Anti-CDH17 antibody, immunoconjugate, and use

Abstract

Provided are a CDH17-targeting antibody, an immunoconjugate, and a use. The CDH17-targeting antibody has high affinity and binding activity, and has a strong endocytosis effect. A CDH17-targeting immunoconjugate has an excellent effect and good safety. The provided CDH17-targeting antibody and / or immunoconjugate has broad prospects for application.

Description

Anti-CDH17 antibodies, immunoconjugates and their uses Technical Field

[0001] This invention relates to the fields of bioengineering and biomedicine, and mainly to an anti-CDH17 antibody or its antigen-binding fragment, which encodes nucleic acid, expression vector and expression cell, preparation method, pharmaceutical composition, immunoconjugate or its pharmaceutically acceptable salt, and their use in treating diseases. Background Technology

[0002] Cadherin 17 (CDH17) is a member of the cadherin superfamily. Cadherins are a class of transmembrane glycoproteins that depend on calcium ions (Ca). 2+ Cadherin mediates cell-cell adhesion and is closely related to normal tissue development and the occurrence of various diseases. The typical cadherin extracellular domain consists of five repeating sequences; the intracellular domain consists of 150-160 amino acids and is highly conserved. CDH17 is similar, but its structure differs slightly. The CDH17 extracellular domain consists of seven repeating sequences EC1-EC7, thus belonging to a subclass of the 7D-cadherin family. It was initially cloned from rat liver in 1994, and its intracellular domain contains 18-20 amino acids. Its function is to maintain the integrity of epithelial tissues as a polypeptide transporter and cell adhesion molecule.

[0003] In normal human cells, CDH17 is highly expressed only in the small intestine and colon, and is not expressed or is expressed at low levels in other tissues such as the liver, heart, and kidneys. In tumor tissues, CDH17 is highly expressed in gastrointestinal, pancreatic, liver, esophageal, bile duct, and colorectal cancers. The tumor-specific high expression of CDH17 makes it a potential therapeutic target and molecular marker for gastrointestinal cancers. There is a huge unmet demand for new drugs in the gastrointestinal cancer market. Globally, gastrointestinal (GI) cancers account for approximately 26.3% of all cancers and cause 35.4% of cancer-related deaths worldwide. East Asia, represented by China, is a high-risk region for gastrointestinal cancers, with incidence and mortality rates far exceeding those of other countries and regions.

[0004] There is still a large unmet need, and there are few drugs targeting CDH17. Therefore, developing drugs targeting this target has broad clinical application and therapeutic prospects. Summary of the Invention

[0005] The present invention provides antibodies or antigen-binding fragments that specifically bind to CDH17, which have: (1) high affinity; (2) excellent antigen-binding ability; and (3) strong endocytosis effect.

[0006] This invention also provides nucleic acid molecules encoding the antibody or antigen-binding fragment, expression vectors, host cells, and methods for preparing the antibody or antigen-binding fragment. This invention further provides immunoconjugates comprising the antibody or antigen-binding fragment, or pharmaceutically acceptable salts thereof, and combinations thereof. The antibody or antigen-binding fragments, immunoconjugates, or pharmaceutically acceptable salts thereof, and combinations thereof described in this invention can be used for the prevention, relief, and / or treatment of diseases and / or conditions.

[0007] On the one hand, the present invention provides an antibody or antigen-binding fragment, characterized in that it can specifically bind to CDH17.

[0008] In some embodiments, the antibody or antigen-binding fragment that specifically binds to CDH17 comprises HCDR1, HCDR2, and HCDR3 of VH as shown in SEQ ID NO: 22.

[0009] In some embodiments, the antibody or antigen-binding fragment that specifically binds to CDH17 comprises LCDR1, LCDR2, and LCDR3 of VL as shown in SEQ ID NO: 23.

[0010] In some embodiments, the antibody or antigen-binding fragment that specifically binds to CDH17 comprises HCDR1, the amino acid sequence of which is shown in SEQ ID NO:24.

[0011] In some embodiments, the antibody or antigen-binding fragment that specifically binds to CDH17 comprises HCDR2, the amino acid sequence of which is shown in SEQ ID NO:25.

[0012] In some embodiments, the antibody or antigen-binding fragment that specifically binds to CDH17 contains HCDR3, the amino acid sequence of which is shown in SEQ ID NO:26.

[0013] In some embodiments, the antibody or antigen-binding fragment that specifically binds to CDH17 comprises LCDR1, the amino acid sequence of which is shown in SEQ ID NO:27.

[0014] In some embodiments, the antibody or antigen-binding fragment that specifically binds to CDH17 comprises LCDR2, the amino acid sequence of which is shown in SEQ ID NO:28.

[0015] In some embodiments, the antibody or antigen-binding fragment that specifically binds to CDH17 comprises LCDR3, the amino acid sequence of which is shown in SEQ ID NO:29.

[0016] In some embodiments, the antibody or antigen-binding fragment that specifically binds to CDH17 may comprise HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3. For example, the amino acid sequence of the antibody or antigen-binding fragment that specifically binds to CDH17 described in this application is shown in SEQ ID NO:24, the amino acid sequence of HCDR2 is shown in SEQ ID NO:25, the amino acid sequence of HCDR3 is shown in SEQ ID NO:26, the amino acid sequence of LCDR1 is shown in SEQ ID NO:27, the amino acid sequence of LCDR2 is shown in SEQ ID NO:28, and the amino acid sequence of LCDR3 is shown in SEQ ID NO:29.

[0017] In some embodiments, the antibody or antigen-binding fragment that specifically binds to CDH17 includes the FR region.

[0018] In some embodiments, the antibody or antigen-binding fragment that specifically binds to CDH17 includes the antibody heavy chain variable region VH and / or the antibody light chain variable region VL.

[0019] In some embodiments, the VH of the antibody or antigen-binding fragment that specifically binds to CDH17 has an amino acid sequence as shown in ID NO: 22 and / or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity compared to it.

[0020] In some embodiments, the VL of the antibody or antigen-binding fragment that specifically binds to CDH17 has an amino acid sequence as shown in SEQ ID NO: 23 and / or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity compared to it.

[0021] In some embodiments, the antibody or antigen-binding fragment that specifically binds to CDH17 comprises VH as shown in SEQ ID NO: 22 and VL as shown in SEQ ID NO: 23, and / or VH and VL having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity compared to them.

[0022] In some embodiments, the antibody or antigen-binding fragment that specifically binds to CDH17 includes a constant region of the antibody heavy chain.

[0023] In some embodiments, the antibody heavy chain constant region is derived from the human IgG heavy chain constant region.

[0024] In some embodiments, the antibody heavy chain constant region is derived from the human IgG1 heavy chain constant region.

[0025] In some embodiments, the antibody or antigen-binding fragment that specifically binds to CDH17 includes a constant region of the antibody light chain.

[0026] In some embodiments, the antibody light chain constant region is derived from the human Igκ constant region.

[0027] In some embodiments, the antibody or antigen-binding fragment includes Fab, Fab', Fv fragments, F(ab')2, F(ab)2, scFv, di-scFv, and / or dAb.

[0028] In some embodiments, the antibody is selected from one or more of the following groups: monoclonal antibody, polyclonal antibody, chimeric antibody, humanized antibody, fully human antibody, natural antibody, engineered antibody, monovalent antibody, multivalent antibody, full-length antibody, antibody fragment, naked antibody, conjugated antibody, etc.

[0029] On the other hand, this application also provides a chimeric antigen receptor comprising a targeting portion, the targeting portion comprising the antibody or antigen-binding fragment described in this application.

[0030] On the other hand, this application also provides polypeptide molecules that contain the antibody or antigen-binding fragment or the chimeric antigen receptor.

[0031] In some embodiments, the polypeptide molecule comprises a fusion protein.

[0032] On the other hand, this application also provides isolated one or more nucleic acid molecules that encode the antibody or antigen-binding fragment, the chimeric antigen receptor, or the polypeptide molecule.

[0033] On the other hand, this application also provides a vector containing the nucleic acid molecule.

[0034] On the other hand, this application also provides a cell comprising the antibody or antigen-binding fragment, the chimeric antigen receptor, the polypeptide molecule, the nucleic acid molecule, or the carrier.

[0035] On the other hand, this application also provides immunoconjugates or pharmaceutically acceptable salts thereof.

[0036] In some embodiments, the immunoconjugate or a pharmaceutically acceptable salt thereof has the structure shown in formula (I).

[0037] Ab-(LD) p (I)

[0038] in:

[0039] Ab is any of the antibody or antigen-binding fragments described above in this invention;

[0040] L stands for connector;

[0041] D represents the payload;

[0042] p is any integer from 1 to 10.

[0043] In some embodiments, the immunoconjugate or a pharmaceutically acceptable salt thereof is an antibody-drug conjugate or a pharmaceutically acceptable salt thereof, and D is a cytotoxic drug.

[0044] In some embodiments, the cytotoxic drug is one or more of the following: a tubulin inhibitor, a DNA intercalator, a DNA topoisomerase inhibitor, an apoptosis inducer, an RNA polymerase inhibitor, a spliceosome inhibitor, or a proteasome inhibitor.

[0045] In some embodiments, the cytotoxic drug tubulin inhibitor is selected from MMAE, MMAF, DM1, DM4, and Tubulysins; the DNA intercalating agent is PBD or IBD; the DNA topoisomerase inhibitor is a topoisomerase I inhibitor, preferably a camptothecin derivative; the apoptosis inducer is a Bcl-xL inhibitor, preferably clezutoclax; the RNA polymerase inhibitor is α-amaminine or β-amaminine; the spliceosome inhibitor is preferably thailanstatin A; and the proteasome inhibitor is selected from carbamycin A and carbamycin B.

[0046] In some embodiments, the cytotoxic drug is a DNA topoisomerase inhibitor, preferably DXD or a derivative of DXD;

[0047] In some embodiments, the DXD or a derivative of DXD is a compound of formula (II), its stereoisomer, or a pharmaceutically acceptable salt thereof.

[0048] Among them, X 1 For O, S, or NH;

[0049] X 2 For -NR 2 - or key, R 2 -H, -C 1-3 Alkyl or 3-6 membered cycloalkyl;

[0050] X 3 It can be -O- or -NH-;

[0051] R 1 -H, -C1-3 Alkyl or 3-6 membered cycloalkyl;

[0052] M is (CR) 3 R 4 ) m -or Among them, R 3 R 4 Each is independently -H, -C 1-5 Alkyl or 3-6 membered cycloalkyl, where m is 1, 2 or 3, and ring A is a 3-6 membered cycloalkane;

[0053] In some implementations, X 1 For O, X 2 For -NH-, R 1 It is -H.

[0054] In some implementations, m is 2; in some implementations, M is -CR. 3 R 4 CH2-, R 3 R 4 One of them is -H, and the other is methyl, isopropyl, or cyclopropyl.

[0055] In some embodiments, D is selected from compounds represented by any of the following structural formulas, their stereoisomers, or pharmaceutically acceptable salts thereof:

[0056] In some embodiments, D is DXD or compound 2A.

[0057] In some implementations, L is -L 4 -L 3 -L 2 -L 1 -;

[0058] in:

[0059] L 1 Each independently serves as a key, Among them, the -NH- end and L 2 Connect one end to D, and the other end to R. a Or R b Each is independently -H or -C 1-3 Alkyl groups, preferably L 1 for

[0060] L 2Each peptide residue is independently composed of 2 to 7 amino acid residues, wherein each amino acid is independently phenylalanine, isoleucine, leucine, isoleucine, tryptophan, valine, methionine, tyrosine, alanine, threonine, histidine, serine, glutamine, arginine, lysine, asparagine, glutamic acid, proline, citrulline, aspartic acid, or glycine, and each amino acid residue is optionally independently divided by one or more halogens, hydroxyl groups, cyano groups, amino groups, or -C groups. 1-5 Alkyl, -C 1-5 Alkoxy or 3-6 membered cycloalkyl substitution, wherein the -NH- terminus is associated with an L-terminus. 3 Connected to L, the other end is connected to L. 1 Linked; preferably, each of the amino acids is independently glycine, phenylalanine, valine, or citrulline; more preferably, L 2 The residues are -glycine residues - phenylalanine residues - glycine residues - (-Gly-Phe-Gly-), -glycine residues - glycine residues - phenylalanine residues - glycine residues - (-Gly-Gly-Phe-Gly-), or -valine residues - citrulline residues - (-Val-Cit-); more preferably, L 2 for

[0061] L 3 Each is independently -(CH2) n1 -C(O)-,-(CH2CH2O) n2 -C(O)- or -(CH2) n3 -W-(CH2) n4 -C(O)-, where the -C(O)- terminal is connected to L 2 Connected to L, the other end is connected to L. 4 Connected, n1, n2, n3, and n4 are each independently an integer from 0 to 8, and W is -O-, -C(O)-NH-, 6-10 aryl, 5-10 aryl heteroalkyl, 3-6 cycloalkyl, or 3-6 heterocyclic group, wherein the 6-10 aryl, 5-10 aryl heteroalkyl, 3-6 cycloalkyl, or 3-6 heterocyclic group is optionally surrounded by one or more halogens, hydroxyl groups, cyano groups, amino groups, or -C 1-5 Alkyl, -C 1-5 Alkoxy or 3-6 membered cycloalkyl substitution; preferably, L 3 for

[0062] L 4 Each independently aC(=O)-b, a-CH2C(=O)-b, where end a is connected to Ab, and end b is connected to L. 3 Connected; preferably

[0063] In some implementations, L 4 for The a-terminus is connected to the thiol group of the cysteine ​​residue in Ab, and the b-terminus is connected to L. 3 Connected.

[0064] In some implementations, L is The left end is connected to Ab, and the right end is connected to D.

[0065] In some implementations, p is 1, 2, 3, 4, 5, 6, 7, or 8.

[0066] In some embodiments, the immunoconjugate or a pharmaceutically acceptable salt thereof has the following structure:

[0067] Among them, R 1 X 1 X 2 M, X 3 L 1 L 2 L 3 L 4 Ab and p are as described above, each independently.

[0068] In some embodiments, the immunoconjugate of formula (III) or a pharmaceutically acceptable salt thereof has the structure shown in formula (IIIa):

[0069] Among them, X 1 R 2 M, L 1 L 2 L 3 L 4 Ab and p are as described above, each independently.

[0070] In some embodiments, the immunoconjugate or a pharmaceutically acceptable salt thereof has the following structure:

[0071] In this context, Ab and p are independent as described above;

[0072] Preferably, p is an integer from 2 to 8; more preferably, p is 8.

[0073] On the other hand, this application also provides compositions of immunoconjugates comprising one or more of the aforementioned immunoconjugates of the present invention or pharmaceutically acceptable salts thereof.

[0074] In some embodiments, the DAR value of the composition of the immunoconjugate is any integer or decimal from 1 to 10; preferably, the DAR value of the composition of the immunoconjugate is any integer or decimal from 2 to 8, more preferably any integer or decimal from 3 to 8, such as 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 5.0, 5.5, 6.0, 6.5, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0 or any value or range thereof.

[0075] On the other hand, this application also provides a pharmaceutical combination comprising any of the foregoing antibodies or antigen-binding fragments, the chimeric antigen receptor, the polypeptide molecule, the nucleic acid molecule, the carrier, the cell, the immunoconjugate, or a pharmaceutically acceptable salt and / or immunoconjugate thereof.

[0076] In some embodiments, the drug combination may further include one or more other therapeutic agents; preferably, the therapeutic agents may be other antibodies, small molecule drugs, immunomodulators, cytotoxic agents, chemotherapeutic agents, or cytokines.

[0077] On the other hand, this application also provides pharmaceutical compositions comprising the antibody or antigen-binding fragment, the chimeric antigen receptor, the polypeptide molecule, the nucleic acid molecule, the carrier, the immunoconjugate or a pharmaceutically acceptable salt thereof, a composition of the immunoconjugate, the pharmaceutical combination and / or the cell, and optionally a pharmaceutically acceptable carrier.

[0078] On the other hand, this application also provides a method for preparing the antibody or antigen-binding fragment, the method comprising culturing the cells under conditions that cause the antibody or antigen-binding fragment to be expressed.

[0079] On the other hand, this application also provides a method for preparing the aforementioned immunoconjugate or a pharmaceutically acceptable salt and / or composition of the immunoconjugate, the method comprising conjugating any of the foregoing antibody or antigen-binding fragments to a payload.

[0080] On the other hand, this application also provides the use of the antibody or antigen-binding fragment, the chimeric antigen receptor, the polypeptide molecule, the nucleic acid molecule, the carrier, the cell, the immunoconjugate or a pharmaceutically acceptable salt thereof, a composition of the immunoconjugate, the pharmaceutical combination and / or the pharmaceutical composition in the preparation of a medicament for the prevention, relief and / or treatment of a disease and / or condition.

[0081] In some implementations, the disease and / or condition includes CDH17-related diseases.

[0082] In some implementations, the disease and / or condition includes tumors.

[0083] In some implementations, the disease and / or condition includes CDH17-related tumors.

[0084] In some embodiments, the disease and / or condition includes gastrointestinal tumors, including colorectal cancer, colorectal adenocarcinoma, gastric cancer, gastric adenocarcinoma, pancreatic cancer, pancreatic adenocarcinoma, etc.

[0085] On the other hand, this application also provides the use of the antibody or antigen-binding fragment, the chimeric antigen receptor, the polypeptide molecule, the nucleic acid molecule, the carrier, the cell, the immunoconjugate or a pharmaceutically acceptable salt thereof, a composition of the immunoconjugate, the pharmaceutical combination and / or the pharmaceutical composition in the prevention, relief and / or treatment of diseases and / or conditions.

[0086] In some implementations, the disease and / or condition includes CDH17-related diseases.

[0087] In some implementations, the disease and / or condition includes tumors.

[0088] In some implementations, the disease and / or condition includes CDH17-related tumors.

[0089] In some embodiments, the disease and / or condition includes gastrointestinal tumors, including colorectal cancer, colorectal adenocarcinoma, gastric cancer, gastric adenocarcinoma, pancreatic cancer, pancreatic adenocarcinoma, etc.

[0090] On the other hand, this application also provides the antibody or antigen-binding fragment, the chimeric antigen receptor, the polypeptide molecule, the nucleic acid molecule, the carrier, the cell, the immunoconjugate or a pharmaceutically acceptable salt thereof, a composition of the immunoconjugate, the pharmaceutical combination and / or the pharmaceutical composition for the prevention and / or treatment of diseases and / or conditions.

[0091] In some implementations, the disease and / or condition includes CDH17-related diseases.

[0092] In some implementations, the disease and / or condition includes tumors.

[0093] In some implementations, the disease and / or condition includes CDH17-related tumors.

[0094] In some embodiments, the disease and / or condition includes gastrointestinal tumors, including colorectal cancer, colorectal adenocarcinoma, gastric cancer, gastric adenocarcinoma, pancreatic cancer, pancreatic adenocarcinoma, etc.

[0095] On the other hand, this application also provides a method for detecting CDH17 in a sample, the method comprising administering the antibody or antigen-binding fragment, the chimeric antigen receptor, the polypeptide molecule, the nucleic acid molecule, the carrier, the cell, the immunoconjugate or a pharmaceutically acceptable salt thereof, a composition of the immunoconjugate, the pharmaceutical combination and / or the pharmaceutical composition.

[0096] On the other hand, this application also provides reagents or kits for detecting CDH17 in samples, which comprise the antibody or antigen-binding fragment, the chimeric antigen receptor, the polypeptide molecule, the nucleic acid molecule, the carrier, the cell, the immunoconjugate or a pharmaceutically acceptable salt thereof, a composition of the immunoconjugate, the pharmaceutical combination and / or the pharmaceutical composition.

[0097] On the other hand, this application also provides the use of the antibody or antigen-binding fragment, the chimeric antigen receptor, the polypeptide molecule, the nucleic acid molecule, the carrier, the cell, the immunoconjugate or a pharmaceutically acceptable salt thereof, a composition of the immunoconjugate, the pharmaceutical combination and / or the pharmaceutical composition in the preparation of a kit for detecting the presence and / or content of CDH17 in a sample.

[0098] Other aspects and advantages of this application will readily be apparent to those skilled in the art from the detailed description below. Only exemplary embodiments of this application are shown and described in the following detailed description. As will be appreciated by those skilled in the art, the content of this application enables them to make modifications to the disclosed specific embodiments without departing from the spirit and scope of the invention to which this application pertains. Accordingly, the descriptions in the accompanying drawings and specification of this application are merely exemplary and not restrictive. Attached Figure Description

[0099] Figure 1 shows the antigen binding detection curve of the chimeric antibody and the CHOK1 engineered cell line expressing huCDH17.

[0100] Figure 2 shows the antigen binding detection curve of the chimeric antibody to the SNU-5 gastric cancer cell line expressing huCDH17.

[0101] Figure 3 shows the antigen binding detection curves of the chimeric antibody and the CHOK1 engineered cell line expressing rhCDH17.

