Anti-CDH17 antibody, conjugate and use thereof

Abstract

An antibody targeting human CDH17, a drug conjugate thereof and the use thereof. Specifically provided are a humanized antibody that specifically binds to human CDH17 and an antibody-drug conjugate targeting human CDH17. The antibody and the antibody-drug conjugate have excellent therapeutic effects on gastric cancer, colorectal cancer and pancreatic cancer, especially colorectal cancer, have excellent PK properties, and are of great significance for developing CDH17-targeted therapy.

Description

Anti-cdh17 antibodies, conjugates, and uses thereof

[0001] The present disclosure claims priority to Chinese Patent Application No. 202410706374.9, filed on May 31, 2024, entitled “Anti-cdh17 antibodies, conjugates, and uses thereof,” and Chinese Patent Application No. 202510504747.9, filed on April 22, 2025, entitled “Anti-cdh17 antibodies, conjugates, and uses thereof,” the entire contents of which are incorporated herein by reference. TECHNICAL FIELD

[0002] The present disclosure relates to the field of antibodies, and in particular, to anti-CDH17 antibodies, conjugates, and uses thereof. BACKGROUND

[0003] Cadherins are a superfamily of Ca 2+ dependent cell adhesion molecules that play an important role in maintaining tissue morphology. Cadherins comprise at least six subfamilies, such as classic Cadherins, atypical Cadherins, desmocollins, desmogleins, protocadherins, etc. The dysregulation of Cadherin expression is often associated with abnormal tissue development, tumor formation, and metastasis. Cadherin-17 (CDH17), also known as hepatocellular carcinoma-associated antigen, is a non-canonical member of the CDH superfamily of calcium-dependent proteins. It was originally cloned from rat liver and preferentially expressed in the intestine. CDH17 is a calcium-dependent single transmembrane glycoprotein that mediates cell adhesion in the intestinal epithelium. The extracellular domain of classic Cadherin is composed of 5 repeating sequences; the intracellular domain consists of 150-160 amino acids, which are highly conserved. Unlike other classic cadherins, CDH17 sequence is formed by seven extracellular cadherin domains and a very short cytoplasmic domain.

[0004] Studies have found that in human normal tissues, the expression of CDH17 is limited to the epithelial cells of the intestinal tract and pancreatic duct. However, in tumor tissues, CDH17 is highly expressed in various digestive tract tumor tissues, such as gastric cancer, colorectal cancer, and pancreatic cancer. Tumor RNA analysis data shows that almost 100% of colorectal cancer tissues highly express CDH17, which is higher than the corresponding normal intestinal tissues. Studies have also reported that about 6% of lung cancer patients' tissues show CDH17 positive staining, while ovarian cancer and liver cancer are 11% and 2%, respectively. These results suggest that CDH17 is a potential therapeutic target in related tumors, particularly digestive tract tumors. SUMMARY

[0005] In a first aspect, the present disclosure provides an antibody-drug conjugate or a pharmaceutically acceptable salt thereof, having a general structure of Pc-(L-D) n wherein,

[0006] D is a cytotoxic drug;

[0007] L is a linker unit;

[0008] Pc is an antibody or an antigen-binding fragment thereof that specifically binds to CDH17; and

[0009] n is a real number from 1 to 16.

[0010] In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain variable region (VL) comprising LCDR1, LCDR2, and LCDR3, and / or a heavy chain variable region (VH) comprising HCDR1, HCDR2, and HCDR3, the LCDR1-3 and / or the HCDR1-3 are selected from the following combinations:

[0011] (1) LCDR1, LCDR2, LCDR3 comprising the sequences set forth in SEQ ID NO. 23, 24, 25; and / or HCDR1, HCDR2, HCDR3 comprising the sequences set forth in SEQ ID NO. 26, 28, 30;

[0012] (2) LCDR1, LCDR2, LCDR3 comprising the sequences set forth in SEQ ID NO. 23, 24, 25; and / or HCDR1, HCDR2, HCDR3 comprising the sequences set forth in SEQ ID NO. 26, 27, 30;

[0013] (3) LCDR1, LCDR2, LCDR3 comprising the sequences set forth in SEQ ID NO. 23, 24, 25; and / or HCDR1, HCDR2, HCDR3 comprising the sequences set forth in SEQ ID NO. 26, 29, 30; or

[0014] (4) the LCDR1-3 or / and the HCDR1-3 comprise a sequence having at least 80% identity compared to each of the CDRs of the LCDR1-3 and HCDR1-3 of any one of the groups (1)-(3), or a sequence that differs by up to 3 insertions, deletions, or substitution mutations; preferably, the at least 80% identity is 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity.

[0015] In some embodiments, the LCDRs and HCDRs of the antibody or antigen-binding fragment thereof are assigned according to the Kabat numbering system.

[0016] In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain variable region (VL) and / or a heavy chain variable region (VH) selected from the following combinations:

[0017] (1) the light chain variable region comprises the sequence set forth in SEQ ID NO. 17, and / or the heavy chain variable region comprises the sequence set forth in any one of SEQ ID NO. 21, 18, 19, 20, 22; or

[0018] (2) the heavy chain variable region and / or the light chain variable region has a sequence that is at least 80% identical to the heavy chain variable region and / or light chain variable region of any one of the above (1) group, or a sequence that differs by up to 3 insertions, deletions, or substitution mutations; preferably, the at least 80% identity is 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity.

[0019] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain constant region sequence and / or a light chain constant region sequence; optionally, the heavy chain constant region and / or light chain constant region is selected from a complete constant region sequence or a fragment thereof, the constant region fragment comprising CH1, a hinge region, CH2, CH3, or Fc; optionally, the heavy chain constant region is selected from a human or murine IgG1, IgG2, IgG3, or IgG4 constant region, and the light chain constant region is selected from a human or murine kappa constant region or lambda constant region; optionally, the antibody or antigen-binding fragment thereof comprises a complete light chain and a complete heavy chain, the light chain consisting of the VL and a light chain constant region having the sequence set forth in SEQ ID NO. 12, and the heavy chain consisting of the VH and a heavy chain constant region having the sequence set forth in SEQ ID NO. 11.

[0020] In some embodiments, the antibody or antigen-binding fragment thereof is: (1) a chimeric antibody or fragment thereof; (2) a humanized antibody or fragment thereof; and / or, (3) a fully human antibody or fragment thereof.

[0021] Preferably, the antibody or antigen-binding fragment thereof is selected from a monoclonal antibody, a polyclonal antibody, a natural antibody, an engineered antibody, a monospecific antibody, a multispecific antibody (e.g., a bispecific antibody), a monovalent antibody, a multivalent antibody, a full-length antibody, an antibody fragment, a naked antibody, a conjugated antibody, a humanized antibody, a fully human antibody, a Fab, a Fab’, a F(ab’)2, a Fd, a Fv, a scFv, a diabody, a nanobody, or an affibody.

[0022] In some embodiments, the antigen binding fragment is selected from one or more of a F(ab)2, Fab', Fab, Fv, scFv, nanobody, or affibody.

[0023] In some embodiments, the cytotoxic drug is selected from a microtubulin inhibitor, a DNA damaging agent, or a DNA topoisomerase inhibitor; the microtubulin inhibitor includes, but is not limited to, dolastatin, auristatin, maytansine, Tubulysins, and cryptomycins; the DNA damaging agent includes, but is not limited to, PBDs; the DNA topoisomerase inhibitor includes, but is not limited to, camptothecins.

[0024] In some embodiments, the cytotoxic drug is a DNA topoisomerase inhibitor.

[0025] In some embodiments, the cytotoxic drug is a compound of Formula (D-I),

[0026] wherein,

[0027] R 1 , R 2 and the atom to which they are attached together form a 5-6 membered heterocyclic ring containing 1 or 2 oxygen atoms as ring atoms, which 5-6 membered heterocyclic ring is optionally substituted with one or more deuterium atoms;

[0028] R 4 is selected from H or C1-C3 alkyl;

[0029] R 5 is selected from H, halogen, CN, OH, NH2, or C1-C3 alkyl;

[0030] R 6 is selected from H or C1-C3 alkyl;

[0031] R 7 is selected from H, C1-C3 alkyl, or C3-C6 cycloalkyl, which C1-C3 alkyl or C3-C6 cycloalkyl is optionally substituted with deuterium, halogen, CN, =0, OH, NH2, or C1-C3 alkyl.

[0032] In some embodiments, the R 1 , R 2 and the atom to which they are attached together form

[0033] In some embodiments, R 4 is H.

[0034] In some embodiments, R 5 is H.

[0035] In some embodiments, R 6 is H.

[0036] In some embodiments, R 7 is cyclopropyl.

[0037] In some embodiments, the compound of Formula (D-I) is selected from

[0038] In some embodiments, the linker unit is covalently attached to the antibody or antigen-binding fragment thereof at the a terminus and covalently attached to the cytotoxic drug D at the b terminus, wherein:

[0039] Ring A is selected from R b1 , R b2 are each independently selected from H, halogen, CN, C1-C6 alkyl, or C3-C6 cycloalkyl, or R b1 , R b2 and the carbon atom to which they are attached together form a C3-C6 cycloalkyl or 4-7 membered heterocyclyl, which is optionally substituted with one or more substituents selected from halogen, CN, =0, C1-C6 alkyl, OH, 0(C1-C6 alkyl), NH2, NH(C1-C6 alkyl), N(C1-C6 alkyl)2, C3-C6 cycloalkyl, and 4-7 membered heterocyclyl;

[0040] m1 is selected from an integer between 2 and 8;

[0041] L a is selected from a chemical bond or covalently attached to L b , R b3 is selected from H or C1-C6 alkyl, m2 is selected from an integer between 1 and 8, R b4 , R b5 are each independently selected from H, halogen, CN, C1-C6 alkyl, C3-C6 cycloalkyl,

[0042] L b is selected from a peptide residue consisting of 1 to 8 amino acids, which is optionally substituted with one or more substituents selected from halogen, CN, =0, C1-C6 alkyl, OH, 0(C1-C6 alkyl), NH2, NH(C1-C6 alkyl), N(C1-C6 alkyl)2, C3-C6 cycloalkyl, and 4-7 membered heterocyclyl.

[0043] In some embodiments, ring A is selected from R b1 , R b2 and the carbon atom to which they are attached together form a 4-7 membered heterocyclyl group, which is optionally substituted with one or more halogen, CN, =0, C1-C6 alkyl, OH, 0(C1-C6 alkyl), NH2, NH(C1-C6 alkyl), N(C1-C6 alkyl)2, C3-C6 cycloalkyl, and 4-7 membered heterocyclyl.

[0044] In some embodiments, ring A is selected from R b1 , R b2 and the carbon atom to which they are attached together form a 4-7 membered heterocyclyl group.

[0045] In some embodiments, ring A is selected from

[0046] In some embodiments, L a is selected from a bond or wherein the a end is covalently attached to L b , R b3 is selected from H or C1-C6 alkyl; R b4 , R b5 one is selected from H and the other is selected from

[0047] In some embodiments, L a is selected from a bond or wherein the a end is covalently attached to L b .

[0048] In some embodiments, the L b is a Gly-Gly-Phe-Gly tetrapeptide residue.

[0049] In some embodiments, m1 is an integer from 2 to 6.

[0050] In some embodiments, m1 is 2, 3, 4, or 5.

[0051] In some embodiments, the linker unit L is wherein the a end is covalently attached to the antibody or antigen binding fragment thereof and the b end is covalently attached to a drug unit.

[0052] In some embodiments, the n is selected from a real number from 1 to 16, for example n is selected from a real number from 2 to 12, for example n is selected from a real number from 4 to 10, for example n is selected from a real number from 3 to 9, for example n is selected from a real number from 4 to 8, for example n is selected from a real number from 6 to 8.

[0053] In some embodiments, n is selected from real numbers from 3 to 9, for example, n is 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, or 9.0.

[0054] In some embodiments, n is selected from real numbers from 6 to 8, for example, n is 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, or 8.0.

[0055] In some embodiments, the antibody-drug conjugate or a pharmaceutically acceptable salt thereof is selected from the following compounds or a pharmaceutically acceptable salt thereof:

[0056] wherein Pc and n are as defined in any of the preceding.

[0057] In a second aspect, the present disclosure provides an antibody or an antigen binding fragment thereof specifically binding to Cadherin-17 (CDH17), wherein the antibody or the antigen binding fragment thereof comprises a light chain variable region and a heavy chain variable region of the following combinations of LCDRs and HCDRs:

[0058] (1) LCDR1, LCDR2 and LCDR3 of the sequences as shown in SEQ ID NO. 23, 24, 25, and HCDR1, HCDR2 and HCDR3 of the sequences as shown in SEQ ID NO. 26, 27, 30;

[0059] (2) LCDR1, LCDR2 and LCDR3 of the sequences as shown in SEQ ID NO. 23, 24, 25, and HCDR1, HCDR2 and HCDR3 of the sequences as shown in SEQ ID NO. 26, 28, 30;

[0060] (3) LCDR1, LCDR2 and LCDR3 as set forth in SEQ ID NO. 23, 24, 25, and HCDR1, HCDR2 and HCDR3 as set forth in SEQ ID NO. 26, 29, 30; or,

[0061] (4) six CDRs having 1, 2, 3 or more amino acid insertions, deletions and / or substitutions or having at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical sequences compared to the sequences of the six CDRs of any one of (1) to (3).

[0062] In some embodiments, the antibody or antigen-binding fragment thereof has a light chain variable region and a heavy chain variable region combination as set forth below:

[0063] (1) the light chain variable region and the heavy chain variable region comprise the sequences set forth in SEQ ID NO. 17 and SEQ ID NO. 18, respectively;

[0064] (2) the light chain variable region and the heavy chain variable region comprise the sequences set forth in SEQ ID NO. 17 and SEQ ID NO. 19, respectively;

[0065] (3) the light chain variable region and the heavy chain variable region comprise the sequences set forth in SEQ ID NO. 17 and SEQ ID NO. 20, respectively;

[0066] (4) the light chain variable region and the heavy chain variable region comprise the sequences set forth in SEQ ID NO. 17 and SEQ ID NO. 21, respectively;

[0067] (5) the light chain variable region and the heavy chain variable region comprise the sequences set forth in SEQ ID NO. 17 and SEQ ID NO. 22, respectively; or

[0068] (6) the light chain variable region comprises a sequence having 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to the light chain variable region set forth in any one of (1) to (5) above, and the heavy chain variable region comprises a sequence having 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to the heavy chain variable region set forth in any one of (1) to (5) above.

[0069] In some embodiments, the antibody or antigen-binding fragment thereof can comprise a light chain and / or a heavy chain constant region sequence.

[0070] In some embodiments, the heavy chain constant region can comprise a heavy chain constant region sequence of a human or murine antibody IgGl, IgG2, IgG3, IgG4, IgA, IgM, IgE, or IgD.

[0071] In some embodiments, the heavy chain constant region comprises a constant region sequence of a human or murine antibody IgGl, IgG2, IgG3, or IgG4, or a sequence having 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to a constant region sequence of a human or murine antibody IgGl, IgG2, IgG3, or IgG4.

[0072] In some embodiments, the heavy chain constant region comprises a heavy chain constant region derived from a human IgGl antibody.

[0073] In some embodiments, the light chain constant region comprises a constant region of a human or murine lambda or kappa chain.

[0074] In another aspect, the present disclosure provides a multispecific antigen binding molecule comprising: (1) a CDH17 antibody or antigen binding fragment thereof as previously described; and (2) an antibody or antigen binding fragment thereof that binds to another antigen other than CDH17, or an antibody or antigen binding fragment thereof that binds to a different epitope of CDH17 than the aforementioned antibody or antigen binding fragment thereof.

[0075] In some embodiments, the other antigen other than CDH17 can comprise: CD3 (preferably CD3e), CD16, NKG2D.

[0076] In some embodiments, the multispecific antigen binding molecule can be bispecific, trispecific, or tetraspecific; preferably, the multispecific antigen binding molecule can be bivalent, trivalent, tetravalent, pentavalent, or hexavalent.

[0077] In another aspect, the present disclosure provides one or more isolated nucleic acid molecules, which can be an isolated form of nucleotides, deoxynucleotides, and / or ribonucleotides of any length, encoding the aforementioned antibody or antigen binding fragment thereof, or the aforementioned multispecific antigen binding molecule.

[0078] In another aspect, the present disclosure provides a vector comprising the aforementioned isolated nucleic acid molecule.

[0079] In another aspect, the present disclosure provides a host cell comprising the aforementioned nucleic acid molecule, or an isolated host cell comprising the aforementioned vector; preferably, the host cell is a eukaryotic cell or a prokaryotic cell; more preferably, the host cell is derived from a mammalian cell, a yeast cell, an insect cell, E. coli, and / or B. subtilis; more preferably, the host cell is selected from Expi293 or CHO cells.

[0080] In another aspect, the present disclosure provides a method of preparing the aforementioned antibody or antigen binding fragment thereof or multispecific antigen binding molecule.

[0081] In another aspect, the present disclosure provides a pharmaceutical composition comprising the aforementioned antibody or antigen binding fragment thereof, the aforementioned multispecific antigen binding molecule or antibody-drug conjugate, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient.

