For the heavy chain of 29H8D3, the candidate germline sequence is IGHV1-18. 01 gene. For the light chain of 29H8D3, the candidate germline sequence is IGKV2-28. 01 gene. In the human germline sequence IGHV1-18 Reversion mutations occurred at A24V, V37M, M48I, R67K, V68A, M70L, T72A, T74K, M81I, R84N, R87T, D89E, and Y95F in the heavy chain framework of IGKV2-28. These mutations were observed in the human germline sequence IGKV2-28. Reversion mutations occurred at Y41F, L51V, D75A, and Q105A in the light chain framework of 01.

[0244] Different combinations of reversion mutation sites were selected to generate the variable regions of humanized antibodies. The sequences of the heavy and light chain variable regions of the 29H8D3 humanized antibody are listed in Table 7A. The VH and VL pairs of individual humanized antibodies are listed in Table 7B. The variable regions of the humanized antibodies were then fused into the constant region of human IgG1 for humanized antibody production and functional characterization.

[0245] Table 7A. Variable region sequence of 29H8D3 humanized antibody (underline indicates CDR; bold / italic indicates reversion mutation) Table 7B. VH and VL pairing of 29H8D3 humanized antibodies For the heavy chain 29D2D7, the candidate phylogenetic sequence is IGHV1-18. 01 gene. For the light chain 29D2D7, the candidate germline sequence is IGKV4-1. 01 gene. In the human germline sequence IGHV1-18 Reversion mutations occurred at R38K, M48I, R67K, V68A, M70L, T72A, T74K, R87T, and D89E in the heavy chain framework of IGKV4-1. These mutations were observed in the human germline sequence IGKV4-1. Reversion mutations occur in V3G, T5S, and P49S of the light chain of 01.

[0246] Different combinations of reversion mutation sites were selected to generate the variable regions of humanized antibodies. The sequences of the heavy and light chain variable regions of the 29D2D7 humanized antibody are listed in Table 8A. The VH and VL pairs of individual humanized antibodies are listed in Table 8B. The variable regions of the humanized antibodies were then fused into the constant region of human IgG1 for antibody production and functional characterization.

[0247] Table 8A. Variable region sequence of 29D2D7 humanized antibody (underline indicates CDR; bold / italic indicates reversion mutation) Table 8B. VH and VL pairing of 29D2D7 humanized antibody For the heavy chain 61C7F12, the candidate phylogenetic sequence is IGHV3-21 01 Gene. For the light chain 61C7F12, the candidate germline sequence is... IGKV1-9 01or IGKV3-20 01 Gene. In the human germline sequence IGHV3-21 A reversion mutation occurs at S49A in the heavy chain framework of 01. This occurs in the human germline sequence IGKV1-9. Reversion mutations occurred at D1Q, Q3V, A44S, L48W, E71S, F72Y, and T73S in the light chain framework of 01. These mutations were observed in the human germline sequence IGKV3-20. The 01 light chain framework does not involve reversion mutations.

[0248] Different combinations of reversion mutation sites were selected to generate the variable regions of humanized antibodies. The sequences of the heavy and light chain variable regions of the 61C7F12 humanized antibody are listed in Table 9A. VH and VL pairings of individual humanized antibodies are listed in Table 9B. The variable regions of the humanized antibodies were then fused into the constant region of human IgG1 for antibody production and functional characterization.

[0249] Table 9A. Variable region sequence of 61C7F12 humanized antibody (underline indicates CDR; bold / italic indicates reversion mutation) Table 9B. VH and VL pairing of 61C7F12 humanized antibodies For the heavy chain 69E3H11, the candidate germline sequence is IGHV1-3. 01 or IGHV1-46 01 gene. For the light chain 69E3H11, the candidate germline sequence is IGKV6-21. 01 or IGKV3-20 01 gene. In human germline sequences IGHV1-3 Reversion mutations occurred at R38K, M48I, R67K, V68A, I70L, R72V, T74K, and R98L in the heavy chain framework of IGHV1. These mutations occurred in the human germline sequence IGHV1-46. Reversion mutations occurred at M70L, V79A, and R87T in the heavy chain framework of IGKV6-21. In the human germline sequence IGKV6-21... Reversion mutations occurred in the K50Y and F72Y sequences within the light chain framework of IGKV3-20 in the human germline sequence. The 01 light chain framework does not involve reversion mutations.

[0250] Different combinations of reversion mutation sites were selected to generate the variable regions of humanized antibodies. The sequences of the heavy and light chain variable regions of the 69E3H11 humanized antibody are listed in Table 10A. VH and VL pairings of individual humanized antibodies are listed in Table 10B. The variable regions of the humanized antibodies were then fused into the constant region of human IgG1 for antibody production and functional characterization.

[0251] Table 10A. Variable region sequence of 69E3H11 humanized antibody (underline indicates CDR; bold / italic indicates reversion mutation) Table 10B. VH and VL pairing of the 69E3H11 humanized antibody. For the heavy chain 143H10E4, the candidate germline sequence is IGHV1-69. 02 gene. For the light chain 143H10E4, the candidate germline sequence is IGKV6-21. 01 or IGKV3-20 01 gene. In the human germline sequence IGHV1-69 Reversion mutations occurred at G27S, S30T, M48I, R67K, V68A, I70L, S84N, and Y95F in the heavy chain framework of IGKV6-21. These mutations were observed in the human germline sequence IGKV6-21. Reversion mutations occurred at L47P, L48W, K50Y, F72Y, and T73S in the light chain framework of 01. These mutations were observed in the human germline sequence IGKV3-20. The 01 light chain framework does not involve reversion mutations.

[0252] Different combinations of reversion mutation sites were selected to generate the variable regions of humanized antibodies. The sequences of the heavy and light chain variable regions of the 143H10E4 humanized antibody are listed in Table 11A. VH and VL pairings of individual humanized antibodies are listed in Table 11B. The variable regions of the humanized antibodies were then fused into the constant region of human IgG1 for antibody production and functional characterization.

[0253] Table 11A. Variable region sequence of 143H10E4 humanized antibody (underline indicates CDR; bold / italic indicates reversion mutation) Table 11B. VH and VL pairing of the 143H10E4 humanized antibody For the heavy chain 152A1D12, the candidate germline sequence is IGHV1-3. 01 gene. For the light chain 152A1D12, the candidate germline sequence is IGKV1-13. 02 or IGKV3-11 01 gene. In human germline sequences IGHV1-3 Reversion mutations occurred at R38K, M48I, R67K, V68A, I70L, and R72V in the heavy chain framework of 01. These mutations occurred in the human germline sequence IGKV1-13. 02 and IGKV3-11 Reversion mutations occurred at L46R, L47W, and F71Y in the heterozygous light chain framework of the 01-generated genotype sequence. In the genotype sequence IGKV3-11... Reversion mutations occur at L47W and I58V in the light chain framework of 01.

[0254] Different combinations of reversion mutation sites were selected to generate the variable regions of humanized antibodies. The sequences of the heavy and light chain variable regions of the 152A1D12 humanized antibody are listed in Table 12A. VH and VL pairings of individual humanized antibodies are listed in Table 12B. The variable regions of the humanized antibodies were then fused into the constant region of human IgG1 for antibody production and functional characterization.