[0102] Figure 4 shows the antigen binding curves of the humanized antibody to the CHOK1 engineered cell line expressing huCDH17.

[0103] Figure 5 shows the antigen binding curves of the humanized antibody to the CHOK1 engineered cell line expressing rhCDH17.

[0104] Figure 6 shows the binding curves of humanized antibodies to AsPC-1 surface antigen.

[0105] Figure 7 shows the binding curves of the humanized antibody and the SNU-16 surface antigen.

[0106] Figure 8 shows the internalization-time curve of humanized antibody on pancreatic cancer AsPC-1 cells.

[0107] Figure 9A shows the internalization MFI-concentration response curve of the 901201 antibody in pancreatic cancer AsPC-1 cells.

[0108] Figure 9B shows the internalized MFI-concentration response curve of antibody 901201 in gastric cancer 23132 / 87 cells.

[0109] Figure 10 shows the HPLC chromatogram of 901201-2A.

[0110] Figure 11 shows the IgG-2A HIC-HPLC detection chromatogram.

[0111] Figure 12 shows the SEC spectra results for 901201-2A.

[0112] Figure 13 shows the IgG-2A SEC spectrum results.

[0113] Figure 14 shows the dose-response curve of the cytotoxic activity of 901201-2A against CL-40 cells.

[0114] Figure 15 shows the dose-response curve of the cytotoxic activity of 901201-2A against 23132 / 87 cells.

[0115] Figure 16 shows the dose-response curve of the cytotoxic activity of 901201-2A against AsPC-1 cells.

[0116] Figure 17 shows the expression detection of CDH17.

[0117] Figure 18 shows the dose-response curves of the cytotoxic activity of 901201-2A against AGS and AGS-CDH17KO cells.

[0118] Figure 19 shows the lateral killing effect curve of 901201-2A on Raji-luc cells.

[0119] Figure 20 shows the lateral killing effect curve of 901201-2A on Raji-luc cells.

[0120] Figure 21 shows the dose-response curve of the killing activity of 901201-2A against 41P CRT tumor organoids.

[0121] Figure 22 shows the dose-response curve of the cytotoxic activity of 901201-2A against 41P CRN in normal colonic organoids.

[0122] Figure 23 shows the antitumor effects of 901201-2A and the control antibody ADC on nude mice subcutaneously transplanted with human colorectal cancer CL-40 cells.

[0123] Figure 24 shows the body weight change curves of different treatment groups in the CL-40 colorectal cancer xenograft model in nude mice.

[0124] Figure 25 shows the antitumor effects of 901201-2A and the control antibody ADC on a human gastric cancer SNU-5 cell subcutaneous xenograft model.

[0125] Figure 26 shows the body weight change curves of each treatment group in the SNU-5 gastric cancer mouse xenograft model.

[0126] Figure 27 shows the growth curve of the AsPC-1-Luci in situ tumor.

[0127] Figure 28 shows the survival curves of AsPC-1 orthotopic tumor-bearing mice.

[0128] Figure 29 shows the effect of the test substance on the body weight of AsPC-1 orthotopic tumor-bearing animals.

[0129] Figure 30 shows the antitumor effects of different treatment groups in the gastric cancer PDX model.

[0130] Figure 31 shows the weight change curves of each treatment group in the gastric cancer PDX model.

[0131] Figure 32 shows the antitumor effects of different treatment groups in the PDX model of colorectal cancer.

[0132] Figure 33 shows the weight change curves of each treatment group in the PDX model of colorectal cancer.

[0133] Figure 34 shows the antitumor effects of different treatment groups in the human pancreatic cancer xenograft subcutaneous tumor PDX model.

[0134] Figure 35 shows the weight change curves of different treatment groups in the human pancreatic cancer xenograft subcutaneous tumor PDX model.

[0135] Figure 36 shows the antitumor effects of different treatment groups in a human gastric cancer xenograft subcutaneous tumor PDX model.

[0136] Figure 37 shows the antitumor effects of different treatment groups in the PDX model of large-volume colorectal cancer.

[0137] Figure 38 shows the weight change curves of different treatment groups in the PDX model of large-volume colorectal cancer. Detailed Implementation

[0138] Terminology Definition

[0139] In this invention, the term "CDH17" is also referred to as "liver-intestine cadherin," "Cadherin-17," etc. "Cadherin 17," "HPT-1," "LI-Cadherin," etc., are members of the cadherin superfamily. The term "CDH17" encompasses "full-length," unprocessed CDH17, and any form of CDH17 produced by cellular processing. In this invention, the term "CDH17" includes full-length wild-type CDH17 and its mutants, fragments, variants, isotypes, and homologs.

[0140] In this invention, the term "isolated" generally refers to a substance or component obtained artificially from its natural state. If an "isolated" substance or component is found in nature, it may be due to an alteration of its natural environment, the isolation of the substance from its natural environment, or both. For example, a certain unisolated polynucleotide or polypeptide may naturally exist in a living animal, and a high-purity identical polynucleotide or polypeptide isolated from this natural state is called isolated. The term "isolated" does not exclude the presence of artificial or synthetic substances, nor does it exclude the presence of other impurities that do not affect the substance's activity. In this invention, the term "isolated antigen-binding protein" generally refers to a protein with antigen-binding ability obtained artificially from its natural state. This "isolated antigen-binding protein" may include an antigen-binding portion and optionally, allow the antigen-binding portion to employ a framework or structural portion that promotes the conformation of the antigen-binding portion to bind the antigen. The antigen-binding protein may include, for example, an antibody-derived protein framework region (FR) or an alternative protein framework region or artificial framework region having a transplanted CDR or CDR derivative. Such frameworks include, but are not limited to, antibody-derived framework regions containing mutations introduced, for example, to stabilize the three-dimensional structure of antigen-binding proteins, and fully synthetic framework regions containing, for example, biocompatible polymers. Examples of antigen-binding proteins include, but are not limited to: human antibodies, humanized antibodies; chimeric antibodies; recombinant antibodies; single-chain antibodies; bifunctional antibodies; trifunctional antibodies; tetrafunctional antibodies; Fab, Fab', Fv fragments, F(ab')2, F(ab)2, scFv, di-scFv, dAb, IgD antibodies; IgE antibodies; IgM antibodies; IgG1 antibodies; IgG2 antibodies; IgG3 antibodies; or IgG4 antibodies and fragments thereof.

[0141] In this invention, the term "antibody" (Ab) refers to an immunoglobulin molecule that specifically binds to or is immunoreactive with a target antigen, including polyclonal, monoclonal, genetically engineered, and other modified forms of antibodies (including, but not limited to, chimeric antibodies, humanized antibodies, fully human antibodies, heterologous conjugates (e.g., bispecific, trispecific, and tetraspecific antibodies, biantibodies, triantibodies, and tetraantibodies, antibody conjugates) and antigen-binding fragments of antibodies (including, for example, Fab', F(ab')2, Fab, Fv, rIgG, and scFv fragments). Furthermore, unless otherwise stated, the term "monoclonal antibody" (mAb) means both complete antibody molecules capable of specifically binding to a target protein and incomplete antibody fragments (e.g., Fab and F(ab')2 fragments, which lack the Fc fragment of the complete antibody (which is cleared more quickly from animal circulation) and therefore lack Fc-mediated effector function (see Wahl et al., J. Nucl. Med. 24:316, 1983; the contents of which are incorporated herein by reference).

[0142] In this invention, the term "antibody" can be derived from any animal, including but not limited to humans and non-human animals. The non-human animals can be selected from primates, mammals, rodents and vertebrates, such as camels, llamas, ostriches, alpacas, sheep, rabbits, mice, rats or cartilaginous fish (e.g., sharks).

[0143] In this invention, the term "specific binding" refers to a binding reaction that determines the presence of an antigen within a heterogeneous population of proteins and other biomolecules, such as those specifically recognized by an antibody or its antigen-binding fragment. Antibodies or their antigen-binding fragments that specifically bind to an antigen will bind with a KD less than 100 nM. For example, antibodies or their antigen-binding fragments that specifically bind to an antigen will bind with a KD up to 100 nM (e.g., between 1 pM and 100 nM). Antibodies or their antigen-binding fragments that do not show specific binding to a particular antigen or its epitope will show a KD greater than 100 nM (e.g., greater than 500 nM, 1 μM, 100 μM, 500 μM, or 1 mM) for that particular antigen or its epitope. Various immunoassays can be used to select antibodies that specifically react with a particular protein or carbohydrate. For example, solid-phase ELISA is conventionally used to select antibodies that specifically react with a protein or carbohydrate. See Harlow & Lane, Antibodies, A Laboratory Manual, Cold Spring Harbor Press, New York (1988) and Harlow & Lane, Using Antibodies, A Laboratory Manual, Cold Spring Harbor Press, New York (1999), which describe the immunoassays and conditions that can be used to determine specific immune reactivity (which are incorporated herein by reference).

[0144] In this invention, the term "single-specific" refers to having one or more binding sites, each binding to the same epitope of the same antigen. In this invention, the term "multi-specific" refers to having at least two antigen-binding sites, each of which binds to a different epitope of the same antigen or a different epitope of a different antigen. Therefore, terms such as "bispecific," "triple-specific," and "quadruple-specific" refer to the number of different epitopes that an antibody / antigen binding molecule can bind to.

[0145] In this invention, the terms "full-length antibody," "intact antibody," and "complete antibody" are used interchangeably, referring to antibodies that have a structure substantially similar to that of natural antibodies.

[0146] In this invention, the term "antigen-binding fragment" refers to one or more antibody fragments that retain the ability to specifically bind to a target antigen. The antigen-binding function of an antibody can be performed by a fragment of a full-length antibody. The antibody fragment can be Fab, F(ab')2, scFv, biantibody, triantibody, affibody, nanobody, aptamer, or domain antibody. Examples of binding fragments encompassing the term "antigen-binding fragment" for antibody include, but are not limited to: (i) Fab fragments, a monovalent fragment consisting of VL, VH, CL, and CH1 domains; (ii) F(ab)2 fragments, a bivalent fragment comprising two Fab fragments linked by disulfide bonds in a hinge region; (iii) Fd fragments consisting of VH and CH1 domains; (iv) Fv fragments consisting of VL and VH domains of an antibody arm; (v) dAbs containing VH and VL domains; (vi) dAb fragments consisting of VH domains (Ward et al., Nature 341:544-546, 1989; the contents of which are incorporated herein by reference); (vii) dAbs consisting of either VH or VL domains; (viii) separate complementarity-determining regions (CDRs); and (ix) combinations of two or more separate CDRs, which may optionally be linked by synthetic linkers. Furthermore, although the two domains VL and VH of the Fv fragment are encoded by independent genes, these two domains can be joined using recombination methods via a linker that enables the formation of a single protein chain in which the VL and VH regions pair to form a monovalent molecule (referred to as single-chain Fv (scFv); see, for example, Bird et al., Science 242:423-426, 1988 and Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883, 1988; the contents of which are incorporated herein by reference). These antibody fragments can be obtained using conventional techniques known to those skilled in the art, and these fragments are screened for use in the same manner as intact antibodies. Antigen-binding fragments can be generated by recombinant DNA techniques, enzymatic or chemical cleavage of intact immunoglobulins, or, in some embodiments, by chemical peptide synthesis procedures known in the art.

[0147] In this invention, the term "CDR," also known as "complementarity-determining region," typically refers to a region within the variable structural domain of an antibody whose sequence is highly variable and / or forms a structurally defining loop. Typically, an antibody comprises six CDRs: three in the VH (HCDR1, HCDR2, HCDR3) and three in the VL (LCDR1, LCDR2, LCDR3). In some embodiments, naturally occurring camel antibodies consisting only of heavy chains can function normally and stably even in the absence of light chains. Antibody CDRs can be determined using various coding systems, such as CCG, Kabat, Chothia, IMGT, and a combination of Kabat / Chothia. These coding systems are known in the art. For example, the CDRs of said antigen-binding proteins can be classified according to the Kabat numbering system.

[0148] In this invention, the term "FR" generally refers to a more conserved portion of the antibody variable domain, which is called the frame region. Typically, the variable domains of the natural heavy and light chains each contain four FR regions, namely four in VH (H-FR1, H-FR2, H-FR3, and H-FR4) and four in VL (L-FR1, L-FR2, L-FR3, and L-FR4).

[0149] In this invention, the terms "variable domain" and "variable region" are used interchangeably and generally refer to a portion of the antibody heavy chain and / or light chain. The variable domains of the heavy and light chains may be referred to as "VH" and "VL" (or "VH" and "VL"), respectively. These domains are typically the most variable parts of the antibody (relative to other antibodies of the same type) and contain antigen-binding sites. In this invention, the term "VH" refers to the variable region of the immunoglobulin heavy chain of the antibody (including the heavy chain of Fv, scFv, or Fab). The term "VL" refers to the variable region of the immunoglobulin light chain (including the light chain of Fv, scFv, dsFv, or Fab).

[0150] In this invention, the term "heavy chain constant region" refers to the carboxyl-terminal portion of the antibody heavy chain, which does not directly participate in the binding of the antibody to the antigen but exhibits effector functions, such as interaction with the Fc receptor, and has a more conserved amino acid sequence relative to the variable domains of the antibody. The "heavy chain constant region" comprises at least one of the following: a CH1 domain, a hinge region, a CH2 domain, a CH3 domain, or a variant or fragment thereof. The "heavy chain constant region" includes a "full-length heavy chain constant region" and a "heavy chain constant region fragment," the former having a structure substantially similar to the natural antibody constant region, while the latter comprises 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 IgE, it also includes a CH4 domain; when the antibody is a heavy chain antibody, it does not include the CH1 domain. Exemplarily, a typical "heavy chain constant region fragment" may be selected from the CH1, Fc, or CH3 domains.

[0151] In this invention, the term "light chain constant region" refers to the carboxyl terminus of the antibody light chain, which does not directly participate in the binding of the antibody to the antigen. The light chain constant region can be selected from a constant κ domain or a constant λ domain.

[0152] In this invention, the term "Fc" refers to the carboxyl-terminal portion of an antibody obtained by papain hydrolysis of an intact antibody, typically comprising the CH3 and CH2 domains of the antibody. The Fc region includes, for example, the native sequence Fc region, the recombinant Fc region, and the variant Fc region. Although the boundaries of the Fc region of the immunoglobulin heavy chain can vary slightly, the Fc region of the human IgG heavy chain is generally defined as extending from the amino acid residue at Cys226 or from Pro230 to its carboxyl terminus. The C-terminal lysine of the Fc region (residue 447 according to the EU numbering system) can be removed, for example, during antibody production or purification, or by recombinant engineering of the nucleic acid encoding the antibody heavy chain; therefore, the Fc region may or may not include Lys447.

[0153] In this invention, the term "humanized antibody" refers to a genetically engineered non-human antibody whose amino acid sequence is modified to increase homology with that of a human antibody. Typically, all or part of the CDR region of a humanized antibody is derived from a non-human antibody (donor antibody), and all or part of the non-CDR region (e.g., the variable region FR and / or constant region) is derived from a human immunoglobulin (receptor antibody). Humanized antibodies generally retain or partially retain the intended properties of the donor antibody, including but not limited to antigen specificity, affinity, reactivity, ability to enhance immune cell activity, and ability to enhance immune responses.

[0154] In this invention, 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 contains a constant region, that constant region is also derived from a human germline immunoglobulin sequence. Fully human antibodies described herein may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced through random or site-specific mutagenesis in vitro or through somatic mutations in vivo). However, "fully human antibody" herein is not intended to include antibodies in which a CDR sequence derived from another mammalian species (e.g., mouse) has been grafted onto a human frame sequence.

[0155] In this invention, the term "naked antibody" refers to an antibody that is not linked, fused, or conjugated to another agent or molecule (e.g., a label or drug), peptide, or polypeptide. In specific embodiments, naked antibodies expressed by mammalian host cells can be glycosylated by the host cell's glycosylation machinery (e.g., glycosylation enzymes). In some embodiments, naked antibodies are not glycosylated when expressed by host cells that do not possess their own glycosylation machinery (e.g., glycosylation enzymes). In some embodiments, naked antibodies are intact antibodies, while in other embodiments, naked antibodies are antigen-binding fragments of intact antibodies, such as Fab antibodies.

[0156] In this invention, the term "conjugated antibody" refers to an antibody that can associate with a pharmaceutically acceptable carrier or diluent, which may be a monoclonal antibody, a chimeric antibody, a humanized antibody, or a human antibody.

[0157] In this invention, the term "monoclonal antibody" refers to an antibody derived from a single clone (including any eukaryotic, prokaryotic, or phage clone), and is not limited to the method of producing the antibody.

[0158] In this invention, the term "biantibody" refers to a bivalent, bispecific antibody that can bind to different epitopes on the same or different antigens.

[0159] In this invention, the term "smallest unit of antibody recognition" refers to the smallest unit of antigen that an antibody can recognize in an antigen-antibody binding reaction.

[0160] In this invention, the term "nanobody" refers to a naturally occurring heavy chain antibody in camels that lacks a light chain. Cloning its variable region yields a single-domain antibody consisting only of the heavy chain variable region, also known as VHH (Variable domain of heavy chain antibody), which is the smallest functional antigen-binding fragment.

[0161] In this invention, the variant can be, for example, a protein or polypeptide that has undergone substitution, deletion, or addition of one or more amino acids in the amino acid sequence of the protein and / or the polypeptide (e.g., an antibody that specifically binds to the CDH17 protein or a fragment thereof). For example, the functional variant may comprise a protein or polypeptide that has undergone amino acid alterations through substitution, deletion, and / or insertion of at least one, such as 1-30, 1-20, or 1-10, or even 1, 2, 3, 4, or 5 amino acids. The functional variant may substantially retain the biological properties of the protein or polypeptide prior to the alteration (e.g., substitution, deletion, or addition). For example, the functional variant may retain at least 60%, 70%, 80%, 90%, or 100% of the biological activity (e.g., antigen-binding capacity) of the protein or polypeptide prior to the alteration. For example, the substitution may be a conserved substitution.

[0162] In this invention, the homolog can be a protein or polypeptide having at least about 85% (e.g., having at least about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 95%, about 96%, about 97%, about 98%, about 99% or higher) sequence homology with the amino acid sequence of the protein and / or the polypeptide (e.g., an antibody that specifically binds to the CDH17 protein or a fragment thereof).

[0163] In this invention, the term "percentage (%) sequence identity" refers to the percentage of amino acid (or nucleotide) residues in a candidate sequence that are identical to those in a reference sequence after sequence alignment and the introduction of vacancies (if necessary) to achieve maximum percentage sequence identity (e.g., vacancies may be introduced in one or both of the candidate and reference sequences for optimal alignment, and non-homologous sequences may be ignored for comparison purposes). Alignment can be performed in a variety of ways well known to those skilled in the art for the purpose of determining percentage sequence identity, such as using publicly available computer software like BLAST, ALIGN, or Megalign (DNASTAIi) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms that require maximum alignment across the full length of the sequences being compared. For example, a reference sequence used for alignment with a candidate sequence may show sequence identity from 50% to 100% across the full length of the candidate sequence or selected portions of consecutive amino acid (or nucleotide) residues of the candidate sequence. The length of a candidate sequence for comparison purposes can be at least 30% (e.g., 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%) of the length of a reference sequence. Molecules are considered identical at that position when a position in a candidate sequence is occupied by the same amino acid (or nucleotide) residue as the corresponding position in the reference sequence.

[0164] In this invention, the term "conservative amino acid" 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 in each of the following groups belong to conserved amino acid residues, and substitutions of amino acid residues within a group constitute substitutions of conserved amino acids:

[0165] (1) Acidic amino acids: Asp (D) and Glu (E);

[0166] (2) Basic amino acids: Lys(K), Arg(R) and His(H);

[0167] (3) Hydrophilic uncharged amino acids: Ser(S), Thr(T), Asn(N) and Gln(Q);

[0168] (4) Aliphatic uncharged amino acids: Gly (G), Ala (A), Val (V), Leu (L) and Ile (I);

[0169] (5) Nonpolar, uncharged amino acids: Cys (C), Met (M), and Pro (P);

[0170] (6) Aromatic amino acids: Phe (F), Tyr (Y) and Trp (W).

[0171] In this invention, the terms "peptide molecule," "polypeptide," and "peptide" are used interchangeably and generally refer to polymers of amino acid residues. The term "fusion protein" generally refers to a polypeptide having at least two parts covalently linked together. Each part can be a polypeptide with different properties. These properties can be biological properties, such as in vitro or in vivo activity. They can also be simple chemical or physical properties, such as binding to target molecules, catalysis of reactions, etc. The two parts can be directly linked by a single peptide bond or through a peptide linker.

[0172] In this invention, the term "nucleic acid molecule" generally refers to a nucleotide, deoxyribonucleotide or ribonucleotide of any length in its isolated form, or an analogue isolated from its natural environment or synthesized artificially.

