Anti-human CDH17 antibody

Abstract

Provided are an anti-CDH17 antibody and an antigen-binding fragment thereof, and an antibody-drug conjugate and a use thereof. A CDH17 antibody-drug conjugate exhibits anti-tumor activity. Also provided are a composition comprising the antibody-drug conjugate and a use of the composition.

Description

Anti-human CDH17 antibodies TECHNICAL FIELD

[0001] The present application relates to the field of biological medicine. Specifically, the present application relates to anti-human CDH17 antibodies. BACKGROUND

[0002] Cadherins are cell adhesion molecules that play an important role in maintaining tissue structure and morphology under normal conditions, and the expression disorder of Cadherin is often associated with diseases, including tissue dysplasia, tumor formation and metastasis.

[0003] Cadherin-17 (CDH17) belongs to a subclass of 7D-cadherin family, and its function is to maintain the integrity of epithelial tissue as a polypeptide transporter and cell adhesion molecule. The structure of Cadherin 17 is different from that of classical Cadherins, including seven cadherin repeat sequences EC1 to EC7 and an intracellular domain of 18-20 amino acids. EC6 of Cadherin 17 (Extracellular 6, extracellular domain 6) contains an RGD motif, which constitutes a key switch for integrin binding and activation.

[0004] CDH17 belongs to type I single transmembrane protein, and Cadherin-16 (CDH16) has a similar structure to CDH17, and also has seven cadherin repeat regions in the extracellular region, which is a protein with high homology to CDH17 in the CDH family.

[0005] CDH17 is expressed in normal tissues in the colon, small intestine, and stomach, and is isolated and hidden between normal cells. CDH17 is highly expressed in colorectal, gastric and pancreatic cancer. Therefore, CDH17 can be used as an ideal target for the development of antibody, bispecific antibody, antibody-drug conjugate and cell therapy fields.

[0006] Currently, there are not many drugs targeting CDH17, and all are in the early clinical or preclinical development stage, of which the two bispecific antibody drugs ARB202 and BI-905711 are the most advanced.

[0007] Therefore, there is a need in the art to develop new antibody drugs targeting CDH17. SUMMARY

[0008] The purpose of the present application is to provide an antibody drug targeting CDH17.

[0009] In a first aspect of the present application, an anti-CDH17 antibody or antigen-binding fragment thereof is provided, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region and the light chain variable region have six complementarity determining regions (CDRs) selected from the group consisting of:

[0010] (1a) three complementarity determining regions of a heavy chain variable region VH-CDRs and three complementarity determining regions of a light chain variable region VL-CDRs defined based on Chothia rules:

[0011] VH-CDR1 as shown in SEQ ID NO: 9,

[0012] VH-CDR2 as shown in SEQ ID NO: 10,

[0013] VH-CDR3 as shown in SEQ ID NO: 11,

[0014] VL-CDR1 as shown in SEQ ID NO: 19,

[0015] VL-CDR2 as shown in SEQ ID NO: 20,

[0016] VL-CDR3 as shown in SEQ ID NO: 21; or

[0017] (1b) three complementarity determining regions of a heavy chain variable region VH-CDRs and three complementarity determining regions of a light chain variable region VL-CDRs defined based on Abm rules:

[0018] VH-CDR1 as shown in SEQ ID NO: 12,

[0019] VH-CDR2 as shown in SEQ ID NO: 13,

[0020] VH-CDR3 as shown in SEQ ID NO: 11,

[0021] VL-CDR1 as shown in SEQ ID NO: 19,

[0022] VL-CDR2 as shown in SEQ ID NO: 20,

[0023] VL-CDR3 as shown in SEQ ID NO: 21; or

[0024] (1c) three complementarity determining regions of a heavy chain variable region VH-CDRs and three complementarity determining regions of a light chain variable region VL-CDRs defined based on Kabat rules:

[0025] VH-CDR1 as shown in SEQ ID NO: 14,

[0026] VH-CDR2 as shown in SEQ ID NO: 24 or 15,

[0027] VH-CDR3 as shown in SEQ ID NO: 11,

[0028] VL-CDR1 as represented by SEQ ID NO: 19,

[0029] VL-CDR2 as represented by SEQ ID NO: 20,

[0030] VL-CDR3 as represented by SEQ ID NO: 21; or

[0031] (1d) three complementarity determining regions of a heavy chain variable region VH-CDRs and three complementarity determining regions of a light chain variable region VL-CDRs defined based on IMGT rules:

[0032] VH-CDR1 as represented by SEQ ID NO: 16,

[0033] VH-CDR2 as represented by SEQ ID NO: 17,

[0034] VH-CDR3 as represented by SEQ ID NO: 18,

[0035] VL-CDR1 as represented by SEQ ID NO: 22,

[0036] VL-CDR2 as represented by SEQ ID NO: 23,

[0037] VL-CDR3 as represented by SEQ ID NO: 21; or

[0038] (2a) three complementarity determining regions of a heavy chain variable region VH-CDRs and three complementarity determining regions of a light chain variable region VL-CDRs defined based on Chothia rules:

[0039] VH-CDR1 as represented by SEQ ID NO: 25,

[0040] VH-CDR2 as represented by SEQ ID NO: 26,

[0041] VH-CDR3 as represented by SEQ ID NO: 27,

[0042] VL-CDR1 as represented by SEQ ID NO: 35,

[0043] VL-CDR2 as represented by SEQ ID NO: 36,

[0044] VL-CDR3 as represented by SEQ ID NO: 37; or

[0045] (2b) three complementarity determining regions of a heavy chain variable region VH-CDRs and three complementarity determining regions of a light chain variable region VL-CDRs defined based on Abm rules:

[0046] VH-CDR1 as set forth in SEQ ID NO: 28,

[0047] VH-CDR2 as set forth in SEQ ID NO: 29,

[0048] VH-CDR3 as set forth in SEQ ID NO: 27,

[0049] VL-CDR1 as set forth in SEQ ID NO: 35,

[0050] VL-CDR2 as set forth in SEQ ID NO: 36,

[0051] VL-CDR3 as set forth in SEQ ID NO: 37; or

[0052] (2c) three complementarity determining regions of a heavy chain variable region VH-CDRs and three complementarity determining regions of a light chain variable region VL-CDRs defined based on the Rabat convention:

[0053] VH-CDR1 as set forth in SEQ ID NO: 30,

[0054] VH-CDR2 as set forth in SEQ ID NO: 40 or 31,

[0055] VH-CDR3 as set forth in SEQ ID NO: 27,

[0056] VL-CDR1 as set forth in SEQ ID NO: 35,

[0057] VL-CDR2 as set forth in SEQ ID NO: 36,

[0058] VL-CDR3 as set forth in SEQ ID NO: 37; or

[0059] (2d) three complementarity determining regions of a heavy chain variable region VH-CDRs and three complementarity determining regions of a light chain variable region VL-CDRs defined based on the IMGT convention:

[0060] VH-CDR1 as set forth in SEQ ID NO: 32,

[0061] VH-CDR2 as set forth in SEQ ID NO: 33,

[0062] VH-CDR3 as set forth in SEQ ID NO: 34,

[0063] VL-CDR1 as set forth in SEQ ID NO: 38,

[0064] VL-CDR2 as set forth in SEQ ID NO: 39,

[0065] a VL-CDR3 as set forth in SEQ ID NO: 37.

[0066] In another preferred embodiment, any one of the above-mentioned amino acid sequences further comprises a derivative sequence in which at least one amino acid is optionally added, deleted, modified and / or substituted, and which is capable of retaining CDH17 binding affinity.

[0067] In another preferred embodiment, the number of the added, deleted, modified and / or substituted amino acids is 1-3, preferably 1-2, more preferably 1.

[0068] In another preferred embodiment, the antibody or antigen-binding fragment thereof is humanized.

[0069] In another preferred embodiment, the amino acid sequence of the heavy chain variable region is as set forth in SEQ ID NO: 1 or 3; and / or the amino acid sequence of the light chain variable region is as set forth in SEQ ID NO: 2 or 4.

[0070] In another preferred embodiment, the amino acid sequence of the heavy chain variable region is as set forth in SEQ ID NO: 1; and the amino acid sequence of the light chain variable region is as set forth in SEQ ID NO: 2.

[0071] In another preferred embodiment, the amino acid sequence of the heavy chain variable region is as set forth in SEQ ID NO: 3; and the amino acid sequence of the light chain variable region is as set forth in SEQ ID NO: 4.

[0072] Preferably, the amino acid sequence of the heavy chain variable region is encoded by a nucleotide sequence as set forth in SEQ ID NO: 47; and the amino acid sequence of the light chain variable region is encoded by a nucleotide sequence as set forth in SEQ ID NO: 49.

[0073] In another preferred embodiment, the amino acid sequence of the heavy chain variable region is as set forth in SEQ ID NO: 5 or 7; and / or the amino acid sequence of the light chain variable region is as set forth in SEQ ID NO: 6 or 8.

[0074] In another preferred embodiment, the amino acid sequence of the heavy chain variable region is as set forth in SEQ ID NO: 5; and the amino acid sequence of the light chain variable region is as set forth in SEQ ID NO: 6.

[0075] In another preferred embodiment, the amino acid sequence of the heavy chain variable region is as set forth in SEQ ID NO: 7; and the amino acid sequence of the light chain variable region is as set forth in SEQ ID NO: 8.

[0076] Preferably, the amino acid sequence of the heavy chain variable region is encoded by the nucleotide sequence set forth in SEQ ID NO: 51; and the amino acid sequence of the light chain variable region is encoded by the nucleotide sequence set forth in SEQ ID NO: 53.

[0077] In another preferred embodiment, the heavy chain of the antibody or antigen binding fragment thereof further comprises a heavy chain constant region.

[0078] In another preferred embodiment, the heavy chain constant region is of human or murine origin.

[0079] In another preferred embodiment, the amino acid sequence of the heavy chain constant region is set forth in SEQ ID NO: 42.

[0080] In another preferred embodiment, the light chain of the antibody or antigen binding fragment thereof further comprises a light chain constant region.

[0081] In another preferred embodiment, the light chain constant region is of human or murine origin.

[0082] In another preferred embodiment, the amino acid sequence of the light chain constant region is set forth in SEQ ID NO: 41.

[0083] In another preferred embodiment, the antibody or antigen binding fragment thereof specifically binds to CDH17.

[0084] In another preferred embodiment, the CDH17 is from human, rat or cynomolgus monkey.

[0085] In another preferred embodiment, the antibody or antigen binding fragment thereof specifically binds to the extracellular domain EC1 or EC3 of CDH17.

[0086] In another preferred embodiment, the antibody is a bispecific antibody, or a single chain antibody (scFv).

[0087] In another preferred embodiment, the antibody is a monoclonal antibody.

[0088] In another preferred embodiment, the antibody is a monospecific, bispecific, or trispecific antibody.

[0089] In another preferred embodiment, the bispecific antibody comprises:

[0090] (1) the anti-CDH17 antibody or antigen binding fragment thereof;

[0091] (2) an antibody or antigen binding fragment thereof that binds to another target.

[0092] In another preferred embodiment, the other target is selected from the group consisting of BCMA, GPRC5D, CD38, CD123, CD19, CD20, CD22, GPC3, TROP-2, Claudin 18.2, EGFR, HER2, HER3, CD3, CD28, 4-1BB, OX40, CD40, CD27, CD47, CTLA4, PD1, PDL1, DLL3, SEZ6, MUC-1, MUC-16, FRa, PSMA, ALPPL2, ALPP, CD228, LIV-1, B7-H1, B7-H3, B7-H6, B7-H7, B7-H4, TIGB6, STEAP1 and STEAP2.

[0093] In a second aspect of the present application, there is provided a recombinant protein comprising:

[0094] (i) an anti-CDH17 antibody or antigen-binding fragment thereof as described in the first aspect of the present application; and

[0095] (ii) an optional tag sequence to facilitate expression and / or purification.

[0096] In another preferred embodiment, the tag sequence comprises a 6His tag.

[0097] In another preferred embodiment, the recombinant protein (or polypeptide) comprises a fusion protein.

[0098] In another preferred embodiment, the recombinant protein is a monomer, a dimer, or a multimer.

[0099] In another preferred embodiment, the recombinant protein is a mono-specific, bi-specific, or tri-specific antibody.

[0100] In another preferred embodiment, the recombinant protein further comprises an additional fusion element (or fusion polypeptide fragment) fused to the element (i).

[0101] In a third aspect of the present application, there is provided a CAR construct, wherein the antigen binding region of the CAR construct comprises a scFv region that specifically binds to CDH17, and the scFv region has a heavy chain variable region and a light chain variable region of an antibody as described in the first aspect of the present application.

[0102] In a fourth aspect of the present application, there is provided a recombinant cell expressing an exogenous CAR construct as described in the third aspect of the present application.

[0103] In another preferred embodiment, the recombinant cell is an immune cell or an IPS cell.