[0255] Table 12A. Variable region sequence of 152A1D12 humanized antibody (underline indicates CDR; bold / italic indicates reversion mutation) Table 12B. VH and VL pairing of the 152A1D12 humanized antibody For the heavy chain 155B11C6, the candidate germline sequence is IGHV1-2. 02 or IGHV1-46 01 gene. For the light chain 155B11C6, the candidate germline sequence is IGKV3-20. 01 or IGKV6-21 01 gene. In the human germline sequence IGHV1-2 Reversion mutations occurred at R38K, M48I, R67K, V68A, M70L, R72A, and T74K in the heavy chain framework of 02. These mutations occurred in the human germline sequence IGHV1-46. Reversion mutations occurred at M70L, R72A, T74K, S85G, and R87T in the heavy chain framework of 01. These mutations were observed in the human germline sequence IGKV3-20. Reversion mutations occurred at A44S, L47P, L48W, I59V, D71S, and F72Y in the light chain framework of 01. These mutations were observed in the human germline sequence IGKV6-21. Reversion mutations occurred at L47P, L48W, K50Y, and F72Y in the light chain framework of 01.

[0256] Different combinations of reversion mutation sites were selected to generate the variable regions of humanized antibodies. The sequences of the heavy and light chain variable regions of the 155B11C6 humanized antibody are listed in Table 13A. VH and VL pairings of individual humanized antibodies are listed in Table 13B. The variable regions of the humanized antibodies were then fused into the constant region of human IgG1 for antibody production and functional characterization.

[0257] Table 13A. Variable region sequence of 155B11C6 humanized antibody (underline indicates CDR; bold / italic indicates reversion mutation) Table 13B. VH and VL pairing of the 155B11C6 humanized antibody. 3.2 Binding characteristics of CDH17 humanized antibody to cells expressing human CDH17 The cell-binding activity of the humanized CDH17 antibody was confirmed in cell-based binding assays using various CDH17-expressing cells, including HEK293-hCDH17, pancreatic cancer cell line AsPC-1, gastric cancer cell line AGS, and colorectal cancer cell line HCT-8. Assays were performed according to the protocol described above.

[0258] As shown in Figures 6-12, the humanized CDH17 antibodies (including 29H8D3-z3, 29H8D3-z6, 29H8D3-z9, 29H8D3-z12, 29D2D7-z19, all humanized 61C7F12 mAb, 69E3H11-z10, 69E3H11-z12, 143H10E4-z3, 143H10E4-z6, 143H10E4-z9, 152A1D12-z13, 152A1D12-z17, 152A1D12-z18, 155B11C6-z3, 155B11C6-z4, and 155B11C6-z7) exhibited cell-binding efficacy comparable to their respective chimeric antibodies.

[0259] To confirm the antigen-binding efficacy, species cross-reactivity, and specificity of the humanized CDH17 antibody, the binding efficacy of one of the humanized antibodies 29H8D3-z12 against recombinant CDH17 proteins from different species and several cadherin superfamily proteins (including human CDH16, CDH9, CDH10, CDH3, and CDH6) was assessed by ELISA.

[0260] To determine the binding ability of humanized mAbs to various antigens, ELISA-based binding assays were performed as follows. In short, the following antigens were used: hCDH17-his protein (custom-made by BioNTech), rhesus monkey CDH17-his (UniProt reference sequence: A0A1D5R2B4, Q23-T784, Kactus, catalog number CDH-RM117), cynomolgus monkey CDH17-hFc (isotype 1, Sino Biopharmaceutical, catalog number 90147-C02H), rat CDH17-his (Sinopharmaceutical, catalog number 80283-R08H), and mouse CDH17-his (Kactus, catalog number CDH-). Human CDH16-his (hCDH16-his, Nearshore Protein Technology Co., Ltd., catalog number CJ16), human CDH9-his (Bepsys, catalog number CA9-H52H6), human CDH10-his protein (Bepsys, catalog number CA0-H52H5), human CDH3-his protein (Kaikai, catalog number CDH-HM103), and human CDH6-his protein (Kaikai, catalog number CDH-HM106) were diluted at 1 μg / mL with 1×ELISA coating buffer (Solepro, catalog number C1050) and adsorbed into the wells of a 96-well microplate overnight at 4°C. After sealing the wells with 2% bovine serum albumin (BSA) to prevent nonspecific binding, humanized CDH17 mAb or an allotype control was titrated at a 4-fold dilution starting at 50 nM and added to wells pre-adsorbed with the antigen. The mixture was incubated at room temperature (RT) for 1 hour. The bound CDH17 mAb was recognized by the detection antibody goat anti-human IgG F(ab')2 conjugated with horseradish peroxidase (HRP) (Jackson Immunologics, catalog number 109-036-097). The substrate of HRP, tetramethylbenzidine (TMB), was added to the wells to visualize the binding signal. After sufficient development, stop solution was added to the wells. The absorbance of the signal was detected at 450 nm using an Envision multi-label plate reader (PerkinElemer). Graphs were generated and statistical analysis was performed using a four-parameter nonlinear regression curve fitted in Graphpad Prism 9 software.

[0261] As shown in Figures 6C-6G, 29H8D3-z12 effectively binds to human, rhesus macaque, and cynomolgus monkey CDH17 isotype 1 protein, but not to rat and mouse CDH17 protein. The EC50 of 29H8D3-z12 antibody binding to human CDH17 (EC50: 0.187 nM) is comparable to that of its parent antibody 29H8D3 (EC50: 0.238 nM, as shown in Table 3). More importantly, the EC50 of 29H8D3-z12 binding to human and rhesus macaque CDH17 protein is comparable (EC50: 0.187 nM vs. 0.163 nM), indicating good cross-reactivity between 29H8D3-z12 and non-human primate (NHP) CDH17 protein, which will facilitate preclinical NHP toxicity assessment. Furthermore, 29H8D3-z12 showed high selectivity for binding to human CDH17, as no binding to human CDH16, CDH9, CDH10, CDH3, and CDH6 proteins was observed (Figures 6H-6L).

[0262] To confirm the binding efficacy of 29H8D3-z12 to the NHP CDH17 antigen, cell-based binding assays were performed using rhesus macaque CDH17 (UniProt reference sequence: A0A1D5R2B4, M1-S832) overexpressed on HEK293 cells (HEK293-rhesus macaque CDH17, internally constructed) and HEK293-cynomolgus macaque CDH17 (isotype 2, custom-made by Giman Biotech in Example 2.7). For cell binding assays, CDH17 mAb or isotype controls were diluted three-fold starting at a concentration of 50 nM in staining buffer (DPBS buffer containing 2% FBS). The antibody dilution was mixed with 5 x 10 4 Cells were incubated in 96-well microplates at 4°C for 30 minutes. The cell-antibody mixture was then washed twice with staining buffer. The solution was then used with Alexa Fluor. ® 647 (Jackson Immunologics, Catalog No. 109-606-098) conjugated goat anti-human IgG Fc secondary antibody was detected at 4°C at a 1:1000 dilution for 30 min to detect antibodies binding to cell surface antigens, followed by thorough washing. The antibody was then analyzed by flow cytometry using an LSR Tortessa flow cytometer. TM Cell analysis was performed using a cell analyzer (BD Biosciences). Data analysis was conducted using Flowjo 10.0 software. Graphs and statistical analyses were generated using three- or four-parameter nonlinear regression curve fitting in Graphpad Prism 9 software.

[0263] As shown in Figures 6M and 6N, 29H8D3-z12 effectively binds to rhesus macaque CDH17 (EC50: 0.225 nM). However, consistent with previous results shown in Figure 5A, 29H8D3-z12 showed no binding to cynomolgus macaque CDH17 isotype 2 protein expressed on HEK293 cells, although binding to recombinant cynomolgus macaque CDH17 isotype 1 protein was observed in Figure 6E. This phenomenon may be due to the low similarity of the predicted EC1 domain of cynomolgus macaque CDH17 isotype 2 to that of cynomolgus macaque CDH17 isotype 1, which has high homology with human CDH17 protein. Overall, 29H8D3-z12 exhibits high specificity and good cross-reactivity, effectively binding to human and rhesus macaque CDH17 proteins expressed on cells.

