Antibody-drug conjugates and uses thereof

Abstract

The present invention relates to a linker unit-cytotoxic small molecule drug, an antibody specifically binding to target mucoprotein 17 (MUC17), an antibody drug conjugate and uses thereof, and a composition and a detection system containing the antibody molecule, the linker unit-cytotoxic small molecule drug and the antibody drug conjugate.

Description

[0001] This application is a divisional application of the Chinese patent application with the application number 202510230523.3, the title of “Anti-Mucin 17 Antibody, Conjugate Thereof and Use Thereof”, which was filed on February 28, 2025. TECHNICAL FIELD

[0002] The present application relates to linker unit-cytotoxic small molecule drugs, antibodies specifically binding to target Mucin 17 (MUC17), antibody drug conjugates thereof and uses thereof, and compositions and detection systems containing the antibody molecules, linker unit-cytotoxic small molecule drugs and antibody drug conjugates. BACKGROUND

[0003] Mucin (MUC) is a high molecular weight glycoprotein produced and secreted by epithelial cells with mucus as the main component. So far, more than twenty Mucin proteins have been found, which are divided into two categories according to the function of Mucin, membrane-bound Mucin and secreted Mucin. Secreted Mucin can form a physical barrier and act as a mucus gel to protect the respiratory and gastrointestinal epithelial cells. Transmembrane Mucin forms a rod-like structure through the O-glycosylation tandem repeat sequence of its extracellular domain, which constructs a protective mucus gel layer.

[0004] MUC17 (Mucin-17) is a transmembrane Mucin encoded by MUC17 gene located at q22 locus of chromosome 7. MUC17 was discovered and identified in 2002, which consists of three parts: transmembrane segment, intracellular segment and extracellular segment. From the amino-terminal end, it is followed by Mucin-like structure (tandem repeat sequence), EGF (epidermal growth factor)-like region, N-glycosylation region, second EGF-like region, hydrophobic transmembrane segment, and cytoplasmic internal shuttle end. MUC17 is combined with cell membrane through the transmembrane region near the carboxyl end, which can be involved in intracellular signal transduction. The extracellular segment determines the spatial structure and immunogenicity of MUC specificity. The expression of MUC17 can enhance the intestinal mucus barrier and promote inflammation healing. In epithelial cells, MUC17 plays a role in signal transduction, maintains luminal structure, provides cell protection, and gives anti-adhesion properties to depolarized cancer cells, thereby making tumor cells lose polarity.

[0005] MUC17 is mainly expressed in the intestinal tract. Studies have found that the expression of MUC17 gradually increases from normal gastric mucosa, intestinal mucosa, to advanced gastric cancer, and its low expression is associated with poor prognosis of gastric cancer. Further studies have shown that the expression of MUC17 is negatively correlated with the degree of tumor differentiation, suggesting that MUC17 can be used as a diagnostic and therapeutic target for gastric cancer.

[0006] MUC17 is a membrane-bound mucin with important biological functions. The expression level of MUC17 is related to various inflammations and tumor cell proliferation. A large number of studies have shown that mucins are overexpressed in different malignant tumors. The close relationship between mucins and tumors and the induced immune response provide important clues for the design of anti-tumor drugs based on mucins. There is still an unmet medical need for diseases or conditions expressing MUC17. SUMMARY

[0007] The present application provides a novel anti-MUC17 antibody or antigen-binding fragment thereof, which has the advantages of high affinity and high specificity for human MUC17. The anti-MUC17 antibody provided by the present application can be used as an independent therapy or in combination with other therapies / or other anti-cancer agents for the treatment of, for example, cancer / tumor. The MUC17 antibody can be used in diagnostics on tissue samples from individuals, particularly individuals who can have MUC17-mediated tumors and / or cancers, or tumors and / or cancers associated with abnormal expression of MUC17, and / or tumors and / or cancers identifiable by abnormal expression of MUC17. The present application also provides nucleic acid molecules encoding the antibody molecules, expression vectors, host cells, and methods for producing the antibody molecules.

[0008] In a first aspect, the present application provides an antibody or antigen-binding fragment thereof capable of specifically binding to MUC17, wherein the antibody or antigen-binding fragment thereof comprises heavy chain complementarity determining regions (HCDRs) and / or light chain complementarity determining regions (LCDRs) selected from the group consisting of:

[0009] (1) 3 CDRs (HCDR1, HCDR2, HCDR3) contained in a VH as shown in SEQ ID NO: 7 or a variant thereof; and / or 3 CDRs (LCDR1, LCDR2, LCDR3) contained in a VL as shown in SEQ ID NO: 8 or a variant thereof; or,

[0010] (2) 3 CDRs (HCDR1, HCDR2, HCDR3) contained in a VH as shown in SEQ ID NO: 17 or a variant thereof; and / or 3 CDRs (LCDR1, LCDR2, LCDR3) contained in a VL as shown in SEQ ID NO: 18 or a variant thereof; or,

[0011] (3) 3 CDRs (HCDR1, HCDR2, HCDR3) contained in a VH as shown in SEQ ID NO: 24 or a variant thereof; and / or 3 CDRs (LCDR1, LCDR2, LCDR3) contained in a VL as shown in SEQ ID NO: 25 or a variant thereof; or,

[0012] (4) the 3 CDRs (HCDR1, HCDR2, HCDR3) contained in a VH as shown in SEQ ID NO: 24 or a variant thereof; and / or the 3 CDRs (LCDR1, LCDR2, LCDR3) contained in a VL as shown in SEQ ID NO: 31 or a variant thereof; or,

[0013] (5) the 3 CDRs (HCDR1, HCDR2, HCDR3) contained in a VH as shown in SEQ ID NO: 34 or a variant thereof; and / or the 3 CDRs (LCDR1, LCDR2, LCDR3) contained in a VL as shown in SEQ ID NO: 35 or a variant thereof; or,

[0014] (6) the 3 CDRs (HCDR1, HCDR2, HCDR3) contained in a VH as shown in SEQ ID NO: 40 or a variant thereof; and / or the 3 CDRs (LCDR1, LCDR2, LCDR3) contained in a VL as shown in SEQ ID NO: 41 or a variant thereof; or,

[0015] (7) the 3 CDRs (HCDR1, HCDR2, HCDR3) contained in a VH as shown in SEQ ID NO: 47 or a variant thereof; and / or the 3 CDRs (LCDR1, LCDR2, LCDR3) contained in a VL as shown in SEQ ID NO: 48 or a variant thereof; or,

[0016] (8) the 3 CDRs (HCDR1, HCDR2, HCDR3) contained in a VH as shown in SEQ ID NO: 47 or a variant thereof; and / or the 3 CDRs (LCDR1, LCDR2, LCDR3) contained in a VL as shown in SEQ ID NO: 53 or a variant thereof; or,

[0017] (9) the 3 CDRs (HCDR1, HCDR2, HCDR3) contained in a VH as shown in SEQ ID NO: 59 or a variant thereof; and / or the 3 CDRs (LCDR1, LCDR2, LCDR3) contained in a VL as shown in SEQ ID NO: 60 or a variant thereof; or,

[0018] (10) the 3 CDRs (HCDR1, HCDR2, HCDR3) contained in a VH as shown in SEQ ID NO: 69 or a variant thereof; and / or the 3 CDRs (LCDR1, LCDR2, LCDR3) contained in a VL as shown in SEQ ID NO: 70 or a variant thereof; or,

[0019] (11) 3 CDRs (HCDR1, HCDR2, HCDR3) contained in a VH as set forth in SEQ ID NO:77, or variants thereof; and / or, 3 CDRs (LCDR1, LCDR2, LCDR3) contained in a VL as set forth in SEQ ID NO:78, or variants thereof; or,

[0020] (12) 3 CDRs (HCDR1, HCDR2, HCDR3) contained in a VH as set forth in SEQ ID NO:87, or variants thereof; and / or, 3 CDRs (LCDR1, LCDR2, LCDR3) contained in a VL as set forth in SEQ ID NO:88, or variants thereof; or,

[0021] (13) 3 CDRs (HCDR1, HCDR2, HCDR3) contained in a VH as set forth in SEQ ID NO:97, or variants thereof; and / or, 3 CDRs (LCDR1, LCDR2, LCDR3) contained in a VL as set forth in SEQ ID NO:98, or variants thereof; or,

[0022] (14) 3 CDRs (HCDR1, HCDR2, HCDR3) contained in a VH as set forth in SEQ ID NO:104, or variants thereof; and / or, 3 CDRs (LCDR1, LCDR2, LCDR3) contained in a VL as set forth in SEQ ID NO:105, or variants thereof; or,

[0023] (15) 3 CDRs (HCDR1, HCDR2, HCDR3) contained in a VH as set forth in SEQ ID NO:114, or variants thereof; and / or, 3 CDRs (LCDR1, LCDR2, LCDR3) contained in a VL as set forth in SEQ ID NO:115, or variants thereof; or,

[0024] (16) 3 CDRs (HCDR1, HCDR2, HCDR3) contained in a VH as set forth in SEQ ID NO:114, or variants thereof; and / or, 3 CDRs (LCDR1, LCDR2, LCDR3) contained in a VL as set forth in SEQ ID NO:119, or variants thereof; or,

[0025] (17) 3 CDRs (HCDR1, HCDR2, HCDR3) contained in a VH as set forth in SEQ ID NO:125, or variants thereof; and / or, 3 CDRs (LCDR1, LCDR2, LCDR3) contained in a VL as set forth in SEQ ID NO:126, or variants thereof; or,

[0026] (18) 3 CDRs (HCDR1, HCDR2, HCDR3) contained in a VH as shown in SEQ ID NO: 135, or variants thereof; and / or, 3 CDRs (LCDR1, LCDR2, LCDR3) contained in a VL as shown in SEQ ID NO: 136, or variants thereof; or,

[0027] (19) 3 CDRs (HCDR1, HCDR2, HCDR3) contained in a VH as shown in SEQ ID NO: 97, or variants thereof; and / or, 3 CDRs (LCDR1, LCDR2, LCDR3) contained in a VL as shown in SEQ ID NO: 140, or variants thereof; or,

[0028] (20) 3 CDRs (HCDR1, HCDR2, HCDR3) contained in a VH as shown in SEQ ID NO: 149, or variants thereof; and / or, 3 CDRs (LCDR1, LCDR2, LCDR3) contained in a VL as shown in SEQ ID NO: 150, or variants thereof; or,

[0029] (21) 3 CDRs (HCDR1, HCDR2, HCDR3) contained in a VH as shown in SEQ ID NO: 158, or variants thereof; and / or, 3 CDRs (LCDR1, LCDR2, LCDR3) contained in a VL as shown in SEQ ID NO: 159, or variants thereof; or,

[0030] (22) 3 CDRs (HCDR1, HCDR2, HCDR3) contained in a VH as shown in SEQ ID NO: 167, or variants thereof; and / or, 3 CDRs (LCDR1, LCDR2, LCDR3) contained in a VL as shown in SEQ ID NO: 168, or variants thereof; or,

[0031] (23) 3 CDRs (HCDR1, HCDR2, HCDR3) contained in a VH as shown in SEQ ID NO: 172, or variants thereof; and / or, 3 CDRs (LCDR1, LCDR2, LCDR3) contained in a VL as shown in SEQ ID NO: 173, or variants thereof; or,

[0032] (24) 3 CDRs (HCDR1, HCDR2, HCDR3) contained in a VH as shown in SEQ ID NO: 179, or variants thereof; and / or, 3 CDRs (LCDR1, LCDR2, LCDR3) contained in a VL as shown in SEQ ID NO: 180, or variants thereof; or,

[0033] (25) the 3 CDRs (HCDR1, HCDR2, HCDR3) contained in a VH as shown in SEQ ID NO: 189, or variants thereof; and / or the 3 CDRs (LCDR1, LCDR2, LCDR3) contained in a VL as shown in SEQ ID NO: 190, or variants thereof; or,

[0034] (26) the 3 CDRs (HCDR1, HCDR2, HCDR3) contained in a VH as shown in SEQ ID NO: 198, or variants thereof; and / or the 3 CDRs (LCDR1, LCDR2, LCDR3) contained in a VL as shown in SEQ ID NO: 199, or variants thereof; or,

[0035] (27) the 3 CDRs (HCDR1, HCDR2, HCDR3) contained in a VH as shown in SEQ ID NO: 204, or variants thereof; and / or the 3 CDRs (LCDR1, LCDR2, LCDR3) contained in a VL as shown in SEQ ID NO: 205, or variants thereof; or,

[0036] (28) the 3 CDRs (HCDR1, HCDR2, HCDR3) contained in a VH as shown in SEQ ID NO: 214, or variants thereof; and / or the 3 CDRs (LCDR1, LCDR2, LCDR3) contained in a VL as shown in SEQ ID NO: 215, or variants thereof;

[0037] (29) the 3 CDRs (HCDR1, HCDR2, HCDR3) contained in a VH as shown in SEQ ID NO: 259, or variants thereof; and / or the 3 CDRs (LCDR1, LCDR2, LCDR3) contained in a VL as shown in SEQ ID NO: 263, or variants thereof;

[0038] (30) the 3 CDRs (HCDR1, HCDR2, HCDR3) contained in a VH as shown in SEQ ID NO: 259, or variants thereof; and / or the 3 CDRs (LCDR1, LCDR2, LCDR3) contained in a VL as shown in SEQ ID NO: 260, or variants thereof;

[0039] (31) the 3 CDRs (HCDR1, HCDR2, HCDR3) contained in a VH as shown in SEQ ID NO: 261, or variants thereof; and / or the 3 CDRs (LCDR1, LCDR2, LCDR3) contained in a VL as shown in SEQ ID NO: 260, or variants thereof;

[0040] (32) the 3 CDRs (HCDR1, HCDR2, HCDR3) contained in a VH set forth in SEQ ID NO: 259, or a variant thereof; and / or the 3 CDRs (LCDR1, LCDR2, LCDR3) contained in a VL set forth in SEQ ID NO: 262, or a variant thereof;

[0041] (33) the 3 CDRs (HCDR1, HCDR2, HCDR3) contained in a VH set forth in SEQ ID NO: 261, or a variant thereof; and / or the 3 CDRs (LCDR1, LCDR2, LCDR3) contained in a VL set forth in SEQ ID NO: 262, or a variant thereof;

[0042] wherein the variant of any one of (1)-(33) has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the sequence from which it is derived, or has one or more (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acid substitutions, deletions, or additions, compared to the sequence from which it is derived; in some embodiments, the substitution is a conservative substitution.

[0043] In some embodiments, the HCDR1, HCDR2, and HCDR3 contained in the heavy chain variable region (VH), and / or the LCDR1, LCDR2, and LCDR3 contained in the light chain variable region (VL) are defined by the Rabat, AbM, Chothia, or IMGT numbering system.

[0044] In embodiments of the antibody of any one of the foregoing, the antibody is a monoclonal antibody.

[0045] In embodiments of the antibody of any one of the foregoing, the antibody is a full-length antibody.

[0046] In some embodiments, the antibody or antigen-binding fragment of the present application is a murine, chimeric, humanized, or fully human antibody.

[0047] In some embodiments, the anti-MUC17 antibody of the present application is a whole antibody, e.g., an IgGl, IgG2, IgG3, IgG4 antibody.

[0048] In another embodiment, the anti-MUC17 antibody of the present application encompasses only the antigen-binding portion thereof, e.g., a Fab, Fab'-SH, Fv, scFv, or (Fab')2 fragment.

[0049] In another aspect, the present application provides an isolated polynucleotide molecule encoding any of the MUC17 antibodies or antigen-binding fragments thereof described herein.

[0050] In another aspect, the present application provides an expression vector comprising the nucleotide molecule described herein. In one embodiment, the vector is a eukaryotic expression vector.

[0051] In another aspect, the present application provides a host cell comprising the expression vector or the nucleotide molecule described herein. In some embodiments, the host cell is prokaryotic, such as E. coli. In other embodiments, the host cell is eukaryotic, such as a 293 cell, a CHO cell, a yeast cell, or a plant cell.

[0052] Further, the present application provides a pharmaceutical composition comprising the anti-MUC17 antibody or antigen-binding fragment thereof described herein, or the polynucleotide molecule described herein, or the expression vector described herein, or the host cell described herein, and a pharmaceutically acceptable pharmaceutical carrier or excipient.

[0053] The present application provides a method of making an anti-MUC17 antibody or antigen-binding fragment thereof described herein, the method comprising expressing the antibody or antigen-binding fragment thereof in a host cell described herein under conditions suitable for expression of the antibody or antigen-binding fragment thereof, and recovering the expressed antibody or antigen-binding fragment thereof from the host cell or culture medium comprising the host cell.

[0054] The anti-MUC17 antibody or antigen-binding fragment thereof or the pharmaceutical composition described herein can also be administered in combination with one or more other therapies, such as therapeutic modalities and / or other therapeutic agents, for the uses described herein, such as for the prevention and / or treatment of the relevant diseases or conditions mentioned herein.

[0055] The present application also provides a pharmaceutical combination comprising the antibody or antigen-binding fragment thereof or the pharmaceutical composition as described herein, and one or more additional therapeutic agents.