[0104] In another preferred embodiment, the immune cell is selected from the group consisting of NK cells, T cells.

[0105] In another preferred embodiment, the recombinant cell is from a human or a non-human mammal (e.g., murine).

[0106] In a fifth aspect of the present application, an immunoconjugate is provided, the immunoconjugate comprising:

[0107] (a) an antibody moiety that is an anti-CDH17 antibody or antigen-binding fragment thereof according to the first aspect of the present application; and

[0108] (b) a conjugated moiety conjugated to the antibody moiety, the conjugated moiety selected from the group consisting of a detectable label, a drug, a toxin, a cytokine, an enzyme, or a combination thereof.

[0109] In another preferred embodiment, the immunoconjugate is an antibody drug conjugate.

[0110] In another preferred embodiment, the antibody moiety is conjugated to the conjugated moiety via a chemical bond or linker.

[0111] In another preferred embodiment, the conjugated moiety is a chemical label or a biological label.

[0112] In another preferred embodiment, the chemical label is an isotope, an immunotoxin, and / or a chemical drug.

[0113] In another preferred embodiment, the biological label is biotin, avidin, or an enzyme label.

[0114] In another preferred embodiment, the conjugated moiety is a drug or a toxin.

[0115] In another preferred embodiment, the drug is a cytotoxic drug.

[0116] In another preferred embodiment, the drug is a nucleic acid drug.

[0117] In another preferred embodiment, the toxin is selected from the group consisting of:

[0118] auristatins (e.g., auristatin E, auristatin F, MMAE, and MMAF), aureusidin, maytansinoid, ricin, ricin A-chain, combrestatin, duocarmycin, dolastatin, doxorubicin, daunorubicin, paclitaxel, cisplatin, cc1065, ethidium bromide, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin, dihydroxy anthracin dione, actinomycin, diphtheria toxin, Pseudomonas exotoxin (PE) A, PE40, abrin, abrin A chain, modeccin A chain, alpha-sarcin, gelonin, mitogellin, retstrictocin, phenomycin, enomycin, curicin, phytolaccin, crotin, calicheamicin, Sapaonaria officinalis inhibitor, glucocorticoid, or a combination thereof.

[0119] In another preferred embodiment, the toxin is a TOP inhibitor.

[0120] In another preferred embodiment, the TOP inhibitor is selected from the group consisting of SN38 or a prodrug thereof, etoposide, tenoposide, daunorubicin, doxorubicin, idarubicin, mitoxantrone.

[0121] In another preferred embodiment, the coupling moiety is a detectable label.

[0122] In another preferred embodiment, the detectable label comprises a radionuclide, the radionuclide comprising:

[0123] (i) a diagnostic isotope selected from the group consisting of Tc-99m, Ga-68, F-18, I-123, I-125, I-131, In-111, Ga-67, Cu-64, Zr-89, C-11, Lu-177, Re-188, or a combination thereof; and / or

[0124] (ii) a therapeutic isotope selected from the group consisting of Lu-177, Y-90, Ac-225, As-211, Bi-212, Bi-213, Cs-137, Cr-51, Co-60, Dy-165, Er-169, Fm-255, Au-198, Ho-166, I-125, I-131, Ir-192, Fe-59, Pb-212, Mo-99, Pd-103, P-32, K-42, Re-186, Re-188, Sm-153, Ra223, Ru-106, Na24, Sr89, Tb-149, Th-227, Xe-133 Yb-169, Yb-177, or a combination thereof.

[0125] In another preferred embodiment, the conjugate is selected from the group consisting of a fluorescent or luminescent marker, a radioactive marker, an MRI (magnetic resonance imaging) or CT (computerized tomography) contrast agent, or an enzyme capable of generating a detectable product, a radionuclide, a biological toxin, a cytokine (such as IL-2, etc.), an antibody, an antibody Fc fragment, an antibody scFv fragment, a gold nanobody particle / nanobody rod, a viral particle, a liposome, a nanobody magnetic particle, a prodrug-activating enzyme (e.g., DT-diaphorase (DTD) or benzylphenyl hydroxylase-like protein (BPHL)), a chemotherapeutic agent (e.g., cisplatin), or any form of nanobody particle, etc.

[0126] In another preferred embodiment, the detection is in vivo or in vitro.

[0127] In another preferred embodiment, the immunoconjugate is used for diagnosis and / or treatment of a tumor expressing CDH17 protein.

[0128] In another preferred embodiment, the immunoconjugate has the following formula:

[0129] wherein:

[0130] nAb is an anti-CDH17 antibody or antigen-binding fragment thereof;

[0131] LU is a linker (also known as a linking group);

[0132] D is a drug;

[0133] and subscript p is a value selected from 1-10.

[0134] In another preferred embodiment, the LU is selected from the group consisting of a maleimidocaproyl (MC), a maleimide (MAL), a succinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC) linker to the antibody moiety and comprises a linker of one or more of valine-citrulline (VC), valine-alanine (VA), glycine-glycine-phenylalanine-glycine (GGFG), alanine-alanine-alanine (AAA), p-aminobenzyloxy carbonyl (PAB), polyethylene glycol (PEG).

[0135] In a sixth aspect of the present application, a pharmaceutical composition is provided, the pharmaceutical composition comprising:

[0136] (i) an anti-CDH17 antibody or antigen-binding fragment thereof according to the first aspect of the present application, a recombinant protein according to the second aspect of the present application, a recombinant cell according to the fourth aspect of the present application, or an immunoconjugate according to the fifth aspect of the present application;

[0137] (ii) a pharmaceutically acceptable carrier.

[0138] In another preferred embodiment, the pharmaceutical composition comprises a single agent, a combination agent, or a synergistic agent.

[0139] In another preferred embodiment, the pharmaceutical composition further comprises other bioactive substances, such as drugs for treating tumors.

[0140] In another preferred embodiment, the pharmaceutical composition is administered by subcutaneous injection, intradermal injection, intramuscular injection, intravenous injection, intraperitoneal injection, microneedle injection, oral administration, or oral and nasal cavity spraying and aerosol inhalation.

[0141] In another preferred embodiment, the pharmaceutical composition is in the form of a liquid, a solid, or a gel.

[0142] In another preferred embodiment, the pharmaceutical composition is a liquid preparation.

[0143] In another preferred embodiment, the pharmaceutical composition is an injection.

[0144] In a seventh aspect of the present application, there is provided a use of an active ingredient selected from the group consisting of an anti-CDH17 antibody or an antigen-binding fragment thereof according to the first aspect of the present application, a recombinant protein according to the second aspect of the present application, a recombinant cell according to the fourth aspect of the present application, or an immunoconjugate according to the fifth aspect of the present application, or a combination thereof, for (a) preparing a detection reagent, a detection plate, or a kit; and / or (b) preparing a drug for preventing and / or treating a CDH17-related disease.

[0145] In another preferred embodiment, the detection reagent, the detection plate, or the kit is used for:

[0146] (1) detecting CDH17 protein in a sample; and / or

[0147] (2) detecting endogenous CDH17 protein in tumor cells or tissues; and / or

[0148] (3) detecting tumor cells expressing CDH17 protein.

[0149] In another preferred embodiment, the detection type includes, but is not limited to, flow detection, cell immunofluorescence detection, enzyme-linked immunosorbent assay detection, immunoblotting detection, etc.

[0150] In another preferred embodiment, the detection reagent, the detection plate, or the kit is used for diagnosing a CDH17-related disease.

[0151] In another preferred embodiment, the drug is used for treating or preventing CDH17-high-expressing tumors, tumor metastasis, or tumor drug resistance.

[0152] In another preferred embodiment, the tumor drug resistance includes: tumor immunotherapy drug resistance, tumor targeted therapy drug resistance, conventional tumor chemotherapy drug resistance, and radiotherapy insensitivity.

[0153] In another preferred embodiment, the CDH17 related disease is a tumor or cancer.

[0154] In another preferred embodiment, the CDH17 related disease includes: tumor occurrence, growth and / or metastasis.

[0155] In another preferred embodiment, the cancer includes solid tumors, blood cancer.

[0156] In another preferred embodiment, the cancer is selected from the group consisting of small intestine cancer, colorectal cancer, gastric cancer, pancreatic cancer, esophageal cancer, ovarian cancer, lung cancer (such as lung adenocarcinoma and non-small cell lung cancer), breast cancer (such as triple negative breast cancer), malignant brain glioma, liver cancer, kidney cancer, bladder cancer, prostate cancer, endometrial cancer, cervical cancer, leukemia, bone marrow cancer, angiosarcoma, or a combination thereof.

[0157] In an eighth aspect of the present application, a polynucleotide is provided, wherein the polynucleotide encodes a polypeptide selected from the group consisting of:

[0158] The anti-CDH17 antibody or antigen binding fragment thereof according to the first aspect of the present application, the recombinant protein according to the second aspect of the present application, or the CAR construct according to the third aspect of the present application.

[0159] In another preferred embodiment, the polynucleotide has a nucleotide sequence as set forth in SEQ ID NO: 48; and / or the polynucleotide has a nucleotide sequence as set forth in SEQ ID NO: 50.

[0160] In another preferred embodiment, the polynucleotide has a nucleotide sequence as set forth in SEQ ID NO: 52; and / or the polynucleotide has a nucleotide sequence as set forth in SEQ ID NO: 54.

[0161] In another preferred embodiment, the polynucleotide includes RNA, DNA or cDNA.

[0162] In a ninth aspect of the present application, a vector is provided, wherein the vector contains the polynucleotide according to the eighth aspect of the present application.

[0163] In another preferred embodiment, the vector includes: bacterial plasmid, bacteriophage, yeast plasmid, plant cell virus, mammalian cell virus such as adenovirus, retrovirus, or other vectors.

[0164] In a tenth aspect of the present application, a host cell comprising the vector or genome of the ninth aspect of the present application or the polynucleotide of the eighth aspect of the present application integrated therein is provided.

[0165] In an eleventh aspect of the present application, a method for detecting (including diagnostic or non-diagnostic) CDH17 in a sample in vitro is provided, comprising the steps of:

[0166] (1) contacting, in vitro, the sample with the anti-CDH17 antibody or antigen binding fragment thereof of the first aspect of the present application, the recombinant protein of the second aspect of the present application or the immunoconjugate of the fifth aspect of the present application;

[0167] (2) detecting whether an antigen-antibody complex is formed, wherein the formation of the complex indicates the presence of CDH17 in the sample.

[0168] In another preferred embodiment, the detection is diagnostic or non-diagnostic.

[0169] In a twelfth aspect of the present application, a method for producing a recombinant polypeptide is provided, comprising:

[0170] (a) culturing the host cell of the tenth aspect of the present application under conditions suitable for expression;

[0171] (b) isolating the recombinant polypeptide from the culture, wherein the recombinant polypeptide is the anti-CDH17 antibody or antigen binding fragment thereof of the first aspect of the present application or the recombinant protein of the second aspect of the present application.

[0172] In a thirteenth aspect of the present application, a method for treating a CDH17 related disease is provided, comprising administering to a subject in need thereof the anti-CDH17 antibody or antigen binding fragment thereof of the first aspect of the present application, the recombinant protein of the second aspect of the present application, the recombinant cell of the fourth aspect of the present application, the immunoconjugate of the fifth aspect of the present application, the pharmaceutical composition of the sixth aspect of the present application, or a combination thereof.

[0173] In another preferred embodiment, the method further comprises administering to the subject in need thereof another drug or therapeutic method for combination therapy.

[0174] In another preferred embodiment, the other drug or therapeutic method comprises an anti-tumor immunotherapy drug, a tumor targeting drug, a tumor chemotherapy drug, or a tumor radiotherapy.

[0175] It should be understood that, in the scope of the present application, each of the technical features of the present application described above and each of the technical features specifically described hereinafter (such as the examples) can be combined with each other to constitute a new or preferred technical scheme. Limited by the length, they are not listed one by one here. BRIEF DESCRIPTION OF DRAWINGS

[0176] The following drawings serve to illustrate the specific embodiments of the present application and are not intended to limit the scope of the present application as defined by the claims.

[0177] Figure 1 shows the detection of CDH17 expression in HEK293T-hCDH17 overexpression cell lines.

[0178] Figure 2A shows the protein affinity detection of anti-human CDH17 humanized antibody with human CDH17-mFc.

[0179] Figure 2B shows the protein affinity detection of anti-human CDH17 humanized antibody with human CDH17-his.

[0180] Figure 3 shows the protein affinity detection of anti-human CDH17 humanized antibody with monkey CDH17-His.

[0181] Figure 4 shows the protein affinity detection of anti-human CDH17 humanized antibody with rat CDH17-His.

[0182] Figure 5 shows the affinity detection of anti-human CDH17 humanized antibody with HEK293T-h.CDH17 overexpression cells.

[0183] Figure 6 shows the affinity detection of anti-human CDH17 humanized antibody with NCI-H716 tumor cells.