[0264] 3.3 via Biacore TM Affinity sequencing of humanized antibodies To explore whether humanized antibodies can maintain their binding kinetics, Biacore was used. TM Affinity sequencing was performed. Antibody (2 μg / ml) was captured using a protein A chip. A single dose was delivered at a flow rate of 30 μL / min. Human CDH17-his protein was injected onto the capture antibody at doses of 100 nM or 400 nM, or two doses (25 nM and 100 nM). Antigen association was allowed for 120-150 s, followed by dissociation for 200-500 s. Experiments were conducted at Biacore. TM Performed on 8K. Using Biacore. TM 8K evaluation software is used for data analysis.

[0265] The results shown in Table 14 indicate that CDH17 humanized antibodies (including 29H8D3-z3, 29H8D3-z6, 29H8D3-z9, 29H8D3-z12, 29H8D3-z17 to 29H8D3-z23, 29D2D7-z19, 61C7F12-z1, 61C7F12-z2, 61C7F12-z3, 61C7F12-z8, 61C7F12-z9, ...) 61C7F12-z10, 69E3H11-z12, 143H10E4-z3, 143H10E4-z6, 143H10E4-z9, 152A1D12-z13, 152A1D12-z17, 152A1D12-z18, 155B11C6-z3, 155B11C6-z4, and 155B11C6-z7) showed affinity comparable to their respective chimeric antibodies.

[0266] Table 14. Affinity ranking results of CDH17 humanized antibodies Single-dose affinity Example 4. Optimization of humanized CDH17 antibody Following antibody humanization, further optimization of the CDR region is typically required to remove potential post-translational modification (PTM) sites, thereby improving the developability of humanized antibodies, including long-term stability, manufacturability, and homogeneity. PTMs (such as deamidation, isomerization, glycosylation, and oxidation) can impair the potency, efficacy, and safety of therapeutic antibodies. Alternatively, in some cases, affinity maturation of humanized antibodies is sought to improve their binding affinity, which may result in enhanced activity.

[0267] 4.1 PTM Removal and Affinity Maturation Design of Humanized CDH17 Antibody In this context, computational tools are used to predict PTM susceptibility sites to facilitate engineered antibodies with better physical and chemical properties.

[0268] Careful examination of the CDR region of the 29D2D7-z19 humanized mAb identified an unpaired cysteine ​​(C) located in CDR3 of the VL region, which may induce antibody heterogeneity during manufacturing. Therefore, an amino acid substitution was performed at this position. The sequence of the mutant variable region of the 29D2D7-z19 humanized mAb is listed in Table 15A, and the mutant CDRs are summarized in Table 15B.

[0269] PTM site prediction for the CDR region of the 29H8D3-z12 humanized mAb identified a potential isomerization site of aspartic acid (D) at CDR2 in the VH region, two potential deamidation sites of asparagine (N) at CDR2 in the VL region, and a potential oxidation site of methionine (M) at CDR3 in the VL region, which may be affected by oxidative stress. Therefore, amino acid substitutions were performed at these positions. The selected sequences for the mutant variable region of the 29H8D3-z12 humanized mAb are listed in Table 15A, and the mutant CDRs are summarized in Table 15B.

[0270] Table 15A. Variable region sequence of CDH17 humanized antibody (underline indicates CDR; bold / italic indicates PTM mutation) Table 15B. Optimized CDRs of heavy and light chains for 29D2D7 and 29H8D3 Alternatively, affinity maturation is performed to enhance the affinity and binding activity of the humanized CDH17 antibody.

[0271] A series of 69E3H11-z12 variants with mutations in the CDR region were displayed on phages. Phage clones exhibiting high binding efficacy to human CHD17 protein and CHD17 expressed on tumor cells were sequenced. The resulting Ab sequences were used to generate mAbs for further characterization. The variable region sequences of affinity-matured 69E3H11-z12 antibodies are summarized in Table 15C.

[0272] Table 15C. Variable region sequence of CDH17 humanized antibody (underline indicates CDR; bold / italic indicates mutation) Table 15D. Optimized light chain CDR1 and CDR2 of 69E3H11 4.2 Binding to cells expressing human CDH17 The cell-binding activity of the PTM-removed and affinity-matured CDH17 humanized antibody was confirmed in cell-based binding assays using colorectal cancer cell lines HCT-8 and LoVo cells, and pancreatic cancer cell line AsPC1 cells. Assays were performed according to the protocol described above.

[0273] As shown in Figures 13A and 13B, all PTM-removed variants of 29D2D7-z19, except for 29D2D7-z19p4, exhibited comparable binding potency to human CDH17 expressed on cells to the parental antibody 29D2D7-z19. As shown in Figures 13C, 13D, and 13E, the PTM-removed variants of 29H8D3-z12p22, 29H8D3-z12p3, 29H8D3-z12p4, and 29H8D3-z12p7 showed comparable human CDH17 binding to the parental antibody 29H9D3-z12 on HCT-8 cells.

[0274] As shown in Figures 13F-13I, all affinity maturation variants of 69E3H11-z12 showed improved binding potency to human CDH17 expressed on LoVo, HCT-8, and AsPC1 cells compared to its parent antibody 69E3H11-z12.

[0275] 4.3 via Biacore TM Ranking by affinity To explore whether humanized antibodies with PTM site removal and affinity maturation can maintain or improve their binding kinetics, Biacore was used. TMTwo dose affinity rankings were performed. The protein A chip was used to capture the antibody (2 μg / ml). Human CDH17-his protein was injected onto the capture antibody at a flow rate of 30 μL / min at 25 nM and 100 nM for 120 s or 150 s. Antigen dissociation was allowed for 200 s. Experiments were conducted at Biacore. TM Performed on 8K. Using Biacore. TM 8K evaluation software is used for data analysis.

[0276] The results shown in Table 16A indicate that the humanized antibodies, including 29D2D7-z19p3, 29D2D7-z19p5, and all PTM-removed 29H8D3-z12, exhibited affinity comparable to their parental humanized antibodies. The binding affinity of all affinity-matured antibodies was increased compared to their parental antibody 69E3H11-z12.

[0277] Table 16A. By Biacore TM Affinity ranking results of CDH17 humanized antibodies To confirm the binding affinity of various antibodies, full-dose affinity measurements were performed using biomembrane interferometry (BLI, ForteBio OCTET®). Briefly, antibodies were captured at a concentration of 100 nM using a protein A sensor. Serially diluted human CDH17-his antigen solutions, starting at 100 nM, were bound to different antibodies for 240 s, followed by dissociation for 600 s. Data were analyzed using OCTET Analysis Studio 12.2.

[0278] As shown in Table 16B, compared with its parent antibody 69E3H11-z12, the binding affinity of the mature antibodies 69E3H11-z12a27 and 69E3H11-z12a42 is improved.

[0279] Table 16B. Results of full-dose affinity measurement of humanized antibodies Example 5. Internalization of CDH17 monoclonal antibody This example characterizes the antibody internalization rate when a CDH17 antibody binds to CDH17 expressed on human tumor cells.