[0082] In some embodiments, the pharmaceutical composition further comprises an additional therapeutic agent, such as another antibody, typically comprising a PD-1 / PD-L1 inhibitor; or a chemotherapeutic drug, for example.

[0083] In another aspect, the present disclosure provides a method of treating a tumor in a mammal, comprising administering to a mammal, preferably a human, in need of such treatment, a therapeutically effective amount of the aforementioned antibody or antigen binding fragment thereof, the aforementioned multispecific antigen binding molecule or the aforementioned antibody-drug conjugate of the general formula Pc-(L-D)n, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof.

[0084] In some embodiments, the method further comprises using an additional therapeutic agent, such as another antibody, typically comprising a PD-1 / PD-L1 inhibitor; or a chemotherapeutic drug, for example.

[0085] In another aspect, the present disclosure provides the use of the aforementioned antibody or antigen binding fragment thereof, the aforementioned multispecific antigen binding molecule or the aforementioned antibody-drug conjugate of the general formula Pc-(L-D)n, or a pharmaceutically acceptable salt thereof, or the aforementioned pharmaceutical composition in the manufacture of a medicament for treating a tumor.

[0086] In some embodiments, the pharmaceutical composition further comprises an additional therapeutic agent, such as another antibody, typically comprising a PD-1 / PD-L1 inhibitor; or a chemotherapeutic drug, for example.

[0087] In another aspect, the present disclosure provides the use of the aforementioned antibody or antigen binding fragment thereof, the aforementioned multispecific antigen binding molecule or the aforementioned antibody-drug conjugate of the general formula Pc-(L-D)n, or a pharmaceutically acceptable salt thereof, or the aforementioned pharmaceutical composition in the manufacture of a medicament for treating a tumor.

[0088] In some embodiments, the pharmaceutical composition further comprises an additional therapeutic agent, such as another antibody, typically comprising a PD-1 / PD-L1 inhibitor; or a chemotherapeutic drug, for example.

[0089] In some embodiments, the tumor is a CDH17-expressing tumor.

[0090] In some embodiments, the tumor is selected from the group consisting of gastric cancer, pancreatic cancer, colorectal cancer, and gastrointestinal stromal tumor.

[0091] In another aspect, the present disclosure provides the antibody or the antigen binding fragment thereof, the multispecific antigen binding molecule or the antibody-drug conjugate, or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition for treating a tumor.

[0092] Beneficial effects: The antibody of the present disclosure has the following advantages:

[0093] 1. The affinity and binding activity of the humanized antibody after modification from murine origin to human origin are higher than those of the positive molecule to the CDH17 target, and the affinity and binding activity of the humanized molecule do not decrease significantly compared with the murine molecule;

[0094] 2. As of the date of completion of the invention, there is no ADC drug for the CDH17 target on the market, only one is in clinical trial I, and the number of CDH17-ADC molecules disclosed is very small. By studying a new ADC targeting CDH17, the in vitro and in vivo efficacy of the ADC molecules for two different drug-linker combinations are better than those of the positive control molecules, which is of great significance for supplementing the treatment of colorectal cancer, gastric cancer, pancreatic cancer and other cancers. BRIEF DESCRIPTION OF DRAWINGS

[0095] FIG. 1A is the expression level of CDH17 of overexpressed cell HEK293T-huCDH17;

[0096] FIG. 1B is the expression level of CDH17 of overexpressed cell HER293T-cyno CDH17;

[0097] FIG. 2A-FIG. 2B are the binding activity of huAb humanized antibody to human hCDH17-his protein detected by ELISA;

[0098] FIG. 2C-FIG. 2D are the binding activity of huAb humanized antibody to monkey cynoCDH17-his protein detected by ELISA;

[0099] FIG. 2E is the binding activity of huAb humanized antibody to human CDH17 at the cellular level detected by FACS;

[0100] FIG. 2F is the binding activity of huAb humanized antibody to monkey CDH17 at the cellular level detected by FACS;

[0101] FIG. 3A is the expression level of CDH17 in tumor cell SNU16;

[0102] FIG. 3B is the binding activity of huAb humanized antibody to human tumor cell SNU16 detected by FACS;

[0103] FIG. 4 is the expression level of CDH17 in tumor cells HT55, T84 and LS1034;

[0104] Figure 5A is the evaluation of the killing activity of ADC molecules ADC-1, ADC-2 and control antibody ADC-3 on tumor cell line HT55;

[0105] Figure 5B is the evaluation of the killing activity of ADC molecules ADC-1, ADC-2 and control antibody ADC-3 on tumor cell line T84;

[0106] Figure 5C is the evaluation of the killing activity of ADC molecules ADC-1, ADC-2 and control antibody ADC-3 on tumor cell line LS1034;

[0107] Figure 6A is the growth curve of human colon cancer HT55 tumor model regulated by ADC molecules ADC-1, ADC-2 and control antibody ADC-3;

[0108] Figure 6B is the effect of ADC molecules ADC-1, ADC-2 and control antibody ADC-3 on the body weight of HT55 tumor-bearing mice;

[0109] Figure 7 is the pharmacokinetics of ADC molecules ADC-1, ADC-2 and control antibody ADC-3 in wild-type Balb / c mice;

[0110] Figure 8A is the regulation result of CDH17-ADC candidate molecules on the tumor volume of T84 tumor-bearing mice;

[0111] Figure 8B is the effect of CDH17-ADC candidate molecules on the body weight change of T84 tumor-bearing mice;

[0112] Figure 9A is the dynamic change of tumor volume of human colorectal cancer PDX model;

[0113] Figure 9B is the monitoring of the body weight change rate of human colorectal cancer PDX model. DETAILED DESCRIPTION

[0114] The advantages and features of the present disclosure will become apparent from the description taken in conjunction with the accompanying drawings. The specific conditions not mentioned in the examples are carried out according to the conventional conditions or the conditions recommended by the manufacturer. The reagents or instruments used are not specified by the manufacturer, and are all conventional products that can be purchased on the market.

[0115] The embodiments of the present disclosure are only exemplary and do not constitute any limitation on the scope of the present disclosure. Those skilled in the art should understand that the details and forms of the technical solutions of the present disclosure can be modified or replaced without departing from the spirit and scope of the present disclosure, and these modifications and replacements all fall within the protection scope of the present disclosure.

[0116] Definitions and explanations of terms

[0117] Unless otherwise indicated, the terms used in the present disclosure have the following meanings, the definitions of the groups and terms recited in the present disclosure, including the definitions as examples, exemplary definitions, preferred definitions, definitions recited in the tables, definitions of specific compounds in the examples, etc., can be combined and incorporated with each other arbitrarily. A particular term should not be considered indefinite or unclear without a specific definition, but should be understood according to the ordinary meaning in the art. When a trade name appears herein, it is intended to refer to its corresponding product or active ingredient thereof.

[0118] In addition, unless otherwise indicated herein, the singular form of a term herein is intended to include the plural form of the term, and the plural form of the term is intended to include the singular form. More specifically, as used in this specification and the appended claims, the singular forms "a," "an" and "the" include plural referents unless expressly identified otherwise.

[0119] The terms "comprise", "include" and "have", which are used herein, are intended to mean the inclusion of the element or elements indicated, but not the exclusion of other elements. It is also to be understood that the description herein using "comprise", "include" and "have" also provides a "consisting of" scheme.

[0120] The term "and / or", when used herein, includes the meaning of "and", "or", and "all or any other combination of the elements linked to the term by the term".

[0121] The term CDH17 (Cadherin-17), also known as "Hepatointestinal cadherin" and "Intestinal peptide transporter HPT-1", belongs to the 7D-cadherin family. CDH17 is often expressed in embryonic, adult intestinal epithelial cells and some pancreatic duct epithelial cells, but there is almost no significant expression in hepatocytes, esophageal epithelial cells and gastric mucosa in healthy people. The characteristics of CDH17 can be described by international patent application WO2008 / 026008, which is incorporated herein by reference in its entirety. The complete amino acid sequence of an exemplary human CDH17 has Genbank accession number NM_004063.

[0122] The term "antigen binding molecule" is used herein in the broadest sense, and refers to a molecule that specifically binds to an antigen. Exemplarily, antigen binding molecules include, but are not limited to, antibodies or antibody mimetics. "Antibody mimetics" refers to organic compounds or binding domains that are capable of specifically binding to an antigen, but are not related to the structure of antibodies. Exemplarily, antibody mimetics include, but are not limited to, affibody, affitin, affilin, designed ankyrin repeat protein (DARPin), aptamer or Kunitz-type domain peptide.

[0123] The term "Affibody" is a small, non-immunoglobulin-based affinity protein that has a triple-stranded β-sheet, a compact globular structure at the molecular level, and the ability to bind to a specific target molecule with high affinity and low immunogenicity.

[0124] The term "antibody" herein includes intact antibodies and any antigen binding fragment (i.e., "antigen binding portion") or single chains thereof. An "antibody" refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, or an antigen binding portion thereof. Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. Each light chain is comprised of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The constant regions of the antibodies can mediate the binding of the immunoglobulin to host tissues or factors including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system.

[0125] The term "variable region" refers to the region of an antibody heavy or light chain that is involved in binding the antibody to an antigen, "heavy chain variable region" is used interchangeably with "VH", "HCVR", and "light chain variable region" is used interchangeably with "VL", "LCVR". The variable domains of the heavy chain and light chain (VH and VL, respectively) of a native antibody generally have similar structures, each domain comprising four conserved framework regions (FRs) and three hypervariable regions (HVRs). A single VH or VL domain can be sufficient to confer antigen binding specificity.

[0126] The term "complementarity determining region" or "CDR" is used interchangeably herein to refer to the hypervariable region of a heavy chain variable region (VH) or light chain variable region (VL) that is responsible for binding antigen, also known as a hypervariable loop (HVL), where the heavy chain variable region CDRs can be abbreviated as HCDRs and the light chain variable region CDRs can be abbreviated as LCDRs. The term "framework region" or "FR region" is used interchangeably herein to refer to those amino acid residues of a heavy chain variable region or light chain variable region that are not included in the CDRs. Typically, a canonical antibody variable region is composed of 4 FR regions and 3 CDR regions in the following order: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

[0127] The CDRs herein can be annotated and defined in ways known in the art, including but not limited to the Kabat, Chothia, IMGT, AbM, Martin, or AHo numbering systems. The CDRs herein include overlaps and subsets of amino acid residues defined in different ways.

[0128] The term "light chain constant region" herein refers to the carboxy-terminal portion of an antibody light chain that is not directly involved in binding of the antibody to an antigen, which is selected from a constant kappa domain or a constant lambda domain. Each of the five classes of Ig can have either a kappa or a lambda chain.

[0129] The term "heavy chain constant region" herein refers to the carboxy-terminal portion of an antibody heavy chain that is not directly involved in binding of the antibody to an antigen, but exhibits effector functions such as interaction with Fc receptors, which has a more conserved amino acid sequence relative to the variable domain of the antibody. The "heavy chain constant region" is selected from a CH1 domain, a hinge region, a CH2 domain, a CH3 domain, or a variant or fragment thereof. The "heavy chain constant region" includes both "full-length heavy chain constant region" and "heavy chain constant region fragment", the former having substantially similar structure as the native antibody constant region, while the latter includes only "a portion of the full-length heavy chain constant region". Exemplarily, a typical "full-length antibody heavy chain constant region" consists of a CH1 domain-hinge region-CH2 domain-CH3 domain; when the antibody is a heavy chain antibody, then it does not include the CH1 domain. Exemplarily, a typical "heavy chain constant region fragment" is selected from an Fc or a CH3 domain. "Immunoglobulin" is classified into five classes according to the different heavy chains, or called isotypes of immunoglobulin, i.e., IgM, IgD, IgG, IgA and IgE, with their corresponding heavy chains being μ chain, δ chain, γ chain, α chain and ε chain, respectively. The same class of Ig can be further divided into different subclasses according to the differences in their hinge region amino acid composition and the number and location of heavy chain disulfide bonds, such as IgG can be divided into IgG1, IgG2, IgG3, IgG4, and IgA can be divided into IgA1 and IgA2.

[0130] The term "Fc" herein refers to the carboxy-terminal portion of an intact antibody that results from papain digestion, which typically comprises the CH3 and CH2 domains of an antibody. Although the boundaries of the Fc region of an immunoglobulin heavy chain might vary slightly, the Fc region of a human IgG heavy chain typically extends from the amino acid residue of Cys226 or from Pro230 to the carboxy-terminus. The C-terminal lysine (residue 447 according to the Kabat numbering system) of the Fc region can be removed, for example, during production or purification of the antibody, or by recombinant engineering of nucleic acid encoding the antibody heavy chain, and therefore the Fc region can or can not include Lys447.

[0131] An "antibody" herein includes alternative protein scaffolds or artificial scaffolds with grafted complementarity determining regions (CDRs) or CDR derivatives. Such scaffolds include antibody-derived scaffolds (which comprise mutations introduced to, for example, stabilize the three-dimensional structure of the antibody) as well as wholly synthetic scaffolds comprising, for example, biocompatible polymers. Such scaffolds can also include non-antibody-derived scaffolds, such as scaffold proteins known in the art to be useful for grafting CDRs.

[0132] An "antibody" can be derived from any animal, including but not limited to humans and non-human animals selected from the group consisting of primates, mammals, rodents, and vertebrates, such as a camelid (e.g., a llama, a guanaco, an alpaca), a sheep, a rabbit, a mouse, a rat, or a chondrichthyan (e.g., a shark).

[0133] The term "specifically binds" herein refers to an antigen binding molecule (e.g., an antibody) that typically binds an antigen and substantially the same antigen with high affinity, but does not bind an unrelated antigen with high affinity. Affinity is typically reflected in the equilibrium dissociation constant (KD), where a lower KD indicates a higher affinity. KD is calculated as follows: KD = Kd / Ka, where Kd is the off-rate and Ka is the on-rate. Equilibrium dissociation constants KD can be measured using methods well known in the art, such as surface plasmon resonance (e.g., Biacore) or equilibrium dialysis. By way of example, high affinity, with respect to an antibody, typically refers to a KD of about 10 -8 M or lower, about 10 -9 M or lower. In some embodiments, an antibody or antigen binding fragment thereof of the present disclosure can bind the human CDH17 with an affinity lower than 10 -8 M. In some embodiments, an antibody or antigen binding fragment thereof of the present disclosure can bind the human CDH17 with an affinity lower than 10

[0134] As used herein, the terms "single domain antibody" (sdAb), "VHH", "nanobody" have the same meaning and are used interchangeably to refer to the variable region of a heavy chain of a cloned antibody, which is constructed to consist of only one heavy chain variable region (VH), which is the smallest antigen-binding fragment with full functionality. The heavy chain variable region (VH) consists of 3 CDR regions and 4 FR regions, arranged in the order of FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 from the amino terminal to the carboxyl terminal.

[0135] The term "minimal recognition unit of an antibody" refers to an antibody containing only one complementarity determining region (CDR) polypeptide, also known as hypervariable region polypeptide, which has a molecular mass of only about 1% of the intact antibody, but still can bind to the corresponding antigen.

[0136] The term "multispecific antibody" refers to an antibody having at least two antigen binding sites, each of which binds to a different epitope of the same antigen or to different epitopes of different antigens. Thus, terms such as "bispecific", "trispecific", "tetraspecific", and the like refer to the number of different epitopes to which an antibody / antigen binding molecule can bind.

[0137] The term "valency" denotes the presence of a defined number of binding sites in an antibody / antigen binding molecule. Thus, the terms "monovalent", "bivalent", "tetravalent" and "hexavalent" denote the presence of one, two, four and six binding sites, respectively, in an antibody / antigen binding molecule.

[0138] An "antigen binding fragment" and "antibody fragment" herein are used interchangeably herein and do not possess all of the structural features of a whole antibody, but only a portion of the whole antibody that is sufficient to retain its ability to bind to an antigen. Illustratively, an "antigen binding fragment" or "antibody fragment" herein includes, but is not limited to, Fab, F(ab')2, Fab', Fab'-SH, Fd, Fv, scFv, diabodies, and single domain antibodies.

[0139] Papain digestion of a whole antibody produces two identical antigen binding fragments, called "Fab" fragments, each containing one heavy and one light chain variable domain, as well as the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. Thus, the term "Fab fragment" herein refers to a light chain fragment comprising the VL and constant domains (CL) of the light chain, and a heavy chain fragment comprising the VH and first constant domain (CH1) of the heavy chain. Fab' fragments differ from Fab fragments by the addition of a few residues at the carboxy terminus of the heavy chain CH1 domain, including one or more cysteines from the antibody hinge region. Fab'-SH is the designation herein for Fab' in which the cysteine residue(s) of the constant domains carry a free thiol group. Pepsin treatment yields an F(ab')2 fragment that has two antigen binding sites (two Fab fragments) and a part of the Fc region.

[0140] An "Fv" fragment is the minimum fragment of IgG and IgM that contains the entire antigen binding site, the Fv fragment has the same binding characteristics as a Fab and a similar three-dimensional structure, the VH and VL chains of the Fv fragment are held together by non-covalent interactions.