[0173] In this invention, the term "vector" generally refers to a nucleic acid delivery vehicle into which a polynucleotide encoding a protein can be inserted, thereby enabling the protein to be expressed. Vectors can be transformed, transduced, or transfected into host cells, allowing the genetic material elements they carry to be expressed within the host cells. For example, vectors may include: plasmids; phage particles; Cosmids; artificial chromosomes such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1-derived artificial chromosomes (PAC); bacteriophages such as λ phage or M13 phage; and animal viruses. Animal viruses used as vectors may include retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomaviruses (such as SV40). A vector may contain multiple elements controlling expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Additionally, the vector may contain a replication initiation site. The carrier may also include components that help it enter the cell, such as viral particles, liposomes, or protein coats, but not only these substances.

[0174] In this invention, the term "cell" generally refers to a single cell, cell line, or cell culture that may be or is already a recipient of a subject plasmid or vector, including the nucleic acid molecules or vectors described in this invention. Cells may include the progeny of a single cell. Due to natural, accidental, or intentional mutations, the progeny may not necessarily be identical to the original parent cell (in terms of the morphology of the total DNA complement or in the genome). Cells may include cells transfected in vitro using the vectors described in this invention. Cells may be bacterial cells (e.g., *Escherichia coli*), yeast cells, or other eukaryotic cells, such as COS cells, Chinese hamster ovary (CHO) cells, CHO-K1 cells, LNCAP cells, HeLa cells, HEK293 cells, COS-1 cells, NSO cells, but are not limited to these cells.

[0175] In this invention, the term "chimeric antigen receptor" (CAR) generally refers to a recombinant polypeptide comprising at least an extracellular domain, a transmembrane region, and an intracellular domain that specifically bind to an antigen or target. For example, a hinge region is included between the extracellular domain and the transmembrane region. For example, the chimeric antigen receptor may include a signal peptide. The binding of the extracellular domain of the CAR to the target antigen on the surface of the target cell leads to CAR clustering and delivers activation stimuli to CAR-containing cells. The CAR redirects the specificity of immune effector cells and triggers proliferation, cytokine production, phagocytosis, and / or production of molecules capable of mediating cell death expressing the target antigen in a major histocompatibility (MHC)-independent manner. For example, the extracellular structure may include the antigen-binding protein described above. For example, the extracellular structure may specifically bind CDH17.

[0176] In this application, the term "immunoconjugate" refers to a conjugate formed by attaching a payload to an antibody or its antigen-binding fragment via a stable linker, thereby allowing the antibody or its antigen-binding fragment to act as a carrier for targeted delivery of the payload to a target site. The term "payload" refers to the active portion conjugated to the antibody or antibody fragment of this invention.

[0177] The immunoconjugates described in this application utilize the antigen-binding protein's recognition of antigens to transport and release a payload near target cells. In this application, the antigen-binding protein can be an antibody or its antigen-binding fragment. For example, the antigen-binding protein can be an IgG antibody (e.g., IgG1 or IgG4 antibody). For example, the antigen-binding protein can bind to CDH17.

[0178] In this application, the immunoconjugate is typically formed by coupling an antigen-binding protein (Ab) to a payload (D) via a linker (L).

[0179] In this application, the payload may be a drug, such as a small molecule drug, a radionuclide, DNA, RNA, an enzyme, or a polypeptide. In this application, the payload may be a small molecule drug, such as a cytotoxic drug.

[0180] In this application, the immune conjugates include, but are not limited to, antibody drug conjugates (ADCs), antibody immunostimulant conjugates (ISACs), antibody oligonucleotide conjugates (AOCs), antibody peptide conjugates (APCs), or antibody radionuclide conjugates (RDCs).

[0181] In this application, preferably, the immunoconjugate is an antibody-drug conjugate, referring to a conjugate formed by linking an antigen-binding protein to a drug via a stable linker, wherein the drug is typically a cytotoxic drug. In this application, the terms "antibody-drug conjugate," "antibody-drug conjugate," "ADC," and "Antibody drug conjugates" are generally used interchangeably. In this application, the payload (D) of the ADC is a cytotoxic drug. In this application, the term "cytotoxic drug" refers to a substance that inhibits or prevents cell function and / or causes cell death or destruction, including toxins and other compounds that can be used for tumor treatment. The cytotoxic drug can kill tumor cells at sufficiently high concentrations.

[0182] This application also includes various deuterated forms of compounds of formula (I). Each available hydrogen atom bonded to a carbon atom can be independently replaced by a deuterium atom. Those skilled in the art can synthesize the deuterated forms of compounds of formula (I) with reference to relevant literature. Commercially available deuterated starting materials can be used in the preparation of the deuterated forms of compounds of formula (I), or they can be synthesized using conventional techniques with deuterating reagents, including but not limited to deuterated boranes, trideuterated borane tetrahydrofuran solutions, deuterated lithium aluminum hydride, deuterated iodoethane, and deuterated iodomethane.

[0183] The term "pharmaceutically acceptable salt" or "medicinal salt" refers to the salt of the immunoconjugate of this application, or the salt of the compound described in this application, which is safe and effective when used in mammals and has the intended biological activity. The ligand-drug conjugate of this application contains at least one amino group and can therefore form a salt with an acid. Non-limiting examples of pharmaceutical salts include: hydrochloride, hydrobromide, hydroiodide, sulfate, hydrogen sulfate, citrate, acetate, succinate, ascorbate, oxalate, nitrate, sorbate, hydrogen phosphate, dihydrogen phosphate, salicylate, hydrogen citrate, tartrate, maleate, fumarate, formate, benzoate, methanesulfonate, ethanesulfonate, benzenesulfonate, and p-toluenesulfonate.

[0184] This application also includes solvates of compounds of formula (I). The term "solvate" refers to an immunoconjugate of this disclosure that forms a pharmaceutically usable solvate with one or more solvent molecules, non-limiting examples of which include water, ethanol, acetonitrile, isopropanol, DMSO, and ethyl acetate.

[0185] In this application, the linker can be any linker capable of conjugating an antibody to an effective payload. In this application, the linker can be any linker used in techniques capable of site-specific conjugation. For example, when the immunoconjugate of this application is an antibody-drug conjugate, the linker is any linker capable of conjugating an antigen-binding protein to a cytotoxic drug.

[0186] In this application, the linker may be a cleavable linker or a non-cleavable linker. For example, the linker may be a non-cleavable linker, an enzyme-cleavable linker, an acid-cleavable linker, a GSH-cleavable reducing linker, an Fe(II)-cleavable linker, a photoresponsive cleavable linker, and / or a bioorthogonal cleavable linker.

[0187] In the immunoconjugates of the present invention, the "linker-payload (LD)" is linked to the antibody (Ab) via conventional coupling methods in the art, including: lysine coupling, reducing disulfide coupling, and directional coupling (Beck A, Reichert JM. Antibody-drug conjugates: Present and future; MAbs, 2014, 6:15-17; McCombs JR, Owen S C. Antibody drug conjugates: design and selection of linker, payload and conjugation chemistry. The AAPS journal, 2015, 17:339-351). Preferably, the "linker-payload (LD)" is coupled to the antibody (Ab) via reducing disulfide coupling, including linkage with one or more thiol groups (thiol groups of cysteine ​​residues) formed after reduction at two sites between the heavy chain.

[0188] As used in this article, This indicates the location where a structural segment connects to other parts of the molecule.

[0189] The term "one or more" means one or more under reasonable conditions, such as two, three, four, five or more.

[0190] The terms “optional” or “optionally” mean that the event or situation described below may, but does not have to, occur, and the description includes the circumstances under which the event or situation may or may not occur.

[0191] The term “substitution” means that any one or more atoms or groups (e.g., hydrogen) on a specified atom or group are replaced by a specified atom or group selected from the specified atom or group, such as hydroxyl, amino, halogen, alkoxy, alkylamine and three to six-membered rings or more.

[0192] The term "alkyl", either on its own or as part of another substituent, refers to an alkyl group having a specified number of carbon atoms (i.e., -C). 1-10This refers to a saturated aliphatic hydrocarbon group (one to ten carbon atoms), which is an uncyclic straight-chain or branched carbon chain (or carbon), or a combination thereof. Examples of saturated hydrocarbon groups include, but are not limited to, homologues and isomers of groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, isobutyl, sec-butyl, (cyclohexyl)methyl, (e.g.) n-pentyl, n-hexyl, n-heptyl, n-octyl, etc. Preferably, it is an alkyl group containing 1 to 12 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12) carbon atoms, more preferably an alkyl group containing 1 to 6 carbon atoms, and even more preferably an alkyl group containing 1 to 3 carbon atoms. For example, -C 1-3 Alkyl refers to methyl, ethyl, n-propyl, and isopropyl.

[0193] The term "cycloalkane" refers to a saturated or partially unsaturated monocyclic or polycyclic ring, and the term "cycloalkyl" refers to a saturated or partially unsaturated monocyclic or polycyclic cyclic hydrocarbon substituent, wherein the cycloalkyl ring contains 3 to 20 carbon atoms, preferably 3 to 12 carbon atoms, more preferably 3 to 8 (e.g., 3, 4, 5, 6, 7, and 8) carbon atoms, and even more preferably 3 to 6 carbon atoms. Non-limiting examples of monocyclic cycloalkyl include cyclopropyl, cyclobutyl, cyclopentyl, etc. Cycloalkyl can be optionally substituted or unsubstituted, and when substituted, the substituent can be substituted at any usable connection point.

[0194] The term "aryl" refers to an all-carbon monocyclic or fused polycyclic group with a fully conjugated π-electron system, including but not limited to phenyl, naphthyl, anthracene, etc., with phenyl being preferred.

[0195] The term "halogen" refers to the six elements belonging to Group 17 (formerly Group VIIA) of the periodic table, including fluorine (F), chlorine (Cl), bromine (Br), iodine (I), astatine (At), and... (Ts).

[0196] The term "heterocyclic" refers to a saturated or partially unsaturated monocyclic, bicyclic, or polycyclic cyclic hydrocarbon with a non-aromatic structure containing 3 to 20 ring atoms, wherein one, two, three, or more ring atoms are selected from N, O, or S, and the remaining ring atoms are C. The term "heterocyclic group" refers to a substituent generated based on the foregoing definition of a heterocyclic group. Exemplary 3-membered heterocyclic groups containing one heteroatom include azirropropyl, oxacyclopropyl, and thioherropropyl. Exemplary 4-membered heterocyclic groups containing one heteroatom include azirrobutyl, oxacyclobutyl, and thioherrobutyl.

[0197] The term "DAR (Drug to Antibody Ratio)" refers to the ratio of the effective payload (D) conjugated to the antibody (Ab) described herein to the amount of the antibody moiety. The DAR value of the immunoconjugates of the present invention can range from 1 to 10, but higher payloads are possible depending on the number of linker sites on the antibody. The DAR value can be calculated as the average DAR of the immunoconjugates in the composition of the immunoconjugates described herein, i.e., the overall ratio (molar ratio) of D conjugated to the Ab moiety described herein in the composition of the immunoconjugates, as determined by detection methods (e.g., by conventional methods such as mass spectrometry, ELISA, electrophoresis, and / or HPLC). In some embodiments of the present invention, the DAR value of the composition of the immunoconjugate is any integer or decimal from 1 to 10, for example 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 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, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10.0 or any value or range during the period.

[0198] In this application, the term "drug combination" generally refers to a combination containing at least two active ingredients / therapeutic agents. In some embodiments, each active ingredient / therapeutic agent may be prepared as an independent formulation (solid, liquid, gel, etc.); in some embodiments, each active ingredient / therapeutic agent may be present in different containers; and may be formulated simultaneously or separately with suitable carriers to form desired formulations as needed; in some embodiments, each active ingredient / therapeutic agent may be from different sources; in some embodiments, each active ingredient / therapeutic agent may be present in the form of a mixture; and in some embodiments, each active ingredient / therapeutic agent may be administered via the same or different routes of administration.

[0199] In this invention, the term "pharmaceutical composition" generally refers to a composition for the prevention / treatment of a disease or condition. The pharmaceutical composition may comprise the antibody or antigen-binding fragment described in this invention, the nucleic acid molecule described in this invention, the carrier described in this invention, and / or the cell described in this invention, and optionally a pharmaceutically acceptable adjuvant. Furthermore, the pharmaceutical composition may also comprise suitable formulations of one or more (pharmaceutically effective) carriers, stabilizers, excipients, diluents, solubilizers, surfactants, emulsifiers, and / or preservatives. The acceptable components of the composition are preferably non-toxic to the recipient at the dosage and concentration used. The pharmaceutical compositions of this invention include, but are not limited to, liquid, freeze-dried, and lyophilized compositions.

[0200] In this invention, the term "pharmaceutically acceptable carrier" generally includes pharmaceutically acceptable carriers, excipients, or stabilizers that are non-toxic to cells or mammals exposed thereto at the doses and concentrations employed. Physiologically acceptable carriers may include, for example, buffers, antioxidants, low molecular weight (less than about 10 residues) peptides, proteins, hydrophilic polymers, amino acids, monosaccharides, disaccharides and other carbohydrates, chelating agents, sugar alcohols, salt-forming anti-charge ions such as sodium; and / or nonionic surfactants.

[0201] In this invention, the terms "subject," "object," and "patient" refer to an organism receiving treatment for a specific disease or condition (such as cancer or an infectious disease) as described herein. Examples of objects and patients include mammals receiving treatment for diseases or conditions (such as proliferative disorders like cancer or infectious diseases), such as humans, primates, pigs, goats, rabbits, hamsters, cats, dogs, guinea pigs, members of the Bovidae family (such as domestic cattle, bison, buffalo, elk, and yaks), sheep, and horses.

[0202] In this invention, the term "treatment" refers to surgical or therapeutic treatment aimed at preventing, slowing (reducing) undesirable physiological changes or lesions in the treated subject, such as the progression of proliferative disorders (e.g., cancer or infectious diseases). Beneficial or desired clinical outcomes include, but are not limited to, symptom relief, disease severity reduction, disease stability (i.e., no worsening), delay or slowing of disease progression, improvement or mitigation of disease status, and remission (whether partial or complete), whether detectable or undetectable. Subjects requiring treatment include those already suffering from the condition or disease, those susceptible to the condition or disease, or those intending to prevent the condition or disease. When the terms slowing, reducing, weakening, mitigating, or alleviating are used, they also include elimination, disappearance, and non-occurrence.

[0203] In this invention, the term "effective amount" refers to a therapeutic amount that, when administered alone or in combination with another therapeutic agent to cells, tissues, or subjects, is effective in preventing or alleviating symptoms of a disease or the progression of that disease. "Effective amount" also refers to an amount of compound sufficient to relieve symptoms, such as treating, curing, preventing, or alleviating an associated medical condition, or increasing the rate at which such symptoms are treated, cured, prevented, or alleviated. When an active ingredient is administered alone to an individual, the therapeutically effective dose refers solely to that ingredient. When a combination is used, the therapeutically effective dose refers to the combined amount of active ingredients that produce a therapeutic effect, regardless of whether they are administered in combination, sequentially, or simultaneously.

[0204] In this invention, the term "tumor" refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all precancerous and cancerous cells and tissues. The terms "cancer" and "tumor" are not mutually exclusive when used herein.

[0205] In this invention, the term "antitumor agent" refers to antitumor drugs, which are a class of drugs for treating tumor diseases, such as chemotherapy drugs and biological agents.

[0206] In this invention, the proteins, polypeptides and / or amino acid sequences involved should also be understood to include at least the following range: variants or homologs that have the same or similar functions as the said protein or polypeptide.

[0207] In this invention, the term "comprising" generally means including, encompassing, containing, or including. In some cases, it also means "as" or "consisting of".

[0208] In this invention, the term "about" generally refers to a variation within a range of 0.5% to 10% above or below a specified value, such as a variation within a range of 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10% above or below a specified value.

[0209] Invention Details

[0210] Antibody or antigen-binding fragment

[0211] The CDR (Complementarity Determinant Region) of an antibody, also known as the complementarity-determining region, is part of the variable region. Amino acid residues in this region can contact antigens or antigenic epitopes. Antibody CDRs can be determined using various coding systems, such as CCG, Kabat, Chothia, IMGT, and a combination of Kabat / Chothia. These coding systems are known in the art and can be found, for example, at http: / / www.bioinf.org.uk / abs / index.html#kabatnum. Those skilled in the art can determine the CDR region using different coding systems based on the antibody's sequence and structure. The CDR region may differ when using different coding systems. In this application, the term CDR encompasses CDR sequences partitioned according to any CDR partitioning method; it also encompasses variants of the CDR, including amino acid sequences with substitutions, deletions, and / or additions of one or more amino acids. For example, 1-30, 1-20, or 1-10 amino acid substitutions, deletions, and / or insertions, or 1, 2, 3, 4, 5, 6, 7, 8, or 9 amino acid substitutions, deletions, and / or insertions; homologs are also included, which can be amino acid sequences having at least about 85% (e.g., having at least about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or higher) sequence homology with the amino acid sequence of the CDR. In some embodiments, the CDR is determined by the KABAT numbering scheme.

[0212] On the one hand, the present invention provides an antibody or antigen-binding fragment that can specifically bind to CDH17.

[0213] In this invention, the antibody or antigen-binding fragment that specifically binds to CDH17 may contain HCDR1, HCDR2 and HCDR3 of VH as shown in SEQ ID NO: 22.

[0214] In this invention, the antibody or antigen-binding fragment that specifically binds to CDH17 may include LCDR1, LCDR2 and LCDR3 of any one of the VLs shown in SEQ ID NO: 23.

[0215] In this invention, the antibody or antigen-binding fragment that specifically binds to CDH17 may contain HCDR1, the amino acid sequence of which is shown in SEQ ID NO:24.

[0216] In this invention, the antibody or antigen-binding fragment that specifically binds to CDH17 may contain HCDR2, the amino acid sequence of which is shown in SEQ ID NO:25.

[0217] In this invention, the antibody or antigen-binding fragment that specifically binds to CDH17 may contain HCDR3, the amino acid sequence of which is shown in SEQ ID NO:26.

[0218] In this invention, the antibody or antigen-binding fragment that specifically binds to CDH17 may contain LCDR1, the amino acid sequence of which is shown in SEQ ID NO:27.

[0219] In this invention, the antibody or antigen-binding fragment that specifically binds to CDH17 may contain LCDR2, the amino acid sequence of which is shown in SEQ ID NO:28.

[0220] In this invention, the antibody or antigen-binding fragment that specifically binds to CDH17 may contain LCDR3, the amino acid sequence of which is shown in SEQ ID NO:29.

[0221] In this invention, the antibody or antigen-binding fragment that specifically binds to CDH17 may comprise HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3. For example, the amino acid sequence of HCDR1 of the antibody or antigen-binding fragment described in this application is shown in SEQ ID NO:24, the amino acid sequence of HCDR2 is shown in SEQ ID NO:25, the amino acid sequence of HCDR3 is shown in SEQ ID NO:26, the amino acid sequence of LCDR1 is shown in SEQ ID NO:27, the amino acid sequence of LCDR2 is shown in SEQ ID NO:28, and the amino acid sequence of LCDR3 is shown in SEQ ID NO:29.

[0222] In this invention, the antibody or antigen-binding fragment that specifically binds to CDH17 may include the FR region.

[0223] In this invention, the antibody or antigen-binding fragment that specifically binds to CDH17 may include the antibody heavy chain variable region VH and / or the antibody light chain variable region VL.

[0224] In this invention, the amino acid sequence of the VH of the antibody or antigen-binding fragment that specifically binds to CDH17 can be as shown in SEQ ID NO: 22 and / or have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with respect to it.

[0225] In this invention, the amino acid sequence of the VL of the antibody or antigen-binding fragment that specifically binds to CDH17 can be as shown in SEQ ID NO: 23 and / or have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity compared to its sequence.

[0226] In this invention, the antibody or antigen-binding fragment that specifically binds to CDH17 may comprise VH as shown in SEQ ID NO: 22 and VL as shown in SEQ ID NO: 23; and combinations of VH and VL having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity compared to them.

[0227] In this invention, the antibody or antigen-binding fragment that specifically binds to CDH17 may include the constant region of the antibody heavy chain.

[0228] In this invention, the antibody heavy chain constant region may be derived from the human IgG heavy chain constant region.

[0229] In this invention, the antibody heavy chain constant region may be derived from the human IgG1 heavy chain constant region.

[0230] In this invention, the antibody or antigen-binding fragment that specifically binds to CDH17 may include the constant region of the antibody light chain.

[0231] In this invention, the antibody light chain constant region may be derived from the human Igκ constant region.

[0232] In this invention, the antigen-binding fragment may include Fab, Fab', Fv fragments, F(ab')2, F(ab)2, scFv, di-scFv and / or dAb.