[0184] Figure 7 shows the affinity detection of anti-human CDH17 humanized antibody with LS1034 tumor cells.

[0185] Figure 8 shows the affinity detection of anti-human CDH17 humanized antibody with DLD-1 tumor cells.

[0186] Figure 9 shows the affinity detection of anti-human CDH17 humanized antibody with MKN45 tumor cells.

[0187] Figure 10 shows the ADCC activity detection of anti-human CDH17 humanized antibody.

[0188] Figure 11 shows the ADCP activity detection of anti-human CDH17 humanized antibody. DETAILED DESCRIPTION

[0189] The present inventors have conducted extensive and in-depth research, and unexpectedly obtained an anti-CDH17 antibody and a humanized antibody thereof with excellent specificity, high affinity and species cross-reactivity through a large number of screenings. The anti-CDH17 antibody of the present application has excellent affinity for CDH17 protein, especially tumor cells expressing CDH17; has species cross-binding activity for human, rat and monkey CDH17 proteins, and can mediate CDH17 endocytosis. On this basis, the present application is completed.

[0190] Terminology

[0191] For easier understanding of the present application, certain technical and scientific terms are defined below. Unless otherwise defined herein, all other technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Before describing the present application, it is to be understood that the present application is not limited to the particular methodology and experimental conditions described, as such methodology and conditions can vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present application will only be limited by the appended claims.

[0192] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. As used herein, the term "about," when used in reference to a recited numerical value, means that the value can vary from the recited value by not more than 1%. For example, as used herein, the expression "about 100" includes all values between 99 and 101 (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0193] The three-letter codes and one-letter codes for amino acids used in the present application are as described in J. Biol. Chem, 243, p3558 (1968).

[0194] As used herein, the term "treatment" refers to the administration of an internal or external therapeutic agent, including the antibodies of the present application directed to respiratory syncytial virus fusion protein (preferably pre-fusion F protein) and compositions thereof, to a patient having one or more symptoms of a disease, where the therapeutic agent is known to have a therapeutic effect on the symptoms. Typically, the patient is administered the therapeutic agent in an amount effective to alleviate one or more symptoms of the disease (therapeutically effective amount).

[0195] As used herein, the term "optional" or "optionally" means that the event or circumstance subsequently described can occur, but that it is not a requirement. For example, "optionally comprising 1-3 antibody heavy chain variable regions" means that the antibody heavy chain variable region of the particular sequence can be, but is not required to be, 1, 2, or 3.

[0196] "Sequence identity" as used herein refers to the extent to which two nucleic acid or two amino acid sequences are identical, when optimally aligned and compared. The sequence identity between a sequence as used herein and a sequence identical thereto can be at least 85%, 90% or 95%, preferably at least 95%. Non-limiting examples include 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%.

[0197] Abbreviations

[0198] CDR: complementarity determining region

[0199] FR: framework region, comprising amino acid residues in a variable region of an antibody other than CDR residues

[0200] VH: variable region of a heavy chain of an antibody

[0201] VL: variable region of a light chain of an antibody

[0202] IgG: immunoglobulin G

[0203] Kabat: immunoglobulin alignment and numbering system as proposed by Elvin A. Kabat

[0204] Chothia: immunoglobulin numbering system as proposed by Chothia et al.

[0205] IMGT: numbering system based on the international immunogenetics information system as initiated by Lefranc et al.

[0206] EC50: half maximal effective concentration, i.e. the concentration causing 50% of the maximal effect

[0207] ELISA: enzyme-linked immunosorbent assay

[0208] FACS: flow cytometry

[0209] PCR: polymerase chain reaction

[0210] Non-CE: non-reducing capillary electrophoresis

[0211] R-CE: reducing capillary electrophoresis

[0212] HRP: horseradish peroxidase

[0213] ADCC: antibody-dependent cell-mediated cytotoxicity

[0214] ADCP: antibody-dependent cell-mediated phagocytosis

[0215] DMEM: Dulbecco's Modified Eagle Medium

[0216] RPMI 1640: Roswell Park Memorial Institute 1640 medium

[0217] FBS: fetal bovine serum

[0218] Antibody

[0219] As used herein, the term "antibody" or "immunoglobulin" refers to a heterotetrameric glycoprotein of about 150,000 daltons having the same structural core features. It is composed of two identical light (L) chains and two identical heavy (H) chains. Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide bonds between the heavy chains of different immunoglobulin isotypes varies. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. At one end of each heavy chain is a variable region (VH) followed by a number of constant regions. At one end of each light chain is a variable region (VL) followed by a constant region; the light chain constant region is attached to the first constant region of the heavy chain, and the light chain variable region is attached to the variable region of the heavy chain. Particular amino acid residues are located at the interface between the variable regions of the light and heavy chains.

[0220] As used herein, the term "variable" refers to the fact that certain portions of the variable regions differ in sequence among antibodies, which pieces vary from one particular antibody to another, and are responsible for the binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the variable regions of antibodies; it is concentrated in three segments called complementarity-determining regions (CDRs) or hypervariable regions both in the light chain and the heavy chain variable regions. The more highly conserved portions of the variable regions are called the framework regions (FRs). The variable regions of the heavy and light chains each comprise four FR regions, largely β-sheet in structure, connected by three CDRs, which form loops that connect, and in some cases form part of, the β-sheet structure. The CDRs in each chain are held together in close proximity by the FR regions and, with the CDRs from the other chain, contribute to the formation of the antigen binding site of antibodies (see Kabat et al., NIH Publ. No. 91-3242, Vol. I, pp. 647- 669 (1991)). The constant regions of the antibodies are not involved directly in binding with the antigen, but exhibit various effector functions that are important in the antibody's role in the immune response.

[0221] The "light chains" of vertebrate antibodies (immunoglobulins) can be assigned to one of two clearly distinct types, called kappa and lambda, based on the amino acid sequences of their constant regions. Depending on the amino acid sequences of their heavy chain constant regions, immunoglobulins can be assigned to different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these can be further divided into subclasses (isotypes) e.g., IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. The heavy chain constant regions of different classes of immunoglobulins are denoted α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known and described generally (see, for example, Basic and Clinical Immunology (1994) Daniel T. O'Sullivan and Prescott R. Stites, eds., 6th ed., Lange Medical Publications, Los Altos, CA; and Fundamental Immunology (1995) Paul V. Halstead, ed., 3rd ed., Raven Press, New York, NY).

[0222] The term "monoclonal antibody" (mAb) as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that can be present. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to conventional polyclonal antibody preparations that typically include different antibodies directed against different determinants, each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, the monoclonal antibodies are advantageous in that they can be synthesized uncontaminated by other immunoglobulins. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method.

[0223] Generally, the antigen binding properties of an antibody can be described by three specific regions on the variable region of both the heavy and light chains, called complementarity determining regions (CDRs), which are interspersed with four framework regions (FRs) whose amino acid sequences are relatively conserved and do not directly participate in binding. The CDRs form loops or "complementarity determining loops" (CDRs) that are held in close proximity by the FR regions, and in which the CDRs on the heavy and light chains interact to form the antigen binding site. The identification of the CDRs can be determined by comparing the amino acid sequences of antibodies of the same class or subclass.

[0224] The term "antigen-binding fragment of an antibody" (or simply "antibody fragment") refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed within the term "antigen-binding fragment of an antibody" include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CHI domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CHI domains; and (iv) a Fv fragment consisting of the VHand VLdomains of a single arm of an antibody. The Fv antibody contains the variable region of the heavy chain and the variable region of the light chain, but not the constant region, and has the smallest antibody fragment with all antigen binding sites. Generally, the Fv antibody also contains a polypeptide linker between the VHand VLdomains, and can form a structure required for antigen binding.

[0225] The present application includes not only intact monoclonal antibodies, but also antibody fragments or fusion proteins of antibodies with other sequences that have immunological activity, such as Fab or (Fab')2 fragments; antibody heavy chains; antibody light chains. Therefore, the present application also includes fragments, derivatives and analogs of the antibodies.

[0226] The term "epitope" or "antigenic determinant" refers to a site on an antigen to which an immunoglobulin or antibody specifically binds. Epitopes usually consist of chemically discrete, spatial conformational regions on an antigen that are composed of at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 contiguous or non-contiguous amino acids. Epitopes can be discontinuous, three-dimensional sites on an antigen recognized by the antibodies or antigen-binding fragments of the present application.

[0227] The terms "specifically binds," "selectively binds," "selectively binds to," and "specifically binds to" refer to the binding of an antibody to an epitope on a predetermined antigen. Typically, an antibody will bind to an antigen with an affinity (KD) of about less than 10 - 7 M, for example, an affinity (KD) of about less than 10 -8 M, 10 -9 M or l0 -10 M or less.

[0228] In the present application, antibodies include murine, chimeric, humanized, or fully human antibodies prepared by techniques well known to those skilled in the art. Recombinant antibodies, such as chimeric and humanized monoclonal antibodies, including both human and non-human portions, can be obtained by standard DNA recombination techniques, and are useful antibodies. A chimeric antibody is a molecule in which different portions are derived from different animal species, such as those having a variable region from a murine monoclonal antibody and a constant region from a human immunoglobulin (see, e.g., U.S. Patent 4,816,567 and U.S. Patent 4,816,397, both of which are incorporated herein by reference in their entireties). Humanized antibodies refer to antibodies derived from non-human species that have one or more complementarity determining regions (CDRs) derived from a non-human species and framework regions derived from a human immunoglobulin molecule (see U.S. Patent 5,585,089, incorporated herein by reference in its entirety). These chimeric and humanized monoclonal antibodies can be prepared using DNA recombination techniques well known in the art.

[0229] In the present application, antibodies can be mono-specific, bi-specific, tri-specific, or more multi-specific.

[0230] As used herein, the term "heavy chain variable region" is used interchangeably with "VH".

[0231] As used herein, the term "light chain variable region" is used interchangeably with "VL".

[0232] The term "CDR" refers to the hypervariable regions within the variable domain of an antibody that primarily contribute to antigen binding. One of the most commonly used definitions of the CDRs is provided by Kabat E.A. et al. (1991) Sequences of proteins of immunological interest. NIH Publication 91-3242). In addition, IMGT (Lefranc, 2003), Chothia (Al-Lazikani, 1997), Abm all provide CDR definition rules, which are well known to those skilled in the art.

[0233] In one aspect, "CDRs" are defined as the complementarity determining region amino acid sequences of an antigen binding protein. They are the hypervariable regions of immunoglobulin heavy and light chains. The variable portion of an immunoglobulin has three heavy and three light chain CDRs (or CDR regions). Thus, "CDRs" as used herein refers to all three heavy chain CDRs, all three light chain CDRs, all heavy and light chain CDRs, or at least two CDRs.

[0234] In the present specification, amino acid residues in variable domain sequences and variable domain regions in full-length antigen binding sequences (e.g., in antibody heavy chain sequences or antibody light chain sequences) are numbered according to the Chothia, AbM, and / or Kabat numbering scheme. Similarly, the terms "CDR", "CDRL1", "CDRL2", "CDRL3", "CDRH1", "CDRH2", "CDRH3" used in the examples follow the Chothia, AbM, and / or Kabat numbering scheme. For more information, see Kabat et al., Sequences of Proteins of Immunological Interest, 4th Ed. U.S. Department of Health and Human Services, National Institutes of Health (1987) and Chothia et al. (1989) Nature 342:877-883. Other numbering schemes for CDR sequences available to one of skill in the art include the "AbM" (University of Bath) and "contact" (University College London) methods.

[0235] The structure and protein folding of an antigen binding protein can mean that other residues are considered part of the CDR sequence, as will be understood by one of skill in the art.

[0236] In the present application, the antibodies of the present application also include conservative variants thereof, which means that a polypeptide is formed by replacing up to 10, preferably up to 8, more preferably up to 5, most preferably up to 3 amino acids of the amino acid sequence of the antibodies of the present application with amino acids of similar or similar properties. These conservative variant polypeptides are preferably generated by amino acid replacement according to Table A.

[0237] Table A

[0238] The present application also provides polynucleotide molecules encoding the above-mentioned antibodies or fragments thereof or fusion proteins thereof. The polynucleotides of the present application can be in the form of DNA or RNA. The DNA form includes cDNA, genomic DNA or artificially synthesized DNA. The DNA can be single-stranded or double-stranded. The DNA can be a coding strand or a non-coding strand.

[0239] Polynucleotides encoding the mature polypeptides of the present application include: a coding sequence encoding only the mature polypeptide; a coding sequence of the mature polypeptide and various additional coding sequences; a coding sequence of the mature polypeptide (and optional additional coding sequences) and non-coding sequences.

[0240] The term "polynucleotide encoding a polypeptide" can be a polynucleotide which includes a polynucleotide which codes for the polypeptide, and also a polynucleotide which further includes additional coding and / or non-coding sequences.