[0141] The term "scFv" (single-chain variable fragment) refers to a single polypeptide chain comprising a VL and a VH domain, wherein the VL and VH are connected by a linker. Such scFv molecules can have the general structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH. Suitable prior art linkers include the group consisting of repeating glycine, serine amino acid sequences or variants thereof. For example, a peptide linker having the amino acid sequence (G4S)n, wherein n is an integer equal to or greater than 1, e.g., n is an integer of 2, 3, 4, 5, 6 or 7, can be used. In some cases, there can also be a disulfide bond between the VH and VL of the scFv, forming a disulfide-bonded Fv (dsFv).

[0142] The term "chimeric antibody" refers to an antibody in which a portion of the light chain or / and the heavy chain is derived from one antibody (which can be derived from a particular species or belong to a particular antibody class or subclass), and the other portion of the light chain or / and the heavy chain is derived from another antibody (which can be derived from the same or a different species or belong to the same or a different antibody class or subclass), but retains the binding activity for the target antigen. For example, the term "chimeric antibody" can include an antibody (e.g., a human murine chimeric antibody) in which the heavy and light chain variable regions of the antibody are from a first antibody (e.g., a murine antibody), while the heavy and light chain constant regions of the antibody are from a second antibody (e.g., a human antibody).

[0143] The term "humanized antibody" refers to a non-human-derived antibody that has been genetically engineered to have an amino acid sequence that is modified to increase homology to a sequence of a human-derived antibody. Typically, a humanized antibody has all or a portion of the CDR regions from a non-human-derived antibody (the donor antibody) and all or a portion of the non-CDR regions (e.g., variable region FRs and / or constant regions) from a human-derived immunoglobulin (the acceptor antibody). A humanized antibody typically retains or partially retains the desired properties of the donor antibody, including but not limited to, antigen specificity, affinity, reactivity, ability to enhance immune cell activity, ability to enhance immune response, etc.

[0144] The term "fully human antibody" refers to an antibody having variable regions in which both the FR and CDR are derived from human germline immunoglobulin sequences. Furthermore, if the antibody comprises a constant region, the constant region is also derived from a human germline immunoglobulin sequence. A fully human antibody herein can include amino acid residues that are not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by in vitro random or site-specific mutagenesis or by in vivo somatic mutation).

[0145] The term "naked antibody" herein refers to an antibody that is not linked to, fused to, or conjugated to another agent or molecule (e.g., a label or a drug), a peptide or a polypeptide. In particular embodiments, a naked antibody expressed by a mammalian host cell can be glycosylated by the glycosylation machinery (e.g., glycosylation enzymes) of the host cell. In certain embodiments, a naked antibody is not glycosylated when expressed by a host cell that does not have its own glycosylation machinery (e.g., glycosylation enzymes). In some embodiments, a naked antibody is an intact antibody.

[0146] The term "conservative amino acid" herein generally refers to amino acids that belong to the same class or have similar characteristics (e.g., charge, side chain size, hydrophobicity, hydrophilicity, main chain conformation, and rigidity). Exemplarily, the amino acids within each of the following groups belong to conservative amino acid residues to each other, and the substitution of the amino acid residues within a group belongs to the substitution of conservative amino acid:

[0147] 1) alanine (A), serine (S), threonine (T);

[0148] 2) aspartic acid (D), glutamic acid (E);

[0149] 3) asparagine (N), glutamine (Q);

[0150] 4) arginine (R), lysine (K), histidine (H);

[0151] 5) isoleucine (I), leucine (L), methionine (M), valine (V); and

[0152] 6) phenylalanine (F), tyrosine (Y), tryptophan (W).

[0153] The term "identity" as used herein can be calculated as follows: to determine the percent "identity" of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., matrices such as PAM250 or GAP can be used with default parameters to determine the alignment). The percent identity is then determined by comparing the amino acid residues or nucleotides at each aligned position, with identical amino acid residues or nucleotides occupying the same position in both sequences. The percent identity between two sequences is therefore the number of identical positions shared by the sequences, taking into account the number and length of any gaps introduced to achieve an optimal alignment.

[0154] The term "antibody drug conjugate" (ADC) refers to an antibody or antigen binding fragment thereof linked to a biologically active drug through a stable linker unit. The linkage can be a covalent bond or a non-covalent interaction (e.g., through electrostatic forces). Various linkers known in the art can be used to form the immunoconjugate.

[0155] The term "DAR" or "drug antibody ratio" refers to the average number of small molecule cytotoxic drugs linked per antibody molecule. In the antibody-drug conjugates of the present disclosure, the DAR is defined by the variable "n", which can be either an integer or can include a decimal.

[0156] The phrase "n is a real number from 1 to 16" as used herein means that n is any real number greater than or equal to 1 and less than or equal to 16.

[0157] The phrase "n is a real number from 1 to 16" as used herein means that n is any real number greater than or equal to 1 and less than or equal to 16. represents a site of attachment.

[0158] The diagrammatic representation of racemic or enantiomerically pure compounds in this article is derived from Maehr, J. Chem. Ed. 1985, 62:114-120. Unless otherwise specified, wedge bonds and virtual wedge bonds are used. The absolute configuration of a solid center is represented by black solid bonds and imaginary bonds. It indicates the relative configuration of a stereocenter (such as the cis-trans configuration of alicyclic compounds).

[0159] The term "stereoisomer" refers to isomers that are produced by different spatial arrangements of atoms in a molecule, including cis-trans isomers, enantiomers and diastereomers.

[0160] The compounds disclosed herein may have asymmetric atoms such as carbon, sulfur, nitrogen, and phosphorus atoms, or asymmetric double bonds, and therefore may exist in specific geometric or stereoisomeric forms. Specific geometric or stereoisomeric forms may be cis and trans isomers, E- and Z-type geometric isomers, (-)- and (+)-enantiomers, (R)- and (S)-enantiomers, diastereomers, (D)- isomers, (L)- isomers, and racemic mixtures thereof or other mixtures, such as mixtures enriched with enantiomers or diastereomers. All such isomers and mixtures thereof are within the scope of the definition of the compounds disclosed herein. Alkyl groups or other substituents may contain additional asymmetric carbon atoms, asymmetric sulfur atoms, asymmetric nitrogen atoms, or asymmetric phosphorus atoms. All such isomers involved in all substituents, and mixtures thereof, are also included within the scope of the definition of the compounds disclosed herein. The compounds containing asymmetric atoms disclosed herein can be isolated in optically active pure form or in racemic form. The optically active pure form can be separated from racemic mixtures or synthesized using chiral starting materials or chiral reagents.

[0161] The term "substituted" refers to the substitution of one or more hydrogen atoms on a specific atom by a substituent, provided that the valence state of the specific atom is normal and the resulting compound is stable. When the substituent is oxo (i.e., =O), it means that two hydrogen atoms are substituted; oxo substitution does not occur on aromatic groups.

[0162] The term "optionally" or "optional" means that the subsequently described event or circumstance can or can not occur, and this description includes instances where the event or circumstance occurs and instances where it does not. For example, an ethyl group "optionally" substituted with a halogen means that the ethyl group can be unsubstituted (CH2CH3), mono-substituted (CH2CH2F, CH2CH2C1, etc.), poly-substituted (CHFCH2F, CH2CHF2, CHFCH2C1, CH2CHC12, etc.), or fully substituted (CF2CF3, CF2CC13, CC12CC13, etc.). It will be understood by those skilled in the art that, for any group containing one or more substituents, no substitution or substitution pattern is introduced that is not

[0163] C m -C n means having an integer number of carbon atoms in the range m-n.

[0164] The term "alkyl" means a hydrocarbon group of formula C n H 2n+1 which alkyl group can be straight-chained or branched. The term "C1-C6alkyl" is to be understood as meaning a straight-chained or branched saturated hydrocarbon group having 1, 2, 3, 4, 5 or 6 carbon atoms. Said alkyl groups include, but are not limited to, methyl, ethyl, propyl, butyl, pentyl, hexyl, isopropyl, isobutyl, sec-butyl, tert-butyl, isopentyl, 2-methylbutyl, 1 -methylbutyl, 1 -ethylpropyl, 1,2-dimethylpropyl, neopentyl, 1,1 -dimethylpropyl, 4-methylpentyl, 3-methylpentyl, 2-methylpentyl, 1 -methylpentyl, 2-ethylbutyl, 1 -ethylbutyl, 3,3-dimethylbutyl, 2,2-dimethylbutyl, 1,1 -dimethylbutyl, 2,3-dimethylbutyl, 1,3-dimethylbutyl or 1,2-dimethylbutyl and the like; the term "C1-C3alkyl" means an alkyl group containing 1 to 3 carbon atoms, for example methyl, ethyl, n-propyl, isopropyl.

[0165] A "C1-C6alkyl" group as described herein can further comprise a "C1-C3alkyl" group.

[0166] The term "cycloalkyl" means a carbocyclic ring which is fully saturated and which exists in a monocyclic, fused ring, bridged ring or spiro ring form, and the like. The term "C3-C6cycloalkyl" is to be understood as meaning a saturated monocyclic, fused ring, spiro ring or bridged ring having 3 to 6 carbon atoms, specific examples including, but not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and the like.

[0167] The term "heterocyclyl" refers to a monocyclic, bicyclic, spiro, or bridged ring radical which is completely saturated or partially saturated, which contains from 1 to 5 heteroatoms or heteroatom groups (i.e., groups of atoms containing heteroatoms) in its ring atoms, said "heteroatoms or heteroatom groups" including, but not limited to, nitrogen (N), oxygen (O), sulfur (S), phosphorus (P), boron (B), -S(=0)2-, -S(=0)-, -P(=0)2-, -P(=0)-, -NH-, -S(=0)(=NH)-, -C(=0)NH-, or -NHC(=0)NH-, and the like. The term "4-7 membered heterocyclyl" refers to a heterocyclyl group having a number of ring atoms which is 4, 5, 6, or 7, and which contains from 1 to 3 heteroatoms or heteroatom groups independently selected from the above-mentioned heteroatoms or heteroatom groups. The term "5-6 membered heterocyclyl" refers to a heterocyclyl group having a number of ring atoms which is 5 or 6, and which contains from 1 to 3 heteroatoms or heteroatom groups independently selected from the above-mentioned heteroatoms or heteroatom groups. Examples of 4-membered heterocyclyl groups include, but are not limited to, azetidinyl, oxetanyl; examples of 5-membered heterocyclyl groups include, but are not limited to, tetrahydrofuranyl, dioxolyl, pyrrolidinyl, imidazolidinyl, pyrazolidinyl, pyrrolinyl, 4,5-dihydrooxazolyl, or 2,5-dihydro-lH-pyrrolyl; examples of 6-membered heterocyclyl groups include, but are not limited to, tetrahydropyranyl, piperidinyl, morpholinyl, dithianyl, thiomorpholinyl, piperazinyl, trithianyl, tetrahydropyridinyl, or 4H-[l,3,4]thiadiazinyl; examples of 7-membered heterocyclyl groups include, but are not limited to, diazepanyl. The "4-7 membered heterocyclyl" can include the range of "4-7 membered heterocycloalkyl", "5-6 membered heterocyclyl", "5-6 membered heterocycloalkyl", and the like.

[0168] The term "halo" or "halogen" refers to fluorine, chlorine, bromine, and iodine.

[0169] The term "nucleic acid" herein includes any compound and / or substance comprising a polymer of nucleotides. Each nucleotide is composed of a base, in particular a purine or pyrimidine base (i.e. cytosine (C), guanine (G), adenine (A), thymine (T) or uracil (U)), a five-carbon sugar (i.e. deoxyribose or ribose), and a phosphate group. Typically, a nucleic acid molecule is described by the sequence of bases, whereby the bases represent the primary structure (linear structure) of the nucleic acid molecule. The sequence of bases is usually denoted 5' to 3'. In the present context, the term nucleic acid molecule encompasses deoxyribonucleic acids (DNA), including, for example, complementary DNA (cDNA) and genomic DNA, ribonucleic acids (RNA), in particular messenger RNA (mRNA), synthetic forms of DNA or RNA, and polymers comprising a mixture of two or more of these molecules. A nucleic acid molecule can be linear or circular. Furthermore, the term nucleic acid molecule includes both the sense and anti-sense strands, as well as single- and double-stranded forms.

[0170] As used herein, the term "vector" includes nucleic acid vectors, such as DNA vectors (e.g., plasmids), RNA vectors, viruses, or other suitable replicons (e.g., viral vectors). A variety of vectors have been developed for the delivery of polynucleotides encoding foreign proteins into prokaryotic or eukaryotic cells. The expression vectors of the present disclosure contain polynucleotide sequences as well as additional sequence elements, e.g., for expressing proteins and / or for integrating these polynucleotide sequences into the genome of a mammalian cell. Certain vectors that can be used to express the antibodies and antibody fragments of the present disclosure include plasmids containing regulatory sequences that direct transcription of the gene, such as promoter and enhancer regions. Other useful vectors for expressing antibodies and antibody fragments contain polynucleotide sequences that enhance the rate of translation of these genes or improve the stability or nuclear export of mRNA produced from transcription of the genes. These sequence elements include, e.g., 5' and 3' untranslated regions, internal ribosome entry sites (IRES), and polyadenylation signal sites in order to direct efficient transcription of the genes carried on the expression vectors. The expression vectors of the present disclosure can also contain a polynucleotide that encodes a marker for selecting cells that contain such a vector. Examples of suitable markers include genes that encode antibiotic (e.g., ampicillin, chloramphenicol, kanamycin, or neomycin) resistance.

[0171] The term "host cell" as used herein refers to a cell into which foreign nucleic acid has been introduced, including the progeny of such a cell. Host cells include "transformants" and "transformed cells," which include both the primary transformed cell and progeny of the primary transformed cell that have been transformed, regardless of the number of passages. Progeny can not be completely identical to the parent cell in nucleic acid content, but can contain mutations. Mutant progeny that have the same function or biological activity as screened or selected for in the initially transformed cell are included herein.

[0172] The term "treatment" refers to a surgical or therapeutic treatment whose purpose is to prevent, slow down (reduce), or halt the progression of an unwanted physiological change or pathological condition, such as cancer, autoimmune disease, and viral infection, in a subject. Beneficial or desired results include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. Subjects in need of treatment include those who have a condition or disease, those who are predisposed to having a condition or disease, or those who are seeking to prevent a condition or disease. When referring to the terms slow down, reduce, diminish, palliate, ameliorate, and the like, the meaning also includes elimination, disappearance, nonoccurrence, and the like.

[0173] The term "effective amount" means the amount of a therapeutic agent, administered alone or in combination with another therapeutic agent, which is effective to prevent or alleviate the symptoms or progression of the disease. The term "effective amount" also refers to the amount of a compound that is sufficient to effect a reduction in a condition, e.g., to treat, cure, prevent or slow the progression of the relevant medical condition, or to increase the rate of treatment, cure, prevention or slowing of these conditions. When the active ingredient is administered individually to an individual, the therapeutically effective dose refers to that ingredient alone. When a combination is used, the therapeutically effective dose refers to the combined amounts of the active ingredients that produce the therapeutic effect, whether administered in combination, serially or simultaneously.

[0174] The term "subject" refers to an organism that receives treatment for a particular disease or condition as described herein. Examples of subjects and patients include mammals such as humans, primates (e.g., monkeys) or non-primate mammals that receive treatment for a disease or condition.

[0175] The amount of a compound of the present disclosure that constitutes a "therapeutically effective amount" will vary depending on the compound, the disease state and its severity, the manner of administration, and the age of the mammal to be treated, but can be determined routinely by one of ordinary skill in the art considering the knowledge in the art and the disclosure.

[0176] The term "pharmaceutically acceptable" refers to those compounds, materials, compositions, and / or dosage forms that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0177] The term "pharmaceutically acceptable salt" refers to pharmaceutically acceptable salts of acids or bases, including salts of compounds with inorganic or organic acids, and salts of compounds with inorganic or organic bases.

[0178] The term "pharmaceutical composition" refers to a mixture of one or more compounds of the present disclosure or salts thereof with a pharmaceutically acceptable excipient. The purpose of a pharmaceutical composition is to facilitate administration of a compound of the present disclosure to an organism.

[0179] The term "pharmaceutically acceptable excipient" refers to those excipients that are not biologically or otherwise undesirable, and that do not interfere with the biological activity of the active compound. Suitable excipients are well known to those skilled in the art, e.g., carbohydrates, waxes, water soluble and / or swellable polymers, hydrophilic or hydrophobic materials, gelatin, oils, solvents, water, and the like.

[0180] The present disclosure also includes isotopically-labeled compounds of the present disclosure which are identical to those recited herein, but for the fact that one or more atoms are replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number usually found in nature. Examples of isotopes that can be incorporated into compounds of the present disclosure include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorous, sulfur, fluorine, iodine, and chlorine, such as 2 H, 3 H, 11 C, 13 C, 14 C, 13 N, 15 N, 15 O, 17 O, 18 O, 31 P, 32 P, 35 S, 18 F, 123 I, 125 I, and 36 Cl, and the like.