[0280] Therefore, the following internalization assay was developed. In this case, CDH17 hyperpancreatic cancer cell line AsPC-1 and CDH17 moderate human colorectal cancer cell lines LoVo and HCT-8 cells were used in this assay. pHAb thiol-reactive dye (Promega, catalog number G9835) is a pH-sensitive dye with very low fluorescence at pH > 7, and fluorescence increases sharply as the solution pH becomes acidic. pHAb thiol-reactive dye has a maleimide group that reacts with thiol groups. This maleimide group conjugates to the antibody after the cysteine ​​disulfide bond in the antibody hinge region is reduced to a thiol group using a reducing agent such as dithiothreitol (DTT) or tris(2-carboxyethyl)phosphine (TCEP). In short, firstly, purified anti-human IgG Fc antibody (Biolegend, catalog number 410701) was labeled with pHAb thiol-reactive dye according to the manufacturer's instructions. Then, the resulting pH-sensitive dye-conjugated secondary antibody (50 nM) was incubated with various CDH17 antibody dilutions (20 nM) in culture medium at a 1:1 volume ratio for 30 minutes at room temperature to produce a 2-fold working solution. Next, 50 μl of the 2-fold working solution was added to a 96-well assay plate, with each well pre-seeded with 50 μl of 2 × 10⁻⁶ antibodies. 4 Tumor cells expressing CDH17. Fluorescence increases dramatically when the antigen-antibody complex is internalized into endosomes or lysosomes at pH values ​​of approximately 6.3 or 4.7. Fluorescence signals are captured and analyzed using the Operetta® CLS™ high-throughput microplate imaging system (PerkinElmer).

[0281] As shown in Figures 14A and 14B, distal membrane EC1 conjugates 29H8D3, 143H10E4, 152A1D12, and 155B11C6, EC2 conjugate 120B10C5, EC3 conjugate 29D2D7, and EC4 conjugates 20C3E8 and 103G6G1 all exhibited efficient internalization rates in both CDH17 high-AsPC-1 cells and CDH17 intermediate-LoVo cells. Conversely, proximal membrane EC6 conjugate 67A11B11 and EC7 conjugates 69E3H11 and 95F2C2F12 showed moderate levels of internalization in both cell lines. EC5 conjugate 16C8 and EC6 conjugate 61C7F12 showed very weak or even undetectable internalization in both CDH17-expressing tumor cell lines. Generally, distal membrane conjugates of CDH17 exhibit superior performance compared to proximal membrane conjugates in terms of antibody internalization rate.

[0282] As shown in Figures 15A-15E, all CDH17 humanized and PTM-removed antibodies induced efficient internalization of antibody-antigen complexes on HCT-8 cells, comparable to their respective parental chimeric antibodies.

[0283] Example 6. Tumor cell killing by CDH17 antibody based on Mc-vc-PABC-MMAE-labeled anti-human IgG secondary antibody CDH17 can serve as a TAA target for ADC drugs due to its high expression levels in GI cancers, such as colorectal and pancreatic cancer. In the following examples, the potency of CDH17 antibodies in ADC-mediated cytotoxicity was evaluated in vitro and in vivo.

[0284] In this example, an established in vitro assay was used to characterize the indirect cytotoxicity of CDH17 monoclonal antibodies against tumor cells mediated by linker-payload conjugated anti-human IgG secondary antibodies.

[0285] In this assay, the established linker-loaded MC-Val-Cit-PABC-monomethyl olistatin E (hereinafter referred to as mc-vc-PABC-MMAE or vc-MMAE, Figure 16A) was explored as a tool linker-loaded molecule. Vc-MMAE consists of a sulfur-reactive maleimide hexanoyl (MC) group, a protease-sensitive Val-Cit dipeptide, a PABC linker, and an MMAE load, which is a mitotic inhibitor that inhibits microtubule polymerization. Briefly, anti-human IgG secondary antibody (Abcam, catalog number ab98616) was first labeled with vc-MMAE and used as the secondary antibody to eliminate tumor cells. The vc-MMAE-labeled anti-human IgG secondary antibody (25 nM) was then incubated with CDH17 antibody dilution (10 nM) in culture medium at room temperature for 30 minutes. Next, the mixture was diluted threefold and added to a 96-well plate at a 1:1 volume ratio, with each well pre-inoculated with 4 × 10⁶ cells / well. 3 AGS cells were collected and incubated in a CO2 incubator at 37°C for 96 hours (hs). Cell viability was assessed by incubating the cell culture with the assay reagents at room temperature for 10 minutes, followed by luminescence detection using an Envision multi-label plate reader (PerkinElemer) and a CellTiter-Glo® luminescent cell viability assay (100 μL, Promega, catalog number G7573).

[0286] As shown in Figures 17A-17C, the distal membrane EC1 conjugates 29H8D3, 143H10E4, 152A1D12, and 155B11C6, the EC2 conjugate 120B10C5, the EC3 conjugate 29D2D7, and the EC4 conjugates 20C3E8 and 103G6G1 showed considerable killing activity against AGS tumor cells. However, the proximal membrane EC5 conjugates 16C8, EC6 conjugates 61C7F12 and 67A11B11, and the EC7 conjugates 69E3H11 and 95F2C2F12 exhibited approximately 100-fold reduced tumor cell killing ability compared to these distal membrane conjugates. This phenomenon is consistent with the higher internalization efficiency of CDH17 antibodies bound to the distal membrane region compared to the proximal region conjugates.

[0287] Example 7. In vitro killing effect of VC-MMAE-labeled CDH17 ADC This example characterizes the cytotoxic efficacy of the vc-MMAE-labeled CDH17 ADC against CDH17-expressing tumor cells.

[0288] In this context, the assay included tumor cell lines with varying CDH17 expression levels. Briefly, the CDH17 ADCs were prepared via a Michael reaction by conjugating the VC-MMAE linker-loador to the thiol group of a reduced CDH17 monoclonal antibody. Following the conjugation and purification steps, the CDH17 ADCs underwent quality control to assess purity, drug-to-antibody ratio (DAR), endotoxin, and free linker-loador. No significant aggregation was observed as all ADCs measured by SEC-HPLC had a purity exceeding 96%. The DAR for each ADC was determined by HIC-HPLC, with calculated DARs for all ADCs ranging from approximately 3.7 to 4.0. Free linker-loador, as measured by RP-HPLC, was undetectable in all CDH17 ADCs.

[0289] Then, the in vitro ADC cytotoxicity assay was developed as follows. MMAE-sensitive AsPC-1, LoVo, and AGS tumor cell lines with high, intermediate, and low levels of CDH17 expression were used, respectively. Briefly, 8 × 10⁸ cells were used... 3 Tumor cells were seeded into 96-well plates and cultured overnight at 37°C in a CO2 incubator. The next day, CDH17 ADC, serially diluted from 30 nM at 2- to 5-fold dilutions, was added to the 96-well plates and incubated with the tumor cells at 37°C in a CO2 incubator for 96 hours. Cell viability was assessed by incubating the cell culture with the assay reagent at room temperature for 10 minutes, followed by luminescence detection using an Envision multi-label plate reader (PerkinElemer) and using CellTiter-Glo reagent (100 μL).

[0290] As shown in Figures 18A-18C, vc-MMAE-labeled CDH17 antibodies, including 29D2D7, 29H8D3, 29H8D3-z3, 143H10E4, 152A1D12, and 155B11C6, exhibited potent cytotoxicity against all three tumor cell lines: CDH17-high AsPC-1, CDH17-medium LoVo, and CDH17-low AGS cells. In contrast, 61C7F12-vc-MMAE consistently showed significantly reduced cytotoxicity against AsPC-1, LoVo, and AGS cells, likely due to the inefficient internalization capacity of the antibody fraction.

[0291] Table 17 summarizes the IC50 values ​​of CDH17 ADC cytotoxicity against various tumor cells.

[0292] Table 17. Cytotoxicity of VC-MMAE-labeled CDH17 ADCs to different cell lines Example 8. In vitro activity of GGFG-DXd-labeled CDH17 ADC This example characterizes the binding, internalization, and killing effects of the GGFG-DXd-labeled CDH17 ADC on tumor cells expressing human CDH17.