[0056] The present application provides a method of preventing or treating a MUC17-mediated disease in a subject in need thereof, comprising administering to the subject a prophylactically or therapeutically effective amount of an antibody or antigen-binding fragment thereof of the present application, a polynucleotide molecule described herein, an expression vector described herein, a host cell described herein, a pharmaceutical composition described herein, or a pharmaceutical combination described herein, the disease or disorder being a MUC17-associated tumor and / or cancer, the tumor and / or cancer being gastric cancer, esophageal cancer, pancreatic cancer, gastroesophageal adenocarcinoma, gastroesophageal junction cancer, gastric cardia cancer, gastric corpus cancer, gastric antrum cancer, liver cancer, bile duct cancer, gallbladder cancer, melanoma, endometrial cancer, kidney cancer, prostate cancer, breast cancer, triple-negative breast cancer, colon cancer, large intestine cancer, lung cancer, bone cancer, skin cancer, head and neck cancer, uterine cancer, cervical cancer, ovarian cancer, and rectal cancer.

[0057] The present application provides use of an antibody or antigen-binding fragment thereof described herein, a polynucleotide molecule described herein, an expression vector described herein, a host cell described herein, a pharmaceutical composition described herein, or a pharmaceutical combination described herein, in the manufacture of a medicament for the treatment and / or prevention of a MUC17-mediated disease or disorder. In one embodiment, the disease or disorder is a MUC17-associated tumor and / or cancer. In one embodiment, the MUC17-associated tumor and / or cancer is selected from gastric cancer, esophageal cancer, pancreatic cancer, gastroesophageal adenocarcinoma, gastroesophageal junction cancer, gastric cardia cancer, gastric corpus cancer, gastric antrum cancer, liver cancer, bile duct cancer, gallbladder cancer, melanoma, endometrial cancer, kidney cancer, prostate cancer, breast cancer, triple-negative breast cancer, colon cancer, large intestine cancer, lung cancer, bone cancer, skin cancer, head and neck cancer, uterine cancer, cervical cancer, ovarian cancer, and rectal cancer.

[0058] In one embodiment, the present application provides an antibody or antigen-binding fragment thereof described herein, a polynucleotide molecule described herein, an expression vector described herein, a host cell described herein, a pharmaceutical composition described herein, or a pharmaceutical combination described herein, for use as a medicament.

[0059] In one embodiment, the present application provides an antibody or antigen-binding fragment thereof described herein, a polynucleotide molecule described herein, an expression vector described herein, a host cell described herein, a pharmaceutical composition described herein, or a pharmaceutical combination described herein, for use in therapy.

[0060] In one embodiment, the present application provides an antibody or antigen-binding fragment thereof described herein, a polynucleotide molecule described herein, an expression vector described herein, a host cell described herein, a pharmaceutical composition described herein, or a pharmaceutical combination described herein for use in treating a MUC17 -associated tumor and / or cancer. In one embodiment, the MUC17 -associated tumor and / or cancer is selected from the group consisting of gastric cancer, esophageal cancer, pancreatic cancer, gastroesophageal adenocarcinoma, gastroesophageal junction cancer, gastric cardia cancer, gastric corpus cancer, gastric antrum cancer, liver cancer, bile duct cancer, gallbladder cancer, melanoma, endometrial cancer, kidney cancer, prostate cancer, breast cancer, triple-negative breast cancer, colon cancer, large intestine cancer, lung cancer, bone cancer, skin cancer, head and neck cancer, uterine cancer, cervical cancer, ovarian cancer, and rectal cancer.

[0061] The present application also provides a kit comprising an anti-MUC17 antibody or antigen-binding fragment thereof of the present application, a polynucleotide molecule described herein, an expression vector described herein, a host cell described herein, a pharmaceutical composition described herein, or a pharmaceutical combination described herein, and instructions for use. The kit can further comprise a suitable container. In certain embodiments, the kit further comprises a device for administration. The kit generally includes a label indicating the intended use or method of use of the contents of the kit. The term "label" includes any writing, recording, or marking on, or associated with, a kit, or any other container or wrapper that provides information concerning the contents of the kit or their intended use.

[0062] The present application provides a method of detecting the presence of MUC17 in a sample using an antibody or antigen-binding fragment thereof described herein or a detection composition comprising the antibody or antigen-binding fragment thereof.

[0063] The present application provides a method of detecting MUC17, wherein a sample to be tested is contacted with an antibody or antigen-binding fragment thereof described herein under conditions that allow the formation of a complex between the antibody or antigen-binding fragment thereof and MUC17.

[0064] In some embodiments, the antibody or antigen-binding fragment thereof described herein is linked to a detectable label.

[0065] In some embodiments, the detectable label is selected from the group consisting of an enzyme (e.g., horseradish peroxidase or alkaline phosphatase), a chemiluminescent reagent (e.g., acridinium esters, luminol and its derivatives, or ruthenium derivatives), a fluorescent dye (e.g., fluorescein or a fluorescent protein), a radionuclide, or biotin.

[0066] In some embodiments, the method for detecting MUC17 further comprises using another reagent (e.g., a secondary antibody) to specifically recognize the antibody or antigen-binding fragment thereof of the present application after the antibody or antigen-binding fragment thereof of the present application is contacted with the sample to be detected. In some embodiments, the other reagent (e.g., a secondary antibody) further comprises a detectable label, such as an enzyme (e.g., horseradish peroxidase or alkaline phosphatase), a chemiluminescent reagent (e.g., acridinium esters, luminol and its derivatives, or ruthenium derivatives), a fluorescent dye (e.g., fluorescein or a fluorescent protein), a radionuclide, or biotin.

[0067] The present application provides a kit comprising the antibody or antigen-binding fragment thereof, the expression vector, the host cell, the polynucleotide segment, or the pharmaceutical composition of the present application.

[0068] The present application provides a detection system comprising the antibody or antigen-binding fragment thereof of the present application. The present application also provides the use of the detection system in a diagnostic method.

[0069] In some embodiments, in the detection system, the antibody or antigen-binding fragment thereof of the present application is free or forms a complex with an antigen (e.g., an antigen present in the sample to be detected). Thus, in some embodiments, the detection system further comprises a complex of the antibody or antigen-binding fragment thereof of the present application bound to an antigen.

[0070] In some embodiments, the antibody or antigen-binding fragment thereof of the present application is linked to a detectable label.

[0071] In some embodiments, the detectable label is selected from an enzyme (e.g., horseradish peroxidase or alkaline phosphatase), a chemiluminescent reagent (e.g., acridinium esters, luminol and its derivatives, or ruthenium derivatives), a fluorescent dye (e.g., fluorescein or a fluorescent protein), a radionuclide, or biotin.

[0072] In some embodiments, the detection system further comprises another reagent (e.g., a secondary antibody) that specifically recognizes the antibody or antigen-binding fragment thereof of the present application. In some embodiments, the other reagent (e.g., a secondary antibody) further comprises a detectable label, such as an enzyme (e.g., horseradish peroxidase or alkaline phosphatase), a chemiluminescent reagent (e.g., acridinium esters, luminol and its derivatives, or ruthenium derivatives), a fluorescent dye (e.g., fluorescein or a fluorescent protein), a radionuclide, or biotin.

[0073] The present application provides a method for detecting tumor growth, comprising using the antibody or antigen-binding fragment thereof of the present application to determine the amount of MUC17 expression in a sample from a patient suspected of having a cancer, and optionally a negative control sample and further optionally a positive control sample.

[0074] The method of detecting tumor growth described herein comprises determining the amount of MUC17 expression in a sample from a patient suspected of having cancer and a negative control sample, further comprising comparing the amount of MUC17 expression between the samples, optionally wherein the amount of expression in the negative control and / or the positive control can be derived from stored data in at least one negative control sample and / or at least one positive control sample obtained in a method of detecting neoplastic growth comprising determining the amount of MUC17 expression, further optionally wherein the amount of expression in the negative control sample and / or the positive control sample can be derived from stored data comprising the average amount of expression in more than one negative control sample and / or more than one positive control sample obtained in a method of detecting neoplastic growth comprising determining the amount of MUC17 expression.

[0075] The sample and control in the method of detection described herein can be a positive expressing cell, a solid tissue sample or a liquid tissue sample.

[0076] The present application provides a method for detecting and / or quantifying MUC17 in a sample, comprising (a) using an antibody according to any one of the preceding embodiments, or produced according to any one of the preceding embodiments, or using a detection system as described above, for determining the amount of MUC17 expression in the sample; and; (b) comparing the amount of MUC17 expression determined in step (a) to (i) a predefined value for the amount of MUC17 expression, (ii) the amount of MUC17 expression determined in a control sample, or (iii) the amount of MUC17 expression determined in a sample obtained from the same source or subject at a previous time point.

[0077] The antibody or antigen-binding fragment thereof targeting human MUC17 provided by the present application has the following advantages:

[0078] (1) binds to target cells expressing human MUC17 with high affinity, with affinity in the range of 10-100 nM, more preferably in the range of 0-10 nM; has cross-binding activity with monkey antigens;

[0079] (2) can enter cells by endocytosis, killing target cells; in some embodiments, the antibody of the present application has high endocytosis efficiency;

[0080] (3) treats, prevents, ameliorates a MUC17-related disorder (e.g., cancer) in a subject, or treats, prevents, ameliorates one or more symptoms of the disease;

[0081] (4) reduces or inhibits tumor growth or progression in a subject;

[0082] (5) induces MUC17-related tumor regression (e.g., long-term regression);

[0083] (6) exhibit cytotoxic activity in MUC17 -associated cells.

[0084] In one aspect of the present application, the present application provides an anti-MUC17 antibody drug conjugate as described in formula (I), isomers thereof, pharmaceutically acceptable salts thereof, or mixtures thereof:

[0085] Ab-(L-D)m[formula (I)]

[0086] wherein,

[0087] L is a linker unit;

[0088] D is a small molecule drug with cytotoxicity;

[0089] m is the average number of L-D units conjugated to Ab, and m is selected from 1-10, preferably 2-8;

[0090] Ab is an anti-MUC17 antibody or an antigen-binding fragment thereof.

[0091] In some embodiments, the small molecule drug D described in the present application is a monomethyl auristatin, a camptothecin, or a maytansinoid.

[0092] In some embodiments, the monomethyl auristatin can be monomethyl auristatin E (MMAE)

[0093]

[0094] or monomethyl auristatin F (MMAF)

[0095]

[0096] In some embodiments, the maytansinoid can be DM1, DM3, or DM4.

[0097] In some embodiments, the camptothecin derivative can be

[0098] In some embodiments, the antibody drug conjugate comprises a plurality of D components, which can be a combination of different therapeutically active substances or pharmaceutically active components, or a combination of the same therapeutically active substances or pharmaceutically active components.

[0099] In some embodiments, the antibody drug conjugate has a drug / antibody ratio (DAR) of 1-15, for example a DAR having a value of about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15.

[0100] In some embodiments, the DAR is an average DAR.

[0101] In some embodiments, the average DAR is 1-15, for example 1-10.

[0102] As a specific embodiment, the average DAR of the antibody drug conjugate of the present application is preferably 2-10, for example 2-8.

[0103] As a specific embodiment, when D is a camptothecin compound, the average DAR of the antibody drug conjugate of the present application is preferably 4-8.

[0104] In some embodiments, the cytotoxin is covalently linked to the anti-MUC17 antibody or antigen-binding fragment thereof by means of a linker, either in a non-site specific manner or in a site specific manner.

[0105] As used herein, "linker", "linker unit", and "linking group" are used interchangeably.

[0106] As used herein, "drug-containing linker" refers to a compound in which a drug, such as a cytotoxic small molecule drug as described for D, is directly or indirectly covalently bonded to a linker.

[0107] In some embodiments, the L is a combination of one or more L's.

[0108] In some embodiments, the L' is selected from the group consisting of a carbonyl, an amino, an amido, an aminoacyl, -(PEG) n -, -(CH2) n -, heteroatom-containing -(CH2) n, -(C=C)-, -(CH=CH)-, -0-, -S-, maleimido (mc), maleimidopropanoyl (MP), methylsulfonylpyrimidinyl, valine-citrulline (val-cit), valine-alanine (val-ala), N-succinimidyl-4-(N-maleimidomethyl)cyclohexane-l-carboxylate (SMCC), alanine-phenylalanine (ala-phe), phenylalanine-lysine (phe-lys), p-aminobenzyloxy carbonyl (PAB), dimethyl ethylenediamine (DMED), N-succinimidyl 4-(2-pyridylthio) valerate (SPP), N-succinimidyl 4-(N-maleimidomethyl)-cyclohexane-l-carboxylate (SMCC), N-succinimidyl 4-(iodo-acetyl) aminobenzoate (SIAB), N-succinimidyl 4-(2-pyridyldithio) butyrate (SPDB), N-succinimidyl 3-(pyrid-2-yl dithio)-propionate (SPDP), glycine-glycine-phenylalanine-glycine (GGFG), acetyl-lysine-valine-citrulline-p-aminobenzyloxy carbonyl (AcLys-vc-PABC).

[0109] In some embodiments, n is independently selected from an integer from 1 to 20.

[0110] As a specific embodiment, when L' is selected from (PEG) n , n is independently preferably an integer from 1 to 16.

[0111] As a specific embodiment, when L' is selected from (CH2) n , n is independently preferably an integer from 1 to 16.

[0112] As a specific embodiment, when L' is selected from (CH2) n , n is independently preferably an integer from 1 to 16; and the heteroatom is selected from N, O or S, and the number of heteroatoms is independently preferably an integer from 1 to 8.

[0113] In some embodiments, L can be J-L1-L2-X-L3.

[0114] In some embodiments, J is selected from

[0115] In some embodiments, L1is selected from a single bond, -(PEG) n , or -(CH2) n , and the -(CH2) n- optionally containing 1-6 heteroatoms selected from N, O, S; wherein n is independently selected from an integer from 1-20. Preferably, L1is selected from -(PEG) n , wherein n is independently 2, 4, 6, or 8.

[0116] In some embodiments, L2is selected from a combination of one or more of a single bond, -O-, -S-, -CH2-, -NH-, -C(O)-; wherein said -CH2- and -NH- can be optionally substituted 1-3 times with C 1～6 alkyl or halogen.

[0117] In some embodiments, X is selected from a single bond or a combination of 1-4 X', said X' is independently selected from an amino acid, such as glycine, alanine, phenylalanine, valine, lysine, and citrulline. In some embodiments, X is selected from a single bond or valine-citrulline (val-cit), valine-alanine (val-ala), alanine-phenylalanine (ala-phe), phenylalanine-lysine (phe-lys), glycine-glycine-phenylalanine-glycine (GGFG).

[0118] In some embodiments, L3is selected from a combination of one or more of a single bond, -O-, -S-, -CH2-, -NH-, -C(O)-, -phenyl-, -cyclopropyl-, -cyclobutyl-, -cyclohexyl-; wherein said -CH2-, -NH-, -phenyl-, -cyclopropyl-, -cyclobutyl-, -cyclohexyl- can be optionally substituted 1-3 times with C 1～6 alkyl, halogen, cyano, or hydroxyl.

[0119] Further, the present application provides three linker unit-small cytotoxic molecule drugs of formula (L-D), as shown in formula Cpd3, formula Cpd5, and formula Cpd6:

[0120]

[0121] In some embodiments, the linker unit-small cytotoxic molecule drugs (Linker-Toxin) of formula Cpd3, formula Cpd5, and formula Cpd6 provided by the present application are suitable for conjugating to antibodies, such as anti-MUC17 antibodies or antigen-binding fragments thereof, her2 antibodies or antigen-binding fragments thereof, with high conjugation efficiency and mild reaction conditions. For example, as demonstrated in the following examples, Cpd3 as a hydrophilic linker-toxin can be used to prepare high DAR value ADCs (DAR about 8) with excellent purity.

[0122] In some embodiments, the present application provides an anti-MUC17 antibody drug conjugate, an isomer thereof, a pharmaceutically acceptable salt thereof, or a mixture thereof, wherein the anti-MUC17 antibody drug conjugate has a structure according to Formula (I-C3), Formula (I-C5), and Formula (I-C6):

[0123]

[0124] wherein,

[0125] m is the average number of attachments, and m is independently selected from 1 to 10, for example, 2 to 8, and in particular, 4 to 8;

[0126] Ab is an anti-MUC17 antibody or antigen binding fragment thereof as previously described herein.

[0127] In some embodiments, the Ab comprises 3 complementarity determining regions (HCDRs) from a heavy chain variable region, HCDR1, HCDR2, and HCDR3, which are the complementarity determining regions (HCDRs) of a heavy chain variable region of an anti-MUC17 antibody or antigen binding fragment thereof as previously described herein.

[0128] In some embodiments, the Ab comprises 3 complementarity determining regions (LCDRs) from a light chain variable region, LCDR1, LCDR2, and LCDR3, which are the complementarity determining regions (LCDRs) of a light chain variable region of an anti-MUC17 antibody or antigen binding fragment thereof as previously described herein.

[0129] In some embodiments, the Ab comprises 3 complementarity determining regions (HCDRs) from a heavy chain variable region and 3 complementarity determining regions (LCDRs) from a light chain variable region, which are the complementarity determining regions (HCDRs) of a heavy chain variable region and the complementarity determining regions (LCDRs) of a light chain variable region of an anti-MUC17 antibody or antigen binding fragment thereof as previously described herein.