[0181] Certain isotopically-labeled compounds of the present disclosure (for example, those 3 H, and 14 C) can be used in compound and / or substrate tissue distribution analysis. Tritiated (i.e., 3 H) and carbon-14 (i.e., 14 C) isotopes are particularly preferred for their ease of preparation and detectability. Positron emitting isotopes such as 15 O, 13 N, 11 C, and 18 F can be used in positron emission tomography (PET) studies to determine substrate occupancy. Isotopically-labeled compounds of the present disclosure can generally be prepared by

[0182] The pharmaceutical compositions of the present disclosure can be adapted for parenteral administration, such as sterile solutions, suspensions, or lyophilized products in suitable unit dosage form. For example, the pharmaceutical compositions of the present disclosure can be in the form of sterile injectable aqueous solutions. Other solvents or solvents systems can be acceptable for the pharmaceutical compositions of the present disclosure when in use, such as water, Ringer's solution, or isotonic sodium chloride solution.

[0183] The pharmaceutical compositions described herein can further comprise a therapeutic antibody or a chemotherapeutic drug, illustratively a PD-1 / PD-L1 inhibitor. The pharmaceutical compositions can be packaged as a combination package in one pharmaceutical package, or the anti-CDH17 antibody or pharmaceutical conjugate thereof described herein can be packaged separately from the other therapeutic agent in different pharmaceutical packages.

[0184] The compounds disclosed herein can be prepared by a variety of synthetic methods known to those skilled in the art, including the specific embodiments listed below, embodiments formed by combining them with other chemical synthetic methods, and equivalent substitutions known to those skilled in the art. Preferred embodiments include, but are not limited to, the embodiments disclosed herein.

[0185] The chemical reactions in the specific embodiments of this disclosure are carried out in a suitable solvent, which must be suitable for the chemical changes of this disclosure and the reagents and materials required therefor. In order to obtain the compounds of this disclosure, it is sometimes necessary for those skilled in the art to modify or select the synthesis steps or reaction flow based on existing embodiments.

[0186] An important consideration in synthetic route planning in this field is the selection of appropriate protecting groups for reactive functional groups (such as amino and carboxyl groups in this disclosure). For example, see Greene's Protective Groups in Organic Synthesis (4th Ed). Hoboken, New Jersey: John Wiley & Sons, Inc. All references cited in this disclosure are incorporated herein by reference in their entirety.

[0187] The term "cancer" in this document refers to or describes a physiological condition in mammals characterized by unregulated cell growth. This definition includes both benign and malignant cancers. The term "tumor" or "tumor" in this document refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues. The terms "cancer" and "tumor" are not mutually exclusive when used herein. Non-limiting examples of cancer include gastric cancer, colorectal cancer, and pancreatic cancer.

[0188] The term "EC50" in this document refers to the half-maximal effective concentration, which includes the antibody concentration that induces a half-range response between baseline and maximum value after a specified exposure time. EC50 essentially represents the antibody concentration in which 50% of its maximum effect is observed, and can be measured by methods known in the art.

[0189] The term "IC50" in this article refers to the half-maximal inhibitory concentration, which is the amount of inhibitor that reduces the reaction rate by half when the inhibitor concentration reaches half of its maximum.

[0190] Example

[0191] Example 1: Preparation method of drug-linker compound

[0192] 1.1 The structure of the drug-linker L1-1 is shown below, and its preparation is described in patent document WO2023217227A1 (Example 39).

[0193] 1.2 Synthesis of drug-linker L3-1

[0194] Synthesis route:

[0195] Step 1: Synthesis of tert-butyl 3-(2-cyclopentathio-5-oxo-7,8-dihydropyrido[4,3-d]pyrimidin-6(5H)-yl)propionate (intermediate 3-2)

[0196] At room temperature, cyclopentylthiol (367 mg) and 2 M sodium hydroxide aqueous solution (1.65 mL) were added sequentially to a solution of compound 3-1 (1.066 g) dissolved in DCM (10 mL). The resulting mixture was stirred at room temperature for 30 min. The mixture was then separated by column chromatography (EA / PE, 0-50%) to obtain the crude title compound, which was used directly in the next reaction.

[0197] Step 2: Synthesis of tert-butyl 3-(2'-(cyclopentylthio)-5'-oxo-2,3,5,6-tetrahydro-5'H-spiro[pyrano-4,8'-pyrido[4,3-d]pyrimidine]-6'(7'H)-yl)propionate (intermediate 3-3)

[0198] Intermediate 3-2 (200 mg) was dissolved in N,N-dimethylformamide (2 mL) at 0 °C under a nitrogen atmosphere. NaH (127.15 mg, 60% effective content) and 1-iodo-2-(2-iodoethoxy)ethane (518.01 mg) were added. The reaction mixture was stirred at 0 °C for 1 h under nitrogen protection. Ice water (2 mL) was then added to the reaction mixture, followed by extraction with ethyl acetate (1 mL × 3). The combined organic phases were dried over anhydrous sodium sulfate, concentrated under reduced pressure, and separated by column chromatography (ethyl acetate / petroleum ether, 0-60%) to give the title compound (110 mg).

[0199] MS m / z (ESI): 448.2 [M+H] + .

[0200] Step 3: Synthesis of tert-butyl 3-(2'-(cyclopentanesulfonyl)-5'-oxo-2,3,5,6-tetrahydro-5'H-spiro[pyrano-4,8'-pyrido[4,3-d]pyrimidine]-6'(7'H)-yl)propionate (intermediate 3-4)

[0201] Intermediate 3-3 (60 mg) was dissolved in anhydrous dichloromethane (1 mL) at 0 °C, and m-chloroperoxybenzoic acid (108.86 mg, 85% purity) was added. The reaction mixture was stirred at 25 °C for 1 h. After the reaction was completed, the solution was concentrated under reduced pressure and separated by column chromatography (ethyl acetate / petroleum ether, 0-55%) to give the title compound (9.8 mg).

[0202] MS m / z (ESI): 480.3 [M+H] + .

[0203] 1 H NMR (400MHz, Chloroform-d) δ = 9.40 (s, 1H), 4.35-4.18 (m, 1H), 4.00 (m, 2H), 3.86-3.72 (m, 6H), 2.68 (t, J= 6.3Hz,2H),2.31-2.23(m,2H),2.18(m,2H),2.06(m,2H),1.93-1.81(m,2H),1.77-1.65(m,4H),1.45(s,9H)

[0204] Step 4: Synthesis of 3-(2'-(cyclopentanyl)-5'-oxo-2,3,5,6-tetrahydro-5'H-spiro[pyran-4,8'-pyrido[4,3-d]pyrimidine]-6'(7'H)-yl)propionic acid (intermediate 3-5)

[0205] Intermediate 3-4 (680 mg) was dissolved in dichloromethane (10 mL), and then trifluoroacetic acid (0.68 mL) was added to the reaction solution. The reaction solution was stirred at 25 °C for 1 hour. After the reaction was complete, the solvent was removed by direct concentration under reduced pressure. The residue was purified by rapid silica gel column chromatography. 12g The title compound (200 mg) was obtained by rapid silica column chromatography with a gradient of 0–53% tetrahydrofuran / petroleum ether at a flow rate of 40 mL / min.

[0206] MS m / z (ESI): 423.8 [M+H] + .

[0207] Step 5: Synthesis of (S)-5-allyloxycarbonyl-1-(9H-fluorene-9-yl)-8,11,14,17,20,23,26,29,32-nonamethyl-3,7,10,13,16,19,22,25,28,31-decaoxo-2-oxa-4,8,11,14,17,20,23,26,29,32-decaazatritetradecane-34-carboxylic acid (intermediates 3-6)

[0208] This compound was synthesized using a peptide solid-phase synthesis method, as follows:

[0209] 1) Dichloromethane was added to a mixture of CTC resin (0.64 mmol / g, 12.5 g) and N-(((9H-fluorene-9-yl)methoxy)carbonyl)-N-methylglycine (2.50 g) under nitrogen protection;

[0210] 2) Add N,N-diisopropylethylamine (DIEA) dropwise and mix and stir for 2 hours;

[0211] 3) Add methanol (13 mL) and mix and stir for 0.5 h;

[0212] 4) Filter and wash three times with N,N-dimethylformamide (DMF);

[0213] 5) Add 20% piperidine / DMF solution and react for 30 min;

[0214] 6) Filter and wash five times with DMF;

[0215] 7) Add the substances listed in the "Ingredients" column of the table below and mix for 30 seconds, then add the substances listed in the "Reagents" column of the table below. The reaction is carried out under nitrogen bubbling conditions for 1 hour, where Fmoc-Sar-OH represents N-(((9H-fluoren-9-yl)methoxy)carbonyl)-N-methylglycine, and Fmoc-Asp-OAll-OH represents (S)-3-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-4-(allyloxy)-4-oxobutyric acid;

[0216] 8) Repeat steps 4-7 to complete the desired peptide synthesis.

[0217] After solid-phase preparation, a 20% solution of 1,1,1,3,3,3-hexafluoroisopropanol in dichloromethane was added to the resulting mixture and the mixture was stirred for 1.5 h. The mixture was then filtered, the filtrate was collected and concentrated, and the resulting mixture was purified by preparative liquid chromatography (A: 0.075% TFA in H2O, B: acetonitrile) to give the title compound (2.15 g).

[0218] MS m / z (ESI): 1035.6 [M+H] + .

[0219] Step 6: Synthesis of (S)-30-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)-1-amino-3,6,9,12,15,18,21,24,27-nonamethyl-1,4,7,10,13,16,19,22,25,28-decaoxo-3,6,9,12,15,18,21,24,27-nonazatrione-31-carboxylic acid allyl ester (intermediate 3-7)

[0220] Intermediate 3-6 (1 g), ammonium chloride (155.09 mg), diisopropylethylamine (499.44 mg), and HATU (728.95 mg) were added to DMF (5 mL), purged three times with nitrogen, and stirred at 25 °C for 3 h. After the reaction was complete, the crude product was purified by reversed-phase high-performance liquid chromatography (RP-HPLC). The title compound (800 mg) was obtained by mixing 80 g of C 18 column with a mobile phase gradient of 0–40% acetonitrile / water at a flow rate of 40 mL / min.

[0221] MS m / z (ESI): 1034.6 [M+H] + .

[0222] Step 7: Synthesis of (S)-30-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)-1-amino-3,6,9,12,15,18,21,24,27-nonamethyl-1,4,7,10,13,16,19,22,25,28-decaoxo-3,6,9,12,15,18,21,24,27-nonazatrione-31-carboxylic acid (intermediates 3-8)

[0223] Intermediate 3-7 (800 mg), tetraphenylphosphine palladium (201.84 mg), and 1,3-dimethylbarbituric acid (108.71 mg) were added to DMF (5 mL), purged three times with nitrogen, and stirred at 25 °C for 4 h. After the reaction was complete, the crude product was purified by reversed-phase high-performance liquid chromatography (RP-HPLC). The title compound (700 mg) was obtained by mixing 80 g of C 18 column with a mobile phase gradient of 0–40% acetonitrile / water at a flow rate of 40 mL / min.

[0224] MS m / z (ESI): 994.9 [M+H] + .

[0225] Step 8: Synthesis of resin-supported (2S,10S,19S)-19-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)-48-amino-10-benzyl-2-cyclopropyl-22,25,28,31,34,37,40,43,46-nonamethyl-6,9,12,15,18,21,24,27,30,33,36,39,42,45,48-pentadecaoxo-3-oxa-5,8,11,14,17,22,25,28,31,34,37,40,43,46-tetradecanoic acid (intermediates 3-9)

[0226] Intermediate 3-8 (700 mg) was added to DMF (10 mL), followed by intermediate L1-25 (400 mg resin plus substrate, approximately 0.18 mmol, prepared according to the method described in Example 22 of WO2025 / 103367), O-benzotriazole-tetramethylurea hexafluorophosphate (336.58 mg), and diisopropylethylamine (205.62 mg). The reaction mixture was purged with nitrogen three times and then reacted at 25 °C with shaking for 1 h. After the reaction, the resin was washed sequentially with methanol (10 mL) and dichloromethane (10 mL), repeated three times. The resin was then filtered to dryness and dried under vacuum to obtain the title compound (420 mg).

[0227] MS m / z (ESI): 1461.4 [M+Na] + .

[0228] Step 9: Synthesis of resin-supported (2S,10S,19S)-19,48-diamino-10-benzyl-2-cyclopropyl-22,25,28,31,34,37,40,43,46-nonamethyl-6,9,12,15,18,21,24,27,30,33,36,39,42,45,48-pentadecaoxo-3-oxa-5,8,11,14,17,22,25,28,31,34,37,40,43,46-tetradecanoic acid (intermediate 3-10)

[0229] Intermediate 3-9 (420 mg) was dissolved in DMF (5 mL), and piperidine (1.25 mL) was added. The reaction solution was placed on a shaker at 25 °C and shaken for 1 h. After the reaction was completed, the resin was washed sequentially with methanol (10 mL) and dichloromethane (10 mL), repeated 3 times. The resin was then filtered to dryness and dried under vacuum to obtain the title compound (400 mg).

[0230] MS m / z (ESI): 1217.4 [M+H] + .

[0231] Step 10: Resin-supported (2S,10S,19S)-48-amino-10-benzyl-19-(3-(2'-(cyclopentanesulfonyl)-5'-oxo-2,3,5,6-tetrahydro-5'H-spiro[pyran-4,8'-pyrido[4,3-d]pyrimidine]-6'(7'H)-yl)propionylamino)-2-cyclopropyl-22,25,28,31,34 Synthesis of 37,40,43,46-Nonmethyl-6,9,12,15,18,21,24,27,30,33,36,39,42,45,48-Decadecyloxo-3-oxa-5,8,11,14,17,22,25,28,31,34,37,40,43,46-Tetraazaoctadecane-1-carboxylic acid (intermediate 3-11)

[0232] Intermediate 3-10 (880 mg) and intermediate 3-5 (393.14 mg) were dissolved in N,N-dimethylformamide (15 mL), and O-benzotriazole-tetramethylurea hexafluorophosphate (453.65 mg) and N,N-diisopropylethylamine (313.82 μL) were added. The reaction solution was placed on a shaker at 25 °C and shaken for 1 hour. After the reaction was completed, the resin was washed sequentially with methanol (10 mL) and dichloromethane (10 mL), repeated 3 times. The resin was then filtered to dryness and dried under vacuum to obtain the title compound (920 mg).

[0233] Step 11: (2S,10S,19S)-48-amino-10-benzyl-19-(3-(2-(cyclopentanesulfonyl)-5'-oxo-2,3,5,6-tetrahydro-5'H-spiro[pyran-4,8'-pyrido[4,3-d]pyrimidine]-6'(7'H)-yl)propionylamino)-2-cyclopropyl-22,25,28,31,34,37 Synthesis of 40,43,46-nonamethyl-6,9,12,15,18,21,24,27,30,33,36,39,42,45,48-pentadecaoxo-3-oxa-5,8,11,14,17,22,25,28,31,34,37,40,43,46-tetradecanoic acid-1-carboxylic acid (intermediate 3-12)

[0234] Intermediate 3-11 (920 mg) was added to a mixed solvent of dichloromethane (8 mL) and 1,1,1,3,3,3-hexafluoroisopropanol (2 mL) and shaken at 25 °C for 0.5 hr. After the reaction was complete, the reaction solution was filtered to remove the resin, and the filtrate was concentrated to dryness under reduced pressure. The residue was directly purified by high performance liquid chromatography (column: Welch Xtimate C18 150*30mm*5um; mobile phase: [phase A: water (0.225% formic acid), phase B: acetonitrile]; B%: 18%-38%, 20 min) to obtain the title compound (65 mg).

[0235] Step 12: (S)-N 4 -(26-amino-3,6,9,12,15,18,21,24-octamethyl-2,5,8,11,14,17,20,23,26-nonaoxo-3,6,9,12,15,18,21,24-octaazahexacosane)-N 1 -((4S,12S)-12-benzyl-4-cyclopropyl-1-((S)-7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxopentano[4,5-g]pyrano[3',4':6,7]inzizo[1,2-b]quinoline-14-yl-2,2-d2)-3,8,11,14,17-pentoxo-5-oxa-2,7,10,13,16-pentazaoctadecane-18-yl)-2-(3-(2'-(cyclopentanesulfonyl)-5'-oxo-2,3,5,6-tetrahydro-5'H-spiro[pyran-4,8'-pyridino[4,3-d]pyrimidinyl]-6'(7'H)-yl)propionylamino)-N 4 Synthesis of methylbutyramide (drug linker L3-1)

[0236] Intermediate 3-12 (65 mg), intermediate 2-11 (22.10 mg), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (15.36 mg), pyridine (9.51 mg), and 1-hydroxybenzotriazole (10.82 mg) were dissolved in N,N-dimethylformamide (3 mL). The mixture was purged with nitrogen three times, and the reaction solution was stirred for 2 hours at 25 °C under nitrogen protection. After the reaction was completed, the reaction solution was directly purified by high performance liquid chromatography (column: Welch Xtimate C18 150*30mm*5um; mobile phase: [phase A: water (0.225% formic acid), phase B: acetonitrile]; B%: 21%-51%, 11 min) to obtain the title compound (35 mg).