[0241] The present application also relates to polynucleotides which hybridize to the above-mentioned sequences and have at least 50%, preferably at least 70%, more preferably at least 80% identity between the two sequences. The present application particularly relates to polynucleotides which hybridize to the polynucleotides of the present application under stringent conditions. In the present application, "stringent conditions" means: (1) hybridization and washing under lower ionic strength and higher temperature, such as 0.2 x SSC, 0.1% SDS, 60°C; or (2) hybridization with the addition of a denaturant, such as 50% (v / v) formamide, 0.1% calf serum / 0.1% Ficoll, 42°C, etc.; or (3) hybridization occurs only when the identity between the two sequences is at least 90%, more preferably 95% or more. Furthermore, the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide.

[0242] The nucleotide full-length sequence of the antibody of the present application or a fragment thereof can be obtained by PCR amplification, recombination or artificial synthesis. One possible method is to synthesize the relevant sequence by artificial synthesis, especially when the length of the fragment is short. Generally, a long fragment can be obtained by first synthesizing a plurality of small fragments and then ligating them together. In addition, the coding sequence of the heavy chain can be fused with an expression tag (such as 6His) to form a fusion protein.

[0243] Once the relevant sequence is obtained, the relevant sequence can be obtained in large quantities by recombination. This is usually done by cloning it into a vector, then transferring it into cells, and then isolating the relevant sequence from the proliferated host cells by conventional methods. The biomolecules (nucleic acids, proteins, etc.) involved in the present application include biomolecules in isolated form.

[0244] At present, the DNA sequence encoding the protein (or fragment thereof, or derivative thereof) of the present application can be obtained entirely by chemical synthesis. The DNA sequence can then be introduced into various existing DNA molecules (or vectors, etc.) and cells known in the art. In addition, mutations can be introduced into the protein sequence of the present application by chemical synthesis.

[0245] The present application also relates to vectors comprising the appropriate DNA sequence described above and an appropriate promoter or control sequence. These vectors can be used to transform appropriate host cells to enable them to express the protein.

[0246] The host cell can be a prokaryotic cell, such as a bacterial cell, preferably a Bacillus subtilis cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell. Representative examples of which are: E. coli, Streptomyces; bacterial cells of Salmonella typhimurium; fungal cells such as yeast; insect cells such as Drosophila S2 or Sf9; animal cells such as CHO, COS7, 293 cells, etc.

[0247] Transformation of host cells with recombinant DNA can be performed using conventional techniques well known to those skilled in the art. When the host is a prokaryote, such as E. coli, the transformation of the host cell can be effected by the use of techniques such as calcium chloride precipitation, which are well known to those skilled in the art. Another method is the use of MgCl2. If necessary, the transformation can also be performed by electroporation. When the host is a eukaryote, the transformation of the host cell can be effected by the use of techniques such as calcium phosphate precipitation, conventional mechanical procedures such as microinjection, electroporation, lipofection, etc.

[0248] The resulting transformant can be cultured in conventional media using conventional procedures to express the polypeptide encoded by the gene of the application. The medium used to culture the host cell is selected to induce production of the polypeptide. After the host cell has been cultured to an appropriate cell density, the selected promoter is induced by the appropriate method, such as temperature shift or chemical induction. The cells are then cultured for an additional period.

[0249] The recombinant polypeptide in the above method can be expressed in the cell, on the cell membrane, or secreted outside the cell. If necessary, the recombinant protein can be isolated and purified by various separation methods using its physical, chemical and other properties. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to: conventional renaturation treatment, treatment with protein precipitants (salting-out method), centrifugation, osmotic lysis, ultra-treatment, ultra-centrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC) and other various liquid chromatography techniques and combinations of these methods.

[0250] The antibody of the application can be used alone or in combination or conjugation with a detectable marker (for diagnostic purposes), a PK (protein kinase) modification moiety or any combination of these.

[0251] Detectable markers for diagnostic purposes include, but are not limited to: fluorescent or luminescent markers, radioactive markers, MRI (magnetic resonance imaging) or CT (computerized tomography) contrast agents, or enzymes capable of producing detectable products.

[0252] Therapeutic agents that can be conjugated or coupled to the antibodies of the present application include, but are not limited to: 1. Radionuclides; 2. Biological toxins; 3. Cytokines such as IL-2 and the like; 4. Gold nanoparticles / nanorods; 5. Viral particles; 6. Liposomes; 7. Magnetic nanoparticles; 8. Drug-activating enzymes (e.g., DT-diaphorase (DTD) or benzyl-hydrolase-like protein (BPHL)); 9. Therapeutic agents (e.g., cisplatin) or any form of nanoparticles and the like.

[0253] Immunoconjugates and antibody-drug conjugates (ADCs)

[0254] The present application also provides immunoconjugates based on the antibodies of the present application. Typically, an immunoconjugate comprises an antibody moiety and a conjugated moiety conjugated to the antibody. The conjugated moiety can be a detectable label, or a therapeutically active drug.

[0255] In one embodiment, the immunoconjugate is an antibody-drug conjugate (ADC).

[0256] Typically, the antibody-drug conjugate comprises the antibody, and an effector molecule conjugated, and preferably chemically conjugated, to the antibody. The effector molecule is preferably a therapeutically active drug. Further, the effector molecule can be one or more of a toxin, a chemotherapeutic drug, a small molecule drug, or a radionuclide.

[0257] The antibody of the present application can be conjugated to the effector molecule via a conjugating agent. Examples of the conjugating agent can be any one or several of a non-selective conjugating agent, a conjugating agent utilizing carboxyl groups, a peptide chain, a conjugating agent utilizing disulfide bonds. The non-selective conjugating agent is a compound that allows the effector molecule and the antibody to form a covalent bond, such as glutaraldehyde and the like. The conjugating agent utilizing carboxyl groups can be any one or several of a conjugating agent of aconitic anhydride (e.g., aconitic anhydride), a conjugating agent of acylhydrazone (conjugation site: acylhydrazone).

[0258] Certain residues on the antibody (e.g., Cys or Lys, etc.) are used to attach a variety of functional groups, including imaging agents (e.g., chromophoric groups and fluorescent groups), diagnostic agents (e.g., MRI contrast agents and radioisotopes), stabilizing agents (e.g., ethylene glycol polymers), and therapeutic agents. The antibody can be conjugated to a functional agent to form an antibody-functional agent conjugate. The functional agent (e.g., a drug, a detection agent, a stabilizing agent) is conjugated (covalently linked) to the antibody. The functional agent can be linked to the antibody directly, or indirectly via a linker.

[0259] Antibodies can be conjugated to drugs to form antibody drug conjugates (ADCs). Typically, an ADC comprises a linker between the drug and the antibody. The linker can be a degradable linker or a non-degradable linker. Degradable linkers are typically susceptible to degradation in the intracellular environment, e.g., the linker is degraded at the target site, thereby releasing the drug from the antibody. Suitable degradable linkers include, for example, enzymatically degradable linkers, including peptide-based linkers that are degradable by intracellular proteases, e.g., lysosomal proteases or endosomal proteases, or saccharide linkers, e.g., glucuronide-containing linkers that are degradable by glucuronidases. Peptide-based linkers can include, for example, dipeptides, e.g., valine-citrulline, phenylalanine-lysine, or valine-alanine. Other suitable degradable linkers include, for example, pH-sensitive linkers (e.g., linkers that hydrolyze at a pH less than 5.5, such as hydrazone linkers) and linkers that are degradable under reducing conditions (e.g., disulfide linker). Non-degradable linkers typically release the drug under conditions in which the antibody is hydrolyzed by proteases.

[0260] The linker has a reactive functional group capable of reacting with certain amino acid residues prior to attachment to the antibody, and the attachment is achieved through the reactive functional group. Thiol-specific reactive functional groups are preferred and include, for example, maleimides, haloamides (e.g., iodo-, bromo-, or chloro-); haloesters (e.g., iodo-, bromo-, or chloro-); halo-methyl ketones (e.g., iodo-, bromo-, or chloro-), benzyl halides (e.g., iodo-, bromo-, or chloro-); vinyl sulfones, pyridyl disulfides; mercury derivatives such as 3,6-bis-(mercurymethyl) dioxane, and counterions are acetate, chloride, or nitrate; and polymethylene dimethylsulfide sulfonate. The linker can include, for example, a maleimide attached to the antibody through a thio-butyrimide.

[0261] The drug can be any cytotoxic, cytostatic, or immunosuppressive drug. In embodiments, the linker attaches the antibody to the drug, and the drug has a functional group that can bond to the linker. For example, the drug can have an amino, carboxyl, thiol, hydroxyl, or keto group that can bond to the linker. In cases where the drug is directly attached to the linker, the drug has a reactive functional group prior to attachment to the linker.

[0262] Useful drug classes include, for example, anti-tubulin drugs, DNA minor groove binding agents, DNA replication inhibitors, alkylating agents, antibiotics, folic acid antagonists, antimetabolites, chemotherapeutic sensitizers, topoisomerase inhibitors, vinca alkaloids, and the like. In the present application, the drug-linker can be used to form the ADC in a single step. In other embodiments, a bifunctional linker compound can be used to form the ADC in a two- or multi-step process. For example, a cysteine residue is reacted with a reactive moiety of the linker in a first step, and in a subsequent step, a functional group on the linker is reacted with the drug, thereby forming the ADC.

[0263] In general, the functional group on the linker is selected to facilitate specific reaction with a suitable reactive group on the drug moiety. As a non-limiting example, an azide-based moiety can be used to specifically react with a reactive alkyne group on the drug moiety. The drug is covalently bound to the linker via a 1,3-dipolar cycloaddition between the azide and alkyne. Other useful functional groups include, for example, ketones and aldehydes (suitable for reaction with hydrazides and alkoxy amines), phosphines (suitable for reaction with azides); isocyanates and isothiocyanates (suitable for reaction with amines and alcohols); and activated esters, such as N-hydroxysuccinimidyl esters (suitable for reaction with amines and alcohols). These and other ligation strategies, such as those described in Bioconjugate Techniques, 2ndEdition (Elsevier), are well known to those skilled in the art. Those skilled in the art will appreciate that for selective reaction of the drug moiety and linker, when a complementary pair of reactive functional groups is selected, each member of the complementary pair can be used on either the linker or the drug.

[0264] Pharmaceutical compositions

[0265] The present application also provides a composition. Preferably, the composition is a pharmaceutical composition, which comprises the above-mentioned antibody or active fragment thereof or fusion protein thereof, and a pharmaceutically acceptable carrier. In general, these substances can be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, wherein the pH is generally about 5-8, preferably the pH is about 6-8, although the pH value can vary depending on the nature of the substance to be formulated and the condition to be treated. The formulated pharmaceutical composition can be administered by conventional routes, including but not limited to intraperitoneal, intravenous, or local administration.

[0266] The pharmaceutical composition of the present application contains a safe and effective amount (e.g., 0.001-99 wt%, preferably 0.01-90 wt%, more preferably 0.1-80 wt%) of the above-mentioned antibody (or conjugate thereof) of the present application and a pharmaceutically acceptable carrier or excipient. Such carriers include, but are not limited to, saline, buffers, dextrose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical preparation should be matched with the administration method. The pharmaceutical composition of the present application can be prepared in the form of a needle, for example, by a conventional method using physiological saline or an aqueous solution containing dextrose and other adjuvants. The pharmaceutical composition such as a needle, a solution is preferably manufactured under sterile conditions. The amount of the active ingredient to be administered is a therapeutically effective amount, for example, about 10 μg / kg body weight to about 50 mg / kg body weight per day. In addition, the polypeptide of the present application can be used with other therapeutic agents.

[0267] When the pharmaceutical composition is used, a safe and effective amount of the antibody or immunoconjugate thereof of the present application is administered to a mammal, wherein the safe and effective amount is usually at least about 10 μg / kg body weight, and in most cases, not more than about 50 mg / kg body weight, preferably the dose is about 10 μg / kg body weight to about 10 mg / kg body weight. Of course, the specific dose should also take into account the administration route, the patient's health condition, etc., which are within the skill of a skilled physician.

[0268] Detection use and kit

[0269] The antibody of the present application can be used for detection applications, for example, for detecting a sample to provide diagnostic information.

[0270] In the present application, the sample (specimen) used includes cells, tissue samples, and biopsy specimens. The term "biopsy" used in the present application should include all kinds of biopsies known to those skilled in the art. Therefore, the biopsy used in the present application can include, for example, a tissue sample prepared by an endoscopic method or a puncture or needle biopsy of an organ.

[0271] The sample used in the present application includes a fixed or preserved cell or tissue sample.

[0272] The present application also provides a kit containing the antibody (or fragment thereof) of the present application, and in a preferred embodiment of the present application, the kit further includes a container, instructions for use, a buffer, etc. In a preferred embodiment, the antibody of the present application can be immobilized on a detection plate.

[0273] The main advantages of the present application include, but are not limited to:

[0274] (1) The antibody of the present application specifically binds to human CDH17, and does not bind to its homologous proteins and other non-target related proteins.