[0233] In this invention, the antibody may be selected from one or more of the following groups: monoclonal antibody, polyclonal antibody, chimeric antibody, humanized antibody, fully human antibody, natural antibody, engineered antibody, monovalent antibody, multivalent antibody, full-length antibody, antibody fragment, naked antibody, conjugated antibody.

[0234] Furthermore, it should be noted that the antibody or antigen-binding fragment described in this application may contain heavy chain and / or light chain sequences with one or more conserved sequence modifications. "Conserved sequence modification" refers to amino acid modifications that do not significantly affect or alter the antibody-binding properties. Such conserved modifications include amino acid substitutions, additions, and deletions. Modifications can be introduced into the antibody or antigen-binding fragment described in this application using standard techniques known in the art, such as point mutations and PCR-mediated mutations. Conserved amino acid substitution involves replacing an amino acid residue with an amino acid residue having a similar side chain. Groups of amino acid residues with similar side chains are known in the art. These amino acid residue groups include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), non-polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). In some embodiments, one or more amino acid residues in the CDR region of the antibody or antigen-binding fragment described in this application may be replaced with other amino acid residues from the same side chain group. Those skilled in the art will recognize that some conserved sequence modifications will not result in the loss of antigen binding.

[0235] Chimeric antigen receptors, polypeptide molecules, nucleic acid molecules, carriers, cells, and drug compositions

[0236] On the other hand, the present invention also provides a chimeric antigen receptor (CAR) that may include a targeting portion that binds to the CDH17 protein.

[0237] On the other hand, this application provides polypeptide molecules that may contain the antibody or antigen-binding fragments described in this application.

[0238] In some embodiments, the polypeptide molecule may comprise a fusion protein. In some embodiments, the polypeptide molecule may be a fusion protein.

[0239] On the other hand, this application provides isolated nucleic acid molecules that can encode the antibody or antigen-binding fragments described in this application. For example, they can be generated or synthesized by: (1) in vitro amplification, for example by polymerase chain reaction (PCR); (2) clonal recombination; (3) purification, for example by enzyme digestion and gel electrophoresis fractionation; or (4) synthesis, for example by chemical synthesis.

[0240] On the other hand, this application provides a vector that may contain the nucleic acid molecule described in this application. Furthermore, the vector may also contain other genes, such as marker genes that allow selection of the vector in appropriate host cells and under appropriate conditions. Additionally, the vector may contain expression control elements that allow the coding region to be correctly expressed in an appropriate host. Such control elements are well known to those skilled in the art and may include, for example, promoters, ribosome binding sites, enhancers, and other control elements regulating gene transcription or mRNA translation. The vector can be transformed, transduced, or transfected into host cells to express the genetic material elements it carries within the host cells. The vector may include, for example, plasmids, granules, viruses, bacteriophages, or other vectors commonly used in, for example, genetic engineering. For example, the vector is an expression vector. Furthermore, the vector may also include components that facilitate its entry into cells, such as viral particles, liposomes, or protein coats, but not only these substances.

[0241] On the other hand, this application provides a cell that may contain the nucleic acid molecules or vectors described in this application. In some embodiments, each or every host cell may contain one or more of the nucleic acid molecules or vectors described in this application. In some embodiments, each or every host cell may contain multiple (e.g., two or more) or more types (e.g., two or more) of the nucleic acid molecules or vectors described in this application. For example, the vectors described in this application may be introduced into the host cell, such as eukaryotic cells, such as cells from plants, fungi, or yeast cells. In some embodiments, the cell may be a bacterial cell (e.g., Escherichia coli), a yeast cell, or other eukaryotic cells, such as COS cells, Chinese hamster ovary (CHO) cells, CHO-K1 cells, LNCAP cells, HeLa cells, 293T cells, COS-1 cells, SP2 / 0 cells, NSO cells, or myeloma cells. The vectors described in this application may be introduced into the host cell by methods known in the art, such as electroporation, lipofectine transfection, lipofectamin transfection, etc.

[0242] On the other hand, this application also provides pharmaceutical compositions that may comprise the antibody or antigen-binding fragment described in this application, the chimeric antigen receptor described in this application, the polypeptide molecule described in this application, the nucleic acid molecule described in this application, the carrier described in this application, the immunoconjugate described in this application or a pharmaceutically acceptable salt thereof, a composition of the immunoconjugate described in this application, a pharmaceutical combination described in this application and / or the cell described in this application, and optionally a pharmaceutically acceptable carrier.

[0243] In this invention, the pharmaceutical composition may further comprise suitable formulations of one or more (pharmaceutically effective) adjuvants, stabilizers, excipients, diluents, solubilizers, surfactants, emulsifiers, and / or preservatives. The acceptable components of the composition are preferably non-toxic to the recipient at the dosage and concentration used. The pharmaceutical compositions of this invention include, but are not limited to, liquid, freeze-dried, and lyophilized compositions.

[0244] In this invention, the pharmaceutical composition may be a bispecific or multispecific molecule comprising the antibody or antigen-binding fragment described in this application. In this invention, among the bispecific or multispecific molecules, the target sites bound to molecules other than those binding to CDH17 may be unrelated to CDH17.

[0245] In this invention, the pharmaceutical composition may be a composition comprising the immunoconjugate described in this application or a pharmaceutically acceptable salt thereof, such as a drug conjugate or a pharmaceutically acceptable salt thereof. In this invention, the pharmaceutical composition may be a composition of the immunoconjugate described in this application.

[0246] In this invention, the pharmaceutical composition may also contain more than one active compound, typically those with complementary activities that do not adversely affect each other. The type and effective amount of such a drug may depend, for example, on the amount and type of antagonist present in the formulation, and on the clinical parameters of the subject.

[0247] In this invention, the pharmaceutically acceptable carrier may include any and all solvents, dispersion media, coatings, isotonic agents and absorption delay agents that are compatible with drug administration, and are generally safe and non-toxic.

[0248] Immunoconjugates or their pharmaceutically acceptable salts

[0249] On the other hand, the present invention provides an immunoconjugate or a pharmaceutically acceptable salt thereof, comprising any of the foregoing antibody or antigen-binding fragments. In this application, the immunoconjugate can specifically bind to CDH17. For example, the immunoconjugate can specifically bind to CDH17-positive target cells.

[0250] In this application, the immune conjugate is formed by coupling an antigen-binding protein (Ab) to a payload (D) via a linker unit (L).

[0251] In this application, the immunoconjugate or its pharmaceutically acceptable salt may have the structure shown in formula (I).

[0252] Ab-(LD) p (I)

[0253] in:

[0254] Ab is any of the aforementioned antibody or antigen-binding fragments;

[0255] L represents the connection unit;

[0256] D represents the payload;

[0257] p is an integer from 1 to 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10.

[0258] In this application, regardless of whether Ab is any of the aforementioned antibodies or antigen-binding fragments, the payload (D) and linker (L) can be any of the following choices:

[0259] 1) In this application, the payload (D) may be a small molecule drug, a radionuclide, DNA, RNA, an enzyme, or a polypeptide. In this application, the payload may be a small molecule drug, such as a cytotoxic drug. In this application, the payload includes, but is not limited to, the payload described in Wang Zhijia, Li Hanxuan, Gou Lantu, Li Wei, Wang Yuxi. Antibody-drug conjugates: Recent advances in payloads J. Acta Pharmaceutica Sinica B, 2023, 13(10): 4025-4059.

[0260] 2) In this application, the connecting unit (L) is a non-disintegrable connecting unit or a disintegrable connecting unit. In this application, the disintegrable connecting unit can break within the target cell and release the payload.

[0261] 3) In this application, each L is independent and may be the same or different; each D is independent and may be the same or different.

[0262] 4) In this application, the immune conjugate may be an antibody-drug conjugate (ADC), an antibody-immunostimulant conjugate (ISAC), an antibody-oligonucleotide conjugate (AOC), an antibody-peptide conjugate (APC), or an antibody-isotope conjugate (RDC).

[0263] 5) In this application, the immunoconjugate may be an ADC, and when the immunoconjugate is an ADC, the payload is a cytotoxic drug. The ADC in this application has one or more of the following properties: (1) high affinity binding to CDH17; (2) high in vitro cytotoxic activity; (3) strong in vivo tumor suppression ability.

[0264] 6) In this application, the ADC is a single-load ADC, a dual-load ADC, or a multi-load ADC.

[0265] 7) In this application, the cytotoxic drug may be a microtubule inhibitor, a DNA intercalator, a DNA topoisomerase inhibitor, an apoptosis inducer, an RNA polymerase inhibitor, a spliceosome inhibitor, or a proteasome inhibitor, etc.

[0266] 8) In this application, the microtubule inhibitor may be an immunotoxin compound (e.g., monomethylolpropamine E (MMAE), monomethylolpropamine F (MMAF)), maytansine compound (e.g., DM1, DM4), tubulolysin compound (e.g., Tubulysins), etc.

[0267] 9) In this application, the DNA intercalating agent may be pyrrolobenzodiazepine. (PBD) or indolebenzodiazepine (IBD).

[0268] 10) In this application, the DNA topoisomerase inhibitor may be a topoisomerase I inhibitor, including camptothecin derivatives, such as hydroxycamptothecin (HCPT) and its derivatives, irinotecan and its derivatives, topotecan and its derivatives, exatecan and its derivatives, etc. In this application, topoisomerase I inhibitors include, but are not limited to, camptothecin, hydroxycamptothecin, 9-aminocamptothecin, SN38 (CAS No. 86639-52-3), irinotecan, ixotecan, topotecan, belotetcan, rubotecan, diflomotecan, lurtotecan, Karenitecin, gimatecan, namitecan, simmitecan, chimmitecan, silatecan, elomotecan, SHR9265 (CAS No. 2414254-51-4), T030 (CAS No. 2356229-14-4), DXD (CAS No. 1599440-33-1), compound 2A, or ixotecan derivatives (or derivatives of DXD) as described in patent PCT / CN2025 / 107799, the entire contents of which are incorporated herein by reference. Compound 2A is... In this application, the DNA topoisomerase inhibitor may be a topoisomerase II inhibitor, such as doxorubicin, doxorubicin, PNU-159682 and its analogues, docalmicin, daunorubicin, mitoxantrone, podophyllotoxin, or etoposide.

[0269] 11) In this application, the apoptosis inducer may be a Bcl-xL inhibitor, such as clezutoclax (CAS No. 1949843-71-3).

[0270] 12) In this application, the RNA polymerase inhibitor may be an amatoxin-like compound, including α-amanitin and β-amanitin.

[0271] 13) In this application, spliceosome inhibitors include thailanstatin and its analogues (e.g., thailanstatin A).

[0272] 14) In this application, the proteasome inhibitors include carbamycin compounds (e.g., carbamycin A, carbamycin B).

[0273] 15) In some embodiments of this application, D is a DNA topoisomerase inhibitor; preferably DXD or a derivative of DXD; more preferably DXD or compound 2A.

[0274] 16) In some preferred embodiments of this application, D is DXD.

[0275] 17) In some preferred embodiments of this application, D is compound 2A.

[0276] 18) In this application, the linking unit may be a cleavable linking unit or a non-cleavable linking unit. For example, the linking unit may be a non-cleavable linking unit, an enzyme-cleavable linking unit, an acid-cleavable linking unit, a GSH-cleavable reducing linking unit, an Fe(II)-cleavable linking unit, a photoresponsive cleavable linking unit, and / or a bioorthogonal cleavable linking unit.

[0277] 19) In some preferred embodiments of this application, L is The left end is connected to Ab, and the right end is connected to D.

[0278] 20) In some preferred embodiments of this application, the LD and the Ab are coupled by a reducing disulfide bond, preferably by a reaction of one or more thiol groups (thiol groups of cysteine ​​residues) formed after reduction at two sites between the heavy chains.

[0279] 21) In some preferred embodiments of this application, L is The left end is connected to Ab, and the right end is connected to D, where D is DXD or compound 2A.

[0280] 22) In some preferred embodiments of this application, the immunoconjugate or its pharmaceutically acceptable salt has the following structure:

[0281] Where p is an integer from 1 to 10.

[0282] 23) In some preferred embodiments of this application, the immunoconjugate or its pharmaceutically acceptable salt has the following structure:

[0283] Where p is an integer from 1 to 10.

[0284] Composition of immune conjugates

[0285] On the other hand, this application provides a composition of an immunoconjugate comprising one or more of the immunoconjugates described above or their pharmaceutically acceptable salts.

[0286] In some embodiments, the DAR value of the immunoconjugate composition is any integer or decimal from 1 to 10, preferably any integer or decimal from 2 to 8, more preferably any integer or decimal from 3 to 8, such as 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 5.0, 5.5, 6.0, 6.5, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0 or any value or range thereof.

[0287] Drug combination

[0288] On the other hand, this application also provides a pharmaceutical combination comprising any of the foregoing antibodies or antigen-binding fragments, the chimeric antigen receptor, the polypeptide molecule, the nucleic acid molecule, the carrier, the cell, the immunoconjugate, or a pharmaceutically acceptable salt and / or immunoconjugate thereof.

[0289] In some embodiments, the drug combination may further include one or more other therapeutic agents; preferably, the therapeutic agents may be other antibodies, small molecule drugs, immunomodulators, cytotoxic agents, chemotherapeutic agents, or cytokines.

[0290] In this application, a pharmaceutical combination can refer to a product resulting from a mixture or combination of more than one active ingredient, and includes fixed and non-fixed combinations of active ingredients. A fixed combination can refer to an active ingredient (e.g., the anti-CDH17 antigen-binding protein described in this invention) and one or more combination partners being administered to a patient simultaneously in a single entity or dose. A non-fixed combination can refer to an active ingredient and one or more combination partners being administered to a patient simultaneously, jointly, or sequentially (without a specific time limit) as separate entities (dosage forms may be independent), and the means of administration may be the same or different, wherein such administration provides a therapeutically effective level of two compounds in the patient's body. The latter also applies to cocktail therapies, such as the administration of three or more active ingredients.

[0291] Preparation method

[0292] On the other hand, the present invention provides a method for preparing the aforementioned antibody or antigen-binding fragment. The method may include culturing the host cells described in this application under conditions that allow the antibody or antigen-binding fragment to bind. For example, this can be achieved by using appropriate culture media, appropriate temperatures, and culture times, methods known to those skilled in the art.

[0293] Any method suitable for producing monoclonal antibodies can be used to generate the antibodies or antigen-binding fragments of this application. For example, animals can be immunized using linked or naturally occurring CDH17 or fragments thereof. Suitable immunization methods can be used, including adjuvants, immunostimulants, repeated booster immunizations, or one or more of these methods. For example, hybridoma preparation methods can be used to obtain spleen cells from immunized mice, fuse them with SP2 / 0 myeloma cells, and screen for hybridoma cell lines using HAT.

[0294] Any suitable form of CDH17 can serve as an immunogen (antigen) for generating non-human antibodies specific to CDH17 and screening for the biological activity of said antibodies. For example, the stimulating immunogen can be full-length mature human CDH17, including natural homodimers or peptides containing one or more epitopes. The immunogen can be used alone or in combination with one or more immunogenicity enhancers known in the art.

[0295] Chimeric human antibodies can be selected from any type of immunoglobulin, including IgM, IgD, IgG, IgA, and IgE. In this application, the antibody can be an IgG antibody, and the IgG1 subtype can be used. The necessary constant domain sequence can be optimized to produce the desired biological activity by screening antibodies using the biological assays described in the examples below. Similarly, any class of light chains can be used in the compounds and methods of this application. For example, the κ chain or a variant thereof can be used in the compounds and methods of this application.

[0296] On the other hand, this application also provides a method for preparing the aforementioned immunoconjugates or pharmaceutically acceptable salts and / or compositions of immunoconjugates, the method comprising conjugating any of the foregoing antibodies or antigen-binding fragments to a payload. In this application, the method may include linking the antigen-binding protein, linker, and payload via a chemical reaction. Suitable methods for preparing immunoconjugates and ADCs of the present invention are, for example, found in US10973924B2 and WO2021 / 115426A1.

[0297] Methods and uses

[0298] On the other hand, the present invention provides the use of the antibody or antigen-binding fragment, the chimeric antigen receptor, the polypeptide molecule, the nucleic acid molecule, the carrier, the cell, the immunoconjugate or a pharmaceutically acceptable salt thereof, a composition of the immunoconjugate, the pharmaceutical combination and / or the pharmaceutical composition in the preparation of a medicament for the prevention and / or treatment of a disease and / or condition.

[0299] On the other hand, the present invention also provides methods for preventing and / or treating diseases and / or conditions, the methods including administering to a subject in need an antibody or antigen-binding fragment of the present application, the chimeric antigen receptor, the polypeptide molecule, the nucleic acid molecule, the carrier, the cell, the immunoconjugate or a pharmaceutically acceptable salt thereof, a composition of the immunoconjugate, the pharmaceutical combination and / or the pharmaceutical composition.

[0300] On the other hand, the antibodies or antigen-binding fragments, the polypeptide molecules, the nucleic acid molecules, the carriers, the cells, the immunoconjugates or their pharmaceutically acceptable salts, the compositions of the immunoconjugates, the pharmaceutical combinations and / or the pharmaceutical compositions of the present invention can be used for the prevention and / or treatment of diseases and / or conditions.

[0301] In this invention, the disease and / or symptom may be a CDH17-related disease and / or symptom.

[0302] In this invention, the disease and / or symptom can be a tumor.

[0303] In this invention, the disease and / or may be a CDH17-related tumor.

[0304] In some embodiments, the disease and / or condition includes gastrointestinal tumors, including colorectal cancer, colorectal adenocarcinoma, gastric cancer, gastric adenocarcinoma, pancreatic cancer, pancreatic adenocarcinoma, etc.

[0305] On the other hand, this application also provides a method for detecting CDH17 in a sample, the method comprising administering the antibody or antigen-binding fragment, the polypeptide molecule, the nucleic acid molecule, the carrier, the cell, the immunoconjugate or a pharmaceutically acceptable salt thereof, a composition of the immunoconjugate, the pharmaceutical combination and / or the pharmaceutical composition.

[0306] In this invention, the method for detecting CDH17 in a sample can be an in vitro method. In this application, the method for detecting CDH17 in a sample can be for non-therapeutic purposes. In this invention, the method for detecting CDH17 in a sample is not a diagnostic method.

[0307] On the other hand, the present invention also provides reagents or kits for detecting CDH17 in samples, comprising the antibody or antigen-binding fragment, the polypeptide molecule, the nucleic acid molecule, the carrier, the cell, the immunoconjugate or a pharmaceutically acceptable salt thereof, a composition of the immunoconjugate, the pharmaceutical combination and / or the pharmaceutical composition.

[0308] On the other hand, the present invention also provides the use of the antibody or antigen-binding fragment, the polypeptide molecule, the nucleic acid molecule, the carrier, the cell, the immunoconjugate or a pharmaceutically acceptable salt thereof, a composition of the immunoconjugate, the pharmaceutical combination and / or the pharmaceutical composition in the preparation of a kit for detecting the presence and / or content of CDH17 in a sample.

[0309] To further illustrate the technical means and effects of this invention, the following description, in conjunction with embodiments and accompanying drawings, provides a further explanation of the invention. It is understood that the specific embodiments described herein are merely illustrative of the invention and not intended to limit it.

[0310] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0311] Example 1: Preparation of mouse monoclonal antibody against human CDH17

[0312] Murine monoclonal antibodies were prepared using hybridoma preparation technology (Kohler et Milstein, Nature, 1975, 256: 495-497). Recombinant human CDH17 protein (Sino Biological, Cat No: 11360-H08H) was used as the primary immunogen, with recombinant monkey CDH17 protein (Sino Biological, Cat No: 90994-C08H) and recombinant mouse CDH17 protein (ACRO, Cat No: CA7-M5256) used alternately as immunogens. The initial immunization used Freund's Adjuvant, Complete (Sigma cat no. F5881, alias: FCA) for emulsification, and subsequent immunizations used Freund's Adjuvant, Incomplete (Sigma cat no. F5006, alias: FICA) for emulsification. Multiple BalB / C, C57BL / 6, ICR, and SJL mice were immunized subcutaneously at multiple sites. After four immunizations, serum titers were measured using ELISA. When the serum titer reached the target value, a pre-fusion booster immunization was performed. Finally, the best mice were selected to obtain spleen cells for fusion with SP2 / 0 myeloma cells. Hybridoma cell lines were selected through HAT screening. BLI and FACS methods were used to screen the hybridoma cell supernatant at the molecular and cellular levels, respectively, to identify monoclonal hybridoma cell lines that specifically bind to human and monkey CDH17. Furthermore, binding activity was screened against the human gastric cancer cell line SNU-5, which highly expresses CDH17. Finally, the best hybridoma cell line was selected for sequence retrieval.