[0130] In some embodiments, the Ab comprises a heavy chain variable region (VH) which is a heavy chain variable region (VH) of an anti-MUC17 antibody or antigen binding fragment thereof as previously described herein.

[0131] In some embodiments, the heavy chain variable region comprises 3 complementarity determining regions (HCDRs) from a heavy chain variable region.

[0132] In some embodiments, the Ab comprises a light chain variable region (VL) which is a light chain variable region (VL) of an anti-MUC17 antibody or antigen binding fragment thereof as previously described herein.

[0133] In some embodiments, the light chain variable region comprises 3 complementarity determining regions (LCDRs) from a light chain variable region.

[0134] In some embodiments, the Ab heavy chain variable region (VH) and light chain variable region (VL) are the heavy chain variable region (VH) and light chain variable region (VL) of the aforementioned anti-MUC17 antibody or antigen-binding fragment thereof of the present application.

[0135] The present application provides an antibody drug conjugate targeting human MUC17, isomers thereof, pharmaceutically acceptable salts thereof, or mixtures thereof, which has the following advantages:

[0136] (1) binds to target cells expressing human MUC17 with high affinity, with affinity ranging from 10-100 nM, more preferably from 0-10 nM; has cross-binding activity with monkey antigens;

[0137] (2) can enter cells by endocytosis, killing target cells; in some embodiments, the ADC of the present application has high endocytosis efficiency;

[0138] (3) treats, prevents, ameliorates a MUC17-related disorder (e.g., cancer) in a subject, or treats, prevents, ameliorates one or more symptoms of the disease;

[0139] (4) reduces or inhibits tumor (MUC17-related tumor) growth or progression in a subject;

[0140] (5) induces MUC17-related tumor regression (e.g., long-term regression);

[0141] (6) exerts cytotoxic activity in MUC17-related cells;

[0142] (7) has a bystander killing activity.

[0143] In another aspect, the present application provides a use of the antibody drug conjugate for (1) preparing a diagnostic reagent; and / or, (2) preparing a medicament for preventing and / or treating a MUC17-related disease or disorder.

[0144] In another aspect, the present application provides a use of an active ingredient selected from the group consisting of: a heavy chain variable region (VH) of an anti-MUC17 antibody or an antigen-binding fragment thereof according to the present application; and / or, a light chain variable region (VL) of an anti-MUC17 antibody or an antigen-binding fragment thereof according to the present application; and / or, a heavy chain of an anti-MUC17 antibody or an antigen-binding fragment thereof according to the present application; and / or, a light chain of an anti-MUC17 antibody or an antigen-binding fragment thereof according to the present application; and / or, an antibody drug conjugate targeting human MUC17 according to the present application, an isomer thereof, a pharmaceutically acceptable salt thereof, or a mixture thereof; for the manufacture of: (a) a medicament for preventing and / or treating a MUC17-related disease or disorder; (b) a method for preventing or treating a MUC17-mediated disease; (c) a detection reagent, a detection plate, or a kit.

[0145] The present application also provides a pharmaceutical composition comprising the anti-MUC17 antibody drug conjugate, an isomer thereof, a pharmaceutically acceptable salt thereof, or a mixture thereof, and a pharmaceutically acceptable carrier or excipient.

[0146] The present application provides a pharmaceutical combination comprising the anti-MUC17 antibody drug conjugate, an isomer thereof, a pharmaceutically acceptable salt thereof, or a mixture thereof, or the pharmaceutical composition, and another therapeutic agent or agents.

[0147] The present application provides a kit comprising the anti-MUC17 antibody drug conjugate, an isomer thereof, a pharmaceutically acceptable salt thereof, or a mixture thereof, or the pharmaceutical composition.

[0148] The present application provides use of the anti-MUC17 antibody drug conjugate, an isomer thereof, a pharmaceutically acceptable salt thereof, or a mixture thereof, or the pharmaceutical composition, or the pharmaceutical combination, or the kit, in the manufacture of a medicament for treating and / or preventing a MUC17-mediated disease or disorder.

[0149] The present application provides the anti-MUC17 antibody drug conjugate, an isomer thereof, a pharmaceutically acceptable salt thereof, or a mixture thereof, or the pharmaceutical composition, or the pharmaceutical combination, or the kit, for use as a medicament.

[0150] The present application provides the anti-MUC17 antibody drug conjugate, an isomer thereof, a pharmaceutically acceptable salt thereof, or a mixture thereof, or the pharmaceutical composition, or the pharmaceutical combination, or the kit, for use in therapy.

[0151] The present application provides an anti-MUC17 antibody drug conjugate, an isomer thereof, a pharmaceutically acceptable salt thereof or a mixture thereof, or a pharmaceutical composition, a pharmaceutical combination or a kit as described herein for use in the treatment of a MUC17 related tumor and / or cancer.

[0152] In one embodiment, the MUC17 related tumor and / or cancer is selected from the group consisting of gastric cancer, esophageal cancer, pancreatic cancer, gastroesophageal adenocarcinoma, gastroesophageal junction cancer, gastric cardia cancer, gastric corpus cancer, gastric antrum cancer, liver cancer, bile duct cancer, gallbladder cancer, melanoma, endometrial cancer, kidney cancer, prostate cancer, breast cancer, triple negative breast cancer, colon cancer, large intestine cancer, lung cancer, bone cancer, skin cancer, head and neck cancer, uterine cancer, cervical cancer, ovarian cancer and rectal cancer.

[0153] The present application provides a method for treating and / or preventing a MUC17 mediated disease or disorder, comprising administering to a subject in need thereof an antibody drug conjugate, an isomer thereof, a pharmaceutically acceptable salt thereof or a mixture thereof, or a pharmaceutical composition as described herein.

[0154] In some embodiments, the MUC17 related and / or MUC17 mediated disease or disorder is a MUC17 related cancer and / or tumor.

[0155] In some embodiments, the MUC17 related cancer and / or tumor is selected from the group consisting of gastric cancer, esophageal cancer, pancreatic cancer, gastroesophageal adenocarcinoma, gastroesophageal junction cancer, gastric cardia cancer, gastric corpus cancer, gastric antrum cancer, liver cancer, bile duct cancer, gallbladder cancer, melanoma, endometrial cancer, kidney cancer, prostate cancer, breast cancer, triple negative breast cancer, colon cancer, large intestine cancer, lung cancer, bone cancer, skin cancer, head and neck cancer, uterine cancer, cervical cancer, ovarian cancer and rectal cancer.

[0156] Further, the present application provides a method for preparing the aforementioned antibody drug conjugate, an isomer thereof, a pharmaceutically acceptable salt thereof or a mixture thereof, comprising: combining an antibody with a linker-toxin compound under conditions sufficient to form an antibody drug conjugate.

[0157] It should be understood that, within the scope of the present application, each of the technical features described above and specifically described hereinafter (e.g. in the examples) can be combined with each other to form new or preferred technical solutions. Due to the limited space, they are not listed one by one here.

[0158] Definitions of terms

[0159] The practice of the present application will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are within the skill of the art.

[0160] For the purposes of the present invention, certain key terms are specifically defined as follows. As used herein, and unless otherwise expressly specified, all technical and scientific terms have the meaning that is commonly understood by one of ordinary skill in the art in the field of the invention. For the purposes of the present invention, reference is made to Current Protocols in Molecular Biology (Ausubel) for definitions and terms of the art. Abbreviations for amino acid residues are the standard three letter and / or one letter codes used in the art to designate one of the 20 commonly occurring L-amino acids. As used herein, including the claims, the singular form "a", "an", and "the" include the corresponding plural references unless the context clearly dictates otherwise.

[0161] The term "about" when used in connection with a numerical value means a range of numerical values that has a lower limit that is 5% less than the specified numerical value and an upper limit that is 5% greater than the specified numerical value.

[0162] The term "and / or" should be understood to mean either one of the items or any combination of the items.

[0163] The term "MUC17" refers to any recombinant or naturally occurring form of mucin 17 (MUC17), variants or homologs thereof that maintain at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the activity of MUC17. The variants or homologs have at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to the entire sequence or a partial sequence (e.g., a 50, 100, 150, or 200 continuous amino acid portion) of a naturally occurring FGFR2 protein.

[0164] The term "antibody" herein is used in the broadest sense and encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments so long as they exhibit the desired antigen-binding activity. An intact antibody will typically comprise at least two full-length heavy chains and two full-length light chains, but can in some cases include fewer chains, such as the naturally occurring heavy chain-only antibodies found in camelids.

[0165] The term "antigen binding molecule" is used in the broadest sense and refers to molecules that specifically bind an antigen. Exemplary antigen binding molecules include, but are not limited to, antibodies or antibody mimetics. An "antibody mimetic" refers to an organic compound or binding domain that is capable of specific binding to an antigen, but is not related to an antibody structure. Exemplary antibody mimetics include, but are not limited to, affibodies, affitins, affilins, designed ankyrin repeat proteins (DARPins), aptamers, or Kunitz domain peptides.

[0166] The term "anti-MUC17 antibody" refers to an antibody molecule that specifically binds to MUC17 and is capable of inhibiting MUC17 activity. An anti-MUC17 antibody is capable of inhibiting MUC17 activity relative to the absence of the MUC17 antibody, for example, by at least partially or completely blocking stimulation of MUC17, reducing, preventing, or delaying activation of MUC17, or inactivating, desensitizing, or down-regulating signal transduction, activity, or amount of MUC17. In some embodiments, the antibody can inhibit MUC17 activity by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more, as compared to a control.

[0167] The terms "MUC17-associated cancer and / or tumor," "MUC17-mediated cancer and / or tumor," or "cancer and / or tumor associated with abnormal expression of MUC1," and the like, can be used interchangeably to refer to various types of tumors that are formed due to abnormal expression, mutation, or malfunction of the MUC17 gene or protein, which leads to abnormal cell proliferation, invasion, metastasis, and the like, and is commonly found in the gastrointestinal tract, pancreas, liver, gallbladder, and the like.

[0168] The term "antibody fragment" refers to a molecule other than an intact antibody that comprises a portion of an intact antibody and that is capable of binding to an antigen bound by the intact antibody. Examples of antibody fragments include but are not limited to Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies; linear antibodies; single-chain antibodies (e.g., scFv); single-domain antibodies; bi- or multi-specific antibodies or fragments thereof; camelid antibodies (heavy chain antibodies); and multispecific antibodies formed from antibody fragments (e.g., bispecific antibodies).

[0169] The term "valency" denotes the presence of a specified number of binding sites in an antibody / antigen-binding molecule. Thus, the terms "monovalent," "bivalent," "tetravalent," and "hexavalent" refer to the presence of one binding site, two binding sites, four binding sites, and six binding sites, respectively, in an antibody / antigen-binding molecule.

[0170] The terms "antigen-binding fragment" and "antibody fragment" are used interchangeably herein and do not possess all of the structural features of an intact antibody, but only a portion of, or a partial variant of, an intact antibody that is capable of binding to an antigen. An "antigen-binding fragment" or "antibody fragment" herein includes, but is not limited to, Fab, Fab', Fab'-SH, F(ab')2, Fd, Fv, scFv, diabodies, and single-domain antibodies.

[0171] The term "chimeric antibody" refers to an antibody in which a portion of the light or / and heavy chain is derived from one antibody (which can be derived from a particular species or belong to a particular antibody class or subclass), and the other portion of the light or / and heavy chain is derived from another antibody (which can be derived from the same or a different species or belong to the same or a different antibody class or subclass), but retains the ability to bind to the target antigen. For example, the term "chimeric antibody" can include an antibody (e.g., a human mouse chimeric antibody) in which the heavy and light chain variable regions of the antibody are from a first antibody (e.g., a murine antibody), while the heavy and light chain constant regions of the antibody are from a second antibody (e.g., a human antibody).

[0172] The term "humanized antibody" refers to a non-human-derived antibody that has been genetically engineered to have an amino acid sequence that is modified to increase homology to the sequence of a human-derived antibody. Typically, a humanized antibody has all or a portion of the CDR regions from a non-human-derived antibody (donor antibody) and all or a portion of the non-CDR regions (e.g., variable region FRs and / or constant regions) from a human-derived immunoglobulin (acceptor antibody). A humanized antibody typically retains or partially retains the desired properties of the donor antibody, including but not limited to, antigen specificity, affinity, reactivity, ability to enhance immune cell activity, ability to enhance immune response, etc.

[0173] The term "fully human antibody" refers to an antibody having variable regions in which both the FRs and CDRs are derived from human germline immunoglobulin sequences. In addition, if the antibody contains a constant region, the constant region also is derived from a human germline immunoglobulin sequence. A fully human antibody herein can include amino acid residues that are not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro, or by somatic mutation in vivo). However, a "fully human antibody" herein does not include antibodies in which CDR sequences derived from the germline of another mammalian species (e.g., mouse) have been grafted onto human framework sequences.

[0174] The term "variable region" or "variable domain" refers to the domain of an antibody heavy or light chain that is involved with binding the antibody to an antigen. The variable domains of the heavy chain and light chain of a native antibody generally have similar structures, with each domain comprising four conserved framework regions (FRs) and three complementary determining regions (CDRs) (see, e.g., Kindt et al. Kuby Immunology, 6thed., W.H. Freeman and Co. p. 91 (2007)). A single VHor VLdomain is sufficient to confer antigen-binding specificity.

[0175] The term "complementarity determining region" or "CDR region" or "CDR" is a region in an antibody variable domain that is hypervariable in sequence and forms structurally defined loops ("hypervariable loops") and / or contains antigen contact residues ("antigen contact points"). CDRs are primarily responsible for binding to an epitope of an antigen. The CDRs in a variable domain are typically referred to as CDR1, CDR2, and CDR3, numbered sequentially from the N-terminus. The precise amino acid sequence boundaries of each CDR in a given variable region amino acid sequence can be determined using any of a number of well-known antibody CDR assignment systems, or combinations thereof, including, for example: Chothia based on the three-dimensional structure of the antibody and the topology of the CDR loops (Chothia et al. (1989) Nature 342: 877-883, Al-Lazikani et al., "Standard conformations for the canonical structures of immunoglobulins", Journal of Molecular Biology, 273, 927-948 (1997)), Kabat based on antibody sequence variability (Kabat et al., Sequences of Proteins of Immunological Interest, 4th Ed., U.S. Department of Health and Human Services, National Institutes of Health (1987)), AbM (University of Bath), Contact (University College London), the international ImMunoGeneTics database (IMGT) (http: / / imgt.cines.fr / ), and North CDR definitions based on affinity propagation clustering with a large number of crystal structures.

[0176] It should be noted that the boundaries of CDRs of a variable region of the same antibody can vary slightly based on the assignment system used. That is, the CDR sequences of the same antibody variable region defined under different assignment systems can vary slightly. Thus, where an antibody is defined in terms of specific CDR sequences as defined herein, the scope of the antibody also encompasses antibodies whose variable region sequences contain the specific CDR sequences recited but whose CDR boundaries differ from the specific CDR boundaries defined herein due to the application of different schemes (e.g., different assignment systems or combinations).

[0177] Unless otherwise indicated, in the application, the term "CDR" or "CDR sequence" encompasses CDR sequences determined in any of the manners described above.

[0178] CDRs can also be determined based on having the same Kabat or AbM numbering position as a reference CDR sequence, such as any of the CDRs exemplified herein. In some embodiments, the CDRs of the antibodies of the application are determined positions according to the Kabat or AbM numbering scheme.

[0179] Unless otherwise indicated, in the application, when referring to residue positions in antibody variable regions and CDRs, including heavy chain variable region residues, the numbering of the positions is according to the Kabat or AbM numbering system.

[0180] As used herein, the term "comprising" or "including," or "having" is intended to mean including but not limited to. In this text, when the term "comprising" or "including" is used, unless otherwise indicated, the meaning is also to be construed as "consisting of. For example, when referring to an antibody variable region "comprising" a particular sequence, an antibody variable region "consisting of the particular sequence is also intended to be covered.

[0181] The term "Fc region" is used to define a C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region. The term includes native sequence Fc regions and variant Fc regions. In some embodiments, a human IgG heavy chain Fc region extends from Cys226, or Pro230, to the carboxy-terminus of the heavy chain. However, the C-terminal lysine (Lys447) of the Fc region can or can not be present (the numbering in this paragraph is according to the EU numbering system, also referred to as EU index, as described in Rabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991).

[0182] The term "conservative amino acid" generally refers to amino acids belonging to the same class or having similar characteristics, such as charge, side chain size, hydrophobicity, hydrophilicity, main chain conformation, and rigidity.

[0183] The term "affinity" or "binding affinity" refers to the intrinsic binding affinity that reflects the interaction between members of a binding pair. The affinity of a molecule X for its partner Y can be generally represented by the equilibrium dissociation constant (KD), which is the ratio of the dissociation rate constant and the association rate constant (kdisand kon, respectively). Affinity can be measured by common methods known in the art. One particular method for measuring affinity is the ForteBio Kinetic Binding Assay herein.

[0184] The term "does not bind" to a protein or cell means that it does not bind to the protein or cell, or does not bind to it with high affinity, i.e., the KDof the binding protein or cell is 1.0 x 10 -6 M or higher, more preferably 1.0 x 10 -5 M or higher, more preferably 1.0 x 10 -4 M or higher, 1.0 x 10 -3 M or higher, more preferably 1.0 x 10 -2 M or higher.