[0275] (2) The antibody of the present application has excellent affinity for CDH17 protein and tumor cells expressing CDH17;

[0276] (3) The antibody of the present application has species cross-binding activity for human, rat and monkey CDH17 proteins, and is suitable for preclinical safety animal studies.

[0277] (4) The antibody of the present application can mediate CDH17 endocytosis, which is helpful for the development of drugs such as ADC.

[0278] (5) The antibody of the present application has good thermal stability, low pH stability and freeze-thaw stability, and is suitable for drug formulation development.

[0279] The present application will be further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present application and not to limit the scope of the present application. The experimental methods in the following examples, if not specified, are generally carried out under conventional conditions, for example, the conditions described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or the conditions recommended by the manufacturer. Unless otherwise specified, percentages and parts are weight percentages and weight parts.

[0280] Example 1 Construction of CDH17 overexpression cell line and protein expression

[0281] 1.1 Construction of HEK293T cells overexpressing CDH17 protein on the surface

[0282] The amino acid sequence of human CDH17 was obtained by database query, and the full-length protein code Q12864 and its extracellular segment domains EC1-EC7 single deletion proteins were constructed into a lentivirus vector according to the protein database of Uniprot, and HEK293T cells were infected with lentivirus to obtain HEK293T-hCDH17, HEK293T-hCDH17, and HEK293T-h.CDH17-△EC1-7 stable cell lines. The positive rate of target expression was determined by FACS detection, which was used for subsequent screening and identification. The results are shown in Figure 1, HEK293T-hCDH17 has high expression of hCDH17, and the positive rate is 99%, which can be used for mouse immunization and antibody evaluation in the later stage; as shown in Table 1, the positive rates of HEK293T-h.CDH17-△EC1-7 cell lines are between 40% and 100%, which can be used for later domain evaluation.

[0283] Table 1. Results of positive rate detection of HEK293-hCDH17 domain deletion cell lines

[0284] 1.2 Expression of proteins

[0285] The genes of protein ectodomain including human CDH17 protein ectodomain (23-787 AA), Cynomolgus CDH17 protein ectodomain (XP_005563762.1) (23-787 AA) and hCDH6 protein ectodomain (P55285) (54-615 AA) were synthesized, and the ectodomain of hCDH7 gene (Yiqi God, HG22474-UT) (28-607 AA) and hCDH20 gene (Yiqi God, HG25413-UT) (60-619 AA) were cloned into mammalian cell expression vectors, respectively, expressed and purified in 293E cells, and used in subsequent experiments.

[0286] 1.3 Purification of proteins

[0287] Purification of mFC and hFc proteins: The cell expression supernatant sample was centrifuged at high speed to remove impurities. The supernatant of recombinant protein containing Fc, chimeric antibody expression supernatant and hybridoma expression supernatant was purified by Protein A column. The supernatant was loaded onto the column at a certain flow rate, and the column was washed with PBS until the A280 reading decreased to the baseline. The target protein was eluted with 100 mM sodium acetate at pH 3.5, neutralized with 2M Tris-HCl at pH 8.0, and concentrated after elution. The sample was divided into PBS ready-to-use. HIS-tag protein purification: The same method as above was used for self-expression. The nickel column (brand: Biaogelong, catalog number: AA0051) was used to elute the sample with a gradient of 20-500 mM imidazole. After PBS ultrafiltration replacement, the sample was divided into PBS ready-to-use.

[0288] Example 2 Preparation of anti-human CDH17 antibody

[0289] Three wild type mouse strains were immunized with human CDH17-mFc protein, h.CDH17 plasmid and HEK293T-hCDH17 cells and used to generate hybridomas. Protein was used to immunize 6 mice by subcutaneous injection with adjuvant (Freund’s, CpG) in multiple sites; plasmid was used to immunize 6 mice by tail vein injection with the addition of helper plasmid, CpG. 3-5 days before fusion, the mice received a boost of 150 million HEK293 cells expressing human CDH17 in two doses. ELISA was used to screen mouse serum for mouse IgG antibodies specific for human CDH17. The immunized mice with positive serum were sacrificed and the spleen and lymph nodes were used as a source of antibody producing cells, which were electrofused and transferred to a humidified 37°C incubator with 5% CO2 and 95% air. After 6 days of growth, the supernatant from each well was assayed for antibodies reactive with CDH17 by ELISA and FACS assay. A total of 73 master clones with high affinity to human CDH17 on cell membrane and low non-specific binding were obtained from three fusions. After subcloning, 72 monoclonal antibodies were selected, purified as described in Example 1.3, and murine antibodies were obtained.

[0290] Example 3 Evaluation of Anti-human CDH17 Murine Antibodies

[0291] 3.1 Evaluation of the affinity of anti-human CDH17 murine monoclonal antibodies by ELISA binding to human CDH17-hFc protein. Evaluation of the species cross-reactivity of anti-human CDH17 murine monoclonal antibodies by ELISA binding to monkey CDH17-hFc protein and rat CDH17-his protein. Evaluation of the non-specific binding of anti-human CDH17 murine monoclonal antibodies to human CDH16 by ELISA binding to human CDH16-his protein.

[0292] The specific experimental steps are as follows: The antigen protein was diluted with CBS coating solution to 1-2 ug / mL, 100 ng / well was added to the enzyme-labeled plate, and incubated at 4°C overnight; the coating solution was removed, and 300 uL / well PBS was washed once, 100 uL / well blocking solution was added, and incubated at 37°C for 2 hours; the murine monoclonal antibody was gradient diluted with PBS (2% BSA), starting at 10 ug / ml, 3-fold dilution, 11 concentration points, 100 uL of the diluted solution was added to the plate, and incubated at 37°C for 2 hours; 300 uL / well PBST was washed three times, 100 uL / well 1:10000 anti-mouse HRP secondary antibody was added, and incubated at 37°C for 1 hour; 300 uL / well PBST was washed five times, then 100 uL of TMB substrate was added to each well, and color developed for 5-20 minutes; 50 uL of 2N HCl was added to each well to stop the reaction, and the OD450nM absorbance value was read on an enzyme-labeled instrument, and the EC50 value was calculated by the four-parameter method.

[0293] 3.2 Evaluation of the binding of anti-human CDH17 murine monoclonal antibodies to tumor cells by FACS. NCI-H716 cells were collected by centrifugation at 500 g for 3 min, and resuspended in PBS (containing 1% BSA). The cells were seeded into a 96-well plate at 3 x 10 5 cells / well, 100 μL of the gradient-diluted test antibody was added to each well, and the mixture was incubated at 4°C for 1 h. The cells were resuspended with 200 μL of pre-cooled PBS, centrifuged at 500 g at 3°C for 5 min, and the supernatant was removed twice. 1 μL of anti-mouse fluorescent secondary antibody (BioLegend, 405307) was added to each well, and the mixture was incubated at 4°C for 1 h. The cells were resuspended with 200 μL of pre-cooled PBS, centrifuged at 500 g at 3°C for 5 min, and the supernatant was removed twice. The cells were resuspended with 100 μL of pre-cooled PBS, and the mixture was subjected to flow cytometry. The higher the signal, the stronger the binding activity of the antibody to the protein on the cell surface. The EC50 value was calculated from the signal value.

[0294] 3.3 Evaluation of the binding of anti-human CDH17 murine monoclonal antibodies to domain-deleted cell lines by FACS. The HEK293T-h.CDH17-△EC1-7 cells were collected by trypsin digestion and centrifugation at 500 g for 3 min, washed three times with pre-cooled PBS, and resuspended in PBS (containing 1% BSA). The cells were seeded into a 96-well plate at 3 x 10 5 cells / well, 100 μL of the gradient-diluted test antibody was added to each well, and the mixture was incubated at 4°C for 1 h. The cells were resuspended with 200 μL of pre-cooled PBS, centrifuged at 500 g at 3°C for 5 min, and the supernatant was removed twice. 1 μL of anti-mouse fluorescent secondary antibody (BioLegend, 405307) was added to each well, and the mixture was incubated at 4°C for 1 h. The cells were resuspended with 200 μL of pre-cooled PBS, centrifuged at 500 g at 3°C for 5 min, and the supernatant was removed twice. The cells were resuspended with 100 μL of pre-cooled PBS, and the mixture was subjected to flow cytometry. The higher the signal, the stronger the binding activity of the antibody to the protein on the cell surface. The EC50 value was calculated from the signal value.

[0295] A total of about 70 murine antibodies were evaluated, and the optimal clones 26H1A8 and 101B4H10 with high NCI-H716 affinity and no non-specific binding to h.CDH16 were selected for antibody variable region sequencing. The evaluation data of the murine antibodies 26H1A8 and 101B4H10 are shown in Table 2.

[0296] The results are shown in Table 2. The monoclonal antibody 26H1A8 has human, murine, and monkey CDH17 species cross-binding activity, and binds to the EC1 region of the domain. The monoclonal antibody 101B4H10 has human and monkey species cross-binding activity, and binds to the EC3 region of the domain. Both monoclonal antibodies do not bind to hCDH16, indicating good specificity.

[0297] Table 2. Evaluation results of anti-human CDH17 murine antibodies

[0298] Example 4 Sequencing of hybridomas of anti-human CDH17 antibodies

[0299] The 26H1A8 and 101B4H10 hybridoma cells were cultured to the logarithmic growth phase, and then the hybridoma cells were counted, 10-100 cells were taken, the cells were lysed using a Single Cell Full Length mRNA-Amplification Kit (Vazyme, N712), and cDNA was obtained by reverse transcription. Then the upstream primers were designed by the adapter on 5'RACE, the downstream primers were designed by the constant region, and the complete light and heavy chain V region fragments were obtained by direct amplification primers for PCR amplification, and then the PCR products were directly or after TA connection for sequencing (Beijing Qikexing). After multiple sequence alignment, the heavy chain variable region sequence (SEQ ID NO: 1) and the light chain variable region sequence (SEQ ID NO: 2) of the murine antibody 26H1A8 were finally confirmed; the heavy chain variable region sequence (SEQ ID NO: 5) and the light chain variable region sequence (SEQ ID NO: 6) of the murine antibody 101B4H10 were finally confirmed.

[0300] Example 5 Humanization of anti-human CDH17 murine antibodies

[0301] The antibody light chain variable region sequence (SEQ ID NO: 2) and the heavy chain variable region sequence (SEQ ID NO: 1) of 26H1A8 were aligned with the sequences in the human Germline database, and the best humanization template was selected by comprehensively considering the similarity of Germline V gene and J gene, the matching degree of CDR segment structure and conformation, the Germline expression and stability, the Germline immunogenicity and other factors. The human Germline template IGHV1-46*01 was selected for the antibody heavy chain, and the human Germline template IGKV1-39*01 was selected for the light chain.

[0302] The antibody light chain variable region sequence (SEQ ID NO: 6) and the heavy chain variable region sequence (SEQ ID NO: 5) of 101B4H10 were aligned with the sequences in the human Germline database, and the best humanization template was selected by comprehensively considering the similarity of Germline V gene and J gene, the matching degree of CDR segment structure and conformation, the Germline expression and stability, the Germline immunogenicity and other factors. The human Germline template IGHV1-69*17 was selected for the antibody heavy chain, and the human Germline template IGKV1-12*01 was selected for the light chain.

[0303] After the template is determined, the CDR of the mouse-derived antibody is grafted onto the corresponding human-derived template, and on this basis, the FR amino acids in the mouse-derived antibody that can maintain the original conformation are calculated by structure analysis to perform back mutation, so as to maintain the affinity. By further simulating the structure before and after humanization, the number of back mutation amino acids is fine-tuned to achieve optimal binding activity, low immunogenicity and high stability. The detailed sequence list of anti-human CDH17 humanized antibodies 26H1A8-hz and 101B4H10-hz is shown in Table 3.

[0304] Table 3 Sequence list of anti-human CDH17 humanized antibodies

[0305] Example 6 Preparation of anti-human CDH17 humanized antibodies

[0306] The heavy chain and light chain variable region amino acid sequences of the anti-human CDH17 humanized monoclonal antibodies 26H1A8-hz, 101B4H10-hz, reference antibody 1 (referred to as BM, the sequence is from patent application: CN 110582513 A, SEQ ID NO: 116 and SEQ ID NO: 117) and reference antibody 2 (referred to as BM-2, the sequence is from patent application: WO2023107558A1, SEQ ID NO: 47 and SEQ ID NO: 48) are added with the heavy chain constant region (SEQ ID NO: 42) and the light chain constant region (SEQ ID NO: 41) respectively to submit to universal biological for codon optimization and gene synthesis, and the expression and purification method is referred to in Example 1.