[0313] Table 1. Hybridoma screening data

[0314] Example 2: Cloning of the variable region gene sequence of the anti-CDH17 antibody

[0315] 2.1 Cloning of variable region genes of antibodies in hybridoma cells

[0316] The preferred hybridomas 31A3 and 60B10 from Example 1 were selected, and total RNA was extracted from mouse hybridoma cells using the QIAGEN RNA Extraction Kit (catalog number: 74181) according to the manufacturer's instructions. Based on the TAKARA 5'RACE technology principle, the cDNA sequence of the variable region of the mouse antibody expressed by the hybridoma cell line was cloned. Using the SMARTer 5'RACE Synthesis Kit (TAKARA, catalog number 634859), gene-specific cDNAs of the heavy and light chain variable regions were synthesized according to the manufacturer's instructions. The 5' and 3' ends of the cDNA sequence were modified with PCR primers designed to add appropriate leader sequences to the heavy and light chain variable region cDNAs, respectively, so that the resulting PCR products could be cloned into existing heavy chain and light chain vectors for recombinant antibody expression using a seamless cloning method. The heavy chain expression vector contains the human IgG1 heavy chain constant region gene sequence, with L234A and L235A (Eu numbering) mutations at CH2 that weaken the antibody ADCC effect; the light chain vector contains the human κ light chain constant region gene sequence. The PCR amplification products of the heavy and light chain variable regions were cloned into the expression vector using an in-fusion cloning reagent (TAKARA, catalog number 638949) to obtain a human-mouse chimeric antibody expression vector, which was then transformed into *E. coli* DH5α competent cells (TAKARA, catalog number 636763). Single colonies were selected and Sanger sequencing was performed to obtain the variable region sequence of the hybridoma clonal antibody.

[0317] Table 2. Sequence List of Mouse Monoclonal Antibodies

[0318] Table 3. CDRs (KABAT) of murine monoclonal antibody molecules

[0319] Example 3: Expression and activity detection of chimeric antibodies

[0320] 3.1 Expression of chimeric antibodies

[0321] The expression vector obtained in Example 2 was amplified by *E. coli*, and sufficient plasmids were prepared using an endotoxin-free plasmid extraction kit (Tiangen Biotech (Beijing) Co., Ltd., catalog number DP117) for transient transfection to express chimeric antibodies. The host cells used for expression were CHO-S cells (Thermo Fisher Scientific, catalog number R80007). The two prepared heavy chain vectors were mixed with the light chain vector and polyetherimide (PEI, Polysciences, catalog number 24765-1) to form liposome complexes, which were then transfected into CHO-S cells and cultured for 5-7 days. The cell culture supernatant was collected by centrifugation and purified using a Protein A affinity chromatography column to obtain human-mouse chimeric antibodies, numbered 901109 and 901134, corresponding to hybridoma clone numbers 31A3 and 60B10, respectively. The control antibody is Anti-human-CDH17 hIgG1 Antibody (GM-52672AB), synthesized based on the CDH17 antibody sequence in BI-905711, hereinafter referred to as BI-CDH17.

[0322] Table 4. Sequence listing of chimeric antibody constant region and control antibody

[0323] 3.2 Affinity Detection of Anti-human CDH17 Chimeric Antibody

[0324] The Biacore (SPR) assay was used to detect the affinity of antibodies for CDH17 from different species, as well as its homologs CDH4 and CDH16. The method was as follows: The Biacore 8k flow cell temperature was set to 25°C. A Series S Sensor Chip Protein A (Cytiva, 29127556) chip was used to capture and detect chimeric antibodies as Ligands. The following proteins were also used: Mouse Cadherin-17 / CDH17 Protein, His Tag (ACRO Biosystems, CA7-M52H5); Recombinant Cynomolgus Cadherin-17 Protein (His Tag) (Sino Biological, 90994-C08H); and Human Cadherin-17 / CDH17 Protein, His Tag (ACRO Biosystems, CA7-M52H5). Biosystems (CA7-H52H3) was diluted to 100 nM, then further diluted 2-fold to create seven concentration gradients for analysis. The flow rate was 30 μL / min, with binding time of 120 s and dissociation time of 300 s. 10 mM Glycine pH 1.5 (Cytiva, BR100354) was used at a flow rate of 50 μL / min for regeneration of 60 s. The kinetic constants were analyzed using the Multicycle Kinetics Using Capture Method model, a 1:1 Binding Model, and Fit local analysis. The results are shown in Table 5.

[0325] Table 5 Affinity of chimeric antibodies Note: NA: Unable to fit the data; NB: No binding.

[0326] 3.3 Detection of binding between anti-human CDH17 chimeric antibody and membrane-expressed antigen

[0327] Collect cultured CDH17-overexpressing cells (CHOK1 cells overexpressing huCDH17 / rhCDH17 / MoCDH17; and SNU-5 tumor cells overexpressing human CDH17). After washing once with PBS, resuspend the cells in an appropriate concentration of Zombie dye and incubate at room temperature for 15 min. Centrifuge at 500g for 3 min, discard the supernatant, and wash twice with FACS buffer. After adjusting the cell density, seed the cells into 96-well U-plates at 2 × 10⁶ cells per well. 4Cells were centrifuged at 500g for 3 min, the supernatant was discarded, and 20 μL of serially diluted antibody or mouse immune serum was added to resuspend the cells. The cells were incubated at room temperature for 30 min. After centrifugation at 500g for 3 min, the supernatant was discarded, and the cells were washed twice with FACS buffer. Then, 20 μL / well of fluorescently labeled secondary antibody (PE-anti-human IgG-Fc) was added, and the cells were incubated at room temperature for 15 min. After centrifugation at 500g for 3 min, the supernatant was discarded, and the cells were washed twice with FACS buffer. The cells were resuspended in FACS buffer, and the signal was detected using flow cytometry. The concentration curve was analyzed, and the results are shown in Table 6 and Figures 1-3.

[0328] Table 6. Binding of chimeric antibodies to CDH17 expressed on cell membranes. Note: NA: Unable to fit the data; NB: No binding.

[0329] 3.4 Detection of endocytosis of anti-human CDH17 chimeric antibodies

[0330] The endocytic activity of anti-human CDH17 chimeric antibodies was assessed using AGS and AsPC-1 cells. AGS or AsPC-1 cells were digested with trypsin (washed once with PBS, approximately 37°C for 3 min), cells were collected and resuspended in culture medium, and the cell density was adjusted to 1 × 10⁻⁶ cells / mL. 6Cells / mL, 100 μL / well were aliquoted into 96-well U-type plates. Antibody was adjusted to the appropriate concentration with culture medium, and 100 μL / well was added to the corresponding cell wells. An equal volume of culture medium was added to the blank control group. Incubation was performed at 37°C for 24 h. After incubation, cells were centrifuged at 500g for 3 min, the supernatant was discarded, and the cells were then digested with trypsin (after washing once with PBS, approximately 37°C, 3 min). After centrifugation to discard the supernatant (room temperature, 500g × 3 min), the cells were washed once with PBS, and 100 μL of an appropriate concentration of Zombie dye was added to each well for resuspending. Incubation was performed at room temperature for 15 min. After incubation, cells were centrifuged to discard the supernatant (room temperature, 500g × 3 min), the cells were washed twice with FACS buffer, and 100 μL of fixative was added to each well for resuspending. Incubation was performed at room temperature for 15 min. After incubation, cells were centrifuged to remove the supernatant (room temperature, 500g × 3 min). For intracellular staining, cells were washed twice with 1× permeabilization buffer, and 100 μL of fluorescent antibody (AF488-anti-human IgG-Fc) diluted to an appropriate concentration with 1× permeabilization buffer was added to each well to resuspend the cells. Cells were incubated at 4℃ for 30 min. For surface staining, cells were washed twice with FACS buffer, and 100 μL of fluorescent antibody (AF488-anti-human IgG-Fc) diluted to an appropriate concentration with FACS buffer was added to each well to resuspend the cells. Cells were incubated at 4℃ for 30 min. After incubation, cells were centrifuged to remove the supernatant (room temperature, 500g × 3 min), washed twice with FACS buffer, and resuspended in FACS buffer. Flow cytometry was used to detect the signal and analyze the endocytosis rate. endocytosis rate = (relative fluorescence intensity of intracellular antibody - relative fluorescence intensity of surface antibody) / relative fluorescence intensity of intracellular antibody (relative fluorescence intensity of intracellular antibody = relative fluorescence intensity of intracellular antibody - average fluorescence intensity of intracellular blank group, relative fluorescence intensity of surface antibody = relative fluorescence intensity of surface antibody - average fluorescence intensity of surface blank group). The results are shown in Table 7.

[0331] Table 7. Ingestion rate of chimeric antibodies

[0332] Example 4: Humanization and Activity Detection of Anti-human CDH17 Antibody

[0333] 4.1 Humanization of anti-human CDH17 antibody

[0334] Humanization of antigen-binding protein 901134 employed a 3D modeling approach: First, the variable region sequence of the murine antigen-binding protein 901134 antibody was used for 3D structure prediction via the Alphafold3 online server (https: / / alphafoldserver.com / ). The structural model with the highest confidence was selected for visualization and modeling analysis in Pymol software. The CDR1, CDR2, and CDR3 of the antibody heavy and light chains were assigned using the Kabat numbering system and labeled with different colors in the software. Then, the variable region sequence of 901134 was aligned and compared with human homologous sequences in the NCBI IgBlast database. The framework regions (FR regions) of the top 10 human sequences with the highest homology were identified as reference sequences for CDR transplantation.

[0335] First, the six FR regions of the light and heavy chains of the 901134 mouse antibody were replaced with the amino acid residues with the highest conservation among multiple human sequences. Then, back mutations were performed on amino acid sequences that might affect antigen binding. Back mutations were defined as amino acids spatially within 3.5 Å of the six CDR regions of 901134; distances were calculated using PyMOL software and visualized for structural verification. Finally, the designed humanized sequences should not affect antibody structural stability and should not introduce deamidation, aspartic acid isomerization, glycosylation, phosphorylation, or sites prone to oxidation. The designed humanized heavy and light chain sequences are as follows: underlined regions are CDR regions (Kabat definition).

[0336] >901201_VH(SEQ ID NO: 22)

[0337] >901201_VL(SEQ ID NO: 23)

[0338] Table 8. Humanized Antibody CDR Sequences (KABAT Definition)

[0339] The humanized heavy chain and light chain variable region genes were synthesized to obtain DNA templates, which were then ligated into mammalian cell expression vectors containing humanized heavy chain IgG1 and Igκ light chain constant regions, respectively. Humanized antibody 901201 was obtained by expression according to the method described in Example 2.

[0340] CH(SEQ ID NO: 17):

[0341] CL (SEQ ID NO: 18):

[0342] 4.2 Affinity and Specificity Detection of Humanized Anti-human CDH17 Antibody

[0343] The affinity of 901201 for CDH17 proteins from different species was systematically detected using Biacore surface plasmon resonance (SPR) technology. Simultaneously, its cross-reactivity with CDH17 congeners (CDH4 and CDH16) was verified. The specific methods are as follows:

[0344] 4.2.1 Affinity testing with CDH17 from different species

[0345] The Biacore 8k instrument was set to a flow cell temperature of 25°C. The Series S Sensor Chip Protein A (Cytiva, catalog number 29127556) chip was used to capture 901201 as a ligand. Human CDH17 protein (ACRO Biosystems, catalog number CA7-H52H3, His tag), monkey CDH17 protein (Sino Biological, catalog number 90994-C08H, His tag), and mouse CDH17 protein (ACRO Biosystems, catalog number CA7-M52H5, His tag) were diluted to 100 nM, and then serially diluted 2-fold to prepare 7 concentration gradients as analytes. The detection parameters were set as follows: flow rate 30 μL / min, binding time 120 s, and dissociation time 300 s; the chip was regenerated for 60 s using 10 mM Glycine (pH 1.5, Cytiva, catalog number BR100354) at a flow rate of 50 μL / min. The experiment employed the "Multicycle Kinetics Using Capture Method" mode, and the kinetic constants were analyzed using a 1:1 Binding Model and the Fit local algorithm.

[0346] 4.2.2 Cross-reactivity detection with CDH4 / CDH16

[0347] The sample chamber and flow cell temperatures of the Biacore 8k instrument were set to 25°C, and the data collection frequency was 10Hz. Protein A chip was used to capture 901201 (approximately 400 RU) as a ligand. Human CDH4 and CDH16 proteins were diluted to 100 nM using HBS-EP+Buffer (pH 7.4) as the diluent. Pure HBS-EP+Buffer was used as a 0-concentration control for background subtraction. Detection parameters were set as follows: analyte flow rate 30 μL / min, binding time 120 s, dissociation time 60 s. The chip was regenerated twice using 10 mM Glycine (pH 1.5) at a flow rate of 50 μL / min for 60 s. The 1:1 Binding Model and Fit Local Kinetics algorithm were used to analyze the detection data.

[0348] Example 4.2 The detection results show (Table 9) that 901201 has a high affinity for human CDH17 protein and cross-binding activity with monkey CDH17 protein; at the same time, the antibody is highly specific and does not cross-react with CDH4 and CDH16 proteins of the CDH17 family, demonstrating good targeting specificity.

[0349] Table 9 Results of Humanized Antibody Affinity and Specificity Detection Note: NA: Unable to fit the data; NB: No binding.

[0350] 4.3 Detection of binding between anti-human CDH17 humanized antibody 901201 and membrane-expressed antigen

[0351] CHOK1 cells, which overexpress CDH17 (overexpressing huCDH17 / rhCDH17 (Rhesus macaque cadherin-17)), and tumor cells overexpressing human CDH17 (SNU-16 human gastric cancer cell line and AsPC-1 human metastatic pancreatic adenocarcinoma cell line) were collected. After washing once with PBS, the cells were resuspended with an appropriate concentration of Zombie stain and incubated at room temperature for 15 min. The cells were centrifuged at 500g for 3 min, the supernatant was discarded, and the cells were washed twice with FACS buffer. After adjusting the cell density, the cells were seeded into 96-well U-plates at 2 × 10⁶ cells per well. 4Cells were centrifuged at 500g for 3 min, the supernatant was discarded, and 20 μL of serially diluted antibody or mouse immune serum was added to resuspend the cells. The cells were incubated at room temperature for 30 min. After centrifugation at 500g for 3 min, the supernatant was discarded, and the cells were washed twice with FACS buffer. Then, 20 μL / well of fluorescently labeled secondary antibody (PE-anti-human IgG-Fc) was added, and the cells were incubated at room temperature for 15 min. After centrifugation at 500g for 3 min, the supernatant was discarded, and the cells were washed twice with FACS buffer. The cells were then resuspended in FACS buffer, and the signal was detected using flow cytometry, and the results were analyzed using concentration curves.

[0352] The test results (Figures 4-7) showed that the humanized anti-human CDH17 antibody 901201 exhibited excellent binding activity to membrane-expressed CDH17 antigen: firstly, it could efficiently bind to CDH17 antigen expressed on the surface of CHOK1 engineered cell line and SNU-16 and AsPC-1 tumor cell lines, demonstrating strong binding ability; secondly, it showed good species cross-reactivity, with its binding ability to membrane-expressed monkey CDH17 being comparable to that of human CDH17, providing experimental support for subsequent evaluation of efficacy and safety in non-human primates.

[0353] 4.4. Rapid endocytosis assay of 901201 antibody in pancreatic cancer AsPC-1 cells

[0354] Detection of endocytosis of humanized antibodies. AsPC-1 cells (ATCC, CRL-1682) were digested with 0.25% Trypsin-EDTA (Gibco, 25200072), collected, centrifuged at 500g for 3 min, and the supernatant was discarded. The cells were resuspended in RPMI 1640 medium (Gibco, A1049101) containing 10% FBS (Gibco, 10099141C), counted by AO / PI (Mengwei, SC1009), and the cell density was adjusted. The cells were then seeded at 100 μL / well into 96-well cell plates (Corning, 7007). The 901201 antibody was diluted to 133.4 nM with complete culture medium and added at 100 μL / well to the above-inoculated cells at preset incubation time points (24 h, 6 h, 3 h, 1 h, 30 min, 15 min, 5 min, 0 min, with the time of centrifugation and discarding of supernatant as min). The cells were then incubated for the corresponding time in a 37℃ CO2 incubator. After incubation, cells were centrifuged at 500g for 3 min, the supernatant was discarded, and the cells were washed twice with PBS (Shanghai Yuanpei, B320KJ). Cells were then digested with 0.25% Trypsin-EDTA (Gibco, 25200072), centrifuged at 500g for 3 min, the supernatant was discarded, and the cells were stained with 1:1000 diluted Zombie violet (BioLegend, 423114) solution at room temperature for 15 min. After washing once with FACS buffer (PBS + 1% BSA, Sangon Biotech, A600332-0100), cells were fixed with Fixation Buffer (BioLegend, 420801) at room temperature for 15 min. After fixation, cells treated with the same antibody were washed twice with FACS buffer and Intracellular Staining Permeabilization Wash Buffer (BioLegend, 421002), and then 1:200 diluted Alexa was added. 488-labeled secondary antibody (Jackson ImmunoResearch, 109-545-098), 100 μL / well, incubated at 4℃ for 30 min. After incubation, the cells were centrifuged at 500g for 3 min, the supernatant was discarded, and the cells were washed twice with FACS Buffer. The cells were then resuspended in FACS Buffer and analyzed using a BD FACS Canto II flow cytometer. The intracellular MFI was calculated according to the formula "Intracellular MFI = Permeabilized MFI - Non-permeabilized MFI". The results (Figure 8) showed that the 901201 antibody reached the endocytic plateau phase in AsPC-1 cells after about 3 hours of incubation, fully demonstrating the rapid advantage of the mediated endocytosis process.

[0355] 4.5. Internalization Concentration Effect Experiment of 901201 Antibody in Pancreatic Cancer AsPC-1 Cells and Gastric Cancer Cell Line 23132 / 87

[0356] Detection of endocytosis of humanized antibodies. CDH17-positive AsPC-1 cells (ATCC, CRL-1682) and gastric cancer cell line 23132 / 87 (Nanjing Kebai Biotechnology) were digested with 0.25% Trypsin-EDTA (Gibco, 25200072), centrifuged at 500g for 3 min, the supernatant was discarded, and the cells were resuspended in RPMI 1640 medium containing 10% FBS. After AO / PI counting, 100 μL / well was seeded into 96-well plates (Corning, 3799) and incubated overnight at 37°C. The 901201 antibody was diluted to 1200 nM using complete culture medium, and the secondary antibody (Jackson ImmunoResearch, 109-005-003) labeled with pHAb Amine Reactive Dye (Promega, G9845) was diluted to 6000 nM. The two were mixed at a 1:1 volume ratio and incubated at room temperature in the dark for 30 min. Then, the mixture was serially diluted 4-fold with complete culture medium, and 100 μL was added to each well. The cells were incubated at 37°C in a CO2 incubator for 24 h. Subsequent treatments included washing with PBS, digestion, Zombie violet staining (1:1000, BioLegend, 423114), fixation, permeabilization, and Alexa staining. Incubation with secondary antibody 488 (1:200, Jackson ImmunoResearch, 109-545-098) followed by flow cytometry analysis. Results showed (Figures 9A-B) that 901201 exhibited strong endocytosis in AsPC-1 and 23132 / 87 cells, showing a concentration gradient effect, and EC... 50 The values ​​are 0.39 nM and 1.37 nM, respectively.

[0357] Example 5 Preparation of 2A toxin

[0358] Ecinotecan mesylate (1.2 g, 2.28 mmol) was added to the reaction flask and dissolved in DMF (60 mL). Under nitrogen protection, DIEA (442.2 mg, 3.42 mmol) and N,N'-carbonyldiimidazole (1.3 g, 7.98 mmol) were added. The reaction was allowed to proceed at room temperature for 1 h, and LCMS monitoring showed complete reaction. Compound 1 (2.16 g, 11.40 mmol) was added, and the reaction was continued at room temperature for 2 h, with LCMS monitoring showing complete reaction. The reaction solution was poured into ice water (300 mL), extracted with EA (600 mL × 3), the organic phases were combined, washed with saturated brine (300 mL), dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude product. The crude product was purified by silica gel column chromatography (PE / EA = 1 / 8) to obtain compound 2' (900 mg).

[0359] Compound 2' (575 mg, 0.74 mmol) was added to the reaction flask, dissolved in DCM (15 mL), and cooled to 0 °C. Then, HCl / Dioxane (0.2 M, 12 mL) solution was added, and the reaction was continued at 0 °C for 10 min. LC-MS showed the reaction was complete. The reaction solution was poured into ice water (100 mL), extracted with DCM (100 mL × 3), and the organic phases were combined. The mixture was washed with saturated brine (100 mL), dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude product. The crude product was purified by pre-HPLC separation and lyophilized to obtain compound 2A (155 mg).