[0185] The term "high affinity" for an IgG antibody refers to a KDof 1.0 x 10 -6 M or lower, preferably 5.0 x 10 -8 M or lower, more preferably 1.0 x 10 -8 M or lower, 5.0 x 10 -9 M or lower, more preferably 1.0 x 10 -9 M or lower. For other antibody subtypes, "high affinity" binding can vary. For example, "high affinity" binding for the IgM subtype refers to a KDof 10 -6 M or lower, preferably 10 -7 M or lower, more preferably 10 -8 M or lower.

[0186] The term "binds" or "specifically binds" as used herein means that the binding is selective for the antigen and can be distinguished from unwanted or non-specific interactions. The ability of an antigen binding site to bind to a particular antigen can be determined by enzyme-linked immunosorbent assay (ELISA) or conventional binding assays known in the art, such as by radioimmunoassay (RIA) or bio-layer interferometry assay or MSD assay or surface plasmon resonance (SPR).

[0187] The term "half maximal effective concentration (EC 50 " refers to the concentration of a drug, antibody, or toxic agent that induces a response that is 50% between the baseline and maximum after a particular exposure time.

[0188] The term "therapeutic agent" encompasses any substance effective in preventing or treating a tumor, e.g., a cancer, including chemotherapeutic agents, cytokines, angiogenesis inhibitors, cytotoxic agents, other antibodies, small molecule drugs, or immunomodulatory agents (e.g., immunosuppressants).

[0189] The term "expression vector" refers to a nucleic acid molecule capable of amplifying another nucleic acid to which it is linked. The term includes vectors that function as self-replicating nucleic acid structures as well as vectors that integrate into the genome of a host cell into which the vector has been introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operably linked. Such vectors are referred to herein as "expression vectors."

[0190] The term "host cell" refers to a cell into which an exogenous nucleic acid has been introduced, including the progeny of such a cell. Host cells include "transformants" and "transformed cells," which include both the primary transformed cell and progeny of the primary transformed cell, regardless of the number of passages. Progeny can not be completely identical to the parent cell both in nucleic acid content and in physical identity, but are still included as long as they have the same function or biological activity as screened or selected for in the initially transformed cell. Mutant progeny are included herein.

[0191] The term "antibody drug conjugate" or "ADC" refers to an antibody or antibody fragment that is covalently coupled to a therapeutically active substance or active pharmaceutical ingredient, such that the therapeutically active substance or active pharmaceutical ingredient is targeted to the binding target of the antibody to exhibit its pharmacological function. The therapeutically active substance or active pharmaceutical ingredient can be a cytotoxin that is capable of killing the cell, preferably a cancer cell, to which the ADC is targeted. The covalent linkage of the therapeutically active substance, active pharmaceutical ingredient, or cytotoxin can be performed in a non-site-specific manner using a linker, or in a site-specific manner.

[0192] The term "site-specific conjugation" refers to a mode of linkage that specifically links a therapeutically active substance or active pharmaceutical ingredient to a particular site of an antibody. In some embodiments, the conjugation is accomplished with the aid of a linker.

[0193] The term "cytotoxic agent" is used interchangeably with "cytotoxin" and refers in the present application to a substance that inhibits or destroys cellular function and / or causes cell death or destruction.

[0194] The terms "linker", "linker unit" and "linker" are used interchangeably in the present application and refer to a chemical module that covalently links the antibody to the therapeutically active substance or active pharmaceutical ingredient in the ADC. In some embodiments, the linker can comprise amino acid residues that link the antigen to the payload. The amino acid residues can form a dipeptide, tripeptide, tetrapeptide, pentapeptide, hexapeptide, heptapeptide, octapeptide, nonapeptide, decapeptide, undecapeptide or dodecapeptide unit. The amino acid residues include naturally occurring ones as well as non-naturally occurring amino acid analogs, such as citrulline or beta-amino acids, such as beta-alanine, or omega-amino acids such as 4-amino-butyric acid.

[0195] According to the classification by property, linkers suitable for use in the present application can be cathepsin-degradable linkers, such as valine-citrulline (val-cit) linkers, cBu-Cit linkers and CX linkers; non-cleavable linkers such as SMCC linkers or MD linkers; acid-sensitive linkers, silicon lipid structured linkers, disulfide-carbamate linkers, MC-GGFG linkers, TRX linkers, galactoside-containing linkers, pyrophosphate linkers, near-infrared sensitive linkers, UV-sensitive linkers such as PC4AP.

[0196] The linkers of the present application can also be a combination of one or more linkers, such as a cathepsin-degradable linker can be combined with other types of linkers to form a new linker. Thus, the "linker" as described in the present application encompasses a single type of linker, or a combination of different types of linkers, as long as it is capable of coupling the antibody of the present application to the drug.

[0197] The term "loading" or "drug loading" or "efficiency loading" refers to the average number of efficiency loadings per antibody within an ADC molecule (the term "efficiency loading" is used interchangeably herein with "therapeutic active or active pharmaceutical ingredient"). The drug loading can range from 1-20 therapeutic active or active pharmaceutical ingredients per antibody. The term "drug / antibody ratio" or "DAR" refers to the ratio of therapeutic active or active pharmaceutical ingredient (D) conjugated to the antibody to the antibody. The ADCs described herein generally have a DAR of 1-20, in certain embodiments, a DAR of 1-8, 2-8, 2-6, 2-5, 2-18, 4-16, 5-12, 6-10, 3-8, 4-6, 6-10, and 2-4. Representative DAR values are 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15, generally denoted as the combination of the letter D and a number, where the number represents the numerical value of the DAR, e.g., D2 represents a drug / antibody ratio with a DAR value of 2. In some embodiments, the DAR is the average DAR, i.e., the overall ratio of small molecule drug moieties (D) conjugated to the Ab moiety to the Ab moiety in the product as measured by detection methods, e.g., by conventional methods such as UV / visible spectroscopy, mass spectrometry, ELISA assays, and HPLC. The DAR can be limited by the number of ligation sites on the antibody. For example, where the ligation site is a cysteine thiol, the antibody can have only one or a few cysteine thiols or can have only one or a few thiols that are sufficiently reactive (through which a drug can be conjugated) to react with a drug.

[0198] The term "treatment" refers to slowing, interrupting, arresting, stopping, reducing, or reversing the progression or severity of an existing symptom, disorder, condition, or disease. Desirable effects of treatment include, but are not limited to, preventing occurrence or recurrence of disease, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, preventing metastasis, decreasing rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis. In some embodiments, the antibodies of the application are used to delay development of a disease or to slow the progression of a disease.

[0199] The term "prevention" includes inhibition of the occurrence or development of a disease or disorder or symptoms of a particular disease or disorder. In some embodiments, a subject with a family history of cancer is a candidate for a prophylactic regimen. Generally, in the context of cancer, the term "prevention" refers to administration of a drug prior to the onset of signs or symptoms of cancer, particularly in a subject at risk for cancer.

[0200] The term "effective amount" refers to the amount or dose of an antibody or conjugate or composition of the application, which, upon single or multiple dose administration to a patient, results in an intended effect in the patient being treated or prevented. The effective amount can be readily determined by the attending physician, as one skilled in the art, by taking into account a variety of factors, such as species; body mass; age and general health of the mammal; specific disease involved; extent or severity of disease; individual patient responses; particular antibody being administered; mode of administration; bioavailability of the formulation administered; chosen route of delivery; and use of other co-therapies.

[0201] The term "therapeutically effective amount" refers to the amount of an antibody or antibody fragment or conjugate or composition thereof effective to achieve the desired therapeutic result at the required dosage and for the required period of time. The therapeutically effective amount of an antibody or antibody fragment or conjugate or composition thereof can vary depending on factors such as the disease state, age, sex, and weight of the individual, and the ability of the antibody or antibody moiety to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of the antibody or antibody fragment or conjugate or composition thereof are outweighed by the therapeutically beneficial effects. A "therapeutically effective amount" preferably inhibits a measurable parameter (e.g., tumor growth rate, tumor volume, etc.) by at least about 20%, more preferably by at least about 40%, even more preferably by at least about 50%, 60%, or 70%, and still more preferably by at least about 80% or 90%, relative to an untreated subject. The ability of a compound to inhibit a measurable parameter (e.g., cancer) can be evaluated in an animal model system predictive of efficacy in humans.

[0202] The term "prophylactically effective amount" refers to the amount of an antibody or antibody fragment or conjugate or composition thereof effective to achieve the desired prophylactic result at the required dosage and for the required period of time. Generally, a prophylactically effective amount will be less than a therapeutically effective amount since a prophylactic dose is used preventatively prior to or at an earlier stage of disease in a subject.

[0203] The term "pharmaceutical composition" refers to a composition that is in a form suitable for its administration to a subject and that contains at least one active agent in an amount that is effective for the intended use of the composition and that does not contain significant amounts of additional ingredients that are toxic to the subject to which the composition is administered.

[0204] The term "in combination with" refers to administration of one or more additional therapeutic agents including simultaneous (co-)administration and consecutive administration in any order.

[0205] The term "pharmaceutically acceptable salt" refers to a salt of a pharmaceutical conjugate of the application which is safe and / or effective for use in a mammal, and which has the desired biological activity, and which can be formed from the antibody pharmaceutical conjugate of the application with an acid.

[0206] The term "pharmaceutically acceptable carrier" refers to a non-toxic carrier, i.e., a carrier that is not deleterious to the subject to which the pharmaceutical formulation or composition is administered.

[0207] The term "subject" includes any human or non-human animal. The term "non-human animal" includes all vertebrates, such as mammals and non-mammals, such as non-human primates, sheep, dogs, cats, horses, cattle, chickens, amphibians, reptiles, etc. Attached Figure Description

[0208] Figure 1 Example 6.1 describes the ELISA detection of the binding of recombinant antibodies M1-M19 to huMucin17-His protein.

[0209] Figure 2 Example 6.1 describes the binding of recombinant antibodies M20-M27 to huMucin17-His protein using ELISA.

[0210] Figure 3 Example 7: Detection of cross-species (monkey) activity binding of candidate antibodies.

[0211] Figure 4 Example 12: Effect of the candidate antibody-drug conjugate on tumor volume in an animal model.

[0212] Figure 5 Results of the antibody endocytosis experiment described in Example 9.

[0213] Figures 6A-6B The FACS binding activity results of the humanized antibody described in Example 13.

[0214] Figure 7 Results of the killing effect of antibody-drug conjugate M21-17-Cpd3 on tumor cells.

[0215] Figures 8A-8C Experimental results of the side-kill effect of antibody-drug conjugate M21-17-Cpd3.

[0216] Figure 9 Results of inhibition of GSU tumor volume by antibody-drug conjugate M21-17-Cpd3.

[0217] Figure 10 Results of inhibition of NUGC4 tumor volume by antibody-drug conjugate M21-17-Cpd3.

[0218] Figure 11 Results of inhibition of SW480 tumor volume by antibody-drug conjugate M21-17-Cpd3.

[0219] Figure 12 SEC map of antibody-drug conjugate Trastuzumab-Cpd3.

[0220] Figure 13: HIC profile of antibody drug conjugate Trastuzumab-Cpd3.

[0221] Figure 14 : RP profile of antibody drug conjugate Trastuzumab-Cpd3. DETAILED DESCRIPTION

[0222] The following examples further illustrate the application, however, it is understood that the examples are described by way of illustration and not by way of limitation and that modifications can be made by those skilled in the art. Where specific conditions are not described in the examples, they are carried out under conventional conditions or as suggested by the manufacturer. Where the reagents or instruments used are not specified by the manufacturer, they are all conventional products that can be obtained commercially.

[0223] In some embodiments, the present application provides a method of making an anti-MUC17 antibody, wherein the method comprises culturing a host cell comprising a nucleic acid encoding an anti-MUC17 antibody or an expression vector comprising the nucleic acid under conditions suitable for the expression of the nucleic acid encoding the anti-MUC17 antibody, and optionally isolating the anti-MUC17 antibody. In a certain embodiment, the method further comprises recovering the anti-MUC17 antibody from the host cell (or host cell culture medium).

[0224] To recombinantly produce the anti-MUC17 antibodies of the present application, a nucleic acid encoding an anti-MUC17 antibody of the present application is first isolated and inserted into a vector for further cloning and / or expression in a host cell. Such nucleic acids are readily isolated and sequenced using conventional procedures, e.g., by employing oligonucleotide probes that are capable of binding specifically to the nucleic acids encoding the anti-MUC17 antibodies of the present application.

[0225] The anti-MUC17 antibodies of the present application, when prepared as described herein, can be purified by known art such as high performance liquid chromatography, ion exchange chromatography, gel electrophoresis, affinity chromatography, size exclusion chromatography, and the like. The actual conditions used to purify a particular protein also depend on factors such as net charge, hydrophobicity, hydrophilicity, and the like, and these will be apparent to those skilled in the art. The purity of the anti-MUC17 antibodies of the present application can be determined by any of a number of well-known analytical methods, including size exclusion chromatography, gel electrophoresis, high performance liquid chromatography, and the like.

[0226] The art describes a number of methods for conjugating cytotoxic agents or other therapeutic agents to antibodies. For example, the conjugation reaction can be effected in the antibody through the amino groups of lysine side chains and the amino groups of the N-terminus, the carboxyl groups of aspartic acid, glutamic acid and the C-terminus, or the activated cysteine sulfhydryl groups.

[0227] The sequence of the DNA molecule of the antibody or fragment thereof of the present application can be obtained by conventional techniques, such as by using a method of PCR amplification or screening of a genomic library. In addition, the coding sequences of the light chain and the heavy chain can be fused together to form a single-chain antibody.

[0228] Once the relevant sequence is obtained, the relevant sequence can be obtained in large quantities by a recombinant method. This is usually to clone into a vector, then into cells, and then to separate the relevant sequence from the proliferated host cells by conventional methods.

[0229] In addition, the relevant sequence can also be synthesized by artificial synthesis method, especially when the length of the sequence fragment is short. Generally, a long sequence fragment can be obtained by synthesizing a plurality of small fragments first and then connecting them.

[0230] Unless specifically indicated to the contrary, the practice of the present application will employ, unless otherwise indicated, conventional methods of the chemical, biochemical, organic, molecular biology, microbiology, recombinant DNA techniques, genetics, immunology, and cell biology within the skill of the art.

[0231] The compounds of the present disclosure can be confirmed by conventional methods well known to those skilled in the art, and if the present disclosure relates to the absolute configuration of the compound, the absolute configuration can be confirmed by conventional means in the art.

[0232] The compounds are named according to the principles of nomenclature in the art or using software naming, and commercially available compounds are named using the supplier's catalog name. When the name of the compound is inconsistent with the structure of the compound, the structure of the compound is used as the standard.

[0233] Example 1: Preparation and identification of raw materials

[0234] 1.1 Preparation and identification of Mucin 17 control antibody

[0235] Preparation of Mucin 17 control antibody: In this application, the anti-Mucin 17 antibody Tab1 is prepared as a positive control antibody according to the sequence disclosed in the patent WO / 2021 / 188851, and is synthesized by GenScript, and the expression plasmid is constructed. The specific heavy chain and light chain sequences of Tab-1 are shown in SEQ ID NO: 218 and SEQ ID NO: 219, respectively. Expi293F (Gibco) cells are transiently transfected, and after 6 days of culture, the supernatant is collected and purified by Protein A affinity column (Cytiva).

[0236] Mucin17 control antibody identification: The activity of the prepared positive control antibody Tab-1 was detected using a purchased Mucin17-His antigen protein (Acro Biosysterm, MU7-H52H3), and the specific method was as follows: Mucin17-His (0.5 μg / mL, 100 μL / well) was coated on a 96-well ELISA plate at 4°C for 16 hours. After washing the plate once, 1% BSA prepared with PBS was used for blocking for 1 hour. After washing the plate 3 times, the control antibody Tab-1 diluted with PBS was added and incubated at room temperature for 1 hour. After washing, the secondary antibody: Anti-Human IgG (H&L) (GOAT) Antibody Peroxidase Conjugated (Rockland, 609-103-123) diluted with PBS was added and incubated at 37°C for 1 hour. After washing 3 times, TMB was added at room temperature (25°C) for 5-20 min. After stopping the color development, the data was read by an enzyme-labeled instrument (Thermo Fisher (Shanghai) Instrument Co., Ltd., Multiskan FC) at OD450. The results showed that the expressed control antibody Tab-1 bound to Mucin17-His protein in a concentration gradient-dependent manner, and the EC 50 was 3.545 ng / ml, indicating that the control antibody had normal anti-Mucin17 activity.

[0237] Table 1: OD450 reading of control antibody Elisa binding detection

[0238] Coated antigen 0.5 pg / mL Muc17-His Primary antibody concentration (ng / mL) Tab 1 1000.000 2.074 333.333 2.024 111.111 2.227 37.037 2.038 12.346 1.730 4.115 1.186 1.372 0.560 0.457 0.253 0.152 0.121 0.051 0.072 0.017 0.057 0.000 0.050 EC50 (ng / ml) 3.545

[0239] wherein the sequences of the heavy chain and the light chain of Tab1 are shown in SEQ ID NO: 218 and SEQ ID NO: 219.