[0293] In this example, another well-validated linker-loador, MC-GGFG-DXd (drutecan, hereinafter referred to as DXd, Figure 16B), was used to evaluate the cytotoxicity efficiency of CDH17 ADCs. MC-GGFG-DXd is an ADC linker-loador conjugate consisting of a maleimide-GGFG peptide linker and a DX-8951 (DNA topoisomerase I inhibitor) derivative (DXd) used in the synthesis of DS-8201, a clinically validated HER2 ADC. CDH17 ADCs were generated by conjugating MC-GGFG-DXd to a CDH17 antibody, followed by quality control of purity, DAR, endotoxin, and free linker-loador. No significant aggregation was observed as all ADCs had a purity exceeding 96% as determined by SEC-HPLC. The DAR of each ADC was determined by RP-HPLC, with calculated DARs for all ADCs ranging from approximately 7.50 to 7.90. Free linker-loador was not detected in any of the CDH17 ADCs as measured by RP-HPLC.

[0294] 8.1 Binding of GGFG-DXd-labeled CDH17 ADC to cells expressing human CDH17 To confirm that the CDH17 ADC retained the binding potency of its parental antibody, the binding potency of GGFG-DXd-labeled CDH17 ADC and its corresponding parental antibody was evaluated in cell-based binding assays using pancreatic cancer cell lines AsPC-1, gastric cancer cell lines AGS and KATO III, and CRC cell line HT-29. In these cell lines, AsPC-1 showed high levels of CDH17 expression, while AGS and HCT-8 showed relatively moderate to low levels of CDH17 expression. Conversely, the KATO III and HT-29 cell lines showed only minimal CDH17 expression. Assays were performed according to the protocol described above.

[0295] As shown in Figures 19A-19D, 29H8D3-z12-DXd and 29D2D7-z19p3-DXd exhibited similar binding potency with their corresponding parental antibodies on the specified cells, even in KATO III and HT-29 cell lines with extremely low CDH17 expression levels. These data indicate that conjugation of these CDH17 mAbs to the linker-payload does not impair their antigen-binding ability.

[0296] 8.2 Internalization of human CDH17-expressing cells by GGFG-DXd-labeled CDH17 ADC Next, to examine whether the CDH17 ADC can mediate the efficient internalization of CDH17, the internalization rate and percentage of the CDH17 ADC and the corresponding naked antibody were evaluated as follows.

[0297] The internalization rate was determined according to the protocol described in Example 5. Alternatively, the percentage of internalization was assessed using FACS. In short, the CDH17 ADC or mAb was reacted with 5 × 10⁻⁶ ppm. 4 Tumor cells expressing CDH17 were incubated in 96-well microplates at 4°C for 30 minutes. The cell-antibody / ADC mixture was then washed three times with staining buffer. The cells were resuspended in complete culture medium and divided into two fractions. One fraction was incubated at 37°C to allow ADC or mAb internalization, while the other fraction was kept on ice as a negative control. After 24 hours, the cell-antibody / ADC mixture was washed three times with staining buffer. The mixture was then incubated with Alexa Fluor. TM 647 (Jackson Immunoassay, Catalog No. 109-606-098) conjugated goat anti-human Fcγ secondary antibody was detected at 4°C at a 1:1000 dilution. Antibodies or ADCs retained on the cell surface were observed for 30 min, followed by thorough washing. The results were analyzed using a flow cytometer (LSR Fortessa). TMCells were analyzed using a cell analyzer (BD Biosciences). Data were analyzed using Flowjo 10.0 software. Graphs were generated using Graphpad Prism 9 software.

[0298] As shown in Figure 20A, both 29H8D3-z12-DXd and 29H8D3-z12 mAb exhibited comparable internalization rates in CDH17-expressing HCT-8 cells within 24 hours. As shown in Figures 20C-20E, in CDH17-expressing AGS, AsPC1, and SW480 cells overexpressing human CDH17 (SW480-hCDH17), 29H8D3-z12-DXd showed an enhanced internalization rate compared to 29H8D3-z12 mAb within 24 hours. Conversely, no internalization was detected in CDH17-negative wild-type SW480 cells (Figure 20F), indicating target-specific internalization mediated by CDH17 mAb and ADC. Regarding the internalization percentage shown in Figure 20B, approximately 95% of 29H8D3-z12-DXd was internalized into HCT-8 cells 24 hours after treatment at 37°C, which is comparable to naked mAb 29H8D3-z12. These data collectively demonstrate the rapid and efficient internalization of CDH17 ADCs when targeting CDH17-expressing tumor cells, and that this ability is unaffected by the ADC conjugation process.

[0299] 8.3 In vitro killing efficacy of GGFG-DXd-labeled CDH17 ADC The following in vitro ADC cytotoxicity assay was developed. DXd-sensitive SK-CO-1, HCT-8, AGS, LS1034, and Caco2 cells, with high and moderate CDH17 expression levels, were selected as target cells. Three other DXd-sensitive cell lines (including the breast cancer cell line MDA-MB-468, and the CRC cell lines SW620 and SW480) lacked native CDH17 expression or had only negligible CDH17 expression levels. To assess the on-target effect of the CDH17 ADC, these three tumor cell lines were forced to overexpress full-length human CDH17 and were also used as target cells.

[0300] Use QIFIT according to the manufacturer's instructions. ® Absolute antigen counts were assessed using a cell surface antigen quantification kit (quantitative analysis kit, Agilent Dako, code K0078). Briefly, CDH17 mAb 69E3H11 with either a mouse IgG2a constant region or a mouse IgG2a isotype control was diluted to 50 nM in staining buffer (DPBS buffer containing 2% FBS). The antibody dilution was then mixed with 5 × 10⁻⁶... 4Cells were incubated in 96-well microplates at 4°C for 30 min. Calibration beads conjugated with high-affinity anti-human CD5 mouse IgG2a antibody (clone CRIS-1) with a defined absolute number were used as standards for quantifying cell surface antigens. The cell-antibody mixture and calibration beads were then washed twice with staining buffer. The mixture was then incubated with Alexa Fluor. TM 647 (Jackson ImmunoResearch, Catalogue No. 115-606-003) Conjugated goat anti-mouse IgG (H+L) secondary antibody was used to detect antibodies or calibration beads binding to cell surface antigens at a dilution of 1:2000 at 4°C for 30 min, followed by thorough washing. The antibody was then analyzed using a flow cytometer (LSR Fortessa). TM Cell analyzers (BD Biosciences) were used to analyze cells and calibration beads. Data were analyzed using Flowjo 10.0 software.

[0301] The results showed that SK-CO-1, LS1034, MDA-MB-468-hCDH17, SW480-hCDH17, and SW620-hCDH17 had high absolute numbers of membrane CDH17 (>1 × 10⁻⁶). 5 / cell), HCT-8, AGS, and Caco2 showed moderate levels of CDH17 (1 × 10⁻⁶ cells). 4 / cell-1 × 10 5 / cells) (Table 18b).

[0302] For in vitro kill assays, in short, 2 × 10 3 Up to 8 × 10 3 Tumor cells were seeded into 96-well plates and cultured overnight at 37°C in a CO2 incubator. The next day, CDH17 ADC, serially diluted 3-fold starting at 30 nM or 50 nM, was added to the 96-well plates and incubated with the tumor cells at 37°C in a CO2 incubator for 5 to 6 days. Cell viability was assessed by incubating the cell culture with the assay reagent at room temperature for 10 minutes, followed by luminescence detection using an Envision multi-label plate reader (PerkinElemer) and using CellTiter-Glo reagent (100 μL).

[0303] As shown in Figures 21A-21D, 29H8D3-DXd (29H8D3-MC-GGFG-DXd) and 29H8D3-z3-DXd (29H8D3-z3-MC-GGFG-DXd) exhibited effective and comparable tumor cell killing against HCT-8 and three types of tumor cells overexpressing human CDH17: SW620, SW480, and MDA-MB-468, indicating the cytotoxic efficiency of CDH17 ADCs against CDH17-expressing tumor cells.