[0360] 1H NMR (400Hz, DMSO-d6): δ7.789-7.761(d,J=11.0Hz,1H),7.307(s,1H),6.640-6.617(d,J=9.0Hz,1H),6. 509(s,1H),5.801-5.781(d,J=8.0Hz,1H),5.425(s,2H),5.377-5.330(m,2H),5.252-5.204(m,1H),4.7 22-4.695(m,1H),3.678-3.662(m,1H),3.404-3.367(m,1H),3.298-3.284(m,1H),3.161(s,2H),2.392( s,3H),2.181-2.115(m,2H),1.881-1.841(m,2H),1.061-1.044(d,J=7.0Hz,3H),0.871(t,J=7.5Hz,3H).

[0361] MS(ESI) m / z 537.2 [M+1].

[0362] Step 1: At 0°C, a solution of NaHCO3 (4.906 g, 58.4 mmol) and Alloc-OSU (11.622 g, 58.4 mmol) in THF (50 mL) was added to a solution of compound 1.1 (4.38 g, 58.4 mmol) in H2O (50 mL). After the addition was complete, the mixture was stirred at room temperature for 1 h. The reaction was monitored by LCMS until completion. The reaction solution was directly concentrated at low temperature. The aqueous phase was extracted with ethyl acetate (80 mL × 2). The organic phases were combined, washed with saturated brine (80 mL × 2), dried over anhydrous sodium sulfate, and concentrated to obtain compound 1.2 (8.4 g), which was directly used in the next step of the reaction.

[0363] Step 2: Under nitrogen protection at 0°C, a THF solution of compound 1.2 (3.24 g, 20.38 mmol) in 10 mL of THF was added to a THF solution of compound 1.2 in 30 mL of [[2-(FMOC-amino)acetamido]methyl]acetate (5 g, 13.57 mmol) and TsOH (1.17 g, 6.79 mmol). The mixture was stirred at this temperature for 0.5 h. After the reaction was monitored by LCMS, the sample was directly loaded onto a wet plate and purified by silica gel column chromatography (55-75% EA / PE) to obtain compound 1.3 (2.2 g).

[0364] Step 3: DEA (7 mL) was added to a DCM solution (14 mL) of compound 1.3 (2.1 g, 4.5 mmol) at room temperature, and the mixture was stirred at room temperature for 1 h. The reaction was monitored by LCMS until it ended. After low-temperature concentration, the mixture was dried by pumping to obtain compound 1.4 (2.4 g), which was directly used in the next step of the reaction.

[0365] Step 4: At 0-5℃, 2,6-dimethylpyridine (1.44 g, 13.47 mmol) and HATU (2.05 g, 5.39 mmol) were added to a DMF (25 mL) solution of compound 1.4 (2.2 g, 8.98 mmol) and (((9H-fluorene-9-yl)methoxy)carbonyl)glycylglycyl-L-phenylalanine (2.25 g, 4.49 mmol). After the addition was complete, the mixture was stirred at room temperature for 1 h. The reaction was monitored by LCMS until completion. The reaction solution was directly purified by pre-HPLC and lyophilized to obtain compound 1.5 (1.7 g).

[0366] Step 5: At room temperature, phenylsilane (475 mg, 4.4 mmol) and Pd(PPh3)4 (507 mg, 0.44 mmol) were added to a THF / MeOH (35 mL, 2 / 1) solution of compound 1.5 (1.6 g, 2.2 mmol). The mixture was stirred at room temperature for 20 min after the addition was complete. The reaction was monitored by LCMS until it was complete. The reaction solution was filtered, purified by pre-HPLC, and lyophilized to obtain compound 1.6 (1 g).

[0367] Step 6: At 0-5℃, DIEA (182 mg, 1.41 mmol) and N,N'-carbonyldiimidazole (183 mg, 1.13 mmol) were added to a DMF (10 mL) solution of eczemacon mesylate (500 mg, 0.942 mmol). The mixture was stirred at room temperature for 1.5 h after the addition was complete. After the reaction was completed as monitored by LCMS, compound 1.6 (364 mg, 0.565 mmol) was added to the reaction solution, and the reaction was continued for 2 h. After the reaction was completed as monitored by LCMS, the pH of the reaction solution was adjusted to 5-6 with acetic acid, and then purified and concentrated by pre-HPLC to obtain compound 1.7 (260 mg).

[0368] Step 7: At room temperature, 1,4-diazabicyclo[2.2.2]octane (106 mg, 0.95 mmol) was added to a DMF (3 mL) solution of compound 1.7 (210 mg, 0.19 mmol), and the mixture was stirred at room temperature for 1.5 h. The reaction was monitored by LCMS until it was complete. The pH of the reaction solution was adjusted to 5-6 with acetic acid, and then purified by pre-HPLC, concentrated, and lyophilized to obtain compound 1.8 (126 mg).

[0369] Step 8: At room temperature, 2,6-dimethylpyridine (31 mg, 0.286 mmol) was added to a DMF (2 mL) solution of compound 1.8 (126 mg, 0.143 mmol) and 2,5-dioxopyrrolidone-1-yl 6-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)hexanoate (44 mg, 0.143 mmol). The mixture was stirred at room temperature for 1 h after the addition was complete. The reaction was monitored by LCMS until it was complete. The pH of the reaction solution was adjusted to 5-6 with acetic acid, and then purified by pre-HPLC, concentrated, and lyophilized to obtain compound 1.9 (115 mg).

[0370] 1H NMR (400MHz, DMSO-d6): δ8.500(t,J=5.0,10.5Hz,1H),8.280-8.253(t,J=5.0,10.5Hz,1H),8.104-7.971(m,3H),7.776(d,J= 11.0Hz,1H),7.305(s,1H),7.216-7.140(m,5H),6.979(s,2H),6.625(d,J=9.0Hz,1H),6.512(s,1H),5.857(d,J=8.0Hz,1H),5 .427-5.205(m,5H),4.600-4.477(m,3H),3.816-3.407(m,9H),3.347-3.264(m,2H),3.141(m,2H),3.043-2.998(m,1H),2.79 8-2.739(m,1H),2.379(s,3H),2.137(m,4H),1.810(m,2H),1.400(m,4H),1.151(m,2H),1.095(m,3H),0.889(t,J=8.5Hz,3H).

[0371] MS(ESI) m / z 1077.4 [M+1] + .

[0372] Example 6: ADC Sample Preparation and Analysis

[0373] 6.1 Coupling Methods

[0374] 1) Preparation of 901201-2A

[0375] The 901201 antibody was displaced into a 20 mM His / His-HCl solution at pH 6.0 (histidine / histidine salt), and a 10-fold molar excess of TCEP reducing agent was added to release free cysteine. The reaction was carried out in a water bath at 25°C for 2 h. Then, a 10-fold molar amount of compound 1.9 was added to the resulting solution, and DMA was added to bring the DMA volume ratio to 10%. The reaction was continued at room temperature for 2 h. Unreacted small molecule drugs were quenched by adding a 10-fold molar amount of N-acetylcysteine ​​to the mixture, and after reacting for 30 min, unreacted small molecule drugs and other impurities were removed using a Zeba™ desalting centrifuge column. The solution was then stored in a 20 mM His / His-HCl solution at pH 6.0 (histidine).

[0376] 2) Preparation of IgG-2A

[0377] IgG antibody (Huabo's self-produced IgG isotype control antibody) was displaced into a 20 mM His / His-HCl pH 6.0 histidine / histidine salt solution, and a 10-fold molar excess of reducing agent TCEP was added to release free cysteine. The reaction was carried out in a water bath at 25°C for 2 h. Then, a 12-fold molar ratio of compound 1.9 was added to the resulting solution, and DMA was added to bring the DMA volume ratio to 10%. The reaction was continued at room temperature for 2 h. Unreacted small molecule drugs were quenched by adding a 10-fold molar ratio of N-acetylcysteine ​​to the mixture, and after reacting for 30 min, unreacted small molecule drugs and other impurities were removed using a Zeba™ desalting centrifuge column. The solution was then stored in a 20 mM His / His-HCl pH 6.0 histidine solution.

[0378] 6.2 HPLC analysis of ADC DAR value

[0379] HIC-HPLC was used to analyze the number of small molecules conjugated to each antibody, i.e., the DAR value, and the results were verified by LC / MS. The HIC-HPLC procedure is as follows: Take an appropriate amount of the prepared ADC solution and analyze it using HIC-HPLC. Calculate the DAR value based on the peak area of ​​each peak at UV280nm. Specific analytical parameters are shown in Table 10.

[0380] Table 10 HIC-HPLC Detection Parameters

[0381] The chromatograms obtained by HCl-HPLC are shown in Figures 10 and 11 below, and the calculated DAR values ​​are shown in Table 11. The results show that the DAR values ​​of both ADC molecules are approximately 8.

[0382] Table 11 HIC-DAR values ​​of the two ADC samples

[0383] 6.3 Size Exclusion Chromatography-HPLC Analysis of ADC Purity

[0384] The antibody purity of each prepared sample was analyzed using SEC-HPLC. The specific method is as follows: an appropriate amount of the prepared ADC solution was taken and analyzed by SEC-HPLC. The SEC-purity value was calculated based on the peak area of ​​each peak at UV280 nm. The analytical conditions are shown in Table 12.

[0385] Table 12 SEC-HPLC Detection Parameters

[0386] The SEC-HPLC chromatogram results are shown in Figures 12 and 13. The single main peak represents the main component of the monoclonal antibody. The calculated SEC monomer purity results are shown in Table 13. The results show that the SEC monomer purity of both ADCs is greater than 95%.

[0387] Table 13 Calculation of SEC monomer purity for two ADC molecules

[0388] Example 7: In vitro killing experiment of 901201-2A against colon cancer cell line CL-40

[0389] The in vitro killing effect of the ADC sample prepared in Example 6 was detected. CL-40 cells (Nanjing Kebai, CBP60565CL-40) were digested with 0.25% Trypsin-EDTA (Gibco, 25200072), collected, centrifuged at 500g for 3 min, the supernatant was discarded, and the cells were resuspended in DMEM / F12 medium (Gibco, 21331020) containing 20% ​​FBS. After AO / PI counting, 100 μL / well was seeded into a 96-well plate (Corning, 3599) and incubated overnight at 37°C. The ADC sample was diluted with complete medium and added at 100 μL / well, and incubated at 37°C for 6 days. Aspirate 100 μL of supernatant, add 50 μL of lysis buffer according to the CellCounting-Lite 2.0 kit (Vazyme, DD1101-02) instructions, incubate at room temperature in the dark for 15 min, then transfer 100 μL to a 96-well black plate (Corning, 3916) and detect the luminescence value using a Molecular Devices Spectra i3Max microplate reader. The relative cell viability (%) was calculated as (sample luminescence value / blank cell luminescence value) × 100%. The results are shown in Figure 14. 901201-2A showed extremely strong cytotoxic activity against CL-40 cells, with an IC50 value of [missing value]. 50 The value was 0.076 nM, indicating that the control antibody ADC had a weak killing effect.

[0390] Example 8: In vitro killing experiment of 901201-2A on gastric cancer cell line 23132 / 87

[0391] Cells of type 23132 / 87 (Nanjing Kebai, CBP6047523132 / 87) were digested with 0.25% Trypsin-EDTA (Gibco, 25200072), collected, centrifuged at 500g for 3 min, and the supernatant was discarded. The cells were resuspended in RPMI 1640 medium containing 10% FBS. After AO / PI counting, 100 μL / well was seeded into 96-well plates and incubated overnight at 37°C. ADC samples were diluted with complete medium and added at 100 μL / well, and incubated at 37°C for 6 days. 100 μL of supernatant was discarded, and 50 μL of Cell Counting-Lite 2.0 lysis buffer (Vazyme, DD1101-02) was added. The cells were incubated at room temperature in the dark for 15 min. 100 μL was then transferred to a 96-well plate, and the luminescence value was detected using a microplate reader. The relative cell viability was calculated, and the results are shown in Figure 15. 901201-2A showed extremely strong killing effect on 23132 / 87 cells, with an IC50 value of [missing information]. 50 The value was 0.073 nM, and the control antibody ADC showed no cytotoxic activity.

[0392] Example 9: In vitro killing experiment of 901201-2A on pancreatic cancer cell line AsPC-1

[0393] AsPC-1 cells (ATCC, CRL-1682) were digested with 0.25% Trypsin-EDTA (Gibco, 25200072), collected, centrifuged at 500g for 3 min, and the supernatant was discarded. The cells were resuspended in RPMI 1640 medium containing 10% FBS, and after AO / PI counting, 100 μL / well was seeded into 96-well plates (Corning, 7007) and incubated overnight at 37°C. Sample 901201-2A was diluted with complete medium and added at 100 μL / well, and incubated at 37°C for 6 days. 100 μL of supernatant was discarded, and 100 μL of lysis buffer was added according to the Cell Counting-Lite 3D kit (Vazyme, DD1102-02) instructions. The cells were incubated at room temperature in the dark for 30 min, and 100 μL was transferred to a 96-well plate for microplate reading. The relative cell viability was calculated, and the results are shown in Figure 16. 901201-2A exhibited extremely strong killing effect on AsPC-1 cells, with an IC50 value of [missing information]. 50 The value was 0.15 nM, indicating that the control antibody ADC had no cytotoxic activity.

[0394] Example 10: Target-specific killing experiment of 901201-2A

[0395] 10.1 Detection of CDH17 expression

[0396] AGS human gastric adenocarcinoma cell line (Nanjing Kebai, CBP60476) and AGS-CDH17KO cells were digested and collected, washed with PBS and resuspended, counted by AO / PI, and 20 μL / well was added to a 96-well plate (BIOFIL, TCP002096). Zombie violet (BioLegend, 423114) diluted 1:500 was added and stained at room temperature for 15 min. After washing twice with FACS Buffer, Anti-CDH17 hIgG1 antibody (Jiman Biotechnology, GM-52672AB) was added and incubated at room temperature for 30 min. After washing, R-Phycoerythrin-labeled secondary antibody (Jackson, 109-115-098) diluted 1:200 was added and incubated at room temperature for 15 min. Flow cytometry analysis was performed, as shown in Figure 17, confirming that AGS cells were CDH17 positive and AGS-CDH17KO cells were negative.

[0397] 10.2 Lethality Experiment

[0398] Both cell types were resuspended in RPMI 1640 medium containing 10% FBS. After AO / PI counting, 100 μL / well was seeded into 96-well plates (Corning, 7007) and incubated overnight at 37°C. The 901201-2A sample was diluted with complete medium at 100 μL / well and incubated for 6 days. The supernatant was discarded, and 100 μL of Cell Counting-Lite 3D lysis buffer (Vazyme, DD1102-02) was added. After incubation for 30 min, the luminescence value was measured. The results are shown in Figure 18. 901201-2A showed an IC50 response to AGS cells. 50 The concentration was 0.042 nM, which significantly tolerated AGS-CDH17KO cells and showed CDH17 target specificity in killing them.

[0399] Example 11: Bypass killing effect experiment of 901201-2A on gastric cancer cell line SNU-16

[0400] SNU-16 cells (Wuhan Shangen, SNL-538, CDH17 positive) were resuspended in RPMI 1640 medium containing 10% FBS, counted by AO / PI (Mengwei, SC1009), and seeded at 50 μL / well in 96-well plates (Corning, 7007). Simultaneously, Raji-luc cells (ELISA, ml-CC2158, CDH17 negative) were treated in the same manner, with 50 μL / well added to the same well, and incubated overnight at 37°C. The next day, the ADC sample was diluted with complete medium and added to the well at 100 μL / well, and incubated for another 6 days. After incubation, 100 μL of supernatant was aspirated from each well, and 100 μL of CellCounting-Lite 3D lysis buffer (Vazyme, DD1102-02) was added. After incubation at room temperature in the dark for 30 min, the luminescence value was measured using a microplate reader to assess Raji cell viability.

[0401] As shown in Figure 19, 901201-2A had no killing effect on CDH17-negative Raji-luc cells cultured alone. However, in a co-culture system of Raji-luc and CDH17-positive SNU-16 cells, it could efficiently kill Raji-luc cells through a side-killing effect, with a corresponding side-killing IC50 value. 50 It is 0.32 nM.

[0402] Example 12: Bypass killing effect experiment of 901201-2A on pancreatic cancer cell line AsPC-1

[0403] CDH17-positive AsPC-1 cells (ATCC, CRL-1682) were resuspended in RPMI 1640 medium containing 10% FBS, and after AO / PI counting, 50 μL / well was seeded into 96-well plates (Corning, 7007). Simultaneously, CDH17-negative Raji-luc cells (ELISA, ml-CC2158) were treated in the same manner, with 50 μL / well added to the same well, and incubated overnight at 37°C. The next day, the 901201-2A sample was diluted with complete medium and added to the well at 100 μL / well, and incubated for another 6 days. After incubation, 100 μL of supernatant was discarded, and 100 μL of Cell Counting-Lite 3D lysis buffer (Vazyme, DD1102-02) was added. The cells were incubated at room temperature in the dark for 30 min before detecting the luminescence value.

[0404] As shown in Figure 20, 901201-2A had no killing effect on CDH17-negative Raji-luc cells cultured alone. However, in the co-culture system of Raji-luc and CDH17-positive AsPC-1 cells, it effectively killed Raji-luc cells through the side-killing effect, with a corresponding side-killing IC50 value. 50It is 0.13 nM.

[0405] Example 13: CDH17-ADC in vitro human colorectal cancer organoid (PDO) killing experiment

[0406] To evaluate the specific killing effect of antibody-drug conjugates (ADCs) on human colorectal cancer organoids, this study established an in vitro efficacy testing system using normal human colorectal organoids (41P-CRN, derived from adjacent normal tissue) and colorectal cancer organoids (41P-CRT) from the same patient as models.

[0407] When organoids reached a diameter of approximately 50 μm, they were seeded into 96-well cell culture plates using a "sandwich method": 50 μL of 50% Matrigel (mixed with PBS 1:1) was placed at the bottom of each well as the bottom layer; 10% Matrigel (mixed with PBS 1:9) containing 50 ± 20 organoids was added to the middle layer; and 200 μL of organoid culture medium was added to the top layer. Drug treatment was initiated on day 1 post-seedling, and the drug-treated medium was replaced on day 3. Live cells were stained with Calcein AM on day 0, and double staining of live / dead cells was performed with Calcein AM / PI on day 6. Images were acquired using Z-stack imaging, and the size of surviving organoids was measured using NIS-Elements AR software. The organoid area change rate was calculated as [Organoid size (%) = (Total area of ​​live organoids in Day 6 detection wells / Total area of ​​live organoids in Day 0 detection wells) / (Total area of ​​live organoids in Day 6 control wells / Total area of ​​live organoids in Day 0 control wells) × 100%], and IC50 plots were generated. 50 Curves were used to assess drug activity.

[0408] The drug concentration gradients were designed as follows: 7 concentration gradients (maximum concentration 2000 nM, 4-fold dilution) for 901201-2A and Isotype IgG-2A, and 7 concentration gradients (maximum concentration 16000 nM, 4-fold dilution) for Payload (2A), with one control group without drug administration for each.

[0409] As shown in Figures 21 and 22, 901201-2A exhibited potent killing activity in colorectal cancer organoids, and its inhibitory activity was significantly better than that of Isotype IgG-2A at the same concentration, and comparable to that of Payload. In normal colorectal organoids, the inhibitory activity of ADC was significantly weaker than that in tumor organoids, demonstrating low toxicity to normal tissues.

[0410] In summary, the ADC prepared in this invention has a specific and potent killing ability against human colorectal cancer organoids, while showing good tolerance to normal colorectal organoids, demonstrating excellent advantages in tumor-targeted killing.

[0411] Table 14 Killing effect of ADC on human organoids in vitro

[0412] Example 14: Efficacy of anti-CDH17 ADC in a mouse model of human colon cancer cell CL40 BALB / c nude xenograft

[0413] The in vivo antitumor effect of the ADC sample prepared in Example 6 was detected. Logarithmically growing human colon cancer CL40 cells were injected at a dose of 1×10⁻⁶. 7 The tumors were resuspended in a 1:1 mixture of PBS and Matrigel and subcutaneously injected into the right rib area of ​​female BALB / cnude mice (Beijing Huafukang Biotechnology Co., Ltd.). When the average tumor volume reached approximately 150 mm³, the mice were randomly divided into four groups (n=6 per group): Group G1 was the PBS solvent control group, Group G2 was given 901201-2A (1 mg / kg), Group G3 was given 901201-2A (5 mg / kg), and Group G4 was given IgG-2A (5 mg / kg). All were administered intravenously once a week for a total of two times (administration volume 10 μL / g, freshly prepared and used).

[0414] During the experiment, mouse tumor volume (calculated as "tumor volume TV = 0.5 × major axis × minor axis 2") and body weight were monitored twice weekly. Statistical analysis of the experimental endpoint was performed using GraphPad Prism 9.0.0 software. The tumor growth inhibition rate (TGITV) was used to evaluate efficacy, calculated as follows: TGITV = (1 - relative tumor volume RTV of treatment group / relative tumor volume RTV of control group) × 100%, where relative tumor volume RTV = experimental endpoint tumor volume Vt / tumor volume V0 at the start of administration. Efficacy is shown in Figure 23 and Table 15. 901201-2A showed significant tumor growth inhibition against CL40 xenografts, and the inhibitory effect was clearly dose-dependent. At the same dose, 901201-2A showed significantly better tumor growth inhibition rate and tumor weight inhibition effect than the control ADC IgG-2A, with the 5 mg / kg group exhibiting potent tumor-killing activity.