[0240] 1.2 Preparation of Mucin17 immunogen

[0241] Mucin 17 extracellular region ((NP_001035194.1) was connected with a mouse Fc fragment (SEQ ID NO: 220), and a plasmid was constructed by commissioning Huaguo Bio (HOEKBIO). Expi293F cells were transiently transfected, and after 6 days of culture, the supernatant was collected, purified by Protein A affinity column, and the eluate was collected. The eluate was replaced by dialysis at 2-8°C for 16 hours, and the antibody in the dialysis bag was collected and stored.

[0242] Antigen identification: Mucin17-Mouse FC (0.5 μg / mL, 100 μL / well) protein was coated on a 96-well ELISA plate, and Tab 1 antibody was used for detection. The specific implementation method is shown in Example 1.1. The results showed that the expressed Mucin17-Mouse FC could be bound by Tab-1 in a concentration gradient-dependent manner, and the EC50 5.299 ng / ml,

[0243]

[0244]

[0245] Example 2: Construction and identification of cell lines overexpressing human and monkey Mucin 17

[0246] Construction of CHO-K1 (ECACC) cell lines overexpressing human and monkey Mucin 17 (hereinafter referred to as huMucin17-CHO K1; cynoMucin17-CHO K1): The coding nucleic acid sequences of human Mucin 17 protein (NP_001035194.1) and monkey Mucin 17 protein (XM_045389603.1) extracellular domain fragment SEQ ID NO:221, 222 were constructed into proprietary lentivirus vector (GenScript Biotech). Then, the lentivirus plasmid was transfected into HEK293T cells (Clontech, 632180) for virus packaging, and after 4 days of culture, the cell culture supernatant was collected. Using the harvested lentivirus supernatant, CHO-K1 cells were transfected, and puromycin was used as a screening pressure. After 3 days of screening, the antigen expression of the cell pool was screened by flow cytometry. After obtaining the positive cell pool, single cell cloning of the positive cell pool was selected by limited dilution or flow sorting. After single clone selection, the cell lines were cultured for another week, and the antigen expression of the cell lines was identified.

[0247] Flow cytometry identification of cell line antigen expression: The logarithmic growth period of the above cell line cells was digested and plated into 96-well plates, washed with FACS buffer (99% DPBS + 1% FBS), and then added with primary antibody Tab 1 (10 ng / ml), incubated at 4°C for 60 min; after washing, the prepared fluorescent secondary antibody PE Goat anti-mouse IgG antibody (BioLegend, Cat. NO: 410708) was added, and incubated at 4°C for 20 min; finally, detection was performed by flow cytometry (BD FACS Celesta). The detection results showed that the huMucin17-CHO K1 and cynoMucin17-CHO K1 cell lines with high surface expression of human and monkey Mucin 17 were obtained.

[0248]

[0249] Example 3: Animal immunization and hybridoma cell screening

[0250] 3.1 Immunization scheme

[0251] Using the Mucin17-His or Mucin17-Mouse Fc protein antigen obtained in Example 2; Mucin17 expression plasmid-DNA antigen; Mucin17 overexpression CHO-K1 cell multiple immunogens, alone or alternately used to immunize different species of mice (Balb / c, SJL, A / J). According to the type of selected antigen, the mice were immunized by subcutaneous / intraperitoneal injection or gene gun injection, once every two weeks, for a total of 3-5 times. One week after the last immunization, the mice were bled for immune titer detection, and finally, the above antigens were used for a booster immunization.

[0252] 3.2 Detection of antibody titers in mouse serum after immunization

[0253] The antigen was diluted to 0.5 μg / mL with coating solution, mixed well, and then added to the designed strip, 100 μL / well, and placed in a 4°C refrigerator overnight for 16 h. The coating solution was discarded, and the plate was washed once and dried. 150 μL of blocking solution (1% BSA prepared with PBS solution) was added to each well, and the enzyme-labeled plate was placed in a 37°C incubator (Shanghai Yiheng Scientific Instrument Co., Ltd.) for 1 h. The enzyme-labeled plate was removed, and the blocking solution was discarded. The mouse serum was gradient-diluted and added to the well plate at 100 μL / well. Incubate in a 37°C incubator for 1 h. Remove the enzyme-labeled plate, discard the inner solution, and wash the plate 3 times. Add 100 μL of diluted enzyme-labeled secondary antibody to each well and incubate in a 37°C incubator for 0.5 h. Remove the enzyme-labeled plate, discard the inner solution, and wash the plate 3 times. After drying, add 100 ul of color developing solution, and react at 25°C for 15 minutes. Add 50 μL of 1M HCl to stop the reaction, and use an enzyme-labeled instrument (Thermo Fisher (Shanghai) Instrument Co., Ltd., Multiskan FC) to read OD450nm. Select mice with an immune titer greater than 512000 for hybridoma fusion.

[0254] 3.3 Hybridoma cell fusion

[0255] Prepare a myeloma cell (SP2 / 0, Chinese Academy of Sciences Cell Bank) suspension, and use a hemocytometer to calculate the total amount of SP2 / 0 cells. Prepare a spleen cell suspension, and use a hemocytometer to calculate the total amount of cells. Mix the myeloma cells and spleen cells in a 1:2 ratio, and perform cell electrofusion. After cell fusion, mix the fused cells with HAT (Sigma) medium to prepare a cell suspension, and then use a multichannel pipette to plate the cells into a 96-well cell plate. Place the fused 96-well cell culture plate in a 5.5% carbon dioxide incubator and incubate at 37°C. After culturing the hybridoma cells, screen the supernatant for parent clones, and screen the cell culture supernatant for protein and cell levels of antigen binding ability. Refer to Examples 1 and 2 for specific screening methods.

[0256] 3.4 Hybridoma subcloning screening

[0257] After the first round of mother clone screening, the screened mother clone cell strains were reserved. The cells in the subcloning wells were gently blown and mixed with a pipette. 20 μl of the cell suspension was taken and mixed with 20 μl of trypan blue solution. 20 μl was taken and counted with a hemocytometer. The cells were spread in a 96-well cell culture plate using 1x HT replacement feeder cell medium by limiting dilution method. The 96-well plate was labeled and placed in a CO2incubator for culture. The subcloned 96-well plate was placed in a 37°C, 5.5% CO2incubator for culture. After 7 days of culture, the supernatant was taken for monoclonal screening. The specific screening method is described in Examples 1 and 2.

[0258] After subcloning screening, 28 antibodies obtained by selection were sequenced, and the sequences of the antibodies are shown in Table 2. The CDR sequences were determined by using the Kabat and AbM definitions of CDR.

[0259] Table 2. Amino acid sequences of CDR regions of 28 antibodies

[0260]

[0261]

[0262]

[0263]

[0264]

[0265]

[0266] Example 4: Antibody construction, expression and purification

[0267] 4.1 Plasmid construction

[0268] The VHand VLcoding sequences in the Fab sequences of the selected 28 monoclonal antibodies were connected to the coding sequence of the heavy chain constant region of human IgG1 (SEQ ID NO: 223) to obtain the heavy chain coding sequence of the human-mouse chimeric antibody, and the VLcoding sequence in the Fab sequence was connected to the coding sequence of the Kappa type of human light chain constant region (CL) (SEQ ID NO: 224) to obtain the light chain coding sequence of the human-mouse chimeric antibody. The coding sequences of the antibody heavy chain and light chain were inserted into a special eukaryotic expression vector plasmid (PCDNA3.4), respectively, transformed into E. coli DH5α, and cultured at 37°C overnight. Endotoxin-free plasmid extraction was performed using an endotoxin-free plasmid extraction kit (Jinsai Biotechnology) to obtain endotoxin-free antibody plasmids for eukaryotic expression.

[0269] 4.2 Expression and purification of antibodies

[0270] Antibody expression: The full-length sequences of the antibodies obtained above were expressed by Expi293F transient expression system (Thermo Fisher, A14635) or ExpiCHO-S transient expression system (Thermo Fisher, A29133), the specific method refers to the corresponding kit operation manual of the supplier.

[0271] Antibody purification: The supernatant was collected and subjected to antibody affinity purification using Protein A Magnetic Beads (Genscript). Protein A Magnetic Beads were added to the supernatant and incubated at room temperature for 2 hours. After incubation, the Protein A Magnetic Beads were equilibrated with Binding buffer (PBS) for 10 column volumes. The Protein A Magnetic Beads were eluted with Elution buffer (0.1M Glycine, pH 2.5) and the eluate was collected. Neutralizing buffer (0.1M Tris pH 9.0) was added to the eluate at a volume of 1 / 10 of the eluate to adjust the pH to neutral. After elution, the Protein A Magnetic Beads were equilibrated with Binding buffer for 10 column volumes. The eluted and neutralized antibodies were loaded into dialysis bags and dialyzed against Dialysis buffer (PBS) at room temperature for 2 hours, and then against Dialysis buffer at 2-8°C for 16 hours. The antibodies were transferred from the dialysis bags to centrifuge tubes and the volume was recorded.

[0272] Example 5: Detection of the physicochemical properties of the antibodies

[0273] In this example, the relative molecular weight and purity of the candidate antibodies were detected by SDS-PAGE and SEC-HPLC.

[0274] Antibody SDS-PAGE identification

[0275] Non-reducing solution preparation: 3μg of each obtained antibody was added to 2x SDS loading buffer, heated at 99°C for 5min, and after cooling to room temperature, centrifuged at 12000rpm for 5min to collect the supernatant.

[0276] Reducing solution preparation: 3μg of each obtained antibody was added to 2x SDS loading buffer and 5mM DTT, heated at 99°C for 5min, and after cooling to room temperature, centrifuged immediately. The supernatant was added to Bis-tris 4-15% gradient gel (Genscript) for gel electrophoresis and protein bands were visualized by Coomassie blue staining.

[0277] The Odyssey 9140 dual-color infrared laser imaging system was used to image protein gels with chromogenic protein bands (decolorized with decolorizing solution until the gel background was transparent), and the purity of reduced and non-reduced bands was calculated using the built-in software.

[0278] The experimental results show that the bands of each antibody on the non-reducing gel are around 150kD, while the bands on the reducing gel are around 55kD and 25kD, respectively, which are in line with the expected size, as shown in Table 3.

[0279] Table 3. Expression levels and physicochemical properties of candidate antibodies

[0280] Clone number Transfection volume (mL) Total amount of antibody (mg) Antibody purity (SDS-PAGE) M1 35 0.36 99％ M2 35 0.35 99％ M3 35 0.21 99％ M4 35 0.07 99％ M5 35 0.11 99％ M6 35 0.64 99％ M7 35 0.12 99％ M8 35 0.32 99％ M9 35 0.43 99％ M10 35 0.56 99％ M11 35 0.09 99％ M12 35 0.10 99％ M13 35 0.08 98％ M14 35 1.18 99％ M15 35 0.49 98％ M16 35 0.14 97％ M17 35 0.50 99％ M18 35 1.51 96％ M19 35 0.32 99％ M20 35 0.26 99％ M21 35 1.01 99％ M22 35 0.36 99％ M23 35 0.72 99％ M24 35 1.00 99％ M25 35 0.23 99％ M26 35 0.50 99％ M27 35 0.29 99％

[0281] Example 6: Detection of Antibody Antigen Binding Activity

[0282] In this embodiment, the binding of the expressed antibody to the human Mucin17 antigen protein huMucin17-His was detected using the ELISA method, and the binding of the expressed antibody to human Mucin17 overexpressing cells huMucin17-CHO K1 was detected using the FACS method.

[0283] 6.1 ELISA-based detection of antibody binding affinity to antigen protein huMucin17-His

[0284] 96-well ELISA plates were coated with 0.5 μg / mL human Mucin17 antigen protein (30 μL / well) and incubated overnight at 4°C. The next day, the plates were washed three times with PBST, blocked with 5% skim milk for 2 h, washed three times with PBST, and then serially diluted (1 μg / mL, 3-fold serial dilution, 9 concentration points) of each antibody and positive control antibody Tab1 were added and incubated for 1 h. After washing three times with PBST, secondary antibody Goat-anti-human Fc-HRP (Abcam, Ab97225) was added and incubated for 30 min. After incubation, the plates were washed six times with PBST and TMB was added for color development. The reaction was stopped at 25°C for 15 min, and the absorbance was read at OD450 using a microplate reader.

[0285] The results are as follows Figures 1-2 As shown, the antibody and antigen protein huMucin17 obtained in this application both have good binding ability.

[0286] 6.2 Detection of antibody binding ability to huMucin17-CHO K1 cells based on FACS

[0287] In this embodiment, the antibody binding activity was evaluated using human Mucin17 overexpressing cells huMucin17-CHO K1.

[0288] Specifically, the following method was used: huMucin17-CHO K1 cells in logarithmic growth phase were prepared into a single cell suspension, the density was adjusted to 2 x 105cells / mL, 50 μL was added to each well of a 96-well round-bottom plate, centrifuged at 4°C and 300 g, and the supernatant was removed. The detection antibody and the positive control antibody Tab 1 were added to the corresponding wells, mixed, and incubated at 4°C for 40 min. After washing the cell mixture after incubation 3 times, 100 μL of diluted secondary antibody Goat F(ab')2 Anti-Human IgG-Fc was added, and incubated at 4°C in the dark for 30 min. After washing 3 times, the mixture was detected by flow cytometry, and the mean fluorescence intensity value (MFI) was read. 6

[0289] The results are shown in Table 4. The tested antibodies had good binding ability to both the overexpression cells and the huMucin17-CHO K1 cells.

[0290] Table 4. FACS detection of binding of recombinant antibodies to huMucin17-CHO K1 cells

[0291]

[0292]

[0293] Example 7: Detection of antibody species cross-reactivity

[0294] In this example, the cross-reactivity of each antibody obtained in the present application to human / monkey species antigens was detected. The antibody prepared in Example 4 and the CHO K1 cell strain expressing monkey Mucin17 were used to determine the binding ability. The specific method is described in Example 6.

[0295] Results Figure 3 As shown in Table 5, all the antibodies obtained in the present application can bind to the cell strain expressing the monkey-derived protein, showing good human / monkey cross-reactivity.

[0296] Example 8: Detection of antibody affinity

[0297] ​In this example, the affinity of the antibody obtained in the application and the positive control antibody Tab-1 to the antigen protein huMucin17-His was detected based on the Biacore device. The running buffer HBS-EP+(10mM HEPES, 150mM NaCl, 3mM EDTA and 0.05% P20, pH 7.4) (BR100826, GE) was used to dilute the anti-Mucin17 antibody sample to 10ug / mL, and the human Mucin17 antigen protein was diluted with HBS-EP+ buffer to a concentration of 0.03125nM-2nM in a 2-fold gradient. The anti-Mucin17 antibody was captured by Protein A biosensor chip (BR100354, GE) with a capture time of 60s and a flow rate of 30uL / min. Then the human Mucin17 sample diluted to different concentrations was injected in turn from zero to the highest concentration, with a binding time of 120s, a dissociation time of 360s and a flow rate of 30uL / min. The reaction signal was detected in real time by Biacore X100 instrument to obtain the binding and dissociation curves. After the dissociation was completed in each experimental cycle, the Protein A biosensor chip was washed and regenerated with 10mM Glycine-HCl (pH 1.5) (29127558, GE) with a regeneration time of 30s and a flow rate of 30uL / min. Parameter fitting: the experiment was run in single cycle mode, with analysis time as the abscissa and response value as the ordinate. After the obtained data was double-reference subtracted, it was fitted by BIAcore X100 (GE) analysis software, and the fitting model used was 1:1 Langmuir binding model to determine the dissociation constant and other affinity indexes. The results are shown in Table 5, and the antibody obtained in the application has higher affinity than the positive control antibody Tab1.

[0298] Table 5. KD value of antibody

[0299] Clone number ka (1 / Ms) kd (1 / s) KD (M) M2 1505992 0.000679 4.51E-10 M11 268485.6 0.001788 6.66E-09 M18 760010.8 0.001679 2.21E-09 M20 1141416 0.001264 1.11E-09 M21 111017.4 0.001368 1.23E-08 M22 1459630 0.00309 2.12E-09 M23 1443877 0.002999 2.08E-09 M24 2927275 0.010404 3.55E-09 M25 1489585 0.002639 1.77E-09 M26 333926.7 0.001269 3.80E-09 M27 843844.7 0.001579 1.87E-09 Tab 1 360533.2 0.002499 6.93E-09

[0300] Example 9: Antibody endocytosis detection

[0301] The ability of the recombinant antibody to be endocytosed by the target cells (huMucin17-CHOK1) was detected by Incucyte (Sartorius, Incucyte S3). The target cells were digested, collected and resuspended with experimental buffer, and the cell suspension was transferred into a 96-well experimental plate at 5000 cells per well. The experimental plate was incubated in a cell incubator (37°C / 5% CO2) overnight. The test / control working solution and the labeling reagent working solution (4x) were prepared with experimental buffer. The test / control working solution and the labeling reagent working solution were mixed at a 1:3 molar ratio, 1:1 volume ratio, and incubated in a cell incubator (37°C / 5% CO2) for 15 minutes to allow full coupling. The coupling mixture working solution was transferred to the corresponding wells of the 96-well experimental plate. The experimental plate was incubated in a cell incubator (37°C / 5% CO2) for a certain period of time, and the Incucyte instrument corresponding to the detection wavelength was used to take pictures at the specified time points.