[0304] As shown in Figures 22A-22D, 29H8D3-z12-DXd (29H8D3-z12-MC-GGFG-DXd) and 29D2D7-z19p3-DXd (29D2D7-z19-MC-GGFG-DXd) exhibited effective and comparable tumor cell killing against CDH17-overexpressing SW480-hCDH17 and MDA-MB-468-hCDH17 cells, as well as CDH17-moderately expressed HCT-8 and AGS cells, indicating the cytotoxic efficiency of CDH17 ADCs against CDH17-overexpressing and CDH17-moderately expressed tumor cells. Furthermore, 29H8D3-z12-DXd also showed dose-dependent cytotoxicity against CDH17-overexpressing SK-CO-1, LD1034, and SW620-hCDH17 cells, as well as CDH17-moderately expressed Caco2 cells (Figures 22E-22H). Conversely, no significant cytotoxicity was observed in CDH17-negative RKO cells (Figure 22I), indicating CDH17 ADC-mediated target-specific cytotoxicity.

[0305] Tables 18A and 18B summarize the IC50 values ​​of CDH17 ADC cytotoxicity against various tumor cells.

[0306] Table 18A. Cytotoxicity characteristics of DXd-labeled CDH17 ADCs in different cell lines Table 18B. Cytotoxicity characteristics of DXd-labeled CDH17 ADCs in different cell lines. 8.4 In vitro bystander kill efficacy of GGFG-DXd-labeled CDH17 ADC To evaluate the bystander killing effect of CDH17-ADC, an in vitro co-culture assay was performed as follows. SW480-hCDH17 cells (1000 cells / well) expressing GFP-positive CDH17 were co-cultured with human CDH17-negative RKO (RFP) cells. +SW480 or MDA-MB-468 cells (2000 cells / well) were co-cultured in 96-well plates in the presence of 1 nM CDH17 ADC or a medium control. In the absence of hCDH17-positive cells, hCDH17-negative cells were seeded at the same number (2000 cells / well) as a negative control to exclude direct killing of human CDH17-negative cells by CDH17 ADC. After 6 days, the absolute number of GFP-positive and GFP-negative cells was counted using the Operetta® CLS™ High Content Analysis System (PerkinElmer). Cell viability was analyzed and graphs were plotted using Graphpad Prism 9 software.

[0307] As shown in Figures 23A-23C, 29H8D3-z12-DXd exhibited effective cytotoxicity against CDH17-positive SW480-hCDH17 cells and CDH17-negative RKO, SW480, and MDA-MB-468 cells in the co-culture system. In contrast, in the absence of CDH17-positive SW480-hCDH17 cells, 29H8D3-z12-DXd showed no killing effect on CDH17-negative RKO, SW480, and MDA-MB-468 cells in the culture system (Figures 23A-23C). These data collectively demonstrate the effective bystander killing effect of CDH17 ADCs in vitro.

[0308] Example 9. In vivo antitumor efficacy of VC-MMAE-labeled CDH17 ADC This example characterizes the in vivo antitumor activity of the vc-MMAE-labeled CDH17 ADC against a cell line-derived xenograft (CDX) mouse model.

[0309] Therefore, MMAE-sensitive colorectal cell line LoVo cells (5 × 10⁻⁶) were used. 6 The tumor was subcutaneously implanted into BALB / c nude mice. When the tumor volume (TV) reached approximately 180 mm... 3 Mice were divided into groups (n = 6 mice / group) and simultaneously administered a single intravenous dose of the specified CDH17 ADC at a dose of 3 mg / kg, with the isotype-vc-MMAE and the mediator group serving as controls. Defined as 0.5 × length × width. 2 Tumor volume was measured 3 times per week. TGI (%) was calculated as follows: TGI (%) = [1 - (mean change in tumor volume in the treatment group on the assessment date) / (mean change in tumor volume in the control group on the assessment date)] × 100.

[0310] like Figure 24A and 24BAs shown, all CDH17 ADCs with the vc-MMAE linker-payload exhibited robust tumor growth inhibition on day 18 post-treatment, with tumor growth index (TGI) ranging from 85% to 95%. In contrast, the non-binding MMAE showed very weak and transient tumor growth inhibition (TGI = 18.96%) at the end of the study. These data collectively demonstrate the antitumor efficacy of vc-MMAE-based CDH17 ADCs in an in vivo CDX mouse model.

[0311] Example 10. In vivo antitumor efficacy of GGFG-DXd-labeled CDH17 ADC in CDX mouse model This example characterizes the in vivo antitumor activity of the GGFG-DXd conjugated CDH17 ADC in a CDX mouse model.

[0312] Therefore, CDH17 expression levels were increased to 1.33 × 10⁻⁶. 5 LS1034 colorectal cell line (5 × 10⁹ cells) 6 The tumor cells were resuspended in DPBS and mixed with Matrigel (Corning®, catalog number 356234) at a 1:1 ratio, then subcutaneously implanted into female CB-17 SCID mice. When the tumor volume (TV) reached approximately 150 mm... 3 Mice were divided into groups (n = 4 mice / group) and simultaneously administered two intravenous doses of 3 mg / kg or 10 mg / kg 29H8D3-z12-DXd on days 1 and 8. The vector group served as a non-treatment control. Defined as 0.5 × length × width. 2 Tumor volume was measured 3 times per week. TGI (%) was calculated as follows: TGI (%) = [1 - (mean change in tumor volume in the treatment group on the assessment date) / (mean change in tumor volume in the control group on the assessment date)] × 100.

[0313] As shown in Figures 25A and 25B, compared with the mediator group, 29H8D3-z12-DXd demonstrated robust dose-dependent tumor growth inhibition on day 25 post-treatment, with tumor growth index (TGI) of 68.45% (for 3 mg / kg) and 106.07% (for 10 mg / kg). Furthermore, no changes in body weight were observed in any treatment group before the end of the study (Figure 25C), indicating the absence of CDH17 ADC treatment-mediated systemic toxicity. These data collectively demonstrate the superior dose-dependent antitumor efficacy of the GGFG-DXd-conjugated CDH17 ADC in an in vivo CDX mouse model.

[0314] Example 11. In vivo antitumor efficacy of GGFG-DXd-labeled CDH17 ADC in a patient-derived xenograft (PDX) mouse model. This example characterizes the in vivo antitumor activity of the GGFG-DXd conjugated CDH17 ADC in a patient-derived xenograft (PDX) mouse model.

[0315] Therefore, a cohort of CRC PDX mouse models with different driver gene mutations (including TP53 and KRAS) or gene amplification (ERBB2) were selected for in vivo efficacy evaluation. Notably, in previous in vivo efficacy studies, one model (Model 1) showed resistance to SOC treatment (a combination of irinotecan, leucovorin, and 5-fluorouracil) (data not shown). Another model (Model 3) has been reported to be resistant to multiple targeted therapies, including irinotecan, and chemotherapy during clinical treatment. It is important to assess whether the CDH17-GGFG-DXd ADC still exhibits antitumor activity in these tumor models with driver gene mutations and chemotherapy resistance.