[0415] In terms of safety, no acute abnormal deaths occurred in any group of mice throughout the experiment. As shown in Figure 24, the model itself caused a trend of weight loss in the mice, while the 901201-2A 5mg / kg group (G3) could alleviate this weight loss to some extent. The weight changes in the other drug-treated groups were not significantly different from those in the solvent control group. No drug-related toxicities such as lethargy and loss of appetite were observed, suggesting that 901201-2A has good in vivo safety at the tested dose.

[0416] In conclusion, the ADC prepared by the present invention has both excellent antitumor efficacy and good safety in in vivo experiments.

[0417] Table 15 Data table of TGITV and tumor weight of 901201-2A against CL40 colon cancer cell xenografts in nude mice

[0418] Example 15 In vivo antitumor experiment of 901201-2A against human gastric cancer SNU-5B xenografts in NDG mice

[0419] Human gastric cancer SNU-5 cells in logarithmic growth phase were resuspended with PBS, and the density was adjusted to 1×10 7 cells / 0.2 mL, and inoculated subcutaneously on the right back of female B-NDG mice (BioMab (Beijing) Pharmaceutical Technology Co., Ltd., production license number: SYXK (Jing) 2020-0007) at a dose of 0.2 mL per mouse. When the average tumor volume of the mice reached 194 mm3, 16 mice were screened according to tumor volume and body weight and randomly divided into groups: Group G1 (PBS vehicle control, 5 mice), G2 (IgG-2A, 10 mg / kg, 5 mice), G3 (901201-2A, 10 mg / kg, 6 mice). All groups were administered by intravenous injection once a week for a total of 2 doses (the administration volume was calculated as 10 μL / g body weight, the drug was prepared immediately before use, the working solution concentration was 1 mg / mL, stored at 2-8°C and used within 4 hours).

[0420] During the experiment, the tumor volume (calculated as "tumor volume TV = 0.5 × long diameter × short diameter²") and body weight of the mice were monitored twice a week. The experiment was ended on the 35th day after grouping and administration. The mice were euthanized with excessive CO2, and the tumors were dissected and photographed. Statistical analysis was performed by GraphPad Prism 9.0.0 software, and the efficacy was evaluated by tumor growth inhibition rate (TGITV). The calculation formula is: TGITV(%) = [1-(Ti-T0) / (Ci-C0)]×100% (Ti: mean tumor volume of the treatment group on day i; T0: mean tumor volume of the treatment group on day 0 of administration; Ci: mean tumor volume of the control group on day i; C0: mean tumor volume of the control group on day 0 of administration)

[0421] It can be seen from the results in Table 16 and Figure 25 that 901201-2A (10 mg / kg) has a potent inhibitory effect on the growth of SNU-5 gastric cancer xenografts: on the 35th day after grouping and administration, the mean tumor volume of 901201-2A group is much lower than that of the vehicle control group (G1) and isotype control ADC group (G2), and the TGITV exceeds 100%, which is significantly better than the other two groups, reflecting specific antitumor activity.

[0422] In terms of safety, no acute abnormal deaths were observed in any group of mice throughout the experiment. Regarding weight changes (Figure 26), the SNU-5 xenograft model itself led to a decrease in weight in the solvent control group (G1) and the isotype control ADC group (G2), while the weight of mice in the 901201-2A (10 mg / kg) group (G3) increased compared to before administration. This suggests that 901201-2A exerts a potent tumor-suppressing effect while also alleviating model-related weight loss, demonstrating good in vivo safety and tolerability.

[0423] Table 16. Data on TGITV and tumor weight of 901201-2A and control ADC in SNU-5 gastric cancer cell B-NDG mouse xenografts.

[0424] Example 16: Efficacy Study of AsPC-1-luci in an Orthotopic Mouse Model of Pancreatic Cancer

[0425] Logarithmic growth phase human pancreatic cancer cells AsPC-1-luci were resuspended in culture medium containing Matrigel to achieve a cell density of 1×10⁻⁶ cells / year. 6 20 μL of the drug was injected orally into the pancreas of female Balb / c nude mice (Zhejiang Vital River Laboratory Animal Technology Co., Ltd.) at a volume of 20 μL per mouse. The tumor fluorescence signal was measured when it reached 3.17 × 10⁻⁶. 8 At approximately 10:00 AM, 24 tumor-bearing mice were randomly divided into three groups based on tumor fluorescence signal values ​​and body weight: Group G1 (control group, 8 mice), Group G2 (IgG-2A, 10 mg / kg, 8 mice), and Group G3 (901201-2A, 10 mg / kg, 8 mice). All groups received the drug via tail vein injection once a week for a total of three times (administration volume calculated at 10 mL / kg body weight; test samples were stored at -80°C; purity was 97%).

[0426] During the experiment, mouse body weight and tumor fluorescence signal were monitored twice weekly (using the PerkinElmer Lumina III small animal in vivo imaging system; D-Luciferin was administered at a dose of 150 mg / kg before imaging, and imaging was performed at a uniform time 10–15 minutes later). Animal survival was continuously observed until Day 89 after drug administration. Tumor growth inhibition rate (TGI) was calculated using statistical analysis to assess efficacy. The formula was: TGI(%) = [1 - (mean tumor fluorescence signal in the treatment group / mean tumor fluorescence signal in the control group)] × 100%

[0427] The experimental results are shown in Table 17 and Figure 27. 901201-2A has a significant inhibitory effect on the growth of AsPC-1-luci pancreatic cancer in situ tumors. Its tumor-suppressing effect is significantly better than that of the control antibody ADC group (IgG-2A) and the PBS control group (Vehicle), which fully demonstrates its strong and specific anti-tumor activity.

[0428] Survival results (Figure 28) showed that all animals in the PBS control group died 47 days after administration; only one animal in the control antibody ADC group survived to 89 days; while all animals in the 901201-2A treatment group survived to 89 days after administration, significantly prolonging the survival of tumor-bearing mice.

[0429] Safety assessment results (Figure 29) showed that no acute deaths occurred in any group of mice throughout the experimental period, and no obvious abnormal signs were observed. Furthermore, no significant weight loss was observed in any group of mice, suggesting that 901201-2A exhibits good in vivo safety and tolerability while exerting its antitumor effect, and did not demonstrate significant drug toxicity.

[0430] Table 17 Tumor data of each treatment group in the AsPC-1 orthotopic tumor model on day 31

[0431] Example 17: In vivo pharmacodynamic study of a human gastric cancer xenograft subcutaneous tumor PDX model.

[0432] Human gastric cancer tumor tissue (PDX model number: YK-STAD-024, Yikang (Beijing) Pharmaceutical Technology Co., Ltd.), measuring 2mm × 2mm × 2mm, was subcutaneously implanted into the right anterior flank of 30 6-8 week old female NCG mice (Jiangsu Jicui Yaokang Biotechnology Co., Ltd.), with one tumor tissue implanted in each mouse. When the tumor volume reached approximately 150mm³ (recorded as PG-D0 on the day of grouping), the mice were randomly divided into 3 groups of 5 mice each: G1 group (PBS solvent control), G2 group (IgG-2A, 10mg / kg), and G3 group (901201-2A, 10mg / kg). All groups were administered the drug intravenously once a week for a total of 2 times (administration volume calculated at 10μL / g body weight).

[0433] During the experiment, the tumor volume of mice was measured twice a week (calculated as “tumor volume TV = 0.5 × major axis × minor axis 2”) and the survival status of mice was recorded. Tumor growth curves were plotted. The experiment ended at PG-D24. Mice were euthanized by excessive CO2 and the tumor tissue was removed and weighed. Statistical analysis was performed using GraphPad Prism software. The efficacy of the drug was evaluated by the tumor growth inhibition rate (TGITV). The calculation formula is: TGITV(%) = [1 - (Ti - T0) / (Ci - C0)] × 100% (Ti: mean tumor volume of PG-D24 in the treatment group, T0: mean tumor volume of PG-D0 in the treatment group; Ci: mean tumor volume of PG-D24 in the control group, C0: mean tumor volume of PG-D0 in the control group).

[0434] The experimental results (Table 18, Figure 30) show that 901201-2A has a strong inhibitory effect on tumor growth in the human gastric cancer PDX model: when the drugs were administered to the groups until PG-D24, the mean tumor volume was much lower than that of the PBS solvent control group (G1) and the isotype control ADC group (G2, IgG-2A), and the tumor inhibition effect was significantly better than the two control groups, demonstrating specific anti-tumor activity.

[0435] Regarding safety (Figure 31), only animal number 8 in group G2 (IgG-2A) died on PG-D24 throughout the experiment. No acute abnormal deaths were observed in any mice in groups G1 and G3 (901201-2A), or in the remaining mice in group G2. In terms of weight changes, no significant weight loss was observed in any group of mice. This suggests that 901201-2A exhibits potent antitumor effects while demonstrating good safety and tolerability in vivo.

[0436] Table 18. Statistical table of tumor weight and tumor growth inhibition rate (TGITV) in mice of different PG-D24 groups.

[0437] Example 18: In vivo pharmacodynamic study of a human colorectal cancer PDX model

[0438] Human colorectal cancer tumor tissue (PDX model number: LD1-2012-200671, moderately differentiated colorectal adenocarcinoma, Shanghai Lidi Biotechnology Co., Ltd.) measuring 3mm×3mm×3mm was subcutaneously implanted into the right subcutaneous tissue of 40 female NU / NU mice (Zhejiang Vital River Laboratory Animal Technology Co., Ltd.) aged 42-62 days, with one tumor tissue implanted in each mouse. When the average tumor volume of the mice reached 138.65mm3 (the day of grouping was recorded as day 0), they were randomly divided into 3 groups of 5 mice each according to the tumor volume: G1 group (PBS, solvent control), G2 group (IgG-2A, 10mg / kg), and G3 group (901201-2A, 10mg / kg). All groups were administered the drug intravenously once a week for a total of 2 times (the administration volume was calculated at 10μL / g body weight, and the test sample was diluted with PBS to prepare a 1mg / mL working solution, which was prepared and used immediately).

[0439] During the experiment, the tumor volume (calculated as “tumor volume TV = 0.5 × major axis × minor axis 2”) and body weight of mice were measured twice a week. Tumor growth curves were plotted and survival status was recorded. The experiment ended on day 31. Mice were euthanized by excessive CO2, and tumor tissue was removed, weighed, and photographed. Statistical analysis was performed using GraphPad Prism software. The efficacy was evaluated by tumor growth inhibition rate (TGI), calculated as follows: TGI(%) = [1 - (Ti - T0) / (Ci - C0)] × 100% (Ti: mean tumor volume on day 31 in the treatment group, T0: mean tumor volume on day 0 in the treatment group; Ci: mean tumor volume on day 31 in the control group, C0: mean tumor volume on day 0 in the control group).

[0440] The experimental results (Table 19, Figure 32) show that 901201-2A (10 mg / kg) has a strong inhibitory effect on tumor growth in a human colorectal cancer PDX model (LD1-2012-200671). After administration to the 31st day, the average tumor volume and tumor weight were much lower than those of the solvent control group (G1) and the isotype control ADC group (G2), and the TGI was significantly higher than those of the two control groups. The tumor inhibition effect was significantly better than that of the two control groups, demonstrating specific anti-tumor activity.

[0441] Regarding safety (Figure 33), no acute abnormal deaths occurred in any group of mice throughout the experiment. As for weight changes, the weight of mice in each group fluctuated within the normal range, and the relative weight change rate showed no significant abnormalities, suggesting that CDH17-ADC exerts a potent tumor-suppressing effect while exhibiting good safety and tolerability in vivo.

[0442] Table 19. Statistics on tumor weight and tumor growth inhibition rate (TGI) of mice in each group on day 31.

[0443] Example 19: In vivo pharmacodynamic study of a human pancreatic cancer xenograft subcutaneous tumor PDX model.

[0444] Human pancreatic cancer tumor tissue (PDX model number: YK-PAAD-003, Yikang (Beijing) Pharmaceutical Technology Co., Ltd.), measuring 2mm × 2mm × 2mm, was subcutaneously implanted into the right anterior flank of 24 6-8 week old female Balb / c nude mice (Beijing Huafukang Biotechnology Co., Ltd.), with one tumor tissue implanted in each mouse. When the average tumor volume reached 160mm³ (recorded as PG-D0 on the day of grouping), the mice were randomly divided into three groups according to tumor volume: G1 group (PBS solvent control, 4 mice), G2 group (901201-2A, 10mg / kg, 5 mice), and G3 group (IgG-2A, 10mg / kg, 5 mice). All groups were administered the drug intravenously once a week for a total of 2 times (administration volume was calculated at 10μL / g body weight; all test samples were freshly prepared using PBS as the solvent, with a working solution concentration of 1mg / mL).

[0445] During the experiment, mouse tumor volume (calculated as "tumor volume TV = 0.5 × major axis × minor axis 2") and body weight were measured twice weekly. Tumor growth curves were plotted and survival status was recorded. The experiment ended at PG-D25. Mice were euthanized using excessive CO2, and tumor tissue was dissected, weighed, and photographed. Statistical analysis was performed using GraphPad Prism software. The efficacy was evaluated using the tumor growth inhibition rate (TGITV), calculated as follows:

[0446] TGITV(%) = [1 - (Ti - T0) / (Ci - C0)] × 100% (Ti: mean PG-D25 tumor volume in the treatment group, T0: mean PG-D0 tumor volume in the treatment group; Ci: mean PG-D25 tumor volume in the control group, C0: mean PG-D0 tumor volume in the control group)

[0447] The experimental results (Table 20, Figure 34) show that 901201-2A (10 mg / kg) has a strong inhibitory effect on the growth of human pancreatic cancer PDX model (YK-PAAD-003): when the drug was administered to the groups up to PG-D25, the mean tumor volume and tumor weight were much lower than those of the PBS solvent control group (G1) and the control drug group (G3, IgG-2A), and the tumor inhibition effect was significantly better than the two control groups, demonstrating specific anti-tumor activity.

[0448] Regarding safety (Figure 35), no acute abnormal deaths occurred in any group of mice throughout the experiment. As for weight changes, the percentage trends of weight change in each group were basically consistent, with no significant abnormal fluctuations, suggesting that 901201-2A exhibits strong antitumor effects while demonstrating good in vivo safety and tolerability.

[0449] Table 20. Statistical table of tumor weight and tumor growth inhibition rate (TGITV) in mice of different PG-D25 groups.

[0450] Example 20: In vivo pharmacodynamic study of a human gastric cancer xenograft subcutaneous tumor PDX model.

[0451] Human gastric cancer tumor tissue (PDX model number: YK-STAD-012, Yikang (Beijing) Pharmaceutical Technology Co., Ltd.) measuring 2mm×2mm×2mm was subcutaneously implanted into the right anterior flank of 24 6-8 week old female Balb / c nude mice (Beijing Huafukang Biotechnology Co., Ltd.), with one tumor tissue implanted in each mouse. When the average tumor volume of the mice reached 150mm3 (recorded as PG-D0 on the day of grouping), they were randomly divided into 3 groups according to tumor volume: G1 group (PBS solvent control, 4 mice), G2 group (901201-2A, 10mg / kg, 5 mice), and G3 group (IgG-2A control antibody ADC, 10mg / kg, 5 mice).

[0452] All groups were administered intravenously, with the following dosing regimens: Group G1 was initially given PBS once a week for a total of 2 weeks (PG-D0 to PG-D27), and subsequently 901201-2A once a week for a total of 2 weeks starting from PG-D28; Group G2 was given 901201-2A once a week for a total of 2 weeks starting from PG-D0; Group G3 was given IgG-2A control antibody ADC once a week for a total of 2 weeks starting from PG-D0, and then again IgG-2A control antibody ADC once a week for a total of 2 weeks starting from PG-D28 (dosing volume was calculated at 10 μL / g body weight, all test samples were freshly prepared using PBS as the solvent, and the working solution concentration was 1 mg / mL).

[0453] During the experiment, the tumor volume (calculated as “tumor volume TV = 0.5 × major axis × minor axis 2”) and body weight of mice were measured twice a week. Tumor growth curves were plotted and survival status was recorded. The experiment ended at PG-D59. Mice were euthanized by excessive CO2, and tumor tissue was removed, weighed, and photographed. Statistical analysis was performed using GraphPad Prism 9.0.0 software. The efficacy was evaluated by tumor growth inhibition rate (TGITV). The calculation formula is: TGITV (%) = (1 - mean RTV of treatment group / mean RTV of control group) × 100% (RTV is the ratio of tumor volume after administration to tumor volume before administration, i.e., relative tumor volume).

[0454] The experimental results (Table 21, Figure 36) show that 901201-2A (10 mg / kg) has a strong inhibitory effect on tumor growth in the human gastric cancer PDX model (YK-STAD-012), and is significantly superior to the control antibody ADC. The specific performance is as follows:

[0455] 1. After the first round of administration, tumor growth in G2 group 901201-2A (10mg / kg) mice was strongly inhibited, and the tumor volume was significantly smaller than that in G1 and G3 groups. Statistical analysis showed that its tumor-inhibiting effect was significant. However, G3 group had no significant inhibitory effect on tumor growth and there was no statistical difference compared with G1 group.

[0456] 2. In cases with large tumor volume, after subsequent treatment with 901201-2A (10mg / kg), the tumor volume in group G1, which had already increased significantly, continued to shrink, verifying that the drug could still exert a significant tumor-suppressing effect even when the tumor volume was large. However, after two rounds of administration, the tumor volume in group G3 continued to increase significantly, and the tumors were eventually euthanized in accordance with animal welfare requirements due to their excessive size, indicating that the tumor-suppressing effect was poor.

[0457] 3. During the long observation period, the tumor volume in group G2 did not show a significant rebound. At the end of the experiment, the tumor weight in group G2 was extremely low, further confirming the potent and long-lasting tumor-suppressing effect of 901201-2A.

[0458] In summary, the ADC prepared in this application has superior efficacy in the treatment of large tumors and exerts a sustained tumor-suppressing effect, demonstrating its outstanding application value and broad application prospects.

[0459] Table 21 Treatment details for each group and tumor volume and weight at both stages

[0460] Example 21: In vivo pharmacodynamic study of a large-volume human colorectal cancer tumor PDX model

[0461] Large-volume tumor models (such as the model with an average volume of 800 mm3 in this experiment) are closer to the actual lesion characteristics of patients with advanced tumors in clinical practice. They can more realistically simulate the complexity of the tumor microenvironment and the difficulty of treatment, and their efficacy data have significantly higher clinical translation reference value. At the same time, Her2 is one of the important therapeutic targets for colorectal cancer, but some patients in clinical practice will develop resistance to Her2-targeted therapy. Exploring drugs targeting other targets (such as CDH17 involved in this model) is expected to provide new treatment options for patients with Her2 resistance and broaden the range of beneficiaries.

[0462] Human colorectal cancer tumor tissue (PDX model number: YK-CRC-022, Her2 Yikang (Beijing) Pharmaceutical Technology Co., Ltd.; CDH17 staining intensity of this tumor tissue was 2+, with a positive tumor cell rate of 82%; Her2 staining intensity was 2+, with a positive tumor cell rate of 90%), measuring 2mm×2mm×2mm, was subcutaneously implanted into the right anterior flank of 28 6-8 week old female NCG mice (Jiangsu Jicui Yaokang Biotechnology Co., Ltd.), with one tumor tissue implanted in each mouse. When the average tumor volume of the mice reached approximately 800mm3 (large volume model) (recorded as PG-D0 on the day of grouping), they were randomly divided into 3 groups of 5 mice each according to the tumor volume: G1 group (PBS solvent control, i.e., vehicle control), G2 group (DS8201, 10mg / kg), and G3 group (901201-2A, 10mg / kg). All groups were administered the medication intravenously once a week for a total of 3 times (the administration volume was calculated at 10 μL / g body weight, and all test samples were prepared and used immediately with PBS as the solvent, and the working solution concentration was 1 mg / mL).

[0463] During the experiment, the tumor volume (calculated as “tumor volume TV = 0.5 × major axis × minor axis 2”) and body weight of mice were measured twice a week. Tumor growth curves were plotted and survival status was recorded. The experiment ended at PG-D25. Mice were euthanized by excessive CO2, and tumor tissue was removed, weighed, and photographed. Statistical analysis was performed using GraphPad Prism software. The efficacy of the drug was evaluated by the tumor growth inhibition rate (TGITV). The calculation formula was: TGITV(%) = [1 - (Ti - T0) / (Ci - C0)] × 100% (Ti: mean tumor volume of PG-D25 in the treatment group, T0: mean tumor volume of PG-D0 in the treatment group; Ci: mean tumor volume of PG-D25 in the control group, C0: mean tumor volume of PG-D0 in the control group).