[0302] The experimental raw data and results were analyzed and exported via Live-Cell Analysis System. The analysis results were expressed as: Total Red Object Integrated Intensity (GCU x pm 2 / Image). The raw data were further analyzed and plotted using Microsoft Office Excel 2016 and GraphPad Prism 6 software. The experimental results are shown in Figure 5 , which show that the tested molecules all have good endocytosis effects, and the antibody endocytosis also increases with time.

[0303] Example 10: Preparation method of linker-toxin

[0304] The preparation of the linkers and toxins used in the present application, such as deruxtecan, GGFG-Dxd, Dxd, GGFG-Exd, Val-Ala-PAB, refers to WO2014057687, WO1997046260, CN101795711, the entire contents of which are incorporated herein by reference.

[0305] The linker-toxin Cpd3 used in the present application and its preparation method are as follows:

[0306]

[0307] Step 1: (9H-fluorene-9-yl)methyl((S)-3-methyl-1-(((S)-1-((4-(((((4-nitrophenoxy)carbonyl)oxy)methyl)phenyl)amino)-1-oxopropyl-2-yl)amino)-1-oxobutyl-2-yl)carbamate (3-1, 2.5 g, 3.6 mmol), (1S,9S)-1-amino-9-ethyl-5-fluoro-9-hydroxy-4-methyl- 1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-10,13-dione (3-2, 1.6 g, 3.6 mmol) and N,N-diisopropylethylamine (965 mg, 7.5 mmol) were dissolved in anhydrous N,N-dimethylformamide (20 mL), and the reaction mixture was stirred at room temperature for 16 hours. The reaction was monitored by LCMS until completion, yielding 4-((S)-2-(S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutyrylamino)propionylamino)benzyl((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamate (3-3). LCMS (ESI) m / z: 977.3 [M+H] + .

[0308] Step 2 Hexahydropyridine (464 mg, 5.4 mmol) was added directly to the reaction solution from step 1, and the reaction solution was stirred at room temperature for 2 hours. The reaction was monitored by LCMS to confirm completion. The reaction solution was then purified by C18 column chromatography to obtain 4-((S)-2-((S)-2-amino-3-methylbutyrylamino)propionylamino)benzyl((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamate (3-4) (1.8 g). LCMS (ESI) m / z: 755.2 [M+H] + .

[0309] Step 3: 1-(9H-fluoren-9-yl)-3-oxo-2,7,10,13,16,19,22,25,28-nonaoxa-4-aza-tri- contane-31-oic acid (3-5, 1.8 g, 2.38 mmol), 4-((S)-2-((S)-2-amino-3-methylbutanoyl- amino)propanoylamino)benzyl ((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl- 10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2- b]quinolin-1-yl)carbamate (3-4, 1.8 g, 2.38 mmol), N,N-diisopropyl ethylamine (614 mg, 4.76 mmol) were dissolved in anhydrous N,N-dimethylformamide (10 mL), then stirred at room temperature for 3 h, the reaction was monitored by LCMS to be completed, to give 4-((33S,36S)-1-(9H-fluoren-9-yl)-33-isopropyl-36-methyl-3,31,34-trioxo- 2,7,10,13,16,19,25,25,28-nonaoxa-4,32,35-triaza-tritriacontane-37-carbonyl)- benzyl ((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15- hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)carbamate (3-6). LCMS (ESI) m / z: 1400.5 [M+H] + .

[0310] Step 4 : To the reaction solution of step 3, hexahydropyridine (409 mg, 4.76 mmol) was directly added, and the reaction solution was stirred at room temperature for 2 h. The raw material was monitored by LCMS to be completed, and the reaction solution was directly purified by C18 column chromatography to give 4-((29S,32S)-1-amino-29-isopropyl-32-methyl-27,30-dioxo-3,6,9,12,15,18,21,24- octaoxa-28,31-diaza-tritriacontane-33-carbonyl)benzyl ((1S,9S)-9-ethyl-5-fluoro-9- hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7] indolizino[1,2-b]quinolin-1-yl)carbamate (3-7) (1.2 g). LCMS (ESI) m / z: 1178.4 [M+H] + .

[0311] Step 5: 4-((29S,32S)-1-amino-29-isopropyl-32-methyl-27,30-dioxo-3,6,9,12,15,18,21,24- octaoxa-28,31-diazatri-33-ylamino)benzyl ((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4- methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)carbamate (3-7, 20 mg, 0.017 mmol), 2-(methylsulfonyl)pyrimidine-5-carboxylic acid (3-8, 13.7 mg, 0.068 mmol), 4-(4,6-dimethoxytriazin-2-yl)-4-methylmorpholine tetrafluoroborate (22.3 mg, 0.068 mmol) were dissolved in anhydrous N,N-dimethylacetamide (3 mL) and the reaction was stirred at room temperature for 16 hours. Purification by C18 column chromatography gave 4-((31S,34S)-31-isopropyl-34-methyl-1-(2-(methylsulfonyl)pyrimidin-5-yl)-1,29,32-trioxo-5,8,11,14,17,20,23,26-octaoxa-2,30,33-triazatri-35-ylamino-benzyl (1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)carbamate (2.3 mg, yield: 10%) as Cpd 3. 1 H NMR (400 MHz, DMSO-d6) δ 9.95 (s, 1H), 9.37 (s, 1H), 9.14 (s, 1H), 8.46 (s, 1H), 8.21 (d, J = 6.8 Hz, 1H), 8.06 (d, J = 7.8 Hz, 1H), 7.89 (d, J = 8.3 Hz, 1H), 7.78 (d, J = 10.9 Hz, 1H), 7.60 (d, J = 8.4 Hz, 2H), 7.36 (d, J = 8.1 Hz, 2H), 7.31 (s, 1H), 6.53 (s, 1H), 5.45 (s, 2H), 5.28 (s, 3H), 5.08 (s, 2H), 4.42 - 4.34 (m, 1H), 4.22 - 4.17 (m, 1H), 3.62 - 3.53 (m, 10H), 3.52 - 3.46 (m, 28H), 2.38 (s, 3H), 2.33 (s, 1H), 2.20 (s, 3H), 2.01 - 1.81 (m, 4H), 1.30 (d, J = 7.0 Hz, 3H), 0.90 - 0.80 (m, 9H).

[0312] The linker-toxin Cpd5 used in the present application and the method for preparing the same are as follows:

[0313]

[0314] Step 1 : 1-(9H-fluoren-9-yl)-3,6-dioxo-2,9-dioxa-4,7-diazoundecanoic acid (5-1, 440 mg, 1.15 mmol), (1S,9S)-1-amino-9-ethyl-5-fluoro-9-hydroxy-4-methyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-10,13-dione (500 mg, 1.15 mmol) were dissolved in anhydrous N,N-dimethylformamide (20 mL), N,N'-dicyclohexylcarbodiimide (308 mg, 1.5 mmol), N-hydroxysuccinimide (132 mg, 1.15 mmol) and N,N-diisopropylethylamine (148 mg, 1.15 mmol) were added under ice bath, and then the reaction solution was stirred at room temperature for 16 hours. The reaction was monitored by LCMS, and the organic phase was concentrated by extraction with ethyl acetate and water, and purified by silica gel column with a system of methanol and dichloromethane (1:20) to obtain (9H-fluoren-9-yl)methyl (2-(((2-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-2-oxoethoxy)methyl)amino)-2-oxoethyl)carbamate (5-3, 650 mg). LCMS (ESI) m / z: 802.3 [M+H] + .

[0315] Step 2: (9H-Fluoren-9-yl)methyl (2-(((2-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl- 10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2- b]quinolin-1-yl)amino)-2-oxoethoxy)methyl)amino)-2-oxoethyl)carbamate (5-3, 650 mg, 0.81 mmol) was dissolved in N,N-dimethylformamide (10 mL), then hexahydropyridine (69 mg, 5.4 mmol) was added, the reaction was stirred at room temperature for 2 hours. LCMS monitoring showed that the reaction was completed, the reaction was directly purified by C18 column chromatography to obtain 2-amino-N-((2-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl- 10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2- b]quinolin-1-yl)amino)-2-oxoethoxy)methyl)acetamide (5-4, 420 mg). LCMS (ESI) m / z: 580.2 [M+H] + .

[0316] Step 3: 2-Amino-N-((2-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo- 2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b] quinolin-1-yl)amino)-2-oxoethoxy)methyl)acetamide (5-4, 420 mg, 0.73 mmol), (((9H-fluoren-9-yl)methoxy)carbonyl)glycylglycyl-L-phenylalanine (5-5, 370 mg, 0.73 mmol) were dissolved in anhydrous N,N-dimethylformamide (10 mL), N,N'-dicyclohexylcarbodiimide (195 mg, 0.95 mmol), N-hydroxysuccinimide (84 mg, 0.73 mmol) and N,N-diisopropylethylamine (94 mg, 0.73 mmol) were added under ice-bath, then the reaction was stirred at room temperature for 16 hours. The reaction was monitored by LCMS, the organic phase was concentrated by extraction with ethyl acetate and water, and purified by silica gel column with methanol and dichloromethane system (1:15) to give (9H-fluoren-9-yl)methyl ((S)-10-benzyl-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-1,6,9,12,15-pentaoxo-3-oxa-5,8,11,14-tetraazahexadec-16-yl)carbamate (5-6, 550 mg). LCMS (ESI) m / z: 1063.5 [M+H] + .

[0317] Step 4: (9H-Fluoren-9-yl)methyl ((S)-10-benzyl-l-(((lS,9S)-9-ethyl-5-fluoro-9-hydroxy-4- methyl-10, 13-dioxo-2,3,9,10,13,15-hexahydro-lH, 12H-benzo[de]pyrano[3',4':6,7]indolizino[l,2-b]quinolin-l-yl)amino)-l,6,9,12,15-pentaoxo-3-oxa-5,8,11,14-tetraazahexadecan-16-yl)carbamate (5-6, 550 mg, 0.52 mmol) was dissolved in N,N-dimethylformamide (5 mL), then hexahydropyridine (44 mg, 0.52 mmol) was added, the reaction was stirred at room temperature for 2 hours. LCMS monitoring showed that the reaction was completed, the reaction was directly purified by C18 column chromatography to obtain ((S)-10-benzyl-l-(((lS,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10, 13-dioxo-2,3,9,10,13,15-hexahydro-lH, 12H-benzo[de]pyrano[3',4':6,7]indolizino[l,2-b]quinolin-l-yl)amino)-l,6,9,12,15-pentaoxo-3-oxa-5,8,11,14-tetraazahexadecan-16-yl)amine (5-7, 400 mg). LCMS (ESI) m / z: 841.4 [M+H] + .

[0318] Step 5: 1-(9H-fluoren-9-yl)-3-oxo-2,7,10,13,16,19,22,25,28-nonaoxa-4-aza-tri- contane-31-oic acid (347 mg, 0.52 mmol), HATU (274 mg, 0.72 mmol) and N,N- diisopropyl ethylamine (124 mg, 0.96 mmol) were dissolved in anhydrous N,N- dimethylformamide (5 mL), then stirred at room temperature for 1 h, then ((S)-10- benzyl-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15- hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)- 1,6,9,12,15-pentaoxo-3-oxa-5,8,11,14-tetraazahexadecan-16-yl)amine (5-7, 400 mg, 0.48 mmol) was added and stirred at room temperature for 16 h, the reaction was monitored by LCMS and the reaction was completed, the reaction solution was directly purified by C18 column chromatography to obtain (9H-fluoren-9-yl)methyl ((S)-10-benzyl-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-1,6,9,12,15,18-hexaoxo-3,21,24,27,30,33,36,39,42-nonaoxa-5,8,11,14,17-pentaazatetra- contan-44-yl)carbamate (5-9, 350 mg). LCMS (ESI) m / z: 1486.6 [M+H] + .

[0319] Step 6: (9H-Fluoren-9-yl)methyl ((S)-10-benzyl-l-(((lS,9S)-9-ethyl-5-fluoro-9-hydroxy-4- methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-lH,12H-benzo[de]pyrano[3',4':6,7]indolizino[l,2-b]quinolin-l-yl)amino)-l,6,9,12,15,18-hexaoxo-3,21,24,27,30,33,36,39,42-nonaoxa-5,8,11,14,17-pentaazatetratetracontan-44-yl)carbamate (5-9, 350 mg, 0.24 mmol) was dissolved in N,N-dimethylformamide (5 mL), then hexahydropyridine (20 mg, 0.24 mmol) was added, the reaction was stirred at room temperature for 2 hours. LCMS monitoring of the reaction was completed, the reaction was directly purified by C18 column chromatography to obtain l-amino-N-((S)-10-benzyl-l-(((lS,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-lH,12H-benzo[de]pyrano[3',4':6,7]indolizino[l,2-b]quinolin-l-yl)amino)-l,6,9,12,15-pentaoxo-3-oxa-5,8,11,14-tetraazahexadecan-16-yl)-3,6,9,12,15,18,21,24-octaoxaheptacosan-27-amide (5-10, 200 mg). LCMS (ESI) m / z: 1264.6 [M+H] + .

[0320] Step 7: 1 -amino- N-((S)-10-benzyl- 1 -((( 1 S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl- 10,13-dioxo-2,3,9,10,13,15-hexahydro-lH, 12H-benzo[de]pyrano[3',4':6,7]indolizino[l,2-b]quinolin-l-yl)amino)- 1,6,9, 12, 15-pentaoxo-3-oxa-5,8, 11, 14-tetraazahexadecan- 16-yl)-3,6,9, 12, 15, 18,21,24-octaoxaheptacosan-27-amide (5-10, 100 mg, 0.079 mmol), 2-(methylsulfonyl)pyrimidine-5-carboxylic acid (64 mg, 0.3 mmol), 2-chloro-4,6-dimethoxy-l,3,5-triazine (53 mg, 0.3 mmol), N-methylmorpholine (182 mg, 1.8 mmol) were dissolved in anhydrous N,N-dimethylacetamide (5 mL), the reaction was stirred at room temperature for 16 hours. Purification by C18 column chromatography to give N-((S)-10-benzyl-l-(((lS,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl- 10,13-dioxo-2,3,9,10,13,15-hexahydro-lH, 12H-benzo[de]pyrano[3',4':6,7]indolizino[l,2-b]quinolin-l-yl)amino)-l,6,9, 12, 15, 18-hexaoxo-3,21,24,27,30,33,36,39,42-nonaoxa-5,8, 11, 14, 17-pentaazatetra-decan-44-yl)-2-(methylsulfonyl)pyrimidine-5-carboxamide (5 mg), Cpd 5. LCMS (ESI) m / z: 1448.6 [M+H] + .

[0321] 1H NMR (400 MHz, DMSO-d6) δ 9.37 (d, J = 2.1 Hz, 2H), 9.12 (s, 1H), 8.63 (s, 1H), 8.51 (d, J = 8.6 Hz, 1H), 8.29 (s, 1H), 8.21 - 8.07 (m, 2H), 7.99 (s, 1H), 7.78 (d, J = 10.9 Hz, 1H), 7.31 (s, 1H), 7.21 (t, J = 15.2 Hz, 5H), 6.52 (s, 1H), 5.60 (s, 1H), 5.42 (s, 2H), 5.20 (s, 2H), 4.64 (d, J = 6.3 Hz, 2H), 4.47 (s, 1H), 4.02 (s, 2H), 3.76 - 3.66 (m, 4H), 3.59 - 3.53 (m, 8H), 3.51 - 3.45 (m, 29H), 2.39 (s, 5H), 2.18 (s, 2H), 2.00 (d, J = 7.9 Hz, 1H), 1.84 (dd, J = 15.4, 7.8 Hz, 2H), 1.33 - 1.20 (m, 5H), 1.12 (d, J = 6.6 Hz, 1H), 0.87 (t, J = 6.3 Hz, 3H).

[0322] The linker-toxin Cpd6 used in the present application and its method of preparation are as follows:

[0323]

[0324] Step 1: ((9H-fluoren-9-yl)methoxy)carbonyl)-L-valyl-L-alanine (6-1, 943 mg, 2.3 mmol), (1S,9S)-1-amino-9-ethyl-5-fluoro-9-hydroxy-4-methyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-10,13-dione (1g, 2.3 mmol) were dissolved in anhydrous N,N-dimethylformamide (20 mL), N,N'-dicyclohexylcarbodiimide (616 mg, 3 mmol), N-hydroxysuccinimide (264 mg, 2.3 mmol) and N,N-diisopropylethylamine (297 mg, 2.3 mmol) were added under ice bath, then the reaction was stirred at room temperature for 16 hours. The reaction was monitored by LCMS, the organic phase was concentrated by extraction with ethyl acetate and water, and purified by silica gel column with methanol and dichloromethane system (1:20) to give (9H-fluoren-9-yl)methyl ((S)-1-(((S)-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate (6-3, 730 mg). LCMS (ESI) m / z: 828.3 [M+H] + .