[0237] 1H NMR (400MHz, DMSO-d6) δ = 9.28 (s, 1H), 8.69-8.55 (m, 2H), 8.30-8.21 (m, 2H), 8.01 (m, 2H), 7.79 (s, 1H), 7.52 (s, 1H), 7.39-7.27 (m, 1H), 7. 26-7.21(m,5H),7.15(s,1H),7.11-7.00(m,1H),6.49(s,1H),5.43(d,J=5.8Hz,4H),4.86-4.70(m,2H),4.70-4.63(m,1H),4.62-4.53(m,1 H),4.52-4.44(m,1H),4.40-4.17(m,10H),4.12-3.89(m,9H),3.86-3.76(m,5H),3.75-3.65(m,9H),3.63-3.52(m,2H),3.51-3.43(m,2H) ,3.00-2.68(m,32H),2.07-1.92(m,6H),1.91-1.80(m,2H),1.71-1.59(m,6H),1.01-0.92(m,1H),0.88(t,J=7.3Hz,3H),0.38-0.27(m,4H)

[0238] MS m / z(ESI): 1014.8 [(M+2) / 2] + .

[0239] The synthesis of intermediate 2-11 is as follows:

[0240] Step 1: Synthesis of 1-(benzo[d][1,3]dioxacyclopenten-5-yl-2,2-d2)ethane-1-one (intermediate 2-5)

[0241] 3',4'-Dihydroxyacetophenone (3 g) was dissolved in anhydrous DMF (25 mL), and deuterated dichloromethane (8.57 g) and potassium carbonate (8.18 g) were added. After the addition was complete, the mixture was heated to 90 °C and stirred for 16 h. The reaction mixture was then added to water (100 mL) and extracted with ethyl acetate (200 mL x 2). The combined organic phases were washed with saturated brine (100 mL) and dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated to dryness under reduced pressure. The residue was purified by column chromatography (ethyl acetate / petroleum ether = 5:1) to give the title compound (2.4 g).

[0242] MS m / z (ESI): 167.1 [M+H] + .

[0243] Step 2: Synthesis of 1-(6-nitrobenzo[d][1,3]dioxacyclopenten-5-yl-2,2-d2)ethane-1-one (intermediate 2-6)

[0244] Intermediate 2-5 (2.4 g) was dissolved in anhydrous acetic acid (10 mL), and concentrated nitric acid (32.50 g, 70% purity) was added dropwise at 0 °C, followed by stirring at 0 °C for 10 min. The mixture was then heated to room temperature and stirred for 1 h. After the reaction was complete, the reaction solution was added dropwise to ice water (200 mL), filtered, and the filter cake was dried to give the title compound (1.9 g).

[0245] MS m / z(ESI): 212.0 [M+H] + .

[0246] 1 H NMR (400MHz, DMSO-d6) δ7.69(s,1H),7.30(s,1H),2.49(s,3H).

[0247] Step 3: Synthesis of N-(6-acetylbenzo[d][1,3]dioxacyclopenten-5-yl-2,2-d2)acetamide (intermediate 2-7)

[0248] Intermediate 2-6 (1.8 g) was dissolved in acetic acid (25 mL), and acetic anhydride (1.84 g) and reduced iron powder (4.76 g) were added. The mixture was stirred at room temperature for 1 h. After the reaction was complete, the mixture was filtered, and the filtrate was concentrated to dryness under reduced pressure. The residue was purified by column chromatography (ethyl acetate / petroleum ether = 5:1) to give the title compound (1.5 g).

[0249] MS m / z(ESI): 224.1 [M+H] + .

[0250] Step 4: Synthesis of N-(6-(2-bromoacetyl)benzo[d][1,3]dioxacyclopenten-5-yl-2,2-d2)acetamide (intermediate 2-8)

[0251] A solution of HBr in acetic acid (2.39 g, 33% purity) was added dropwise to a solution of intermediate 2-7 (1.45 g) in anhydrous acetic acid (25 mL), and then Br was added dropwise. 2( 1.07 g was added dropwise, and the mixture was stirred at room temperature for 1 h. After the reaction was complete, the reaction solution was concentrated to dryness under reduced pressure. The residue was added to water (50 mL), extracted with ethyl acetate (50 mL * 2), the organic phase was washed with saturated brine (50 mL), dried over anhydrous sodium sulfate, filtered, and the residue was purified by column chromatography (ethyl acetate / petroleum ether = 5:1) to give the title compound (1.3 g).

[0252] MS m / z (ESI): 302.1 [M+H] + .

[0253] Step 5: Synthesis of 1-(6-aminobenzo[d][1,3]dioxacyclopenten-5-yl-2,2-d2)-2-chloroethane-1-one (intermediate 2-9)

[0254] Intermediate 2-8 (1.2 g) and concentrated hydrochloric acid (144.82 mg) were dissolved in ethanol (15 mL), and the reaction mixture was stirred at 60 °C for 16 h. After the reaction was completed, the reaction mixture was concentrated to dryness under reduced pressure, and the residue was purified by high performance liquid chromatography (YMC-Actus Triart C18 column, 5 μm silica, 30 mm diameter, 150 mm length; water (containing 0.05% NH4HCO3) and a mixture of acetonitrile with decreasing polarity as the eluent (acetonitrile gradient ratio 40%-50%), to give the title compound (577 mg).

[0255] MS m / z(ESI): 216.0 [M+H] + .

[0256] Step 6: Synthesis of (S)-14-(chloromethyl)-7-ethyl-7-hydroxy-10,13-dihydro-11H-[1,3]dioxacyclopenteno[4,5-g]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-8,11(7H)-dione-2,2-d2 (intermediate 2-10)

[0257] Intermediate 2-9 (100.0 mg) and (S)-4-ethyl-4-hydroxy-7,8-dihydro-1H-pyrano[3,4-f]indazine-3,6,10(4H)-trione (109.87 mg) were dissolved in toluene (1 mL) and acetic acid (1 mL), and pyridinium p-toluenesulfonic acid salt (5.24 mg) was added. The reaction mixture was stirred at 100 °C for 16 h. After the reaction was completed, the mixture was cooled to room temperature and concentrated to dryness under reduced pressure. Ethanol (5 mL) was added, and the mixture was stirred at 25 °C for 0.5 h. The mixture was filtered, and the filter cake was washed with ethanol (5 mL * 2) to obtain the title compound (100.0 mg).

[0258] MS m / z(ESI): 443.0 [M+H] + .

[0259] Step 7: Synthesis of (S)-14-(aminomethyl)-7-ethyl-7-hydroxy-10,13-dihydro-11H-[1,3]dioxacyclopenteno[4,5-g]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-8,11(7H)-dione-2,2-d2 (intermediate 2-11)

[0260] Intermediate 2-10 (100.00 mg) was dissolved in anhydrous ethanol (1.5 mL) and anhydrous DMF (1.5 mL), and hexamethylenetetramine (94.97 mg) was added. The reaction mixture was stirred at 50 °C for 6 h. After the reaction was completed, the reaction mixture was concentrated to dryness under reduced pressure, and the residue was purified by high performance liquid chromatography (column: Boston Green ODS150*30 mm*5 μm; mobile phase: [A: water (formic acid), B: acetonitrile]; B%: 0%-30%, 12 min) to give the title compound (25.0 mg).

[0261] MS m / z(ESI): 424.0 [M+H] + .

[0262] Example 2: Preparation and purification methods of recombinant protein and control antibody

[0263] 2.1 Design and expression of recombinant proteins

[0264] Construction of CDH17 recombinant protein: Using the human CDH17 protein (UniProt: Q12864) as a template sequence, a tagged fusion protein was designed, cloned into the pTT5 vector (Ubibio, VT2202), and the hCDH17-his plasmid was constructed. The disclosed antigen and detection protein were transiently expressed in Expi293F cells (Gibco, A14527). The preparation method of the cynomolgus monkey CDH17 recombinant protein is the same as that of the human recombinant protein. The cynomolgus monkey CDH17 sequence is from NCBI: XP_005563762.2. The specific sequence information of the recombinant protein is shown in Table 1.

[0265] Nickel column purification of recombinant proteins: Cell expression supernatant samples were centrifuged at high speed to remove impurities. The nickel column was equilibrated with 20 mM PBS + 500 mM NaCl solution and washed with 2-5 column volumes. The culture supernatant was loaded onto a Ni affinity chromatography column (GE Healthcare), and the UV absorbance (A280 nm) was monitored using a UV detector. The column was washed with equilibration buffer until the A280 reading returned to baseline. Gradient elution was then performed with equilibration buffers containing 10 mM, 20 mM, 40 mM, 90 mM, 250 mM, and 500 mM imidazole, and each elution peak was collected. The fraction containing the target protein was identified based on the SDS-PAGE gel image. The collected elution product containing the target protein was concentrated and further purified using Superdex 200 (GE) gel chromatography with PBS as the mobile phase to remove aggregates and other protein peaks. The elution peak of the target product was then collected. The obtained protein was identified by electrophoresis, peptide mapping, and LC-MS before being aliquoted and used. The proteins purified using this method include human CDH17-His and monkey CDH17-His.

[0266] Table 1. Human and monkey CDH17-His recombinant protein sequences

[0267] 2.2 Design and expression of control antibodies

[0268] The control antibodies used in this disclosure are all derived from published patents. The hu646, hu653, and hu663 antibodies are derived from the published patent WO2023107558A1 (CDH17-646-h7, CDH17-653-h43, and CDH17-663-h7, respectively, in WO2023107558A1). Unless otherwise specified, the hu646, hu653, and hu663 control antibodies are all recombinantly expressed using the human IgG1+κ subtype (CH and Cκ sequences are shown in Table 2). The negative control anti-FITC-hIgG1 antibody (sequence shown in Table 2) is derived from the literature J Biol Chem. 1990 Jan 5; 265(1):133-8.

[0269] The expression and purification process of the control antibody was as follows: The antibody sequence gene was synthesized and cloned into the expression vector pTT5, then transiently transfected into Expi293F cells (purchased from Gibco, A14527). After culturing at 37°C for 7 days on a shaker, the cell supernatant was collected for Protein A antibody purification. The Protein A affinity column was washed with 3-5 column volumes of 0.1M NaOH, followed by 3-5 column volumes of pure water. The column was equilibrated with 3-5 column volumes of 1×PBS (pH 7.4) buffer. The cell supernatant was loaded at a low flow rate for binding, controlling the flow rate to maintain a retention time of approximately 1 min or longer. After binding, the column was washed with 3-5 column volumes of 1×PBS (pH 7.4) until the UV absorbance returned to baseline. Samples were eluted using a 50 mM citrate / sodium citrate buffer (pH 3.0-3.5). Elution peaks were collected based on UV detection. The eluted products were temporarily stored by rapidly adjusting the pH to 5-6 with 1 M Tris-HCl (pH 8.0). Solution replacement of the eluted products could be performed using methods well-known to those skilled in the art, such as ultrafiltration concentration using an ultrafiltration tube followed by solution replacement to the desired buffer system, or desalting using size exclusion columns such as G-25, or removing aggregate components from the eluted products using a high-resolution size exclusion column such as Superdex 200 to improve sample purity. The resulting control antibodies were named hu646-hIgG1, hu653-hIgG1, and hu663-hIgG1. Specific antibody sequence information is shown in Table 2.

[0270] Table 2. Control antibody sequence listing

[0271] Example 3: Construction of stable cell lines

[0272] The nucleotide sequence encoding the full-length amino acid sequence of human CDH17 (Uniprot ID: Q12864, as shown in Table 3) was cloned into the vector pCMV3-untagged (Sinochem, Cat: CV011) and a plasmid was prepared. HEK293T cells were transfected with the plasmid. The 3000 Transfection Kit (purchased from Invitrogen, catalog number: L3000-015) was used to selectively culture cells in DMEM medium containing 100 μg / mL hygromycin for 2 weeks. Monoclonal cells were seeded into 96-well plates using the limiting dilution method and cultured at 37°C with 5% (v / v) CO2. After approximately 2 weeks, a subset of wells were selected for amplification. The amplified clones were screened by flow cytometry to detect antibodies against human CDH17 antibody (huAb-H3aL3-hIgG1, self-produced; preparation method and sequence are described in sections 4.3 and 4.4 of Example 4) and anti-human IgG (H+L) antibody (Jackson, catalog number: 109-605-088). Cell lines with good growth, high fluorescence intensity, and monoclonal expression were selected for further amplification and cryopreservation in liquid nitrogen. The resulting cell line was named HEK293T-huCDH17, and its expression status as detected by FACS is shown in Figure 1A.

[0273] The nucleotide sequence encoding the full-length amino acid sequence of cynomolgus monkey CDH17 (NCBI ID: >XP_005563762.2, as shown in Table 3) was cloned into the vector pCMV3-untagged (Sinochem, Cat:CV011) and a plasmid was prepared. HEK293T cells were transfected with the plasmid and selectively cultured for 2 weeks in DMEM medium containing 100 μg / mL hygromycin. Single-clone cells were seeded into 96-well plates using the limiting dilution method and cultured at 37°C with 5% (v / v) CO2. After approximately 2 weeks, a subset of single-clone wells were selected for amplification. The amplified clones were screened by flow cytometry to detect human CDH17 antibody and anti-human IgG (H+L) antibody. Cell lines with good growth, high fluorescence intensity, and single clones were selected for further expansion and cryopreservation in liquid nitrogen. The resulting cell line was named HEK293T-cyno CDH17, and its expression was analyzed by FACS, as shown in Figure 1B.

[0274] Table 3. Full-length sequences of human and monkey CDH17 sequences

[0275] Example 4: Antibody Humanization Design

[0276] 4.1 Humanization design of antibodies

[0277] By comparing the IMGT (http: / / imgt.cines.fr) human antibody heavy and light chain variable region germline gene database and MOE (Molecular Operating Environment) software, germline genes of the heavy and light chain variable regions with high homology to murine antibodies were selected as templates. The CDRs of the murine antibodies were transplanted into the corresponding human templates, forming variable region sequences in the order FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Reversion mutations and / or hotspot mutations were performed as needed. In this embodiment, the CDR sequences were divided according to the Kabat numbering system.

[0278] Referring to the method in section 2.2 of Example 2, the murine antibody PTA001_A4 (derived from US9181339B2, which is incorporated herein by reference) was recombinantly expressed using the human IgG1+κ subtype (CH and Cκ sequences are shown in Table 2, SEQ ID NO.11 and SEQ ID NO.12, respectively), and named PTA001_A4-IgG1.

[0279] The humanized light chain templates for PTA001_A4 are IGKV2-40*01 and IGKJ4*01, and the humanized heavy chain templates are IGHV1-69-2*01 and IGHJ6*01. The CDR of the murine antibody PTA001_A4 was transplanted into the corresponding human templates to obtain the humanized antibody huAb of PTA001_A4. Its variable region sequence is shown below:

[0280] huAb HCVR (VH-CDR graft, IGHV1-69-2*01):

[0281] huAb LCVR (VL-CDR graft, IGKV2-40*01):

[0282] 4.2 Design of Reverse Mutations and Hotspot Mutations for PTA001_A4 Humanized Antibody

[0283] As needed, key amino acids in the FR region sequence of the PTA001_A4 humanized antibody were reverse-mutated to ensure the original affinity. Meanwhile, given the presence of a high-risk, easily modified site DG in the CDR region of the PTA001_A4 heavy chain, amino acid mutations were performed on DG using computer simulation based on the antibody structure to eliminate the risk of molecular modification. The specific mutation design is shown in Table 4 (reverse mutations are listed in natural numbering order).

[0284] Table 4. Humanized antibody reverse mutation and hotspot mutation design for PTA001_A4

[0285] Note: Graft represents inserting a mouse antibody CDR into the human FR region sequence; Graft+F29L means mutating F at position 29 of Graft to L, and so on.

[0286] 4.3 PTA001_A4 humanized antibody

[0287] By combining the reversion mutations and hotspot mutations of the humanized PTA001_A4 in Table 4 above, a variety of humanized PTA001_A4 antibodies were finally obtained (see Table 5 for details).

[0288] Table 5. Amino acid sequences corresponding to PTA001_A4 humanized antibody

[0289] Note: huAbH1L3 indicates that the PTA001_A4 humanized antibody huAb has a light chain variable region as described in L3 and a heavy chain variable region as described in H1, and so on.

[0290] The amino acid sequences of the variable regions of the humanized heavy and light chains are shown in Table 6.

[0291] Table 6. Amino acid sequence of the variable region of the PTA001_A4 humanized antibody reversion mutation.

[0292] According to the Kabat numbering system, the CDR sequence analysis results of the heavy and light chain variable regions of the PTA001_A4 humanized antibody are shown in Table 7.

[0293] Table 7. Kabat analysis results of the CDR sequence of the heavy and light chain variable region of the PTA001_A4 humanized antibody.