[0316] CDH17 expression levels in these PDX tumor tissues derived from CRC patients were confirmed by immunohistochemical (IHC) staining of formalin-fixed, paraffin-embedded (FFPE) sections, using a specific CDH17 mAb (Abogen, catalog number ab183318) as the primary antibody and a rabbit IgG mAb (Abogen, catalog number ab172730) as an isotype control. The intensity and extent of CDH17 expression were assessed using a histochemical scoring system (H score). Expression grades were classified as 0 (H score <1), 1+ (H score 1-99), 2+ (H score 100-199), and 3+ (H score 200-300). PDX tumor samples with high (3+) or moderate (2+) CDH17 expression levels were selected and passaged in host mice. When the tumors grew to approximately 500-800 mm... 3 At that time, the tumor sample was cut into small pieces (approximately 3 mm). 3 The drug (45-60 mg) was subcutaneously injected into female NU / NU nude mice (Vital River). When the tumors grew to an appropriate size, the tumor-bearing mice were randomly divided into a treatment group and a control group (n = 4 mice / group), with an average tumor volume of approximately 200 mm. 3 Simultaneously, a single, two, or three-dose intravenous administration of the indicated CDH17 ADC at 10 mg / kg was administered, with the mediator treatment serving as a negative control. Defined as 0.5 × length × width. 2Tumor volume was measured twice weekly. TGI (%) was calculated as follows: TGI (%) = [1 - (mean change in tumor volume in the treatment group on the assessment date) / (mean change in tumor volume in the control group on the assessment date)] × 100. Relative body weight change (%) (RCBW) was calculated as follows: RCBW (%) = (BW) / (R² - R²) / (R² - R²) / (R² - R²) i - BW0) / BW0× 100, BW i BW0 is the average body weight on the assessment day, and BW0 is the average body weight at the start of the dose.

[0317] like Figure 26A As shown, the CDH17 ADC conjugated with the GGFG-DXd linker-payload exhibited robust tumor growth inhibition or even regression in all four CRC PDX models with different ranges of CDH17 expression levels and different dosage regimens. At the end of the study, the tumor growth index (TGI) in the treatment groups ranged from 99% to 113%. Figure 26A Notably, this significant antitumor efficacy of CDH17 ADCs was observed not only in SOC-resistant PDX models but also in PDX models with driver gene mutations or amplifications, indicating that the efficacy of CDH17-ADCs is independent of background gene mutation status or previous response to chemotherapy drugs (even irinotecan, which is also a camptothecin derivative, similar to DXd). Furthermore, no weight loss was observed during treatment. Figure 26B This indicates that these ADCs have no obvious toxicity. These data collectively demonstrate the robust antitumor efficiency of the CDH17-GGFG-DXd ADC in the CRC PDX mouse model.

[0318] Next, to understand whether CDH17-GGFG-DXd ADC can effectively and dose-dependently inhibit tumor growth in PDX mouse models with a single-dose regimen, three of the four PDX mouse models shown in Figure 26 were selected for further in vivo efficacy studies.

[0319] This study followed the protocol described above. When the tumor grew to approximately 500-800 mm... 3 At that time, the tumor sample was cut into small pieces (approximately 3 mm). 3 The drug (45-60 mg) was subcutaneously injected into female NU / NU nude mice (Vitol Dermatologice). When the tumors grew to an appropriate size, the tumor-bearing mice were randomly divided into a treatment group and a control group (n = 4 mice / group), with an average tumor volume of approximately 180-200 mm. 3 Simultaneously, a single intravenous dose of 3 mg / kg or 10 mg / kg of 29H8D3-z12-DXd was administered, with the carcass treatment serving as a negative control. Defined as 0.5 × length × width.2 Tumor volume was measured twice a week. TGI (%) was calculated as follows: TGI (%) = [1 - (mean change in tumor volume in the treatment group on the assessment date) / (mean change in tumor volume in the control group on the assessment date)] × 100.

[0320] like Figure 27 As shown, a single dose of 29H8D3-z12-DXd demonstrated robust dose-dependent tumor growth inhibition in all three CRC PDX mouse models with varying CDH17 expression levels. A single dose of 3 mg / kg of 29H8D3-z12-DXd achieved tumor growth inhibition, with a TGI ranging from 54.5% to 79.0% on day 21 in the treatment groups. A single dose of 10 mg / kg of 29H8D3-z12-DXd achieved even enhanced tumor growth inhibition, with a TGI ranging from 98.1% to 100.2% on day 21 in the treatment groups. These data collectively demonstrate the efficacy and dose-dependent tumor growth inhibition of the 29H8D3-z12-DXd ADC in CRC PDX mouse models.

[0321] The scope of this disclosure is not limited to the particular embodiments described, which are intended as a single illustration of various aspects of this disclosure, and any functionally equivalent compositions or methods are within the scope of this disclosure. It will be apparent to those skilled in the art that various modifications and variations can be made to the methods and compositions of this disclosure without departing from the spirit or scope of this disclosure. Therefore, this disclosure is intended to cover such modifications and variations, provided they fall within the scope of the appended claims and their equivalents.

[0322] All publications and patent applications mentioned in this specification are incorporated herein by reference to the same extent that each individual publication or patent application is specifically and individually indicated to be incorporated by reference.

Claims

1. An antibody or antigen-binding fragment thereof specific to human cadherin 17 (CDH17) protein, wherein the antibody or antigen-binding fragment comprises a heavy chain variable region (VH) and a light chain variable region (VL), the heavy chain variable region comprising heavy chain complementarity-determining regions HCDR1, HCDR2, and HCDR3, and the light chain variable region comprising light chain complementarity-determining regions LCDR1, LCDR2, and LCDR3, wherein HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 each comprise the following amino acid sequences: The following are the SEQ ID NOs: 49, 50, 51, 52, 239-241, 53, 242-244, and 54.

2. The antibody or antigen-binding fragment thereof according to claim 1, wherein the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 or LCDR3 comprises post-translational modification (PTM) site removal, wherein the PTM site comprises one or more of an oxidation site, an unpaired cysteine ​​residue site, a deamidation site, a glycosylation site, a disulfide formation site, an N-terminal pyroglutamate formation site and a high-mannose glycosylation site.

3. The antibody or its antigen-binding fragment according to claim 2, wherein... (a) The HCDR1 contains the amino acid sequence of SEQ ID NO:49, the HCDR2 contains the amino acid sequence of SEQ ID NO:50, the HCDR3 contains the amino acid sequence of SEQ ID NO:51, the LCDR1 contains the amino acid sequence selected from the group consisting of SEQ ID NO:239-241, the LCDR2 contains the amino acid sequence selected from the group consisting of SEQ ID NO:53 and 242-244, and the LCDR3 contains the amino acid sequence of SEQ ID NO:54; or (b) The HCDR1 contains the amino acid sequence of SEQ ID NO:49, the HCDR2 contains the amino acid sequence of SEQ ID NO:50, the HCDR3 contains the amino acid sequence of SEQ ID NO:51, the LCDR1 contains the amino acid sequence selected from the group consisting of SEQ ID NO:52 and 239-241, the LCDR2 contains the amino acid sequence selected from the group consisting of SEQ ID NO:242-244, and the LCDR3 contains the amino acid sequence of SEQ ID NO:

54.

4. The antibody or antigen-binding fragment thereof according to any one of claims 1-3, wherein the antibody is a chimeric antibody or a humanized antibody.

5. The antibody or its antigen-binding fragment according to claim 1, wherein: The VH contains an amino acid sequence selected from the group consisting of SEQ ID NO: 55, 149-152 and 154, and the VL contains an amino acid sequence selected from the group consisting of SEQ ID NO: 56, 156-158, 160 and 232-238.