[0325] Step 2: (9H-Fluoren-9-yl)methyl ((S)-1-(((S)-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl- 10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2- b]quinolin-1-yl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate (6-3, 730 mg, 0.88 mmol) was dissolved in N,N-dimethylformamide (10 mL), then hexahydropyridine (75 mg, 0.88 mmol) was added, the reaction was stirred at room temperature for 2 hours. LCMS monitoring showed that the reaction was completed, the reaction was directly purified by C18 column chromatography to obtain (S)-2-amino-N-((S)-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo- 2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)- 1-oxopropan-2-yl)-3-methylbutanamide (6-4, 450 mg). LCMS (ESI) m / z: 606.2 [M+H] + .

[0326] Step 3: 1-(9H-fluoren-9-yl)-3-oxo-2,7,10,13,16,19,22,25,28-nonaoxa-4-azatriacontan-31-oic acid (592 mg, 0.89 mmol), HATU (422 mg, 1.11 mmol) and N,N-diisopropyl ethylamine (143 mg, 1.11 mmol) were dissolved in anhydrous N,N-dimethylformamide (5 mL), stirred at room temperature for 1 h, then (S)-2-amino-N-((S)-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-1-oxopropan-2-yl)-3-methylbutanamide (450 mg, 0.74 mmol) was added and stirred at room temperature for 4 h, the reaction was monitored by LCMS and the reaction was completed, the reaction solution was directly purified by C18 column chromatography to give (9H-fluoren-9-yl)methyl ((29S,32S)-33-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-29-isopropyl-32-methyl-27,30,33-trioxo-3,6,9,12,15,18,21,24-octaoxa-28,31-diazatriacontyl)carbamate (6-5, 400 mg). LCMS (ESI) m / z: 1251.6 [M+H] + .

[0327] Step 4: (9H-Fluoren-9-yl)methyl ((29S,32S)-33-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4- methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-29-isopropyl-32-methyl-27,30,33-trioxo- 3,6,9,12,15,18,21,24-octaoxa-28,31-diazatriacontan-1-yl)carbamate (6-5, 400 mg, 0.32 mmol) was dissolved in N,N-dimethylformamide (5 mL), then hexahydropyridine (27 mg, 0.32 mmol) was added, the reaction was stirred at room temperature for 2 hours. LCMS monitoring showed that the raw material was completely reacted, the reaction was directly purified by C18 column chromatography to obtain 1-amino-N-((S)-1-(((S)-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)-3,6,9,12,15,18,21,24-octaoxa-heptacosan-27-ylamide (6-6, 210 mg). LCMS (ESI) m / z: 1029.6 [M+H] + .

[0328] Step 5: 1 -Amino- N-((S)- 1 -(((S)- 1 -((( 1 S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl- 10,13-dioxo-2,3,9,10,13,15-hexahydro-lH, 12H-benzo[de]pyrano[3',4':6,7]indolizino[l,2-b]quinolin-l-yl)amino)-l-oxopropan-2-yl)amino)-3-methyl- 1 -oxobutan-2-yl)-3,6,9, 12, 15, 18,21,24-octaoxa-heptatriacontan-27-amide (6-6, 100 mg, 0.097 mmol), 2-(methylsulfonyl)pyrimidine-5-carboxylic acid (78 mg, 0.39 mmol), 2-chloro-4,6-dimethoxy-l,3,5-triazine (68 mg, 0.39 mmol) and N-methylmorpholine (59 mg, 0.582 mmol) were dissolved in anhydrous N,N-dimethylacetamide (5 mL) and the reaction was stirred at room temperature for 16 hours. Purification by C18 column chromatography gave N-((29S,32S)-33-(((lS,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl- 10,13-dioxo-2,3,9,10,13,15-hexahydro-lH, 12H-benzo[de]pyrano[3',4':6,7]indolizino[l,2-b]quinolin-l-yl)amino)-29-isopropyl-32-methyl-27,30,33-trioxo-3,6,9, 12, 15, 18,21,24-octaoxa-28,31-diazatriacontan-yl)-2-(methylsulfonyl)pyrimidine-5-carboxamide (11.5 mg), Cpd 6. LCMS (ESI) m / z: 1213.6 [M+H] +

[0329] 1H NMR (400 MHz, DMSO-d6) δ 9.36 (s, 2H), 9.13 (s, 1H), 8.41 (d, J = 8.9 Hz, 1H), 8.10 (d, J = 6.9 Hz, 1H), 7.86 (d, J = 8.4 Hz, 1H), 7.79 (d, J = 11.0 Hz, 1H), 7.30 (s, 1H), 6.53 (s, 1H), 5.54 (s, 1H), 5.42 (s, 2H), 5.24 (d, J = 18.9 Hz, 1H), 5.11 (d, J = 19.0 Hz, 1H), 4.32 - 4.21 (m, 1H), 4.17 - 4.06 (m, 1H), 3.58 - 3.52 (m, 8H), 3.48 (s, 25H), 3.44 (s, 6H), 2.40 (s, 3H), 2.29 (dd, J = 13.7, 7.1 Hz, 1H), 2.13 (s, 2H), 1.91 (ddd, J = 23.2, 22.6, 7.6 Hz, 4H), 1.27 (d, J = 7.0 Hz, 3H), 0.87 (t, J = 7.4 Hz, 3H), 0.83 - 0.72 (m, 6H)

[0330] Example 11: MUC17-ADC preparation and validation

[0331] The antibody stock solution was taken out from the freezer (-80 °C), after thawing, it was added into a centrifuge tube, then PB (20 mM, pH 7.2, 751 uL) buffer, DTPA-3Na (10 mM, 375 uL), TCEP (10 mM, 94 uL) solution were added, and the reaction was carried out at 25 °C for 2 hours. Then the linker-toxin as described in Example 10, such as Cpd3, was added, and the reaction was continued at 25 °C for 2 hours. After the reaction was completed, it was added into an ultrafiltration centrifuge tube (30 KD), and dialyzed against His / His-HCl (10 mM, pH 5.5) to replace, to obtain the ADC stock solution (DAR: 7.5-8.5, SEC purity ≥ 95%).

[0332] The linker-toxin used in the above experiment is Cpd3 described in Example 10, as an example. The antibody stock solution can be any one of M1-M27 clones; the linker-toxin can be a combination of common linkers and camptothecin compounds in the art, including but not limited to deruxtecan, Cpd3, Cpd5, Cpd6, etc.

[0333] The above experiment was carried out using other linker-toxins with clone M26. Each ADC sample was validated, and the test results are shown in Table 6 below.

[0334] Table 6. ADC sample test results

[0335]

[0336] Example 12: ADC drug activity detection

[0337] Animal efficacy evaluation experiments were performed using Balb / c nude mice (source: Shanghai Lingchang) weighing 18-20 g. After the animals arrived, they were housed in separate cages, with 5 animals per cage, in a barrier environment (independent air supply system). All cages, bedding, and drinking water were sterilized before use. After 7 days of acclimation, 10*10 6 GSU tumor cells (Riken, RCB2278) were mixed with Matrigel at a ratio of 1:1 and inoculated subcutaneously on the right upper back of the mice.

[0338] A body weight meter was used to measure body weight, and an electronic digital caliper (manual caliper, OMC Fontana) was used to measure the long diameter and short diameter of the tumor, and the tumor volume (mm 3 ) was calculated. Tumor volume (mm 3 ) = 1 / 2 x long diameter (mm) x [short diameter (mm)] 2 was measured twice a week. After 7 days of inoculation, when the average tumor volume reached 279mm 3 , 36 experimental animals were randomly divided into 6 groups, and the test ADC drug or control PBS was administered intravenously according to the following doses on the same day.

[0339] Experiment group one: PBS control group (Vehicle Single), single dose of ADC at the same dose.

[0340] Experiment group two: M21-Deruxtecan, 3 mg / kg, single dose.

[0341] Experiment group three: M26-Deruxtecan, 3 mg / kg, single dose.

[0342] Experiment group four: M26-Cpd 3, 3 mg / kg, single dose.

[0343] Experiment group five: M26-Cpd 5, 3 mg / kg, single dose.

[0344] Experiment group six: M26-Cpd 6, 3 mg / kg, single dose.

[0345] All data are expressed as mean values. All data were analyzed using Microsoft Excel 2016. The tumor inhibition rate in mice is shown in Table 7 below.

[0346] TGI (Tumor Growth Inhibition) refers to the tumor growth inhibition rate. TGI is usually used to evaluate the effect of a certain treatment or intervention on tumor growth. Specifically, TGI can be calculated by the following formula:

[0347] TGI = (1 - tumor weight of treatment group / tumor weight of control group) x 100%.

[0348] In this application, the TGI of other groups is calculated based on the experimental group.

[0349] Table 7. Tumor inhibition rate of ADC in mice

[0350]

[0351] The experimental results are shown in Figure 4 and Table 7, indicating that M21 and M26 antibody conjugated with Deruxtecan or Cpd3, Cpd5 can significantly inhibit tumor growth. Among them, M26-Cpd3 ADC has better effect on inhibiting tumor growth than M26-Deruxtecan ADC, and M26-Cpd5 ADC has similar effect on inhibiting tumor growth as M26-Deruxtecan ADC. No weight loss was observed in all experimental animals during the experiment.

[0352] Example 13: Humanization of chimeric antibodies and characterization of humanized antibodies

[0353] A. Humanization of chimeric antibodies

[0354] Humanization method: Model the parent antibody structure by computer-aided homology modeling. Select a human natural germ line sequence with high homology to the parent sequence. Design a humanized antibody using CDR grafting technology. Simply put, graft the CDRs of the parent antibody into the human natural germ line sequence to obtain a humanized chimeric antibody of the parent antibody. Compare the different amino acid residues of the chimeric antibody and the parent antibody, and judge the key points that will affect the subsequent affinity according to the classic residues, interaction loop region, core region, mutation hotspots, etc. Select appropriate sites for back mutation to obtain a humanized antibody. Randomly pair the heavy and light chains of the humanized antibody for antibody production and identification.

[0355] Humanized antibody screening: According to the above humanization design principles and the method disclosed in Example 4, the chimeric antibody M21 was subjected to humanized sequence design. The VH coding sequence of the humanized antibody was connected with the coding sequence of the heavy chain constant region of human IgG1 to obtain the heavy chain coding sequence, and the VL coding sequence of the humanized antibody was connected with the coding sequence of the Kappa subtype of the human light chain constant region to obtain the light chain coding sequence, and then the coding sequences of the heavy chain and the light chain were inserted into the eukaryotic expression vector PCDNA3.4 respectively, and combined expression, purification and detection were carried out. Finally, the humanized antibodies M21-01, M21-02, M21-03, M21-04, M21-05, M21-06, M21-07, M21-08, M21-09, M21-10, M21-11, M21-12, M21-13, M21-14, M21-15, M21-16, M21-17, M21-18 were obtained.

[0356] B: Characterization of humanized antibodies

[0357] B.1 ELISA: Coat 96-well ELISA plates with 1 μg / mL of human Mucin17 antigen protein (100 μL / well) at 4°C overnight. The next day, wash the plates 3 times with PBST and block with 1% BSA in PBST for 1 h. After washing the plates 3 times with PBST, add each humanized antibody at a gradient dilution (10 nM, 5-fold gradient dilution, 7 concentration points) and incubate at 37°C for 1 h. Then wash 3 times with PBST, add secondary antibody anti-human IgG-HRP (Sigma, Cat#A 0170) and incubate for 45 min. After incubation, wash the plates 3 times with PBST, add TMB and develop. Develop at 25°C for 15 min, stop the reaction by adding 1 M HCl, and read the absorbance at OD450 by an enzyme labeler. The ELISA results are shown in Table 8 below.

[0358] Table 8. ELISA results of humanized antibodies

[0359] Antibody number EC50 (nM) Span Top M21-01 0.04 2.84 2.96 M21-02 0.04 2.80 2.92 M21-03 0.04 2.81 2.93 M21-04 0.06 2.83 2.95 M21-05 0.03 2.82 2.94 M21-06 0.04 2.85 2.98 M21-07 0.04 2.85 2.96 M21-08 0.04 2.80 2.91 M21-09 0.03 2.78 2.90 M21-10 0.07 2.82 2.94 M21-11 0.06 2.81 2.92 M21-12 0.04 2.81 2.97 M21-13 0.05 2.86 2.96 M21-14 0.04 2.81 2.93 M21-15 0.05 2.82 2.93 M21-16 0.05 2.80 2.91 M21-17 0.05 2.81 2.92 M21-18 0.04 2.84 2.95

[0360] B.2 FACS binding activity: HEK293 cells with high expression of huMucin17 protein were constructed by conventional methods. Logarithmic growth period huMucin17-HEK293 cells were prepared into a single cell suspension, and the density was adjusted to 2 x 10 6Cells were incubated with 100 nM of antibody at 4°C for 30 min, washed twice, and incubated at 4°C and / or 37°C for 0 / 2 / 4 / 6 hours. Cells were fixed with 2% PFA on ice for 10 min, washed twice, and incubated with fluorescent secondary antibody (Alexa 488 Goat Anti-Human IgG, Fc gamma fragments specific, Jackson, Cat#109-545-098) on ice for 1 hour in the dark. FACS analysis was performed, and the results are shown in Table 9 below. The results show that all humanized antibodies exhibited significant internalization. Figures 6A-6B

[0361] B.3 Endocytosis activity experiment: GSU cells (Riken, RC2278) were incubated with primary antibody (100 nM) at 4°C for 30 min, washed twice, and incubated at 4°C and / or 37°C for 0 / 2 / 4 / 6 hours. Cells were fixed with 2% PFA on ice for 10 min, washed twice, and incubated with fluorescent secondary antibody (Alexa 488 Goat Anti-Human IgG, Fc gamma fragments specific, Jackson, Cat#109-545-098) on ice for 1 hour in the dark. FACS analysis was performed, and the results are shown in Table 9 below. The results show that all humanized antibodies exhibited significant internalization.

[0362] Table 9. Endocytosis activity results of humanized antibodies

[0363]

[0364] B.4 BiaCore affinity detection: BIAcore 8K was used to perform affinity analysis according to the method disclosed in Example 8, with a flow cell and sample chamber temperature of 25°C. The results are shown in Table 10 below.

[0365] Table 10. Affinity detection results of humanized antibodies

[0366]

[0367] Example 14: Binding of humanized antibodies or antibody drug conjugates to tumor cells

[0368] A. Construction of antibody drug conjugates of humanized antibodies

[0369] ​Using clone M21-17 and Linker-Toxin, antibody drug conjugates of humanized antibody were prepared according to the method of Example 11.

[0370] The linker-toxin used in the above experiment is Cpd3 described in Example 10, as an example. The linker-toxin can be a combination of a linker commonly used in the art and a camptothecin compound, including but not limited to deruxtecan, Cpd3, Cpd5, Cpd6, etc.

[0371] B. Verification and characterization of antibody drug conjugates

[0372] Each ADC sample was verified, and the results are shown in the table below.

[0373]

[0374] C. Binding of humanized antibodies and their conjugates to tumor cells

[0375] Experimental method: Logarithmic growth phase GSU cells (Riken, RC2278) or NUGC4 cells (Nanjing Kebai Biotechnology Co., Ltd.) were prepared into a single cell suspension, and the density was adjusted to 2x10 6 cells / mL, 100 μL per well was added to a 96-well round-bottom plate, centrifuged at 4°C, 300g and the supernatant was removed. The corresponding wells were added with the antibody to be tested, ADC, or positive control AMG199 antibody (commercially available), mixed and incubated at 4°C for 30 min. The cell mixture after incubation was washed 3 times, and 100 μL of diluted secondary antibody Goat Anti-Human IgG (Jackson 109-136-098) was added, incubated at 4°C in the dark for 30 min, washed 3 times, and then detected by flow cytometry to read the mean fluorescence intensity value (MFI).

[0376] The experimental results show that the maximum MFI value of the candidate molecule binding to the cells is higher than that of the positive control molecule on the tumor cell lines detected. Table 11 shows the binding data of humanized antibodies and their ADCs to tumor cells (GSU and NUGC4).

[0377] Table 11. Binding activity of target antibodies to tumor cells

[0378]

[0379] Example 15: In vitro killing study of antibody drug conjugates on tumor cells

[0380] Experimental method: Take the high expression MUC17 CHO-K1 cells, tumor cells (NUGC4, GSU and SW480 cells, among which GSU cells express the highest amount of MUC17, MUCG4 expresses an intermediate amount of MUC17, and SW480 expresses a relatively low amount of MUC17) in the logarithmic growth phase, and adjust the cell suspension to a cell density of 1.5x10 4 / ml to prepare a cell working solution, and add 100 μL per well to a 96-well cell culture plate. Place the cell culture plate in a 37°C, 5% CO2 cell incubator and incubate overnight. Add the ADC sample M21-17-Cpd3 to be tested to the corresponding wells, and incubate in the cell incubator for 5 days. Place the cell culture plate at room temperature for 10 min, and equilibrate to room temperature. Add 75 uL of a luminescence cell viability detection kit solution (Promega) to each well. Mix at room temperature for 2 min using an orbital shaker, and incubate at room temperature for 10 min. Use an enzyme-labeled instrument to read the plate and read the fluorescence signal value, and calculate the cell viability.

[0381] Experimental results: The in vitro cell killing ability of the candidate ADC molecule is positively correlated with the expression amount of MUC17 molecules on the cell surface. The higher the expression amount, the higher the killing effect of the ADC. The cell killing results of the antibody drug conjugate M21-17-Cpd3 in the three tumor cell lines are shown in Figure 7 .