[0464] This experiment used a large-volume PDX model with an average tumor volume of 800 mm³, which more closely reflects the clinical characteristics of patients with advanced-stage cancer, and its efficacy evaluation results have higher clinical translational value. The experimental results showed (Table 22, Figure 37) that 901201-2A (10 mg / kg) had a strong inhibitory effect on the growth of this large-volume colorectal cancer PDX model: when the drugs were administered to the groups up to PG-D25, the mean tumor volume and tumor weight were much lower than those of the PBS solvent control group (G1) and the positive control drug group (G2, DS8201), and the TGITV reached 62%, which was significantly higher than that of the DS8201 group (39%). The tumor inhibition effect was significantly better than that of DS8201, demonstrating superior specific antitumor activity.

[0465] Regarding safety (Figure 38), no abnormal deaths occurred in any group of mice throughout the experiment. As for weight changes, the weight change trend of the 901201-2A group mice was basically consistent with that of the control group, with no significant abnormal fluctuations, suggesting that 901201-2A exerts a potent tumor-suppressing effect in large-volume tumor models while exhibiting good in vivo safety and tolerability.

[0466] Table 22 Statistical table of tumor weight and tumor growth inhibition rate (TGITV) in mice of different PG-D25 groups

Claims

An antibody or antigen-binding fragment, characterized in that, It can specifically bind to CDH17. The antibody or antigen-binding fragment according to claim 1 is characterized in that, The antibody or antigen-binding fragment comprises: (a) HCDR1, HCDR2 and HCDR3 of VH as shown in SEQ ID NO: 22; and / or (b) LCDR1, LCDR2 and LCDR3 of VL as shown in SEQ ID NO:

23. The antibody or antigen-binding fragment according to claim 1 or 2 is characterized in that, The amino acid sequence of HCDR1 is shown in SEQ ID NO:

24. The antibody or antigen-binding fragment according to any one of claims 1-3 is characterized in that, The amino acid sequence of HCDR2 is shown in SEQ ID NO:

25. The antibody or antigen-binding fragment according to any one of claims 1-4 is characterized in that, The amino acid sequence of HCDR3 is shown in SEQ ID NO:

26. The antibody or antigen-binding fragment according to any one of claims 1-5 is characterized in that, It contains LCDR1, the amino acid sequence of which is shown in SEQ ID NO:

27. The antibody or antigen-binding fragment according to any one of claims 1-6 is characterized in that, It contains LCDR2, the amino acid sequence of which is shown in SEQ ID NO:

28. The antibody or antigen-binding fragment according to any one of claims 1-7 is characterized in that, It contains LCDR3, the amino acid sequence of which is shown in SEQ ID NO:

29. The antibody or antigen-binding fragment according to any one of claims 1-8 is characterized in that, The amino acid sequence of HCDR1 is shown in SEQ ID NO:24, the amino acid sequence of HCDR2 is shown in SEQ ID NO:25, the amino acid sequence of HCDR3 is shown in SEQ ID NO:26, the amino acid sequence of LCDR1 is shown in SEQ ID NO:27, the amino acid sequence of LCDR2 is shown in SEQ ID NO:28, and the amino acid sequence of LCDR3 is shown in SEQ ID NO:

29. The antibody or antigen-binding fragment according to any one of claims 1-9 is characterized in that, The antibody or antigen-binding fragment includes the FR region. The antibody or antigen-binding fragment according to any one of claims 1-10 is characterized in that, It contains the antibody heavy chain variable region VH and / or the antibody light chain variable region VL. The antibody or antigen-binding fragment according to any one of claims 1-11 is characterized in that, The amino acid sequence of the VH is as shown in ID NO: 22 and / or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with the aforementioned sequence. The antibody or antigen-binding fragment according to any one of claims 1-12 is characterized in that, The VL has an amino acid sequence as shown in SEQ ID NO: 23 and / or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with the aforementioned sequence. The antibody or antigen-binding fragment according to any one of claims 1-13 is characterized in that, The antibody or antigen-binding fragment that specifically binds to CDH17 comprises VH as shown in SEQ ID NO: 22 and VL as shown in SEQ ID NO: 23, and / or VH and VL having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity compared to them. The antibody or antigen-binding fragment according to any one of claims 1-14 is characterized in that, The antibody or antigen-binding fragment includes the constant region of the antibody heavy chain. The antibody or antigen-binding fragment according to claim 15 is characterized in that, The antibody heavy chain constant region is derived from the human IgG heavy chain constant region. The antibody or antigen-binding fragment according to claim 15 or 16 is characterized in that, The antibody heavy chain constant region is derived from the human IgG1 heavy chain constant region. The antibody or antigen-binding fragment according to any one of claims 1-17 is characterized in that, The antibody or antigen-binding fragment includes the constant region of the antibody light chain. The antibody or antigen-binding fragment according to claim 18 is characterized in that, The antibody light chain constant region is derived from the human Igκ constant region. The antibody or antigen-binding fragment according to any one of claims 1-19 is characterized in that, The antibody or antigen-binding fragment includes Fab, Fab', Fv fragments, F(ab')2, F(ab)2, scFv, di-scFv and / or dAb. The antibody or antigen-binding fragment according to any one of claims 1-20 is characterized in that, The antibody is selected from one or more of the following groups: monoclonal antibody, polyclonal antibody, chimeric antibody, humanized antibody, fully human antibody, natural antibody, engineered antibody, monovalent antibody, multivalent antibody, full-length antibody, antibody fragment, naked antibody, conjugated antibody. A chimeric antigen receptor, characterized in that, The targeting portion comprises the antibody or antigen-binding fragment of any one of claims 1-21. A polypeptide molecule, characterized in that, The polypeptide molecule comprises the antibody or antigen-binding fragment of any one of claims 1-21 or the chimeric antigen receptor of claim 22. One or more isolated nucleic acid molecules encoding an antibody or antigen-binding fragment as described in any one of claims 1-21, a chimeric antigen receptor as described in claim 22, or a polypeptide molecule as described in claim 23. A carrier comprising the nucleic acid molecule of claim 24. Cells comprising any one of the antibodies or antigen-binding fragments of claims 1-21, the chimeric antigen receptor of claim 22, the polypeptide molecule of claim 23, the nucleic acid molecule of claim 24, or the vector of claim 25. Immunoreconjugates or their pharmaceutically acceptable salts, characterized in that, It comprises the antibody or antigen-binding fragment as described in any one of claims 1-21. The immunoconjugate or a pharmaceutically acceptable salt thereof according to claim 27 is characterized in that, The immunoconjugate or its pharmaceutically acceptable salt has the structure shown in formula (I). Ab-(LD) p (Ⅰ) in: Ab is the antibody or antigen-binding fragment as described in any one of claims 1-21; L stands for connector; D represents the payload; p is any integer from 1 to 10. The immunoconjugate or a pharmaceutically acceptable salt thereof according to claim 28 is characterized in that, The immunoconjugate or its pharmaceutically acceptable salt is an antibody-drug conjugate or its pharmaceutically acceptable salt, and D is a cytotoxic drug. The immunoconjugate or a pharmaceutically acceptable salt thereof according to claim 29 is characterized in that, The cytotoxic drug is one or more of the following: tubulin inhibitor, DNA intercalator, DNA topoisomerase inhibitor, apoptosis inducer, RNA polymerase inhibitor, spliceosome inhibitor, or proteasome inhibitor. The immunoconjugate or a pharmaceutically acceptable salt thereof according to claim 29 or 30, wherein the cytotoxic microtubule inhibitor is selected from MMAE, MMAF, DM1, DM4, Tubulysins; and / or The DNA intercalating agent is PBD or IBD; and / or The DNA topoisomerase inhibitor is a topoisomerase I inhibitor, preferably a camptothecin derivative; and / or The apoptosis inducer is a Bcl-xL inhibitor, preferably clezutoclax; and / or The RNA polymerase inhibitor is α-amaminine or β-amaminine; and / or The spliceosome inhibitor is preferably thailanstatin A; and / or The proteasome inhibitor is selected from carbamycin A and carbamycin B. The immunoconjugate or a pharmaceutically acceptable salt thereof according to any one of claims 29-31 is characterized in that, The cytotoxic drug is a DNA topoisomerase inhibitor, preferably DXD or a derivative of DXD. The immunoconjugate or a pharmaceutically acceptable salt thereof according to claim 32 is characterized in that, The DXD or its derivatives are compounds of formula (II), their stereoisomers, or pharmaceutically acceptable salts thereof. Among them, X 1 For O, S, or NH; X 2 For -NR 2 - or key, R 2 -H, -C 1-3 Alkyl or 3-6 membered cycloalkyl; X 3 It can be -O- or -NH-; R 1 -H, -C 1-3 Alkyl or 3-6 membered cycloalkyl; M is (CR) 3 R 4 ) m -or Among them, R 3 R 4 Each is independently -H, -C 1-5 Alkyl or 3-6 membered cycloalkyl, where m is 1, 2 or 3, and ring A is 3-6 membered cycloalkane. The immunoconjugate or a pharmaceutically acceptable salt thereof according to claim 33 is characterized in that, The X 1 For O, X 2 For -NH-, R 1 It is -H. The immunoconjugate or a pharmaceutically acceptable salt thereof according to claim 33 or 34 is characterized in that, m is 2; preferably, M is -CR 3 R 4 CH2-, R 3 R 4 One of them is -H, and the other is methyl, isopropyl, or cyclopropyl. The immunoconjugate or a pharmaceutically acceptable salt thereof according to any one of claims 28-35 is characterized in that, The D is selected from compounds represented by any of the following structural formulas, their stereoisomers, or pharmaceutically acceptable salts thereof: The immunoconjugate or a pharmaceutically acceptable salt thereof according to any one of claims 28-36 is characterized in that, D represents DXD or compound 2A. The immunoconjugate or a pharmaceutically acceptable salt thereof according to any one of claims 28-37 is characterized in that, L is -L 4 -L 3 -L 2 -L 1 -; in: L 1 Each independently serves as a key, Among them, the -NH- end and L 2 Connect one end to D, and the other end to R. a Or R b Each is independently -H or -C 1-3 Alkyl groups, preferably L 1 for L 2 Each peptide residue is independently composed of 2 to 7 amino acid residues, wherein each amino acid is independently phenylalanine, isoleucine, leucine, isoleucine, tryptophan, valine, methionine, tyrosine, alanine, threonine, histidine, serine, glutamine, arginine, lysine, asparagine, glutamic acid, proline, citrulline, aspartic acid, or glycine, and each amino acid residue is optionally independently divided by one or more halogens, hydroxyl groups, cyano groups, amino groups, or -C groups. 1-5 Alkyl, -C 1-5 Alkoxy or 3-6 membered cycloalkyl substitution, wherein the -NH- terminus is associated with an L-terminus. 3 Connected to L, the other end is connected to L. 1 Linked; preferably, each of the amino acids is independently glycine, phenylalanine, valine, or citrulline; more preferably, L 2 The residues are -glycine residues - phenylalanine residues - glycine residues - (-Gly-Phe-Gly-), -glycine residues - glycine residues - phenylalanine residues - glycine residues - (-Gly-Gly-Phe-Gly-), or -valine residues - citrulline residues - (-Val-Cit-); more preferably, L 2 for L 3 Each is independently -(CH2) n1 -C(O)-,-(CH2CH2O) n2 -C(O)- or -(CH2) n3 -W-(CH2) n4 -C(O)-, where the -C(O)- terminal is connected to L 2 Connected to L, the other end is connected to L. 4 Connected, n1, n2, n3, and n4 are each independently an integer from 0 to 8, and W is -O-, -C(O)-NH-, 6-10 aryl, 5-10 aryl heteroalkyl, 3-6 cycloalkyl, or 3-6 heterocyclic group, wherein the 6-10 aryl, 5-10 aryl heteroalkyl, 3-6 cycloalkyl, or 3-6 heterocyclic group is optionally surrounded by one or more halogens, hydroxyl groups, cyano groups, amino groups, or -C 1-5 Alkyl, -C 1-5 Alkoxy or 3-6 membered cycloalkyl substitution; preferably, L 3 for L 4 Each independently aC(=O)-b, a-CH2C(=O)-b, where end a is connected to Ab, and end b is connected to L. 3 Connected; preferably The immunoconjugate or a pharmaceutically acceptable salt thereof according to claim 38 is characterized in that, L 4 for The a-terminus is connected to the thiol group of the cysteine ​​residue in Ab, and the b-terminus is connected to L. 3 Connected. The immunoconjugate or a pharmaceutically acceptable salt thereof according to any one of claims 28-39 is characterized in that, L is The left end is connected to Ab, and the right end is connected to D. The immunoconjugate or a pharmaceutically acceptable salt thereof according to any one of claims 28-40 is characterized in that, The value of p is 1, 2, 3, 4, 5, 6, 7 or 8. The immunoconjugate or a pharmaceutically acceptable salt thereof according to any one of claims 28-41 is characterized in that, The immunoconjugate or its pharmaceutically acceptable salt has the following structure: Among them, R 1 X 1 X 2 M, X 3 L 1 L 2 L 3 L 4 Ab and p are each independently claimed according to any one of claims 33-41. The immunoconjugate or a pharmaceutically acceptable salt thereof according to claim 42 is characterized in that, The immunoconjugate of formula (III) or its pharmaceutically acceptable salt has the structure shown in formula (IIIa): Among them, X 1 R 2 M, L 1 L 2 L 3 L 4 Ab and p are each independently claimed according to any one of claims 33-41. The immunoconjugate or a pharmaceutically acceptable salt thereof according to any one of claims 28-43 is characterized in that, The immunoconjugate or its pharmaceutically acceptable salt has the following structure: Wherein, Ab and p are each independently as described in claim 28; preferably, p is an integer from 2 to 8; more preferably, p is 8. The composition of the immunoconjugate is characterized in that, It comprises one or more of the immunoconjugates as described in any one of claims 27-44 or their pharmaceutically acceptable salts. The composition of the immunoconjugate according to claim 45 is characterized in that, The DAR value of the composition of the immunoconjugate is any integer or decimal from 1 to 10; preferably, the DAR value of the composition of the immunoconjugate is any integer or decimal from 2 to 8, more preferably any integer or decimal from 3 to 8, such as 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 5.0, 5.5, 6.0, 6.5, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0 or any value or range thereof. The drug combination is characterized by, It comprises an antibody or antigen-binding fragment of any one of claims 1-21, a chimeric antigen receptor of claim 22, a polypeptide molecule of claim 23, a nucleic acid molecule of claim 24, a carrier of claim 25, a cell of claim 26, an immunoconjugate of any one of claims 27-44 or a pharmaceutically acceptable salt thereof, and / or an immunoconjugate of claim 45 or 46. The drug combination according to claim 47 is characterized in that, The drug combination may also include one or more other therapeutic agents optionally; preferably, the therapeutic agents may be other antibodies, small molecule drugs, immunomodulators, cytotoxic agents, chemotherapeutic agents or cytokines. A pharmaceutical composition comprising an antibody or antigen-binding fragment of any one of claims 1-21, a chimeric antigen receptor of claim 22, a polypeptide molecule of claim 23, a nucleic acid molecule of claim 24, a carrier of claim 25, a cell of claim 26, an immunoconjugate or a pharmaceutically acceptable salt thereof of any one of claims 27-44, a composition of an immunoconjugate of claim 45 or 46, and / or a pharmaceutical combination of claim 47 or 48, and optionally a pharmaceutically acceptable carrier. A method for preparing an antibody or antigen-binding fragment according to any one of claims 1-21, the method comprising culturing the cells of claim 26 under conditions that cause the antibody or antigen-binding fragment to be expressed. A method for preparing an immunoconjugate or a pharmaceutically acceptable salt thereof, or a composition of an immunoconjugate according to any one of claims 27-44 and / or claims 45 or 46, the method comprising conjugating an antibody or antigen-binding fragment of any one of claims 1-21 to a payload. The use of any antibody or antigen-binding fragment of claim 1-21, the chimeric antigen receptor of claim 22, the polypeptide molecule of claim 23, the nucleic acid molecule of claim 24, or the carrier of claim 25, the cell of claim 26, the pharmaceutical composition of claim 49, the immunoconjugate or a pharmaceutically acceptable salt thereof of any one of claims 27-44, the composition of the immunoconjugate of claim 45 or 46, and / or the pharmaceutical combination of claim 47 or 48 in the preparation of a medicament for the prevention, relief, and / or treatment of diseases and / or conditions. The use according to claim 52 is characterized in that, The diseases and / or conditions mentioned include CDH17-related diseases. The use according to claim 52 or 53 is characterized in that, The diseases and / or conditions mentioned include tumors. The use according to any one of claims 52-54 is characterized in that, The diseases and / or conditions mentioned include CDH17-related tumors. The use according to any one of claims 52-55 is characterized in that, The diseases and / or conditions mentioned include tumors of the digestive tract, including colorectal cancer, colorectal adenocarcinoma, gastric cancer, gastric adenocarcinoma, pancreatic cancer, and pancreatic adenocarcinoma. Use of the antibody or antigen-binding fragment of any one of claims 1-21, the chimeric antigen receptor of claim 22, the polypeptide molecule of claim 23, the nucleic acid molecule of claim 24, or the carrier of claim 25, the cell of claim 26, the pharmaceutical composition of claim 49, the immunoconjugate or a pharmaceutically acceptable salt thereof of any one of claims 27-44, the composition of the immunoconjugate of claim 45 or 46, and / or the pharmaceutical combination of claim 47 or 48 in the prevention, relief, and / or treatment of diseases and / or conditions. The use according to claim 57 is characterized in that, The diseases and / or conditions mentioned include CDH17-related diseases. The use according to claim 57 or 58 is characterized in that, The diseases and / or conditions mentioned include tumors. The use according to any one of claims 57-59 is characterized in that, The diseases and / or conditions mentioned include CDH17-related tumors. The use according to any one of claims 57-60 is characterized in that, The diseases and / or conditions mentioned include tumors of the digestive tract, including colorectal cancer, colorectal adenocarcinoma, gastric cancer, gastric adenocarcinoma, pancreatic cancer, and pancreatic adenocarcinoma. The antibody or antigen-binding fragment of any one of claims 1-21, the chimeric antigen receptor of claim 22, the polypeptide molecule of claim 23, the nucleic acid molecule of claim 24, or the carrier of claim 25, the cell of claim 26, the pharmaceutical composition of claim 49, the immunoconjugate or a pharmaceutically acceptable salt thereof of any one of claims 27-44, the composition of the immunoconjugate of claim 45 or 46, and / or the pharmaceutical combination of claim 47 or 48 are used for the prevention and / or treatment of diseases and / or conditions. According to the use described in claim 62, the disease and / or condition includes CDH17-related diseases. According to the use described in claim 62 or 63, the disease and / or condition includes tumors. For the use according to any one of claims 62-64, the disease and / or condition includes CDH17-related tumors. The use according to any one of claims 62-65 is characterized in that, The diseases and / or conditions mentioned include tumors of the digestive tract, including colorectal cancer, colorectal adenocarcinoma, gastric cancer, gastric adenocarcinoma, pancreatic cancer, and pancreatic adenocarcinoma. A method for detecting CDH17 in a sample, characterized in that, The method comprises administering an antibody or antigen-binding fragment of any one of claims 1-21, a chimeric antigen receptor of claim 22, a polypeptide molecule of claim 23, a nucleic acid molecule of claim 24, or a carrier of claim 25, a cell of claim 26, a pharmaceutical composition of claim 49, an immunoconjugate or a pharmaceutically acceptable salt thereof of any one of claims 27-44, a composition of an immunoconjugate of claim 45 or 46, and / or a pharmaceutical combination of claim 47 or 48. A reagent or kit for detecting CDH17 in a sample, comprising an antibody or antigen-binding fragment as described in any one of claims 1-21, a chimeric antigen receptor as described in claim 22, a polypeptide molecule as described in claim 23, a nucleic acid molecule as described in claim 24, or a carrier as described in claim 25, a cell as described in claim 26, a pharmaceutical composition as described in claim 49, an immunoconjugate or a pharmaceutically acceptable salt thereof as described in any one of claims 27-44, a composition of an immunoconjugate as described in claim 45 or 46, and / or a pharmaceutical combination as described in claim 47 or 48. The use of the antibody or antigen-binding fragment of any one of claims 1-21, the chimeric antigen receptor of claim 22, the polypeptide molecule of claim 23, the nucleic acid molecule of claim 24, or the carrier of claim 25, the cell of claim 26, the pharmaceutical composition of claim 49, the immunoconjugate or a pharmaceutically acceptable salt thereof of any one of claims 27-44, the composition of the immunoconjugate of claim 45 or 46, and / or the pharmaceutical combination of claim 47 or 48 in the preparation of a kit for detecting the presence and / or content of CDH17 in a sample.

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