[0294] 4.4 Construction, Expression, and Purification of Humanized Full-Length Antibodies

[0295] PCR primers were designed to construct the VH / VL gene fragments of each humanized antibody, which were then homologously recombinated with the vector to construct the full-length expression vector of the humanized antibody. The huAb humanized antibodies were expressed in human IgG1 form (heavy chain constant region and light chain constant region sequences are shown in Table 2). After plasmid construction, Expi293F cells were transiently transfected. The supernatant was collected by centrifugation after 7 days, and the antibodies were purified according to the purification method described in section 2.2 of Example 2.

[0296] Example 5: Determination of antibody affinity

[0297] The binding strength between antibodies and antigens was detected using a BIAcore 8K instrument and the Protein A capture method. First, Protein A was immobilized onto a CM4 chip (GE, BR-1005-34) using an amino-coupling method. Following the instructions of the Amine Coupling Kit (GE, BR100633), HBS-EP + pH 7.4 was used as the mobile phase. After mixing NHS and EDC, the chip was activated for approximately 600 seconds. Protein A was then diluted to 50 μg / mL with 10 mM sodium acetate at pH 4.5 and injected for 600 seconds. Finally, the remaining activation sites were blocked with ethanolamine. Then, a multi-cycle kinetic method was used to determine the affinity between the antibody and the antigen. In each cycle, the antibody to be tested was first captured using a Protein A chip, and then a single concentration of antigen protein was injected. The binding and dissociation processes of the antibody and antigen protein were recorded. Finally, the chip was regenerated using Glycine pH 1.5. The mobile phase was HBS-EP+ (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.05% surfactant P2O), the flow rate was 30 μL / min, the regeneration time was 30 s, and the detection temperature was 25 °C. Finally, the data were analyzed according to a 1:1 binding model to fit the antibody-antigen binding kinetic parameters, including the binding rate constant Ka, the dissociation rate constant Kd, the equilibrium dissociation constant KD, and the maximum binding signal Rmax.

[0298] The binding rate (Ka), dissociation rate (Kd), and binding affinity (KD) of each humanized huAb antibody and control antibody to human and monkey CDH17 protein are shown in Table 8.

[0299] Table 8. Affinity of antibody to CDH17 protein as determined by SPR (Biacore).

[0300] Example 6: Identification of antibody binding activity

[0301] 6.1 ELISA detection of antibody binding to human and monkey CDH17 protein

[0302] Human CDH17-his protein was diluted to a final concentration of 1 μg / mL with PBS, and then 50 μl was added to each well of a 96-well ELISA plate and incubated overnight at 4°C. The next day, the plate was washed twice with PBST, and blocking buffer [PBS + 2% (w / w) BSA] was added and blocked at room temperature for 2 hours. The blocking buffer was discarded, and 50 μl of antibody (50 nM, serially diluted 4-fold), positive control antibody, and negative control antibody were added to each well. After incubation at 37°C for 1 hour, the plate was washed three times with PBST. HRP (horseradish peroxidase)-labeled secondary antibody (purchased from Merck, catalog number: AP113P) was added, and after incubation at 37°C for 1 hour, the plate was washed five times with PBST. 50 μl of TMB substrate was added to each well, and after incubation at room temperature for 10 minutes, 50 μl of stop buffer (1.0 M HCl) was added to each well. OD was read using an ELISA plate reader (Multimode Plate Reader, EnSight, purchased from Perkin Elmer). 450nm The numerical values ​​and the binding activity of the antibody to human CDH17-his protein are shown in Figures 2A and 2B. The results showed that the negative control anti-FITC-hIgG1 (from the literature J Biol Chem. 1990 Jan 5; 265(1):133-8) did not bind to CDH17, while the humanized antibodies of huAb could effectively bind to human CDH17-his protein.

[0303] Monkey cyno CDH17-his protein was diluted with PBS to a final concentration of 2 μg / mL, and then 50 μl was added to each well of a 96-well ELISA plate and incubated overnight at 4°C. The binding activity of CDH17 antibody to cyno CDH17-his protein was analyzed using the same detection method as in section 4.1 of Example 4. The results showed that each humanized antibody of huAb could effectively bind to cyno CDH17-his protein (Figures 2C-2D).

[0304] 6.2 Flow cytometry (FACS) assay for antibody binding to CDH17 at the cellular level

[0305] Expand the desired cell line to the logarithmic growth phase, digest and collect the cells, and pipette the cells into a single-cell suspension. After cell counting, centrifuge and resuspend the cell pellet in FACS buffer (PBS + 2% fetal bovine serum) to 2 × 10⁻⁶. 6 Add 100 μl of cells per well to a 96-well FACS plate, centrifuge, discard the supernatant, add 50 μl of the antibody sample to each well, mix well, and incubate at 4°C for 1 hour. Wash three times with PBS buffer by centrifugation, and add 50 μl of Alexa antibody sample to each well. 647AffiniPure Goat Anti-Human IgG, Fcγfragment-specific labeled secondary antibody (purchased from Jackson, catalog number: 109-605-098) was incubated at 4°C for 1 hour. After washing three times with PBS buffer, the cells were resuspended in 100 μl PBS and the results were detected and analyzed using FACS. Data analysis was performed using FlowJo software to obtain the mean fluorescence density (MFI) of the cells. Further analysis was performed using GraphPad Prism8 software for data fitting. The results are shown in Figure 2E. Each humanized huAb antibody specifically bound to human HEK293T-huCDH17 cells, with significantly better binding ability than hu653-hIgG1 and hu663-hIgG1.

[0306] The binding ability of each humanized huAb antibody to monkey CDH17 cells was detected using the same method. The results are shown in Figure 2F. All humanized huAb antibodies can effectively bind to HEK293T-cynoCDH17 cells.

[0307] 6.3 Flow cytometry (FACS) assay for the binding of CDH17 antibody to tumor cells

[0308] The expression of CDH17 in gastric cancer cells SNU16 (Nanjing Kebai, CBP60502) was detected using the method and antibodies described in Example 2. The results are shown in Figure 3A. The cell-binding activity of each humanized antibody was detected as follows: SNU16 cells were cultured in T-175 flasks to the logarithmic growth phase. The culture medium was removed, and the cells were washed twice with PBS buffer. The cells were digested with digestion solution, and then digestion was terminated with complete culture medium. The cells were then pipetted into a single-cell suspension. After cell counting, the cells were centrifuged, and the cell pellet was resuspended in FACS buffer (PBS + 10% fetal bovine serum) to a concentration of 2 × 10⁻⁶. 6 Add 100 μl of cells per well to a 96-well FACS plate, centrifuge, discard the supernatant, add 50 μl of the antibody sample (200 nM as the starting concentration, serially diluted 4-fold) per well, mix well, and incubate at 4°C for 1 hour. Wash three times with PBS buffer by centrifugation, and add 50 μl of Alexa antibody to each well. 647AffiniPure Goat Anti-Human IgG, Fcγfragment-specific labeled secondary antibody (purchased from Jackson, catalog number: 109-605-098) was incubated at 4°C for 1 hour. After washing three times with PBS buffer, the cells were resuspended in 100 μl of PBS and the results were detected and analyzed using FACS (FACS Canto™, purchased from BD). Data analysis was performed using FlowJo software to obtain the mean fluorescence density (MFI) of the cells, followed by data fitting and EC50 calculation using GraphPad Prism8 software. As shown in Figure 3B and Table 9, each humanized antibody of huAb specifically binds to SNU16 cells.

[0309] Table 9. Antibody binding to SNU16 cells

[0310] Example 7: Preparation method of antibody-drug conjugate

[0311] This embodiment describes three methods for preparing ADCs, and their structures are shown in Table 10:

[0312] Table 10 ADC Structure

[0313] 7.1 Coupling reaction of ADC-1 and negative control ADC-1-iso

[0314] The huAb-H3aL3-hIgG1 and Anti-FITC-hIgG1 were dialyzed to PBS phosphate buffer (pH 7.4). 30 molar equivalents of 10 mM tris(2-carboxyethyl)phosphine solution (TCEP, Thermo Scientific #77720) were added to the antibody solution, and the mixture was incubated at 4°C for 17 h to reduce the antibody. 15 molar equivalents of drug-linker L1-1 (the structure and preparation of drug-linker L1-1 are described in Example 1) were dissolved in DMSO and added to the reaction system. The reaction mixture was coupled at 25°C for 4 h. The reaction product was eluted using a Capto SPimRes cation exchanger (Cytiva) gradient to a pH 5.5 sodium acetate + NaCl solution to remove unreacted free small molecule toxins. Finally, the mixture was dialyzed overnight to 559 buffer (10 mM sodium acetate, 9% Sucrose, pH 5.5). The purity and DAR values ​​of the ADC products were analyzed using SEC and LC-MS methods. The obtained ADC and antibody were named ADC-1 and ADC-1-iso, respectively.

[0315] 7.2 Coupling reaction of ADC-2 and negative control ADC-2-iso

[0316] The huAb-H3aL3-hIgG1 and Anti-FITC-hIgG1 were dialyzed into PBS phosphate buffer. 30 molar equivalents of 10 mM tris(2-carboxyethyl)phosphine solution (TCEP, Thermo Scientific #77720) were added to the antibody solution, and the mixture was incubated at 4°C for 17 h to reduce the antibody. 15 molar equivalents of drug-linker L3-1 (the structure and preparation of drug-linker L3-1 are described in Example 1) were dissolved in DMSO and added to the reaction system. The reaction solution was coupled at 25°C for 4 h. The reaction product was eluted using a Capto SPImpRes cation exchanger (purchased from Cytiva) gradient elution to a pH 5.5 sodium acetate + NaCl solution to remove unreacted free small molecule toxins. Finally, the product was dialyzed overnight to a 559 buffer (10 mM sodium acetate, 9% Sucrose, pH 5.5). The purity and DAR value of the ADC product were analyzed using SEC and LC-MS methods. The resulting ADC antibodies were named ADC-2 and ADC-2-iso.

[0317] 7.3 Coupling reaction of ADC-3 and negative control ADC-3-iso

[0318] The hu663-hIgG1 and Anti-FITC-hIgG1 were dialyzed into PBS phosphate buffer. 2.8 molar equivalents of 10 mM tris(2-carboxyethyl)phosphine solution (TCEP, Thermo Scientific #77720) were added to the antibody solution, and the mixture was incubated at 4°C for 17 h to reduce the antibody. 10 molar equivalents of the drug-linker compound (MC-VC-PAB-MMAE, MCE, HY-15575) were dissolved in DMSO and added to the reaction system. The reaction mixture was incubated at 4°C for 6 h. The reaction product was eluted through a gradient on an SP column, with the buffer changed to pH 5.5 sodium acetate + NaCl solution to remove unreacted free small molecule toxins. Finally, the product was dialyzed overnight to 559 buffer (10 mM sodium acetate, 9% Sucrose, pH 5.5). The purity and DAR value of the ADC products were analyzed using SEC and LC-MS methods. The obtained ADC antibodies were named ADC-3 and ADC-3-iso.

[0319] 7.4 ADC Sample Purity Analysis and DAR Value Determination

[0320] SEC Purity Analysis: The SEC-HPLC method was used to analyze the protein samples, characterize the molecular size uniformity of the recombinant protein, and determine the purity of the recombinant protein. The HPLC system used was an Agilent 1260, the column was a TSKgel G3000SWXL (purchased from Tosoh Bioscience), the mobile phase was 200 mM phosphate buffer, pH 7.0, the detection temperature was 25℃, the flow rate was 0.5 mL / min, the detection wavelength was 280 nm, the target protein loading was 50 μg, and the analysis time was 40 min.

[0321] DAR value determination: The DAR value of ADC molecules was measured using ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS). First, the ADC molecule to be tested was treated with PNGase F (NEB#P0705L) to remove the N-sugar modification, and then treated with dithiothreitol (DTT, Sigma#646563) and incubated at 37℃ for 1 h to reduce it to light and heavy chains. Then, it was analyzed using a Thermo Vanquish UHPLC-Q Exactive Plus mass spectrometry system. 2 μg of protein was injected into a Waters ACQUITY Protein BEH size-exclusion column. The mobile phase was an aqueous solution containing 0.1% formic acid, 0.05% TFA, and 25% acetonitrile. The flow rate was 0.2 mL / min, and the analysis time was 30 min. The mass spectrometer was a Thermo Q Exactive Plus. The main mass spectrometry parameters were: spray voltage 3.8 kV, capillary heating temperature 300℃, sheath gas flow rate 35 arb, and precursor ion scan range 800-3000. Finally, the mass spectrometry data was analyzed using the Biopharma Finder software. 4.1 The Respect algorithm was used for deconvolution processing to calculate the molecular weight information of the light and heavy chain mass spectrometry peaks and the mass spectrometry response signals of each component, thereby calculating the DAR value of the ADC sample to be tested. The coupling purity and DAR value detection data for each sample are shown in Table 11.

[0322] Table 11 DAR value and SEC purity of ADC samples

[0323] Example 8: In vitro proliferation inhibition test of ADC on tumor cells

[0324] Human colon cancer cells HT55 (Nanjing Kebai, CBP60012), T84 (Nanjing Kebai, CBP60021T84), and LS1034 (Nanjing Kebai, CBP60013LS1034) were selected for ADC killing assay of tumor cells. The expression level of CDH17 in each tumor cell type is shown in Figure 4 (transformation and detection methods are as described in Example 3). All ADC samples were derived from Example 6; ADC-1 and ADC-2 were candidate molecules, and ADC-3 was a control molecule.

[0325] Cells in the logarithmic growth phase were digested and plated. The inhibitory activity of ADCs on the proliferation of HT55, T84, and LS1034 cells was detected using Cell-Titer Glo reagent (Promega, G7573). HT55 cells (2000 cells per well), T84 cells (2000 cells per well), and LS1034 cells (1500 cells per well) were seeded in 96-well plates and cultured at 37°C and 5% CO2 for 24 hours. ADC molecules were diluted with the corresponding cell culture medium to an initial concentration of 300 nM, and then serially diluted 10-fold with culture medium to obtain 8 concentrations. 200 μL of the prepared ADC solution was transferred to each well of the 96-well plate to a final concentration of 0-300 nM. After adding the ADC solution to be tested, the plates were cultured at 37°C and 5% CO2. HT55, T84, and LS1034 cells were cultured for 6 days, after which Cell-Titer Glo reagent was added, and cell viability was detected.

[0326] Data analysis: Calculate %inhibition (inhibition rate) and fit IC50. 50 % inhibition = 1 - 100% × (Signal-Bottom) / (Top-Bottom). Signal refers to the signal value of the ADC sample group, Bottom refers to the signal value of the sample without cells but with the same volume of culture medium, and Top refers to the signal value of the sample without ADC but with cells.

[0327] Experimental Results: Under the experimental conditions, the ADC candidate molecules ADC-1 and ADC-2 both exhibited good inhibitory activity against the proliferation of the three cell types. The results are shown in Table 12 and Figures 5A-5C. ADC-1 and ADC-2 showed significantly better tumor cell killing ability than the control antibody ADC-3.

[0328] Table 12 Inhibitory effect of ADCs on endogenous tumor cell proliferation Note: " / " indicates that IC cannot be fitted. 50 .

[0329] Example 9: In vivo assay of ADC's inhibitory effect on tumor cell proliferation

[0330] CDH17-positive human colon cancer cells HT55 were selected to establish in vivo mouse models (Balb / c-nude, female, 6-8 weeks old, Vitallix) and these models were used to evaluate the in vivo antitumor efficacy of candidate molecules.

[0331] Human colon cancer cells HT55 were seeded as day 0 of this experiment. On day 0, colon cancer cells (sourced from Nanjing Kebai Biotechnology Co., Ltd., CBP60012) that had been expanded to the required number and were in the logarithmic growth phase (confluence approximately 80%, with the culture medium replaced the day before seeding) were collected and seeded. First, the culture medium in the cell culture flask was removed, and the cells were washed twice with phosphate-buffered saline (PBS, Hyclone, SH30256.01). Then, an appropriate amount of 0.25% trypsin digestion solution (Yeasen, #40127ES60) was added, and the bottom of the flask was gently shaken to ensure the trypsin digestion solution evenly covered the cell surface. The flask was then placed at 37°C for 7 minutes for digestion. Finally, a solution containing 10% fetal bovine serum (Fetal Bovine Serum) was added. The digestion reaction was terminated with complete medium (serum, FBS, Gibco, #10091-148 / 2418958P). Cells adhering to the bottom of the flask were gently blown away from the culture flask. The digested cell suspension was collected into a 50mL centrifuge tube, centrifuged at 350g for 5 minutes, and a suitable amount of serum-free RPMI-1640 medium (Sigma, #R8758-500mL) was used to resuspend the cells. The cells were then filtered through a 70μm sieve. 500μL of the cell suspension was used for cell counting using a cell counter (Beckman Coulter, SIC-TP-573). Finally, based on the cell count results, the cell density was adjusted to 100 × 10⁶ cells / mL using serum-free medium. 6 Cells / mL were placed on ice and transferred through a transfer window to the SPF animal room for inoculation and modeling. Before inoculation, the above cell suspension was mixed with Matrigel (Corning Biotech, #356237) in an equal ratio, and 100 μL of the above cell mixture was subcutaneously injected into the right axilla of each mouse.