[0382] Example 16: Bystander killing effect of antibody drug conjugate on tumor cells

[0383] This example investigates whether the ADC has a bystander killing effect. The bystander killing effect refers to the killing effect of the ADC drug on cells around or adjacent to target antigen-positive cells.

[0384] Mix the antigen high-expression cells (positive cells: NUGC4 or GSU) and negative cells (MDA-MB-468 (source: Chinese Academy of Sciences Cell Bank) or HCC827 cells (source: Nanjing Kebai Biological)) at a cell density of 1x10 5 / ml, respectively, to prepare a mixed cell working solution, and add 1 ml of the cell working solution per well to a 12-well cell culture plate, and incubate at 37°C, 5% CO2 overnight. Add the ADC sample M21-17-Cpd3 to be tested to the corresponding wells, and incubate in the cell incubator for 5 days. Digest and centrifuge the cells, resuspend the cells, count the cells, transfer the cells to a 96-well plate for flow cytometry analysis, count the number of viable cells of the two types of cells, and use GraphPad software to plot a bar graph.

[0385] The side-killing effect of M21-17-Cpd3 was detected by using target point negative cells to co-culture with positive cells, detecting the survival number of negative cells, and the results were shown in Figure Figures 8A-8C As shown in Figure 17, the results showed that the antibody drug conjugate of the application had a side-killing effect, wherein the molecule M21-Dxd refers to the antibody conjugate formed after the M21 antibody is coupled with the toxin linker (Dxd) of DS8201.

[0386] Example 17: Anti-tumor effect verification of humanized ADC on human gastric cancer GSU xenograft tumor model

[0387] The anti-tumor activity of the antibody drug conjugate M21-17-Cpd3 of the application was detected by using the experimental method as described in Example 12.

[0388] The experimental design is as follows:

[0389] Experimental group one: PBS control group, single administration according to the same dose of ADC.

[0390] Experimental group two: M21-17-Cpd3, 0.3 mg / kg, single administration.

[0391] Experimental group three: M21-17-Cpd3, 1 mg / kg, single administration.

[0392] Experimental group four: M21-17-Cpd3, 10 mg / kg, single administration.

[0393] Experimental group five: M21-17-Cpd3, 30 mg / kg, single administration.

[0394] Experimental group six: M21-17-Cpd3, 10 mg / kg, QW*3.

[0395] All data are expressed as mean. The mouse in vivo pharmacodynamic indexes are shown in Table 12.

[0396] Table 12. Humanized ADC in vivo pharmacodynamics (GSU cells)

[0397] Experimental group TGI (%) T / C (%) PRR IR (%) 1 - - - - 2 10.63 89.37 0 / 6 8.58 3 48.87 51.13 0 / 6 41.03 4 81.41 18.59 3 / 6 76.73 5 96.45 3.55 6 / 6 96.25 6 99.17 0.83 6 / 6 97.83

[0398] Note: Complete response (CR) refers to tumor volume (TV) = 0 mm 3; Partial response (PR) refers to at least 30% reduction in tumor volume relative to baseline; Complete Response Rate (CRR) refers to the proportion of subjects in which the tumor is completely eliminated, i.e. CR / number of animals used in the experiment; Partial Response Rate (PRR) refers to the proportion of subjects in which the tumor is partially reduced after treatment, i.e. PR / CR / number of animals used in the experiment; TGI refers to tumor growth inhibition rate; T / C refers to relative tumor proliferation rate, T / C(%) = T / C x 100 (T: average tumor volume of the treatment group; C: average tumor volume of the solvent control group); IR refers to tumor weight inhibition rate, IR(%) = [1-(average tumor weight of the treatment group / average tumor weight of the solvent control group)] x 100.

[0399] The experimental results show that a single dose of 0.3-30 mg / kg M21-17-Cpd3 can dose-dependently inhibit the growth of GSU transplanted tumors; the tumor volume detection data of each experimental group are shown in Table 2. Figure 9 No weight loss was observed in all experimental animals during the experiment.

[0400] Example 18: Anti-tumor effect verification of humanized ADC on human gastric cancer NUGC-4 transplanted tumor model

[0401] The anti-tumor activity of the antibody drug conjugate M21-17-Cpd3 of the present application was detected using the experimental method described in Example 12.

[0402] The experimental design is as follows:

[0403] Experimental group one: PBS control group, single dose according to the same dose of ADC.

[0404] Experimental group two: M21-17-Cpd3, 1 mg / kg, single dose.

[0405] Experimental group three: M21-17-Cpd3, 3 mg / kg, single dose.

[0406] Experimental group four: M21-17-Cpd3, 10 mg / kg, single dose.

[0407] Experimental group five: M21-17-Cpd3, 30 mg / kg, single dose.

[0408] Experimental group six: M21-17-Cpd3, 10 mg / kg, QW*3.

[0409] All data are expressed as mean values. The in vivo pharmacodynamic indicators of mice are shown in Table 13.

[0410] Table 13. Pharmacodynamics of humanized ADC in mice (NUGC4 cells)

[0411] Experimental group TGI (%) T / C (%) PRR IR (%) 1 - - - - 2 38.33 61.67 0 / 6 36.74 3 49.92 50.08 0 / 6 48.25 4 74.20 25.80 0 / 6 70.90 5 96.24 3.76 6 / 6 94.20 6 96.14 3.86 6 / 6 93.37

[0412] Note: Complete response (CR) refers to tumor volume (TV) = 0 mm 3 ; Partial response (PR) refers to at least 30% reduction in tumor volume relative to baseline; Complete Response Rate (CRR) refers to the proportion of subjects whose tumors were completely eliminated, i.e. CR / number of animals used in the experiment; Partial Response Rate (PRR) refers to the proportion of subjects whose tumors were partially reduced after treatment, i.e. PR / CR / number of animals used in the experiment; TGI refers to tumor growth inhibition rate; T / C refers to relative tumor proliferation rate, T / C (%) = T / C x 100 (T: average tumor volume of the treatment group; C: average tumor volume of the solvent control group); IR refers to tumor weight inhibition rate, IR (%) = [1- (average tumor weight of the treatment group / average tumor weight of the solvent control group)] x 100.

[0413] The experimental results show that a single dose of 1-30 mg / kg M21-17-Cpd3 can dose-dependently inhibit the growth of NUGC-4 transplanted tumors; the tumor volume detection data of each experimental group are shown in Table 13. Figure 10

[0414] Example 19: Anti-tumor effect verification of humanized ADC on human colon cancer SW480 transplanted tumor model

[0415] The anti-tumor activity of the antibody drug conjugate M21-17-Cpd3 of the present application was detected using the experimental method described in Example 12. SW480 cells were purchased from ATCC, CCL-228.

[0416] The experimental design is as follows:

[0417] Experimental group one: PBS control group, single dose according to the same dose of ADC.

[0418] Experimental group two: M21-17-Cpd3, 3 mg / kg, single dose.

[0419] Experimental group three: M21-17-Cpd3, 10 mg / kg, single dose.

[0420] Experimental group four: M21-17-Cpd3, 30 mg / kg, single dose.

[0421] Experimental group five: M21-17-Cpd3, 3 mg / kg, QW*3.​

[0422] Experiment group six: M21-17-Cpd3, 10 mg / kg, QW*3.

[0423] All data are expressed as mean. The mouse in vivo efficacy index is shown in Table 14 as follows.

[0424] Table 14. Humanized ADC in vivo efficacy (SW480 cells)

[0425] Experimental group TGI (%) T / C (%) PRR IR (%) 1 - - - - 2 44.76 55.24 0 / 6 41.39％ 3 63.03 36.97 0 / 6 62.72％ 4 88.74 11.26 0 / 6 87.40％ 5 26.09 73.91 0 / 6 27.25％ 6 80.07 19.93 1 / 6 77.72％

[0426] Note: Complete response (CR) refers to tumor volume (TV) = 0 mm 3 ; Partial response (PR) refers to at least 30% reduction in tumor volume relative to baseline; Complete Response Rate (CRR) refers to the proportion of subjects whose tumors were completely eliminated, i.e. CR / number of animals used in the experiment; Partial Response Rate (PRR) refers to the proportion of subjects whose tumors were partially reduced after treatment, i.e. PR / CR / number of animals used in the experiment; TGI refers to tumor growth inhibition rate; T / C refers to relative tumor proliferation rate, T / C(%) = T / C x 100 (T: average tumor volume of the treatment group; C: average tumor volume of the solvent control group); IR refers to tumor weight inhibition rate, IR(%) = [1- (average tumor weight of the treatment group / average tumor weight of the solvent control group)] x 100.

[0427] The experimental results show that a single dose of 3-30 mg / kg M21-17-Cpd3 can dose-dependently inhibit the growth of SW480 transplanted tumors; the tumor volume detection data of each experimental group are shown in Table 14 as follows. Figure 11

[0428] Example 20: Verification of plasma stability of linker-toxin Cpd3

[0429] The tested ADC molecule M21-17-Cpd3 (concentration 0.2 mg / mL) was incubated in different species plasma (human / monkey / rat / mouse) at 37°C for 21 days, and the concentration of free drug (e.g. Cpd3 or Dxd) in plasma was detected at different time points by LC-MS / Ms. The control sample of this example is DS-8201 (commercially available). Table 15 below shows the experimental parameters of M21-17-Cpd3 (analyte Cpd3) and the reference DS-8201 (analyte Dxd).

[0430] ​The release rate (%) refers to the release rate of free drug in plasma after incubation of the ADC and plasma for a certain time, and the release rate of free drug in plasma is calculated according to the ratio of the content of free drug in plasma to the total amount of drug in the ADC.

[0431] Table 14. Plasma stability of ADC

[0432]

[0433] The experimental results show that the humanized antibody M21-17 conjugated with Cpd3 (DAR8) has good plasma stability and stability in different species of plasma. Cpd3 shows very high stability in humans and monkeys, and is superior to the linker-toxin Dxd of the control DS-8201.

[0434] Example 21: Verification of conjugation properties of linker-toxin Cpd3

[0435] Cpd3 was used to conjugate with her2 antibody Trastuzumab, and the conjugation efficiency and quality standards of the antibody drug conjugate were verified.

[0436] Using commercially available her2 antibody Trastuzumab, a conventional antibody drug conjugation preparation method similar to Example 11 was used, the antibody Trastuzumab was exchanged into 20mM His, pH 6.0 buffer, and the concentration was detected by ultraviolet spectrophotometer to be 16.95mg / mL. A new TCEP (5mmol / L) stock solution was prepared with 20mM His, pH 6.0. In an EP tube, His, antibody, and TCEP were added in sequence, and the final reaction concentration of the antibody was 6.0mg / mL, and the amount of TCEP added was 10 molar equivalents (relative to the concentration of the antibody), and incubated at 25°C for 2h (for example, using 4.8mg / mL of the original antibody solution, 450.8ul of His, 283.19ul of antibody, and 65.97ul of TCEP were added in sequence); after incubation at 25°C for 1h, the buffer of the reaction mixture was replaced with 20mM His pH 6.0 using a desalting column to remove small molecule drugs that were not conjugated, and stored at 4°C. The final sample was concentrated and detected for the absorption of ADC and LP (linker-toxin) at 280nm and 365nm. The final sample was sterile filtered with a 0.22um filter membrane to obtain Trastuzumab-Cpd3. The prepared antibody drug conjugate was characterized as follows.

[0437] Sample analysis:

[0438] 1. SEC analysis: 100% buffer A at 0.8 ml / min for 20 min. (Buffer: 100 mM PB, 15% IPA, pH 6.8).

[0439] 2. Reverse phase analysis (RP) of ADC: 27% - 50% buffer B at 1.0 ml / min for 40 min. (Buffer A: 0.05% TFA, water; Buffer B: 0.05% TFA, acetonitrile).

[0440] 3. Hydrophobic interaction analysis (HIC) of ADC: 0% - 100% buffer B at 0.5 ml / min for 35 min (Buffer A: 1.5 M (NH4)2SO4, 25 mM PB, pH 7.0; Buffer B: 25 mM PB, 20% IPA, pH 7.0).

[0441] 4. Free small molecule determination (C18) of ADC: UV full wavelength scanning of linker-toxin (L-D) with UV spectrophotometer to determine the maximum absorption wavelength of L-D other than 280 nm; standard curve of L-D was prepared with gradient dilution, 10 ul of each concentration was loaded, and the final loading amount was 1 ug, 0.5 ug, 0.3 ug, 0.1 ug, 0.05 ug, 0.02 ug, respectively, while the absorption of multiple wavelengths was detected. (Buffer A: 0.05% TFA, water; Buffer B: 0.05% TFA, acetonitrile).

[0442] 5. Endotoxin detection: (1) standard curve preparation: endotoxin standard was dissolved with 1 ml of water for injection and vortexed for 10 min to prepare 10 EU / ml. The standard was gradient diluted with water for injection to 1 EU / ml, 0.1 EU / ml, 0.01 EU / ml, and a positive reference of 0.5 EU / ml was set; (2) sample treatment: the sample was diluted 20 times with water for injection and vortexed for 30 s; (3) loading: the standard, positive reference, and test sample were sequentially added to a sterile, enzyme-free, and pyrogen-free 96-well plate, 100 ul of limulus reagent was added to another plate, 100 ul of the test sample was taken with a gun and added to the detection plate containing limulus reagent, and two replicate wells were set for each sample; (4) detection: the wavelength at 405 nm was dynamically detected for 90 min.

[0443] The experimental results are shown in Table 15 and Figures 12-14 The prepared Her-2-Cpd3 ADC had a SEC purity of 98%, an RP-DAR value of 7.9, a free toxin content of <5%, a recovery rate of 67%, an HIC-DAR value of 7.95, and an endotoxin of 0.01 EU / mg.

[0444] Table 15. ADC sample test results

[0445]

[0446] Experimental conclusion: Cpd3 as linker-toxin, its coupling efficiency is high, the reaction condition is mild. Thus, Cpd3 as hydrophilic linker-toxin can be used to prepare high DAR value of ADC, while having excellent purity.

[0447] Numerous modifications to the present application will be apparent to those skilled in the art in view of the foregoing description. Aspects of the present application can be used separately or in any combination. Such modifications are intended to fall within the scope of the appended claims. Each of the references cited herein (including all patents, patent applications, journal articles, books and any other publications) is incorporated by reference in its entirety.

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Claims

1. A linker unit-cytotoxic small molecule drug selected from the group consisting of: an isomer thereof, a pharmaceutically acceptable salt thereof, or a mixture thereof.

2. The antibody drug conjugate formed by conjugating the linker unit-cellular toxic small molecule, its isomer, its pharmaceutically acceptable salt or mixture thereof of claim 1 with an antibody, wherein, The antibody is selected from the group consisting of a MUC17 antibody or an antigen binding fragment thereof, a HER2 antibody or an antigen binding fragment thereof.

3. The antibody drug conjugate of claim 2, wherein the linker unit-cytotoxic small molecule drug is an isomer thereof, a pharmaceutically acceptable salt thereof, or a mixture thereof.

4. The antibody drug conjugate of claim 2, wherein the linker unit-cytotoxic small molecule drug is The antibody is a MUC17 antibody or a HER2 antibody.

5. A pharmaceutical composition comprising the linker unit-cytotoxic small molecule drug of claim 1, an isomer thereof, a pharmaceutically acceptable salt thereof, or a mixture thereof, or the antibody drug conjugate of any one of claims 2 to 4, and a pharmaceutically acceptable excipient.

6. Use of the linker unit-cytotoxic small molecule drug of claim 1, an isomer thereof, a pharmaceutically acceptable salt thereof, or a mixture thereof, or the antibody drug conjugate of any one of claims 2 to 4, in the manufacture of a medicament for treating a cancer and / or a tumor selected from the group consisting of: gastric cancer, esophageal cancer, pancreatic cancer, gastroesophageal adenocarcinoma, gastroesophageal junction cancer, gastric cardia cancer, gastric corpus cancer, gastric antrum cancer, liver cancer, bile duct cancer, gallbladder cancer, melanoma, endometrial cancer, kidney cancer, prostate cancer, breast cancer, triple negative breast cancer, colon cancer, large intestine cancer, lung cancer, bone cancer, skin cancer, head and neck cancer, uterine cancer, cervical cancer, ovarian cancer, and rectal cancer.

7. A method of making the linker unit-cytotoxic small molecule, isomers thereof, pharmaceutically acceptable salts thereof, or mixtures thereof of claim 1, wherein, The method comprises The compound of formula (3-7) is reacted with 2-(methylsulfonyl)pyrimidine-5-carboxylic acid to give Cpd 3, Cpd 5 and Cpd 6 of claim 1, respectively. The compound of formula (6-6) is reacted with 2-(methylsulfonyl)pyrimidine-5-carboxylic acid 8. The method of claim 7, wherein, The reaction is carried out in anhydrous N,N-dimethylacetamide in the presence of 2-chloro-4,6-dimethoxy-l,3,5-triazine and N-methylmorpholine. The reaction is carried out in anhydrous N,N-dimethylacetamide in the presence of 2-chloro-4,6-dimethoxy-l,3,5-triazine and N-methylmorpholine.

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