6. The antibody or antigen-binding fragment thereof according to claim 5, wherein the VH and VL respectively comprise the following amino acid sequences: (a) SEQ ID NO: 149 and 156; (b) SEQ ID NO: 149 and 157; (c) SEQ ID NO: 149 and 158; (d) SEQ ID NO: 150 and 156; (e) SEQ ID NO: 150 and 157; (f) SEQ ID NO: 150 and 158; (g) SEQ ID NO: 151 and 156; (h) SEQ ID NO: 151 and 157; (i) SEQ ID NO: 151 and 158; (j) SEQ ID NO: 152 and 156; (k) SEQ ID NO: 149 and 160; (l) SEQ ID NO: 152 and 160; (m) SEQ ID NO: 154 and 156; (n) SEQ ID NO: 154 and 160; (o) SEQ ID NO: 152 and 232; (p) SEQ ID NO: 152 and 233; (q) SEQ ID NO: 152 and 234; (r) SEQ ID NO: 152 and 235; (s) SEQ ID NO: 152 and 236; (t) SEQ ID NO: 152 and 237 or (u) SEQ ID NO: 152 and 238.

7. An antibody or antigen-binding fragment thereof specific to human CDH17 protein, wherein the antibody or antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising heavy chain complementarity-determining regions HCDR1, HCDR2, and HCDR3, and the light chain variable region comprising light chain complementarity-determining regions LCDR1, LCDR2, and LCDR3. in: The HCDR1, HCDR2, and HCDR3 groups are selected from antibody 69E3H11 in Table 2A, or from the CDR group derived from antibody 69E3H11 in Table 2A, wherein one, two, or three amino acids are added, deleted, and / or substituted in one or more of these CDRs. The LCDR1, LCDR2 and LCDR3 group is selected from antibody 69E3H11 in Table 2B, or from the CDR group derived from antibody 69E3H11 in Table 2B, wherein one, two or three amino acids are added, deleted and / or substituted in one or more of these CDRs.

8. An antibody or antigen-binding fragment thereof that is specific to human CDH17 protein, wherein the antibody or antigen-binding fragment thereof competes with an antibody or antigen-binding fragment thereof according to any one of the preceding claims.

9. The antibody or antigen-binding fragment thereof according to any one of the preceding claims, wherein the antibody or antigen-binding fragment thereof is selected from the group consisting of full-length antibody, Fab, Fab', F(ab')2, Fd, Fv, single-chain Fv (scFv), single-chain antibody, disulfide-linked Fv (sdFv), nanobody, domain antibody, isolated CDR and divalent domain antibody.

10. The antibody or antigen-binding fragment thereof according to any one of the preceding claims further comprises a heavy chain constant region, a light chain constant region, an Fc region, or a combination thereof.

11. The antibody or antigen-binding fragment thereof according to claim 9, wherein the light chain constant region is a κ or λ chain constant region.

12. The antibody or antigen-binding fragment thereof according to any one of claims 1-11, wherein the antibody or antigen-binding fragment thereof belongs to the isotype of IgG, IgM, IgA, IgE or IgD.

13. The antibody or antigen-binding fragment thereof according to claim 12, wherein the isotype is IgG1, IgG2, IgG3 or IgG4.

14. An antibody-drug conjugate comprising an antibody or an antigen-binding fragment thereof according to any one of claims 1-13, and a drug optionally linked to the antibody or the antibody-binding fragment thereof via a linker.

15. The antibody-drug conjugate of claim 14, wherein the linker is connected to the antibody or its antigen-binding fragment according to any one of claims 1-13 via chemical or enzymatic conjugation.

16. The antibody-drug conjugate of claim 14, wherein the adapter is a cleavable adapter or an uncleavable adapter.

17. The antibody-drug conjugate of claim 16, wherein the cleavable linker is a chemically sensitive linker or an enzyme-cleavable linker; wherein the uncleavable linker comprises a thioether or maleimide hexanoyl (MC).

18. The antibody-drug conjugate of claim 17, wherein the chemically sensitive linker is a pH-sensitive linker or a glutathione-sensitive disulfide linker; wherein the enzyme-cleavable linker is a peptide-based linker or a β-glucuronide linker.

19. The antibody-drug conjugate of claim 14, wherein the linker comprises 4-(N-maleimidemethyl)cyclohexane-1-carboxylic acid succinimide ester (SMCC), sulfonyl-SMCC, p-carboxycyclohexylmethylmaleimide, maleimide hexanoyl (MC)-valine-citrulline (VC)-p-aminobenzyloxycarbamoyl (PABC), CL2A, maleimide hexanoyl (MC)-glycine-glycine-phenylalanine-glycine (GGFG), MC, or maleimide propionyl (MP)-PEG8-valine-alanine (VA)-PABC.

20. The antibody-drug conjugate according to any one of claims 14-19, wherein the drug is selected from the group consisting of cytotoxins, therapeutic peptides, and polypeptides.

21. The antibody-drug conjugate of claim 20, wherein the drug is selected from the group consisting of: olritamines, maytansines, benzodiazepines, tubulolysins, pyroxine, camptothecin, chalcogenide, eczemab, irinotecan (SN38), doxorubicin, anthracycline, pyrrolobenzodiazepines (PBD), TLR agonists, STING agonists, Pseudomonas aeruginosa exotoxin PE38, diphtheria toxin, Staphylococcus aureus enterotoxin A / E-120, antibacterial antibiotics, Shiga toxin, ricin, and urease.

22. The antibody-drug conjugate of claim 21, wherein the drug is monomethylolpropionate E (MMAE), monomethylolpropionate F (MMAF), maytansine, methanotrophin (DM1), rivanine (DM4), tubulolysin A, DX-8951f, DXd, 7-ethyl-10-hydroxycamptothecin (SN-38), DGN462, ambroxol 269, anthramycin, SG3199 / SCX, IRDye®700DX, TLR7 / 8 agonist, diABZI STING agonist-2, or any derivative thereof.

23. The antibody-drug conjugate according to any one of claims 14-22, wherein the drug and the adapter are collectively referred to as ozomicin, virdotin, malfotin, entansine, soravtansine, rivansine, metansine, delutecan, gavitan or tecillin.

24. The antibody-drug conjugate of claim 23, wherein the drug and the adapter together comprise (a) Vidodrine with Formula I: (Mc-vc-PABC-MMAE); (b) Drotecon with Formula II: (Mc-GGFG-DXd); (c) Ozomi stars with Formula III: ； (d) Mafaltin with Formula IV: ； (e) Entansine with formula V: ； (f) Govitan with Formula VI: ;or (g) Ticillin with Formula VII: 。 25. A composition comprising an antibody or an antigen-binding fragment thereof according to any one of claims 1-13 or an antibody-drug conjugate according to any one of claims 14-24, and a pharmaceutically acceptable carrier.

26. An isolated cell comprising one or more polynucleotides encoding an antibody or an antigen-binding fragment thereof according to any one of claims 1-13.

27. A polynucleotide encoding one or more chains of an antibody or an antigen-binding fragment thereof according to any one of claims 1-13.

28. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1-13, the antibody-drug conjugate according to any one of claims 14-24, or the composition according to claim 25 in the manufacture of a medicament for treating cancer in patients in need.

29. The antibody or antigen-binding fragment thereof according to any one of claims 1-13, the antibody-drug conjugate according to any one of claims 14-24, or the composition according to claim 25, for use in treating cancer in patients in need.

30. The use according to claim 28, or the antibody or antigen-binding fragment thereof according to any one of claims 1-13, the antibody-drug conjugate according to any one of claims 14-24, or the composition according to claim 25, wherein the cancer is selected from the group consisting of bladder cancer, breast cancer, colorectal cancer, endometrial cancer, esophageal cancer, head and neck cancer, kidney cancer, leukemia, liver cancer, lung cancer, lymphoma, melanoma, pancreatic cancer, prostate cancer, and thyroid cancer.

31. A method for detecting CDH17 expression in a sample, the method comprising contacting the sample with an antibody or antigen-binding fragment according to any one of claims 1-13 under conditions in which the antibody or antigen-binding fragment thereof is bound to CDH17, and detecting binding indicating CDH17 expression in the sample.

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