[0332] After HT55 inoculation of colon cancer cells, tumor growth was monitored. When the mean overall tumor volume was 110 mm, the tumor growth was monitored. 3Around 1000 mice with suitable tumor volume were randomly divided into groups of 6. Each group received the corresponding drug via tail vein administration according to the protocol, and tumor volume and mouse weight changes were measured. The specific administration protocol is shown in Table 13. PBS served as the negative control group, ADC-1-iso, ADC-3-iso, and ADC-2-iso were isotype controls for different ADCs, ADC-1 and ADC-2 were candidate ADC molecules, and ADC-3 was the positive control ADC molecule. The candidate ADC molecules and positive control molecules were administered at the same dose as the isotype controls to explore the efficacy of low-dose drugs. Since the mice in the negative control PBS group also showed a significant decrease in body weight, it was speculated that the model might exhibit cachexia. Therefore, starting from the fifth day after drug treatment, all mice were fed experimental animal complete nutrition gel (J10001, Ruide Biotechnology) to ensure the drug efficacy monitoring time window.

[0333] Table 13. Dosage regimens for ADC candidate molecules in HT55 colon cancer-bearing mice.

[0334] The formula for calculating the tumor inhibition rate is as follows: Tumor inhibition rate (%) = [1 - (V t (Treatment group) - V0(Treatment group)) / (V t (Negative control group) - V0(Negative control group))]×100%, where V0 is the average tumor volume at the time of grouping, V t This represents the average tumor volume at a single measurement after treatment. Relative change in mouse body weight: RCBW (%) = (BW) i –BW0) / BW0×100%, where BW i BW0 is the body weight after the start of drug administration, and BW0 is the body weight at the time of the first drug administration.

[0335] The results are shown in Figures 6A and 6B and Table 14. On day 20 after administration, the isotype control showed no tumor-suppressing effect, while all tested drug groups showed good efficacy, with ADC-1 and ADC-2 showing significantly better efficacy than ADC-3. During the monitoring period, the relative change rate of mouse body weight in each treatment group was not significantly different from that in the negative control PBS group, suggesting that tumor-bearing mice tolerated the test substances well.

[0336] Table 14. Efficacy of ADC candidate molecules in HT55 colon cancer-bearing mice. Notes: a. Mean ± SD; b. Comparisons between experimental groups were calculated using one-way ANOVA followed by Tukey's method. The p-values ​​on day 20 are shown in the table (ns: not significant, *: P < 0.05, **: P < 0.01, ***: P < 0.001, ****: P < 0.0001); c. "-" indicates no evaluation.

[0337] Example 10: Pharmacokinetics of ADC in Mice

[0338] To compare the pharmacokinetic differences among different antibodies, this example used SPF-grade female wild-type Balb / c mice, 6-8 weeks old, weighing approximately 18-20g, with 3 mice per group. Each mouse was given a single tail vein injection of 1 mg / kg of the test compound to compare their pharmacokinetic differences. The samples used in this example were the three compounds (ADC-1, ADC-2, and ADC-3) prepared in Example 6.

[0339] Mice were fed a standard diet, with no restrictions on food or water. The drug was diluted with physiological saline. Blood was collected from the orbital rim at time points: before drug administration, and 0.25 hours, 2 hours, 8 hours, 24 hours, 72 hours, 120 hours, 168 hours, 240 hours, 336 hours, 504 hours, and 672 hours after drug administration. Blood samples were collected in micro-volume collection tubes and allowed to stand for approximately 30 minutes. The tubes were then centrifuged at 12,000 rpm for 5 minutes at 4°C. Serum was separated into low-adsorption centrifuge tubes, labeled with the compound code and time point, and stored at -80°C before analysis.

[0340] The methods for detecting naked antibody and ADC blood drug concentrations are as follows: For the naked antibody (TA) coated with hCDH17-his protein, the concentration of the compound in serum was determined using an indirect enzyme-linked immunosorbent assay (ELISA). For the ADC coated with hCDH17-his protein, biotinylated anti-small molecule antibody was added, and the concentration of the compound in serum was determined using a sandwich ELISA. Pharmacokinetic parameters were calculated based on the blood drug concentrations of each animal at different time points; specific results are shown in Table 15 and Figure 7.

[0341] The results show that the compounds ADC-1 and ADC-2 prepared in this disclosure exhibit good pharmacokinetic performance and similar pharmacokinetic behavior. The half-lives of ADC-1 and ADC-2 were 201.6 h and 249.32 h, respectively, significantly higher than the 44.78 h of the control group ADC-3. The terminal exposures of ADC-1 and ADC-2 were 1693.87 h*μg / mL and 1645.53 h*μg / mL, respectively, significantly higher than the 431.86 h*μg / mL of the control group ADC-3.

[0342] Table 15 Pharmacokinetic parameters of wild-type Balb / c mice Note: "TA" indicates naked antibody

[0343] Example 11: Efficacy experiment of CDH17-ADC candidate molecule in inhibiting tumor growth in human colon cancer T84 tumor-bearing mice

[0344] 1) Experimental objective:

[0345] The purpose of this experiment was to evaluate the antitumor effect of the CDH17-ADC candidate molecule on the human colon cancer CDX animal model T84 and to compare it with the positive molecule ADC-3.

[0346] 2) Laboratory animals:

[0347] Balb / c nude mice, female, 6-8 weeks old, provided by Beijing Vital River Laboratory Animal Technology Co., Ltd., SPF grade.

[0348] 3) Test instruments:

[0349] CO2 incubator: Thermo Fisher Scientific, 3111

[0350] Cell counter: Beckman Coulter, Vi-cell™ XR, 383556

[0351] Centrifuge: Eppendorf, Centrifuge 5810R

[0352] Electronic balance (for weighing mice): Changshu Shuangjie Testing Instrument Factory, T1000

[0353] Vernier calipers: Guanglu Measurement, digital display vernier calipers

[0354] Thermostatic water bath: Changzhou Aohua Instrument Co., Ltd., HH-4

[0355] Fully Automatic Animal Asphyxiation Machine: Suzhou Fengshi Experimental Animal Equipment Co., Ltd., FSZZ-2A

[0356] 4) Reagent preparation:

[0357] Phosphate-buffered saline (PBS): Hyclone, SH30256.01

[0358] DMEM medium: Gibco, 10569-010

[0359] Fetal bovine serum (FBS): Gibco, A5669701

[0360] Penicillin / Streptomycin (P / S): Gibco, 10091-148

[0361] Matrigel: Corning, 356234

[0362] 0.25% pancreatic enzyme digestion solution: Yeasen, 40127ES60

[0363] 5) Experimental Procedure:

[0364] Human colon cancer T84 cells (10×10)6 100 μL of cells were injected subcutaneously into the right rib area of ​​female Balb / c nude mice (purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.). Eight days after cell inoculation, the tumor grew to approximately 137 mm. 3 Approximately 60 mice were selected based on tumor volume and weight after removing mice with excessively large or small tumors, and then divided into 10 experimental groups of 6 mice each. The day of drug administration was defined as day 0. Tumor volume was measured twice weekly, and data were recorded. Specific grouping information is shown in Table 16 below.

[0365] Table 16 Dosing and Grouping Note: N: Number of animals; IV: Tail vein injection; Single dose: Single administration. Dosage volume: Adjust the dosage volume according to the body weight of the tumor-bearing rat (10 μL / g).

[0366] 6) Data processing:

[0367] Tumor volumes in each group of animals are expressed as mean ± standard deviation (Mean ± SEM), where SEM = SD / SQRT(n), and N = number of animals in the experimental group. Graphpad Prism 8.0 or later software was used for plotting. Two-way ANOVA was used for statistical analysis. For data at each time point, pairwise comparisons between groups were performed using Tukey analysis, and P < 0.05 was considered statistically significant. The tumor inhibition rate was calculated as: TGI (%) = [1 - (T i -T0) / (V i -V0)]×100%, T i T0 represents the average tumor volume at a single measurement after drug administration in the treatment group, while V0 represents the average tumor volume at the first drug administration in the treatment group, and V represents the average tumor volume at the first drug administration in the control group. i The average tumor volume is the result of a single measurement taken after the control group received the drug.

[0368] The formula for calculating tumor volume is: V = 0.5a × b 2 a and b represent the long and wide diameters of the tumor, respectively;

[0369] Simultaneously calculate the relative change in mouse body weight after administration: RCBW(%) = (BW) / (RCB ... i -BW0) / BW0×100%, BW i BW0 represents the body weight at a specific weighing after administration, while BW0 represents the body weight at the time of the first administration.

[0370] 7) Experimental results:

[0371] The results of tumor volume changes in each treatment group are shown in Table 17 and Figure 8A. On day 22 of administration, compared with the PBS control group, ADC-1-iso (10 mg / kg, Single dose), ADC-3-iso (10 mg / kg, Single dose), and ADC-2-iso (10 mg / kg, Single dose) all showed weak or no tumor-suppressing effects, with tumor inhibition rates of 10.37%, 13.33%, and -0.55%, respectively. ADC-1 (10 mg / kg, Single dose) and ADC-2 (10 mg / kg, Single dose) both showed significant tumor-suppressing effects, and their efficacy was significantly better than that of the positive molecule ADC-3 (10 mg / kg, Single dose) group (p < 0.0001), with tumor inhibition rates of 108.20%, 109.34%, and 41.53%, respectively. ADC-1 (3 mg / kg, Single dose) and ADC-2 (3 mg / kg, Single dose) also showed significant tumor-suppressing effects. All tested drugs (3 mg / kg, Single dose) showed significant inhibitory activity against tumor growth, and their efficacy was significantly superior to the positive molecule ADC-3 (3 mg / kg, Single dose) group (p < 0.0001), with tumor inhibition rates of 50.95%, 56.06%, and 2.78%, respectively. During treatment with the tested drugs, no significant weight loss was observed in any group of animals, indicating good safety of the tested drugs. The results of weight changes in mice are shown in Figure 8B.

[0372] Table 17. The therapeutic effects of CDH17-ADC candidate molecules on subcutaneous xenografts in T84 mice. Note: a. Mean ± SD; b. The comparisons among the 10 groups in this experiment were calculated using two-way ANOVA and then analyzed using Tukey's method. The p-values ​​for Day 22 are shown in the table (ns: not significant, *: P < 0.05, **: P < 0.01, ***: P < 0.001, ****: P < 0.0001); c. "-" indicates no evaluation.

[0373] Example 12: Efficacy experiment of CDH17-ADC candidate molecule in inhibiting tumor growth in a human colorectal cancer PDX model.

[0374] 1) Experimental objective:

[0375] To evaluate the in vivo antitumor efficacy of the candidate drug CDH17-ADC in a mouse model of human colorectal cancer patient-derived xenografts (PDX).

[0376] 2) Laboratory animals:

[0377] Female BALB / c nude mice aged 6-8 weeks (weighing 18-22 grams) were purchased from Chengdu Jinkai Biotechnology Co., Ltd. and housed in an SPF-grade environment (independent ventilation cage system). All animals had free access to food and water.

[0378] 3) Test instruments:

[0379] Tumor inoculation needle: Cadence, catalog number 7928, lot number W006128 / 1

[0380] Vernier calipers: Mitutoyo, model 500-171-20

[0381] Analytical balance: Mettler Toledo, model XS205DU

[0382] Electronic scale: Changshu Shuangjie, model JJ300Y

[0383] Matrix adhesive: YESEN Biotechnology, Product No. 40183ES10, Batch No. C3315050

[0384] HBSS buffer: BasalMedia, catalog number B420KJ, lot number D211224

[0385] Antibiotic-Antifungal Agent: BasalMedia, Catalog No. S120JV, Lot No. F121104

[0386] PBS buffer: HyClone, catalog number SH30256.01, lot number AK30787934

[0387] 4) Experimental procedure:

[0388] In this experiment, the colorectal cancer PDX model was derived from patient samples who had received multiple lines of therapy (including bevacizumab, pyrotinib, capecitabine, cetuximab, XELOX, and FOLFIRI). The PDX mouse model was established by subcutaneously inoculating passaged tumor tissue blocks into female BALB / c nude mice. The model was established when the tumor volume reached 148 mm². 3 At approximately 10:00 AM, the animals were randomly divided into 6 groups (6 animals in each group) and given a single intravenous injection of the following: PBS control group, 10 mg / kg ADC-3, 3 mg / kg ADC-3, 10 mg / kg ADC-2, 3 mg / kg ADC-2, and 1 mg / kg ADC-2.

[0389] 5) Data processing:

[0390] Tumor volumes in each group of animals are expressed as mean ± standard deviation (Mean ± SEM), where SEM = SD / SQRT(n), and N = number of animals in the experimental group. Graphpad Prism 8.0 or later software was used for plotting. Two-way ANOVA was used for statistical analysis. For data at each time point, pairwise comparisons between groups were performed using Tukey analysis, and P < 0.05 was considered statistically significant. The tumor inhibition rate was calculated as: TGI (%) = [1 - (T i -T0) / (V i -V0)]×100,T i T0 represents the average tumor volume at a single measurement after drug administration in the treatment group, while V0 represents the average tumor volume at the first drug administration in the treatment group, and V represents the average tumor volume at the first drug administration in the control group. i This represents the average tumor volume at a single measurement after the control group received the drug. The formula for calculating tumor volume is: V = 0.5a × b 2 a and b represent the long and wide diameters of the tumor, respectively; simultaneously, the relative change in mouse body weight after drug administration was calculated as: RCBW(%) = (BW) / (BW) i –BW0) / BW0×100%, BW i BW0 represents the body weight at a specific weighing after administration, while BW0 represents the body weight at the time of the first administration.

[0391] 6) Test Results

[0392] The experimental results are shown in Table 18, Figure 9A, and Figure 9B. On day 28 post-treatment, compared to the blank control group (PBS): ADC-3: both the 10 mg / kg and 3 mg / kg dose groups showed moderate but statistically significant tumor-suppressing effects. The tumor growth inhibition rate (TGI) was 78.04% and 77.52%, respectively. ADC-2 groups: the 10 mg / kg, 3 mg / kg, and 1 mg / kg dose groups showed significant dose-dependent tumor-suppressing effects, with TGI values ​​of 106.21%, 95.26%, and 43.51%, respectively. The tumor-suppressing effect of the 10 mg / kg ADC-2 dose group was significantly better than that of the equivalent dose of ADC-3 (p<0.05), and 3 / 6 (50%) of the mice in the 10 mg / kg ADC-2 group achieved complete remission (CR). All treatment groups were well-tolerated, and there was no significant difference in body weight change compared to the blank control group.

[0393] Table 18. Efficacy evaluation of CDH17-targeted antibody-drug conjugates in a PDX mouse model of colorectal cancer. Note: a. Mean ± SD; b. Comparisons among the 6 groups in this experiment were calculated using two-way ANOVA and then analyzed using Tukey's method. The p-values ​​for Day 28 are shown in the table (ns: not significant, *: P < 0.05, **: P < 0.01, ***: P < 0.001, ****: P < 0.0001); c. "-" indicates no evaluation.

Claims

An antibody-drug conjugate (ADC) or a pharmaceutically acceptable salt thereof, having the general structure of Pc-(L-D) n wherein, D is a cytotoxic drug; L is a linker unit; Pc is an antibody or antigen-binding fragment thereof that specifically binds to CDH17; and n is a real number from 1 to 16. The antibody-drug conjugate or pharmaceutically acceptable salt thereof according to claim 1, wherein, The antibody or antigen-binding fragment thereof comprises a light chain variable region (VL) comprising LCDR1, LCDR2 and LCDR3, and / or a heavy chain variable region (VH) comprising HCDR1, HCDR2 and HCDR3, the LCDR1-3 and / or the HCDR1-3 are selected from the following combinations: (1) LCDR1, LCDR2, LCDR3 comprising the sequences set forth in SEQ ID NO. 23, 24, 25; and / or HCDR1, HCDR2, HCDR3 comprising the sequences set forth in SEQ ID NO. 26, 28, 30; (2) LCDR1, LCDR2, LCDR3 comprising the sequences set forth in SEQ ID NO. 23, 24, 25; and / or HCDR1, HCDR2, HCDR3 comprising the sequences set forth in SEQ ID NO. 26, 27, 30; (3) LCDR1, LCDR2, LCDR3 comprising the sequences set forth in SEQ ID NO. 23, 24, 25; and / or HCDR1, HCDR2, HCDR3 comprising the sequences set forth in SEQ ID NO. 26, 29, 30; or (4) the LCDR1-3 or / and the HCDR1-3 comprise a sequence having at least 80% identity, or a sequence having up to 3 insertions, deletions or substitution mutations, compared to each of the CDRs of the LCDR1-3 and HCDR1-3 of any one of the groups (1)-(3); preferably, the at least 80% identity is 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity. The antibody-drug conjugate or pharmaceutically acceptable salt thereof according to Claim 1 or 2, wherein, The antibody or antigen-binding fragment thereof comprises a light chain variable region (VL) or / and a heavy chain variable region (VH), the light chain variable region and / or the heavy chain variable region is selected from the following combinations: (1) the light chain variable region comprises the sequence set forth in SEQ ID NO. 17, or / and the heavy chain variable region comprises the sequence set forth in any one of SEQ ID NO. 21, 18, 19, 20, 22; or (2) the heavy chain variable region or / and the light chain variable region has a sequence having at least 80% identity, or a sequence having up to 3 insertions, deletions or substitution mutations, compared to the heavy chain variable region or / and the light chain variable region of any one of the group (1) above; preferably, the at least 80% identity is 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity. The antibody-drug conjugate or pharmaceutically acceptable salt thereof according to any one of claims 1 to 3, wherein, The antibody or antigen-binding fragment thereof comprises a heavy chain constant region sequence and / or a light chain constant region sequence; optionally, the heavy chain constant region and / or the light chain constant region is selected from a complete constant region sequence or a fragment thereof, the constant region fragment comprising CH1, a hinge region, CH2, CH3 or Fc; optionally, the heavy chain constant region is selected from the constant region of human or murine IgG1, IgG2, IgG3 or IgG4, and the light chain constant region is selected from human or murine kappa constant region or lambda constant region; optionally, the antibody or antigen-binding fragment thereof comprises a complete light chain and a complete heavy chain, the light chain consisting of the VL and a light chain constant region having the sequence as shown in SEQ ID NO. 12, and the heavy chain consisting of the VH and a heavy chain constant region having the sequence as shown in SEQ ID NO.