[0307] Example 7 Quality detection of anti-human CDH17 humanized antibodies

[0308] 7.1 Purity detection of humanized antibodies

[0309] The purity of the humanized antibodies is identified by SEC, and the detection method is as follows:

[0310] Instrument: Waters Alliance e2695 HPLC;

[0311] Column: Thermo MabPac SEC-1, 5um, 7.8*300mm;

[0312] Mobile phase: 61mmol / L Na2HPO4, 39mmol / L NaH2PO4, 200mmol / L NaCl, 5% IPA;

[0313] Instrument parameters: sample chamber temperature: 8℃; column temperature: 30℃; flow rate: 0.5ml / min; sample amount: 20ug; detection wavelength: 280nm; isocratic operation: 30min.

[0314] 7.2 Hydrophobicity detection of humanized antibodies

[0315] Hydrophobicity of humanized antibodies was detected by HIC, the detection method was as follows: hydrophobicity detection was performed on Agilent HPLC device using TOSOH Tskgel Buty-NPR (2.5), 4.6*100) of TOSOH Corporation, mobile phase A was 1.5M (NH4)2SO4, mobile phase B was 25mM Na2HPO4 (pH = 7.0) + 25% IPA. The instrument parameters were set as follows: sample chamber temperature: 8°C, column temperature: 30°C, flow rate: 0.5 mL / min, detection wavelength: 280 nm. The sample to be tested was diluted with mobile phase A to a final concentration of 1 mg / mL, and 20 uL was injected for gradient elution. The elution gradient was as follows:

[0316] The detection results are shown in Table 4, SEC of 26H1A8-hz and 101B4H10-hz is >95%, indicating good purity properties; 26H1A8-hz and 101B4H10-hz are weakly combined with the hydrophobic chromatographic column, and the HIC retention time is short, and the hydrophilicity is good.

[0317] Table 4. Quality detection results of anti-human CDH17 humanized antibodies

[0318] Example 8 Tm value determination of anti-human CDH17 humanized antibodies

[0319] The Tm value of humanized antibodies was determined by DSF to reflect the thermal stability of the antibodies. The experimental steps were as follows: the antibody sample to be tested was diluted with PBS to 1 mg / mL; the dye SYPRO Orange dye (Thermo #56651) was diluted with ddH2O to 40x; the reaction system: sample 12.5 uL + 40x dye 2.5 uL + ddH2O 5 uL; film sealing, transient centrifugation; Q-PCR detection, Q-PCR parameter setting: Target (ROX), program (25°C, 3 min; 1% rate, 95°C; 95°C, 2 min). The experimental results showed that the Tm of 26H1A8-hz and 101B4H10-hz humanized antibodies was 69.2°C and 67.2°C respectively, indicating that they had good thermal stability.

[0320] Example 9 Affinity evaluation of anti-human CDH17 humanized antibodies

[0321] The affinity of anti-human CDH17 humanized antibodies was evaluated by ELISA with hCDH17-His or hCDH17-mFc protein. hCDH17-His or hCDH17-mF was diluted to 100 ng / well with CBS coating solution, 4°C overnight; remove the coating solution, add 100 uL / well blocking solution, 37°C for 2 hours; dilute the antibody to be tested with PBS (containing 2% BSA), 50 ug / ml start, 3 times dilution, 12 concentration points, add 100 uL / well to the enzyme-labeled plate, 37°C for 2 hours; remove the supernatant, wash three times with 300 uL / well PBST, add 100 ul / 10000-1:20000 anti-human HRP secondary antibody (Jackson Immuno Research, 109-035-088) per well, 37°C for 1 hour; remove the secondary antibody, wash five times with 300 uL / well PBST, then add 100 uL TMB substrate per well, color for 5-20 minutes; add 50 uL 2N HCl per well to stop the reaction, and read the OD450nM absorbance value with an MD enzyme-labeled instrument, and calculate the EC50 value by the four-parameter method.

[0322] The experimental results are shown in Figures 2A and 2B and Table 5, and the anti-human CDH17 humanized antibody 101B4H10-hz and 26H1A8-hz are comparable to the control antibody BM in terms of affinity to hCDH17-his or hCDH17-mFc, but better than the control antibody BM-2.

[0323] Table 5. Anti-CDH17 humanized antibody protein affinity test results

[0324] Example 10 Dynamic affinity evaluation of anti-human CDH17 humanized antibodies

[0325] To detect the dynamic affinity of anti-human CDH17 humanized antibodies, the Fortebio device was used to detect the binding rate and dissociation rate of the antibodies. 26H1A8-hz and 101B4H10-hz were diluted to 5 ug / mL with PBST, and the antibody molecules were captured by ProA biosensor chip; hCDH17-his antigen (described in Example 1) was diluted with PBST starting from 1800 nM, 2 times dilution, 5 concentration points, and 0 concentration point was set as the analyte; set the program, put in the sensor plate and sample plate, and detect the reaction signal in real time to obtain the binding and dissociation curves. After each test cycle, the biosensor chip was cleaned and regenerated with 20 mM glycine solution (pH 1.7); the data was fitted with a 1:1 model to obtain the affinity value.

[0326] The experimental results are shown in Table 6. 26H1A8-hz and 101B4H10-hz showed high affinity to hCDH17-his, E-08 and E-09, respectively.

[0327] Table 6. Results of dynamic affinity detection of anti-CDH17 humanized antibodies and hCDH17-his

[0328] Example 11 Evaluation of species affinity of anti-human CDH17 humanized antibodies

[0329] The species cross-reactivity of anti-human CDH17 humanized monoclonal antibodies was evaluated by ELISA with monkey CDH17-His or rat CDH17-his protein. The specific experimental method is shown in Example 9.

[0330] The results are shown in FIG. 3 and Table 7. Anti-human CDH17 humanized antibodies 26H1A8-hz and 101B4H10-hz showed good monkey cross-reactivity with the control antibody BM, while the monkey cross-reactivity of the control antibody BM-2 was slightly weaker. The results are shown in FIG. 4 and Table 7. Anti-human CDH17 humanized antibodies 26H1A8-hz and the control antibody BM2 showed good rat cross-reactivity, while 101B4H10-hz and the control antibody BM showed no rat cross-reactivity.

[0331] Table 7. Results of protein affinity detection of anti-CDH17 humanized antibodies

[0332] Example 12 Evaluation of specificity of anti-human CDH17 humanized antibodies

[0333] The non-specific binding of the anti-human CDH17 humanized monoclonal antibody to the homologous proteins hCDH6-his (see Example 1 for details), hCDH7-his (see Example 1 for details), hCDH9-his (ACRO, CA9-H52H6), hCDH10-his (ACRO, CA0-H52H5), hCDH12-his (Yiqi God, 10317-H08H), hCDH16-his (Yiqi God, 10915-H08H), and hCDH20-his (see Example 1 for details) was evaluated by ELISA. The specific experimental method is as follows: the homologous protein antigen was diluted to 1 ug / mL with CBS coating solution, 100 ul / well was added to the enzyme-labeled plate, and it was incubated at 4°C overnight; 300 uL / well PBS was washed once, 100 uL / well blocking solution was added, and it was incubated at 37°C for 2 hours; the humanized monoclonal antibody was diluted with PBS (2% BSA) to a concentration of 20 ug / ml, 5 ug / ml, and 1 ug / ml, and 100 uL of the diluted solution was added to the plate and incubated at 37°C for 2 hours; the supernatant was removed, each well was washed with 300 uL of PBST for three times, 100 uL of 1:10,000-1:20,000 anti-human HRP secondary antibody was added to each well, and it was incubated at 37°C for 1 hour; the secondary antibody was removed, each well was washed with 300 uL of PBST for five times, then 100 uL of TMB substrate was added to each well, and it was colored at room temperature for 5-20 minutes; 50 uL of 2N HCl was added to each well to stop the reaction, and the OD450nM reading was read by the enzyme-labeled instrument.

[0334] The experimental results show that the anti-human CDH17 humanized antibodies 26H1A8-hz and 101B4H10-hz do not bind to the homologous proteins hCDH6, hCDH7, hCDH9, hCDH10, hCDH12, hCDH16, and hCDH20 (data not shown), and have excellent specificity.

[0335] Example 13 Evaluation of the tumor affinity of the anti-human CDH17 humanized antibody

[0336] To detect the affinity of anti-human CDH17 humanized antibodies 26H1A8-hz and 101B4H10-hz to different expression abundance cells, FACS was used to detect the binding to HEK293T-h.CDH17 cells, NCI-H716, LS1034, and DLD-1 and MKN45 tumor cells. The CDH17 high expression NCI-H716 tumor cells were collected by centrifugation at 500g for 3 minutes; the HEK293T-h.CDH17 cells, CDH17 medium expression LS1034, DLD-1, and MKN45 tumor cells were collected by EDTA digestion and centrifugation at 500g for 3 minutes, and the cells were resuspended with FACS buffer (PBS containing 1% BSA), and the tested antibodies were added in gradient dilution of FACS buffer, incubated at 4°C for 1 hour, washed five times with pre-cooled PBS, and then 1 μL of anti-human fluorescent secondary antibody (BioLegend, item number 410712) was added to each well, incubated at 4°C for 1 hour, resuspended after washing three times with pre-cooled PBS, and detected on a flow cytometer. The EC50 value was calculated by the fluorescence signal value.

[0337] The experimental results are shown in FIGS. 5 to 9 and Table 8: on HEK293T-h.CDH17 overexpression cells, 26H1A8-hz and 101B4H10-hz have similar affinity to BM and BM2; on MKN45 tumor cells, 26H1A8-hz and 101B4H10-hz have weaker affinity than BM, but stronger affinity than BM2; on LS1034 tumor cells, 26H1A8-hz and 101B4H10-hz have similar affinity to BM; on NCI-716 tumor cells, 26H1A8-hz and 101B4H10-hz have slightly weaker affinity than BM; and on DLD-1 tumor cells, 26H1A8-hz and 101B4H10-hz have stronger affinity than BM. Overall, 26H1A8-hz and 101B4H10-hz both have strong cell affinity.

[0338] Table 8. Anti-CDH17 humanized antibody cell affinity detection results

[0339] Example 14 Anti-human CDH17 humanized antibody binding domain detection

[0340] The binding domain of the anti-human CDH17 human monoclonal antibody was evaluated by FACS. The HEK293T-h.CDH17-ΔEC1-7 cells were collected by centrifugation, resuspended with PBS (containing 1% BSA), 3×10 5Cells were plated in 96-well V-bottom plates, and the diluted antibody was added. After incubation at 4°C for 1 h, the cells were washed three times with pre-cooled PBS, and 1 μL of anti-human secondary antibody was added to each well. After incubation at 4°C for 0.5 h, the cells were washed three times with pre-cooled PBS, resuspended, and detected by flow cytometry. The binding domain of the humanized monoclonal antibody to human CDH17 was determined.

[0341] The experimental results showed that 26H1A8-hz and 101B4H10-hz were bound to the EC1 and EC3 domains of human CDH17 protein, respectively.

[0342] Example 15 Evaluation of endocytosis activity of anti-human CDH17 humanized monoclonal antibodies

[0343] The endocytosis activity of the anti-human CDH17 humanized monoclonal antibodies was detected by FACS. The experimental procedure was as follows: HEK293T-h.CDH17 cells were collected and incubated with the antibody to be detected at 4°C for 60 min. After washing with PBS, a portion was incubated with a secondary antibody, and the fluorescence intensity was measured as “F0” after detection. The remaining cells incubated with the antibody to be detected were incubated at 37°C for 2 h. After incubation, the cells were washed with PBS and then incubated with a secondary antibody before detection. The fluorescence intensity was measured as “FX”.

[0344] The endocytosis rate at a certain time point was calculated as follows: [1-(FX sample to be detected-FX isotype control) / (F0 sample to be detected-F0 isotype control)]*100.

[0345] The experimental results showed that the endocytosis rates of the anti-human CDH17 humanized antibodies 26H1A8-hz, 101B4H10-hz, the control antibody BM, and the control antibody BM-2 were 32.80%, 34.68%, 14.51%, and 17.05%, respectively. The endocytosis activities of 26H1A8-hz and 101B4H10-hz were better.

[0346] Example 16 Evaluation of thermal stability, low pH stability, and freeze-thaw stability of anti-human CDH17 humanized antibodies

[0347] To evaluate the thermal stability, low pH stability, and freeze-thaw stability of the anti-human CDH17 humanized antibodies.

[0348] 1) Thermal stability: The anti-human CDH17 humanized antibody 26H1A8-hz and 101B4H10-hz samples were ultrafiltrated and replaced into PBS (pH = 7.4) at a concentration of 10 mg / mL. After filtration and sterilization, 0.5 mL was placed in each tube, and the samples were placed at 37°C for 0, 7, and 14 days, respectively. The SEC and CE purity of the samples were then detected by HPLC.

[0349] 2) Freeze-thaw stability: freeze-thaw 4 times, 8 times at -80°C, then detect the SEC purity of the sample by HPLC device;

[0350] 3) Low PH stability: replace the sample of 26H1A8-hz and 101B4H10-hz to be detected into NaAc-Ac (PH = about 3.3) by ultrafiltration, save for 4h, then neutralize with 2M Tris, PH is about 7.0, then detect the SEC and CE purity of the sample by HPLC device.

[0351] The experimental results are shown in Table 9 below, and 26H1A8-hz and 101B4H10-hz have good stability after high temperature acceleration, repeated freeze-thaw and low PH treatment.

[0352] Table 9. Stability study of anti-CDH17 humanized antibody

[0353] Example 17 Evaluation of antibody-dependent cell-mediated cytotoxicity (ADCC) activity of anti-human CDH17 humanized antibody

[0354] The ADCC activity of anti-human CDH17 humanized monoclonal antibody was detected by luciferin reporter system. The experimental steps are as follows: collect HEK293T-h.CDH17 cells by digestion and centrifugation, resuspend the cells in DMEM+1% FBS, and then plate 50000 cells per well; collect Jurkat-NFAT-CD16a cells by centrifugation, resuspend the cells in RPMI1640+1% FBS, and then plate 100000 cells per well; add gradient-diluted antibody to be detected, with a final concentration of 50ug / ml, 4-fold dilution, and incubate at 37°C for 5 hours; add 50uL Bio-Lite-Luciferase Assay Buffer (Nanjing Novizhan, DD1201-02) to each well, and read by enzyme marker (MD, i3x). CCR8 humanized antibody and HEK293T-CCR8 cells are used as system positive control (reference patent application: WO2024008110A1).

[0355] The experimental results are shown in Figure 10, and BM, 26H1A8-hz and 101B4H10-hz have no obvious ADCC activity.

[0356] Example 18 Evaluation of antibody-dependent cell-mediated phagocytosis (ADCP) activity of anti-human CDH17 humanized antibody

[0357] HEK293T-h.CDH17 cells were collected by trypsin digestion and centrifugation, 50000 cells were plated per well after resuspending the cells with DEME + 1% FBS; Jurkat-NFAT-H131 cells were collected by centrifugation, 100000 cells were plated per well after resuspending the cells with RPMI1640 + 1% FBS; the gradient-diluted antibody to be detected was added, the final concentration was 50ug / ml, 3-fold dilution, 37℃ incubation for 5 hours; 50uL Bio-Lite-Luciferase Assay Buffer was added per well, and the reading was taken by an enzyme marker (MD, i3x).

[0358] The experimental results are shown in Figure 11, and BM, 26H1A8-hz and 101B4H10-hz all have no obvious ADCP activity.

[0359] All documents mentioned in the present application are incorporated herein by reference as if each document were individually incorporated. In addition, it is to be understood that the application can be carried out by specifically different embodiments and by equivalent means without departing from the scope of the appended claims. Accordingly, other embodiments are within the scope of the following claims.

[0360] SEQUENCE LISTING

Claims

1. An anti-CDH17 antibody or antigen-binding fragment thereof, comprising a heavy chain variable region and a light chain variable region, wherein, The heavy chain variable region and the light chain variable region have six complementarity determining regions (CDRs) selected from the group consisting of: (1a) three complementarity determining regions of a heavy chain variable region VH-CDRs and three complementarity determining regions of a light chain variable region VL-CDRs defined based on Chothia rules: a VH-CDR1 as set forth in SEQ ID NO: 9, a VH-CDR2 as set forth in SEQ ID NO: 10, a VH-CDR3 as set forth in SEQ ID NO: 11, a VL-CDR1 as set forth in SEQ ID NO: 19, a VL-CDR2 as set forth in SEQ ID NO: 20, a VL-CDR3 as set forth in SEQ ID NO: 21; or (1b) three complementarity determining regions of a heavy chain variable region VH-CDRs and three complementarity determining regions of a light chain variable region VL-CDRs defined based on Abm rules: a VH-CDR1 as set forth in SEQ ID NO: 12, a VH-CDR2 as set forth in SEQ ID NO: 13, a VH-CDR3 as set forth in SEQ ID NO: 11, a VL-CDR1 as set forth in SEQ ID NO: 19, a VL-CDR2 as set forth in SEQ ID NO: 20, a VL-CDR3 as set forth in SEQ ID NO: 21; or (1c) three complementarity determining regions of a heavy chain variable region VH-CDRs and three complementarity determining regions of a light chain variable region VL-CDRs defined based on Kabat rules: a VH-CDR1 as set forth in SEQ ID NO: 14, a VH-CDR2 as set forth in SEQ ID NO: 24 or 15, a VH-CDR3 as set forth in SEQ ID NO: 11, a VL-CDR1 as set forth in SEQ ID NO: 19, a VL-CDR2 as set forth in SEQ ID NO: 20, a VL-CDR3 as set forth in SEQ ID NO: 21; or (1d) three complementarity determining regions of a heavy chain variable region VH-CDRs and three complementarity determining regions of a light chain variable region VL-CDRs defined based on IMGT rules: a VH-CDR1 as set forth in SEQ ID NO: 16, a VH-CDR2 as set forth in SEQ ID NO: 17, a VH-CDR3 as set forth in SEQ ID NO: 18, a VL-CDR1 as set forth in SEQ ID NO: 22, a VL-CDR2 as set forth in SEQ ID NO: 23, a VL-CDR3 as set forth in SEQ ID NO: 21; or (2a) three complementarity determining regions of a heavy chain variable region VH-CDRs and three complementarity determining regions of a light chain variable region VL-CDRs defined based on Chothia rules: a VH-CDR1 as set forth in SEQ ID NO: 25, a VH-CDR2 as set forth in SEQ ID NO: 26, a VH-CDR3 as set forth in SEQ ID NO: 27, a VL-CDR1 as set forth in SEQ ID NO: 35, a VL-CDR2 as set forth in SEQ ID NO: 36, a VL-CDR3 as set forth in SEQ ID NO: 37; or a VL-CDR3 as set forth in SEQ ID NO: 37; or (2b) three complementarity determining regions of a heavy chain variable region VH-CDRs and three complementarity determining regions of a light chain variable region VL-CDRs defined based on the Abm convention: a VH-CDR1 as set forth in SEQ ID NO: 30, a VH-CDR2 as set forth in SEQ ID NO: 40 or 31, a VH-CDR3 as set forth in SEQ ID NO: 27, a VL-CDR1 as set forth in SEQ ID NO: 35, a VL-CDR2 as set forth in SEQ ID NO: 36, a VL-CDR3 as set forth in SEQ ID NO: 37; or (2c) three complementarity determining regions of a heavy chain variable region VH-CDRs and three complementarity determining regions of a light chain variable region VL-CDRs defined based on the Kabat convention: a VH-CDR1 as set forth in SEQ ID NO: 30, a VH-CDR2 as set forth in SEQ ID NO: 40 or 31, a VH-CDR3 as set forth in SEQ ID NO: 27, a VL-CDR1 as set forth in SEQ ID NO: 35, a VL-CDR2 as set forth in SEQ ID NO: 36, a VL-CDR3 as set forth in SEQ ID NO: 37; or (2d) three complementarity determining regions of a heavy chain variable region VH-CDRs and three complementarity determining regions of a light chain variable region VL-CDRs defined based on the IMGT convention: a VH-CDR1 as set forth in SEQ ID NO: 32, a VH-CDR2 as set forth in SEQ ID NO: 33, a VH-CDR3 as set forth in SEQ ID NO: 34, a VL-CDR1 as set forth in SEQ ID NO: 38, a VL-CDR2 as set forth in SEQ ID NO: 39, a VL-CDR3 as set forth in SEQ ID NO:

37.

2. The antibody or antigen-binding fragment thereof of claim 1, wherein, Any one of the amino acid sequences further comprises a derivative sequence in which at least one amino acid is optionally added, deleted, modified and / or substituted, and which is capable of retaining CDH17 binding affinity.

3. The antibody or antigen-binding fragment thereof of claim 2, wherein, The number of the added, deleted, modified and / or substituted amino acids is 1-3, preferably 1-2, more preferably 1.

4. The antibody or antigen-binding fragment thereof of claim 1, wherein, The antibody or antigen binding fragment thereof is humanized.

5. The antibody or antigen-binding fragment thereof of claim 1, wherein, The amino acid sequence of the heavy chain variable region is as set forth in SEQ ID NO: 1 or 3; and / or the amino acid sequence of the light chain variable region is as set forth in SEQ ID NO: 2 or 4.

6. The antibody or antigen-binding fragment thereof of claim 1, wherein, The amino acid sequence of the heavy chain variable region is as set forth in SEQ ID NO: 1; and the amino acid sequence of the light chain variable region is as set forth in SEQ ID NO:

2.

7. The antibody or antigen-binding fragment thereof of claim 1, wherein, The amino acid sequence of the heavy chain variable region is as set forth in SEQ ID NO: 3; and the amino acid sequence of the light chain variable region is as set forth in SEQ ID NO:

4. Preferably, the amino acid sequence of the heavy chain variable region is encoded by a nucleotide sequence as set forth in SEQ ID NO: 47; and the amino acid sequence of the light chain variable region is encoded by a nucleotide sequence as set forth in SEQ ID NO:

49.

8. The antibody or antigen-binding fragment thereof of claim 1, wherein, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 5 or 7; and / or the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 6 or 8.

9. The antibody or antigen-binding fragment thereof of claim 1, wherein, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 5; the amino acid sequence of the light chain variable region is shown in SEQ ID NO:

6.

10. The antibody or antigen-binding fragment thereof of claim 1, wherein, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 7; the amino acid sequence of the light chain variable region is shown in SEQ ID NO:

8. Preferably, the amino acid sequence of the heavy chain variable region is encoded by the nucleotide sequence shown in SEQ ID NO: 51; and the amino acid sequence of the light chain variable region is encoded by the nucleotide sequence shown in SEQ ID NO:

53.

11. The antibody or antigen-binding fragment thereof of claim 1, wherein, The heavy chain of the antibody or antigen-binding fragment thereof further comprises a heavy chain constant region.

12. The antibody or antigen-binding fragment thereof of claim 11, wherein, The heavy chain constant region is of human or murine origin.

13. The antibody or antigen-binding fragment thereof of claim 11, wherein, The amino acid sequence of the heavy chain constant region is shown in SEQ ID NO:

42.

14. The antibody or antigen-binding fragment thereof of claim 1, wherein, The light chain of the antibody or antigen-binding fragment thereof further comprises a light chain constant region.

15. The antibody or antigen-binding fragment thereof of claim 14, wherein, The light chain constant region is of human or murine origin.

16. The antibody or antigen-binding fragment thereof of claim 14, wherein, The amino acid sequence of the light chain constant region is shown in SEQ ID NO:

41.

17. The antibody or antigen-binding fragment thereof of claim 1, wherein the amino acid sequence of the heavy chain of the antibody is shown in SEQ ID NO: 43; and / or the amino acid sequence of the light chain of the antibody is shown in SEQ ID NO: 44; Preferably, the amino acid sequence of the heavy chain of the antibody is encoded by the nucleotide sequence shown in SEQ ID NO: 48; and / or the amino acid sequence of the light chain of the antibody is encoded by the nucleotide sequence shown in SEQ ID NO:

50.

18. The antibody or antigen-binding fragment thereof of claim 1, wherein, the amino acid sequence of the heavy chain of the antibody is shown in SEQ ID NO: 45; and / or the amino acid sequence of the light chain of the antibody is shown in SEQ ID NO: 46; Preferably, the amino acid sequence of the heavy chain of the antibody is encoded by the nucleotide sequence shown in SEQ ID NO: 52; and / or the amino acid sequence of the light chain of the antibody is encoded by the nucleotide sequence shown in SEQ ID NO:

54.

19. The antibody or antigen-binding fragment thereof of claim 1, wherein, The antibody or antigen-binding fragment thereof specifically binds to CDH17.

20. The antibody or antigen-binding fragment thereof of claim 19, wherein, The CDH17 is from human, rat or cynomolgus monkey.

21. The antibody or antigen-binding fragment thereof of claim 1, wherein, The antibody or antigen-binding fragment thereof specifically binds to the extracellular domain EC1 or EC3 of CDH17.

22. The antibody or antigen-binding fragment thereof of claim 1, wherein, The antibody is a bispecific antibody, or a single chain antibody (scFv).

23. The antibody or antigen-binding fragment thereof of claim 1, wherein The antibody is a monoclonal antibody.

24. The antibody or antigen-binding fragment thereof of claim 1, wherein, The antibody is a monospecific, bispecific, or trispecific antibody.

25. The antibody or antigen-binding fragment thereof of claim 1, wherein, The bispecific antibody comprises: (1) the anti-CDH17 antibody or antigen-binding fragment thereof; (2) an antibody or antigen-binding fragment thereof that binds to another target.