11. The antibody-drug conjugate or pharmaceutically acceptable salt thereof according to any one of claims 1 to 4, wherein, The antibody or antigen-binding fragment thereof is (1) a chimeric antibody or fragment thereof; (2) a humanized antibody or fragment thereof; and / or, (3) a fully human antibody or fragment thereof; preferably, the antibody or antigen-binding fragment thereof is selected from a monoclonal antibody, a polyclonal antibody, a natural antibody, an engineered antibody, a monospecific antibody, a multispecific antibody (e.g., a bispecific antibody), a monovalent antibody, a multivalent antibody, a full-length antibody, an antibody fragment, a naked antibody, a conjugated antibody, a humanized antibody, a fully human antibody, a Fab, a Fab', a F(ab')2, a Fd, a Fv, a scFv, a diabody, a nanobody or an affibody; more preferably, the antigen-binding fragment is selected from one or more of F(ab')2, Fab', Fab, Fv, scFv, nanobody or affibody. The antibody-drug conjugate or pharmaceutically acceptable salt thereof according to any one of claims 1 to 5, wherein, The cytotoxic drug is selected from a microtubulin inhibitor, a DNA damaging agent or a DNA topoisomerase inhibitor; the microtubulin inhibitor includes but is not limited to dolastatins, auristatins, maytansines, tubulysins and cryptomycins; the DNA damaging agent includes but is not limited to PBDs; the DNA topoisomerase inhibitor includes but is not limited to camptothecins. The antibody-drug conjugate or pharmaceutically acceptable salt thereof according to claim 6, wherein, The cytotoxic drug is a DNA topoisomerase inhibitor. The antibody-drug conjugate or pharmaceutically acceptable salt thereof according to claim 7, wherein, the cytotoxic drug is a compound of formula (D-I), wherein, R 1 , R 2 with the atom it is attached to, form a 5-6 membered heterocyclic ring, said 5-6 membered heterocyclic ring contains 1 or 2 oxygen atoms as ring atoms, said 5-6 membered heterocyclic ring is optionally substituted with one or more deuterium atoms; R 4 selected from H or C1-C3alkyl; R 5 selected from H, halogen, CN, OH, NH2, or Ci-C3alkyl; R 6 selected from H or C1-C3alkyl; R 7 is selected from H, C1-C3 alkyl, or C3-C6 cycloalkyl, said C1-C3 alkyl or C3-C6 cycloalkyl optionally substituted with deuterium, halogen, CN, =0, OH, NH2, or C1-C3 alkyl. The antibody-drug conjugate or pharmaceutically acceptable salt thereof according to claim 8, wherein, R 1 R 2 with the atom to which they are attached, form a The antibody-drug conjugate or pharmaceutically acceptable salt thereof according to claim 8, wherein, R 4 is H. The antibody-drug conjugate or pharmaceutically acceptable salt thereof according to claim 8, wherein, R 5 is H. The antibody-drug conjugate or pharmaceutically acceptable salt thereof according to claim 8, wherein, R 6 is H. The antibody-drug conjugate or pharmaceutically acceptable salt thereof according to claim 8, wherein, R 7 is cyclopropyl. The antibody-drug conjugate or pharmaceutically acceptable salt thereof according to any one of claims 8 to 13, wherein, The compound of formula (D-I) is selected from The antibody-drug conjugate or pharmaceutically acceptable salt thereof according to any one of claims 1 to 5, wherein, The linker unit is the a end is covalently linked to the antibody or antigen-binding fragment thereof, and the b end is covalently linked to a cytotoxic drug, wherein: Ring A is selected from R b1 , R b2 each independently is selected from H, halogen, CN, C1-C6 alkyl or C3-C6 cycloalkyl, or R b1 , R b2 and the carbon atom to which they are attached together form a C3-C6 cycloalkyl or 4-7 membered heterocyclyl, said C3-C6 cycloalkyl or 4-7 membered heterocyclyl being optionally substituted with one or more substituents selected from halogen, CN, =0, C1-C6 alkyl, OH, O(C1-C6 alkyl), NH2, NH(C1-C6 alkyl), N(C1-C6 alkyl)2, C3-C6 cycloalkyl and 4-7 membered heterocyclyl; m1 is selected from an integer from 2 to 8; L a selected from a chemical bond or Its * end and L b Connection, R b3 Selected from H or C1-C6 alkyl groups, m2 is selected from integers 1 to 8, R b4 R b5 Each is independently selected from H, halogen, CN, C1-C6 alkyl, C3-C6 cycloalkyl, L b a peptide residue consisting of 1 to 8 amino acids, which is optionally substituted with one or more substituents selected from the group consisting of halogen, CN, =0, C1-C6alkyl, OH, 0(C1-C6alkyl), NH2, NH(C1-C6alkyl), N(C1-C6alkyl)2, C3-C6cycloalkyl, and 4-7 membered heterocyclyl. The antibody-drug conjugate or pharmaceutically acceptable salt thereof according to claim 15, wherein, Ring A is selected from R b1 , R b2 and the carbon atom to which they are attached together form a 4-7 membered heterocyclyl group, which is optionally substituted with one or more halogen, CN, =0, C1-C6alkyl, OH, 0(C1-C6alkyl), NH2, NH(C1-C6alkyl), N(C1-C6alkyl)2, C3-C6cycloalkyl, and 4-7 membered heterocyclyl substituents. The antibody-drug conjugate or pharmaceutically acceptable salt thereof according to claim 15, wherein, Ring A is selected from R b1 , R b2 with the carbon atom to which they are attached forming a 4-7 membered heterocyclyl. The antibody-drug conjugate or pharmaceutically acceptable salt thereof according to claim 16 or 17, wherein, Ring A is selected from The antibody-drug conjugate or pharmaceutically acceptable salt thereof according to claim 15, wherein, L a is a chemical bond or one of R b R b3 R b4 R b5 one of R The antibody-drug conjugate or pharmaceutically acceptable salt thereof according to claim 19, wherein, L a selected from a chemical bond or Its end with L b connected. The antibody-drug conjugate or pharmaceutically acceptable salt thereof according to claim 15, wherein, The L b is a Gly-Gly-Phe-Gly tetrapeptide residue. The antibody-drug conjugate or pharmaceutically acceptable salt thereof according to claim 15, wherein, m1 is an integer from 2 to 6. The antibody-drug conjugate or pharmaceutically acceptable salt thereof according to claim 22, wherein, m1 is 2, 3, 4 or 5. The antibody-drug conjugate or pharmaceutically acceptable salt thereof according to any one of claims 15 to 23, wherein, The linker unit L is the a end is covalently linked to the antibody or antigen-binding fragment thereof, and the b end is covalently linked to a drug unit. The antibody-drug conjugate or pharmaceutically acceptable salt thereof according to any one of claims 1 to 5, wherein, n is selected from a real number from 1 to 16, for example, n is selected from a real number from 2 to 12, for example, n is selected from a real number from 4 to 10, for example, n is selected from a real number from 3 to 9, for example, n is selected from a real number from 4 to 8, for example, n is selected from a real number from 6 to 8. The antibody-drug conjugate or pharmaceutically acceptable salt thereof according to claim 25, wherein, n is a real number selected from 3 to 9, for example, n = 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, or 9.

0. The antibody-drug conjugate or pharmaceutically acceptable salt thereof according to claim 26, wherein, n is a real number selected from 6 to 8, for example, n is 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9 or 8.

0. The antibody-drug conjugate or pharmaceutically acceptable salt thereof according to any one of claims 1 to 27, wherein, The antibody-drug conjugate or pharmaceutically acceptable salt thereof is selected from the following compounds or a pharmaceutically acceptable salt thereof: An antibody or antigen-binding fragment thereof that specifically binds to hepatocellular carcinoma-associated antigen (HCCAA), wherein, The antibody or its antigen-binding fragment comprises a light chain variable region and a heavy chain variable region of a combination of LCDRs and HCDRs with the following sequences: (1) LCDR1, LCDR2 and LCDR3 of the sequences shown in SEQ ID NO.23, 24 and 25, and HCDR1, HCDR2 and HCDR3 of the sequences shown in SEQ ID NO.26, 27 and 30; (2) LCDR1, LCDR2 and LCDR3 of the sequences shown in SEQ ID NO.23, 24 and 25, and HCDR1, HCDR2 and HCDR3 of the sequences shown in SEQ ID NO.26, 28 and 30; (3) LCDR1, LCDR2, and LCDR3 of the sequences shown in SEQ ID NO. 23, 24, and 25, and HCDR1, HCDR2, and HCDR3 of the sequences shown in SEQ ID NO. 26, 29, and 30; or, (4) A sequence of six CDRs having 1, 2, 3 or more amino acid insertions, deletions and / or substitutions compared to the sequence of any one of the six CDRs described in (1) to (3), or having at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher of the same sequence. The antibody or antigen-binding fragment thereof of claim 29, wherein, The antibody or its antigen-binding fragment has the following combination of light chain variable regions and heavy chain variable regions: (1) The light chain variable region and the heavy chain variable region respectively contain the sequences shown in SEQ ID NO.17 and SEQ ID NO.18; (2) The light chain variable region and the heavy chain variable region respectively contain the sequences shown in SEQ ID NO.17 and SEQ ID NO.19; (3) The light chain variable region and the heavy chain variable region respectively contain the sequences shown in SEQ ID NO.17 and SEQ ID NO.20; (4) the light chain variable region and the heavy chain variable region comprise the sequences set forth in SEQ ID NO. 17 and SEQ ID NO. 21, respectively; (5) the light chain variable region and the heavy chain variable region comprise the sequences set forth in SEQ ID NO. 17 and SEQ ID NO. 22, respectively; or (6) the light chain variable region comprises a sequence that is 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical to the light chain variable region set forth in any one of (1) to (5) above, and the heavy chain variable region comprises a sequence that is 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical to the heavy chain variable region set forth in any one of (1) to (5) above. The antibody or antigen binding fragment thereof of claim 29 or 30, wherein, The antibody or antigen-binding fragment thereof can comprise a light chain and / or heavy chain constant region sequence; preferably, the heavy chain constant region can comprise a heavy chain constant region sequence of a human or murine antibody IgGl, IgG2, IgG3, IgG4, IgA, IgM, IgE or IgD. The antibody or antigen binding fragment thereof of claim 31, wherein, The heavy chain constant region comprises a heavy chain constant region derived from a human IgGl antibody. The antibody or antigen binding fragment thereof of claim 31, wherein, The light chain constant region comprises a constant region of a human or murine lambda or kappa chain. A multispecific antigen-binding molecule comprising: (1) an antibody or antigen-binding fragment thereof that specifically binds CDH17 according to any one of claims 29 to 33; and (2) an antibody or antigen-binding fragment thereof that binds to an antigen other than CDH17, or an antibody or antigen-binding fragment thereof that binds to an epitope of CDH17 that is different from the antibody or antigen-binding fragment thereof according to any one of claims 29 to 33. The multispecific antigen-binding molecule of claim 34, wherein, The other antigen other than CDH17 comprises: CD3 (preferably CD3s), CD16, NKG2D. The multispecific antigen-binding molecule of claim 34 or 35, wherein The multispecific antigen-binding molecule can be bispecific, trispecific or tetraspecific; preferably, the multispecific antigen-binding molecule can be bivalent, trivalent, tetravalent, pentavalent or hexavalent. An isolated nucleic acid molecule, which can be of any length, in isolated form, comprising nucleotides, deoxynucleotides, and / or ribonucleotides, wherein, The nucleic acid molecule encodes the antibody or antigen-binding fragment thereof according to any one of claims 29 to 33, or the multispecific antigen-binding molecule according to any one of claims 34 to 36. A carrier, wherein, The vector comprises the isolated nucleic acid molecule according to claim 37. A host cell, wherein, The host cell comprises the nucleic acid molecule according to claim 37, or the vector according to claim 38; preferably, the host cell is a eukaryotic cell or a prokaryotic cell; more preferably, the host cell is derived from a mammalian cell, a yeast cell, an insect cell, E. coli and / or B. subtilis; more preferably, the host cell is selected from Expi293 or CHO cells. A method of preparing the antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 28, the antibody or antigen-binding fragment thereof according to any one of claims 29 to 33, or the multispecific antigen-binding molecule according to any one of claims 34 to 36. A pharmaceutical composition, wherein, The antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 28, the antibody or antigen-binding fragment thereof according to any one of claims 29 to 33, or the multispecific antigen-binding molecule according to any one of claims 34 to 36, and a pharmaceutically acceptable excipient. The pharmaceutical composition of claim 41, wherein, The pharmaceutical composition further comprises other therapeutic agents, such as other antibodies, typically comprising PD-1 / PD-L1 inhibitors; or such as chemotherapeutics. A method of treating a tumor in a mammal, comprising administering to a mammal, preferably a human, in need of such treatment, a therapeutically effective amount of the antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 28, the antibody or antigen-binding fragment thereof according to any one of claims 29 to 33, the multispecific antigen-binding molecule according to any one of claims 34 to 36, or the pharmaceutical composition according to claim 41 or 42. The method of claim 43, wherein, The method further comprises administering an additional therapeutic agent, such as other antibodies, typically comprising PD-1 / PD-L1 inhibitors; or such as chemotherapeutics. The method according to claim 43, wherein the tumor is a CDH17-expressing tumor; preferably, the tumor is selected from the group consisting of gastric cancer, pancreatic cancer, colorectal cancer, and gastrointestinal stromal tumor. Use of the antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 28, the antibody or antigen-binding fragment thereof according to any one of claims 29 to 33, the multispecific antigen-binding molecule according to any one of claims 34 to 36, or the pharmaceutical composition according to claim 41 or 42, for the manufacture of a medicament for the treatment of a tumor. Use according to claim 46, wherein, The pharmaceutical composition further comprises other therapeutic agents, such as other antibodies, typically comprising PD-1 / PD-L1 inhibitors; or such as chemotherapeutics. Use according to claim 46, wherein, The tumor is a CDH17-expressing tumor; preferably, the tumor is selected from the group consisting of gastric cancer, pancreatic cancer, colorectal cancer, and gastrointestinal stromal tumor. Use of the antibody-drug conjugate of general formula Pc-(L-D)n or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 28, the antibody or antigen-binding fragment thereof according to any one of claims 29 to 33, the multispecific antigen-binding molecule according to any one of claims 34 to 36, or the pharmaceutical composition according to claim 41 or 42, for the treatment of a tumor. Use according to claim 49, wherein, The pharmaceutical composition further comprises other therapeutic agents, such as other antibodies, typically comprising PD-1 / PD-L1 inhibitors; or such as chemotherapeutics. Use according to claim 49, wherein, The tumor is a CDH17-expressing tumor; preferably, the tumor is selected from the group consisting of gastric cancer, pancreatic cancer, colorectal cancer, and gastrointestinal stromal tumor. The tumor is a CDH17-expressing tumor; preferably, the tumor is selected from the group consisting of gastric cancer, pancreatic cancer, colorectal cancer, and gastrointestinal stromal tumor. The antibody-drug conjugate of any one of claims 1 to 28 or a pharmaceutically acceptable salt thereof, the antibody or antigen-binding fragment thereof of any one of claims 29 to 33, or the multispecific antigen-binding molecule of any one of claims 34 to 36, or the pharmaceutical composition of claim 41 or 42 for use in the treatment of a tumor; preferably, the pharmaceutical composition further comprises another therapeutic agent, such as another antibody, typically a PD-1 / PD-L1 inhibitor; or such as a chemotherapeutic drug. The composition of claim 52, wherein, The tumor is a CDH17-expressing tumor; preferably, the tumor is selected from the group consisting of gastric cancer, pancreatic cancer, colorectal cancer, and gastrointestinal stromal tumor.

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