26. The antibody or antigen-binding fragment thereof of claim 25, wherein, the other target is selected from the group consisting of BCMA, GPRC5D, CD38, CD123, CD19, CD20, CD22, GPC3, TROP-2, Claudin 18.2, EGFR, HER2, HER3, CD3, CD28, 4-1BB, OX40, CD40, CD27, CD47, CTLA4, PD1, PDL1, DLL3, SEZ6, MUC-1, MUC-16, FRa, PSMA, ALPPL2, ALPP, CD228, LIV-1, B7-H1, B7-H3, B7-H6, B7-H7, B7-H4, TIGB6, STEAP1, and STEAP2.

27. A recombinant protein comprising: (i) an anti-CDH17 antibody or antigen-binding fragment thereof according to any one of claims 1-26; and (ii) optionally, a tag sequence to facilitate expression and / or purification.

28. The recombinant protein of claim 27, wherein, The tag sequence comprises a 6His tag.

29. The recombinant protein of claim 27, wherein, The recombinant protein (or polypeptide) comprises a fusion protein.

30. The recombinant protein of claim 27, wherein, The recombinant protein is a monomer, a dimer, or a multimer.

31. The recombinant protein of claim 27, wherein, The recombinant protein is a monospecific, bispecific, or trispecific antibody.

32. The recombinant protein of claim 27, wherein, The recombinant protein further comprises an additional fusion element (or fusion polypeptide fragment) fused to the element (i).

33. A CAR construct, wherein the antigen-binding region of the CAR construct comprises a scFv region that specifically binds to CDH17, and the scFv region has a heavy chain variable region and a light chain variable region of an antibody according to any one of claims 1-26.

34. A recombinant cell, wherein the cell expresses an exogenous CAR construct according to claim 33.

35. The recombinant cell of claim 34, wherein The recombinant cell is an immune cell or an IPS cell.

36. The recombinant cell of claim 34, wherein The immune cell is selected from the group consisting of an NK cell, a T cell.

37. The recombinant cell of claim 34, wherein The recombinant cell is from a human or a non-human mammal (e.g., a murine).

38. An immunoconjugate, wherein the immunoconjugate comprises: (a) an antibody moiety that is an anti-CDH17 antibody or antigen-binding fragment thereof according to any one of claims 1-26; and (b) a conjugated moiety conjugated to the antibody moiety, wherein the conjugated moiety is selected from the group consisting of a detectable label, a drug, a toxin, a cytokine, an enzyme, or a combination thereof.

39. The immunoconjugate of claim 38, wherein The immunoconjugate is an antibody drug conjugate.

40. The immunoconjugate of claim 38, wherein The antibody moiety is conjugated to the conjugated moiety via a chemical bond or a linker.

41. The immunoconjugate of claim 38, wherein The conjugated moiety is a chemical label or a biological label.

42. The immunoconjugate of claim 41, wherein The chemical label is an isotope, an immunotoxin, and / or a chemical drug.

43. The immunoconjugate of claim 41, wherein The biological label is biotin, avidin, or an enzyme label.

44. The immunoconjugate of claim 41, wherein The conjugated moiety is a drug or a toxin.

45. The immunoconjugate of claim 44, wherein the linker is of the formula: ###0002### wherein R is H, alkyl, aryl, or heteroaryl. 5 The drug is a cytotoxic drug.

46. The immunoconjugate of claim 44, wherein The drug is a nucleic acid drug.

47. The immunoconjugate of claim 38, wherein The toxin is selected from the group consisting of: auristatins (e.g., auristatin E, auristatin F, MMAE, and MMAF), aureusidin, maytansinoid, ricin, ricin A-chain, combrestatin, duocarmycin, dolastatin, doxorubicin, daunorubicin, paclitaxel, cisplatin, cc1065, ethidium bromide, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin, dihydroxy anthracin dione, actinomycin, diphtheria toxin, Pseudomonas exotoxin (PE) A, PE40, abrin, abrin A chain, modeccin A chain, alpha-sarcin, gelonin, mitogellin, retstrictocin, phenomycin, enomycin, curicin, phytolaccin, crotin, calicheamicin, Sapaonaria officinalis inhibitor, glucocorticoid, or a combination thereof.

48. The immunoconjugate of claim 38, wherein The toxin is a TOP inhibitor.

49. The immunoconjugate of claim 48, wherein The TOP inhibitor is selected from the group consisting of SN38 or a prodrug thereof, etoposide, tenoposide, daunorubicin, doxorubicin, idarubicin, mitoxantrone.

50. The immunoconjugate of claim 38, wherein The conjugation moiety is a detectable label.

51. The immunoconjugate of claim 50, wherein The detectable label comprises a radionuclide, the radionuclide comprising: (i) a diagnostic isotope selected from the group consisting of Tc-99m, Ga-68, F-18, I-123, I-125, I-131, In-111, Ga-67, Cu-64, Zr-89, C-11, Lu-177, Re-188, or a combination thereof; and / or (ii) a therapeutic isotope selected from the group consisting of Lu-177, Y-90, Ac-225, As-211, Bi-212, Bi-213, Cs-137, Cr-51, Co-60, Dy-165, Er-169, Fm-255, Au-198, Ho-166, I-125, I-131, Ir-192, Fe-59, Pb-212, Mo-99, Pd-103, P-32, K-42, Re-186, Re-188, Sm-153, Ra223, Ru-106, Na24, Sr89, Tb-149, Th-227, Xe-133 Yb-169, Yb-177, or a combination thereof.

52. The immunoconjugate of claim 38, wherein The conjugate is selected from the group consisting of a fluorescent or luminescent label, a radioactive label, an MRI (magnetic resonance imaging) or CT (computerized tomography) contrast agent, or an enzyme capable of producing a detectable product, a radionuclide, a biological toxin, a cytokine (such as IL-2, etc.), an antibody, an antibody Fc fragment, an antibody scFv fragment, a gold nanobody particle / nanobody rod, a viral particle, a liposome, a nanobody magnetic particle, a prodrug-activating enzyme (e.g., DT-diaphorase (DTD) or biphenyl hydrolase-like protein (BPHL)), a chemotherapeutic agent (e.g., cisplatin), or any form of nanobody particle, etc.

53. The immunoconjugate of claim 38, wherein The detection is in vivo or in vitro.

54. The immunoconjugate of claim 38, wherein The immunoconjugate is used for diagnosis and / or treatment of a tumor expressing CDH17 protein.

55. The immunoconjugate of claim 38, wherein The immunoconjugate has the following formula: wherein: nAb is an anti-CDH17 antibody or antigen-binding fragment thereof; LU is a linker (also known as a linking group); D is a drug; and subscript p is a value selected from 1-10.

56. The immunoconjugate of claim 55, wherein LU is selected from the group consisting of a maleimidocaproyl (MC), a maleimide (MAL), a succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) linker, and a linker comprising one or more of valine-citrulline (VC), valine-alanine (VA), glycine-glycine-phenylalanine-glycine (GGFG), alanine-alanine-alanine (AAA), p-aminobenzyloxy carbonyl (PAB), and polyethylene glycol (PEG) attached to the antibody moiety.

57. A pharmaceutical composition comprising: (i) an anti-CDH17 antibody or antigen-binding fragment thereof of any one of claims 1-26, a recombinant protein of any one of claims 27-32, a recombinant cell of any one of claims 34-37, or an immunoconjugate of any one of claims 38-56; (ii) a pharmaceutically acceptable carrier.

58. The pharmaceutical composition of claim 57, wherein, The pharmaceutical composition comprises a single drug, a compound drug, or a synergistic drug.

59. The pharmaceutical composition of claim 57, wherein, The pharmaceutical composition further comprises other biologically active substances, such as drugs for treating tumors.

60. The pharmaceutical composition of claim 57, wherein, The pharmaceutical composition is administered by subcutaneous injection, intradermal injection, intramuscular injection, intravenous injection, intraperitoneal injection, microneedle injection, oral administration, or oral and nasal cavity spraying and aerosol inhalation.

61. The pharmaceutical composition of claim 57, wherein, The pharmaceutical composition is in a liquid, solid, or gel state.

62. The pharmaceutical composition of claim 57, wherein, The pharmaceutical composition is in a liquid state.

63. The pharmaceutical composition of claim 57, wherein, The pharmaceutical composition is an injection.

64. Use of an active ingredient selected from the group consisting of an anti-CDH17 antibody or antigen-binding fragment thereof of any one of claims 1-26, a recombinant protein of any one of claims 27-32, a recombinant cell of any one of claims 34-37, or an immunoconjugate of any one of claims 38-56, or a combination thereof, for (a) preparing a detection reagent, a detection plate, or a kit; and / or (b) preparing a drug for preventing and / or treating a CDH17-related disease.

65. The use of claim 64, wherein the compound is administered in a daily dose of about 0.1 to about 100 mg / m2. The detection reagent, detection plate, or kit is used for: (1) detecting CDH17 protein in a sample; and / or (2) detecting endogenous CDH17 protein in a tumor cell or tissue; and / or (3) detecting a tumor cell expressing CDH17 protein.

66. The use of claim 64, wherein the compound is administered in a daily dose of about 0.1 to about 100 mg / kg. The detection types include, but are not limited to, flow detection, cellular immunofluorescence detection, enzyme-linked immunosorbent detection, immunoblot detection, etc.

67. The use of claim 64, wherein the compound is administered in a daily dose of about 0.01 to 100 mg / kg. The detection reagent, detection plate or kit is used for diagnosing CDH17 related diseases.

68. The use of claim 64, wherein the compound is ###0010### The drug is used for treating or preventing CDH17 high expression tumor, tumor metastasis, or tumor drug resistance.

69. The use of claim 64, wherein the composition is administered to the subject in an amount sufficient to reduce the subject's risk of developing a disease or disorder associated with the at least one genetic variant. The tumor drug resistance includes tumor immunotherapy drug resistance, tumor targeted therapy drug resistance, conventional tumor chemotherapy drug resistance, and radiotherapy insensitivity.

70. The use of claim 64, wherein the composition is administered to the subject in an amount sufficient to reduce the subject's risk of developing a disease or disorder associated with the at least one genetic variant. The CDH17 related disease is a tumor or cancer.

71. The use of claim 64, wherein the composition is administered to the subject in an amount sufficient to reduce the subject's risk of developing a disease or disorder associated with the at least one genetic variant. The CDH17 related disease includes tumor occurrence, growth and / or metastasis.

72. The use of claim 64, wherein, The cancer includes solid tumors and blood cancers.

73. The use as described in claim 64, characterized in that, The cancer is selected from the group consisting of small intestine cancer, colorectal cancer, gastric cancer, pancreatic cancer, esophageal cancer, ovarian cancer, lung cancer (such as lung adenocarcinoma and non-small cell lung cancer), breast cancer (such as triple negative breast cancer), malignant brain glioma, liver cancer, kidney cancer, bladder cancer, prostate cancer, endometrial cancer, cervical cancer, leukemia, bone marrow cancer, angiosarcoma, or a combination thereof.

74. A polynucleotide encoding a polypeptide selected from the group consisting of: The anti-CDH17 antibody or antigen-binding fragment thereof of any one of claims 1-26, the recombinant protein of any one of claims 27-32, or the CAR construct of claim 33.

75. The polynucleotide of claim 74, wherein The polynucleotide includes RNA, DNA or cDNA.

76. A vector comprising the polynucleotide of claim 74 or 75.

77. The vector of claim 76, wherein The vector includes bacterial plasmid, bacteriophage, yeast plasmid, plant cell virus, mammalian cell virus such as adenovirus, retrovirus, or other vectors.

78. A host cell comprising the vector of claim 76 or 77 or genome integrated with the polynucleotide of claim 74 or 75.

79. An in vitro method of detecting CDH17 in a sample, the method comprising the steps of: (1) in vitro, contacting the sample with the anti-CDH17 antibody or antigen-binding fragment thereof of any one of claims 1-26, the recombinant protein of any one of claims 27-32, or the immunoconjugate of any one of claims 38-56; (2) detecting whether an antigen-antibody complex is formed, wherein the formation of the complex indicates the presence of CDH17 in the sample.

80. The method of claim 79, wherein, The detection includes diagnostic or non-diagnostic.

81. A method of producing a recombinant polypeptide, the method comprising: (a) culturing the host cell of claim 78 under conditions suitable for expression; (b) isolating the recombinant polypeptide from the culture, the recombinant polypeptide being the anti-CDH17 antibody or antigen-binding fragment thereof of any one of claims 1-26 or the recombinant protein of any one of claims 27-32.

82. A method of treating a CDH17-associated disease, the method comprising: administering to a subject in need thereof an anti-CDH17 antibody or antigen binding fragment thereof of any one of claims 1-26, a recombinant protein of any one of claims 27-32, a recombinant cell of any one of claims 34-37, an immunoconjugate of any one of claims 38-56, or a pharmaceutical composition of any one of claims 57-63, or a combination thereof.

83. The method of claim 82, wherein, The method further comprises administering to the subject in need thereof other drugs or treatment methods for combination therapy.

84. The method of claim 83, wherein, The other drugs or treatment methods comprise anti-tumor immunotherapy drugs, tumor targeting drugs, tumor chemotherapy drugs, tumor radiotherapy.

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