Anti-CDH17 antibody-drug conjugate and use thereof

Abstract

Disclosed in the present application are an anti-CDH17 antibody-drug conjugate and the use thereof. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof of the present application comprises an anti-CDH17 antibody or an antigen-binding fragment thereof, a linker unit L and a cytotoxic drug M. Further disclosed in the present application are a preparation method for the antibody-drug conjugate or the pharmaceutically acceptable salt thereof, and a pharmaceutical composition containing the same. The antibody-drug conjugate of the present application has an extremely strong inhibitory activity against the proliferation of tumor cells both in vivo and in vitro.

Description

An anti-CDH17 antibody-drug conjugate and its application Technical Field

[0001] This application belongs to the field of biomedicine, specifically relating to an anti-CDH17 antibody-drug conjugate and its application. Background Technology

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

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

[0004] CDH17 is a type I single transmembrane protein. Cadherin-16 (CDH16) has a similar structure to CDH17 and also has 7 cadherin repeat regions in the extracellular space. It is a protein in the CDH family that has a high homology with CDH17.

[0005] CDH17 is expressed in normal tissues, specifically in the colorectal and small intestines, where it is isolated and hidden among normal cells. CDH17 is highly expressed in colorectal cancer, gastric cancer, and pancreatic cancer. Therefore, CDH17 can serve as an ideal target for the development of antibodies, bispecific antibodies, antibody-drug conjugates, and cell therapy. Summary of the Invention

[0006] To address the technical problems of low inhibitory activity against tumors in existing CDH17-targeting antibody drugs, this application discloses an anti-CDH17 antibody-drug conjugate and its applications. The anti-CDH17 antibody-drug conjugate of this application exhibits extremely strong inhibitory activity against tumor cell proliferation both in vitro and in vivo.

[0007] To solve the above-mentioned technical problems, one of the technical solutions provided in this application is: an antibody-drug conjugate or a pharmaceutically acceptable salt thereof, wherein the antibody-drug conjugate or a pharmaceutically acceptable salt thereof comprises an anti-CDH17 antibody or its antigen-binding fragment, a linker unit L, and a cytotoxic drug M;

[0008] The anti-CDH17 antibody or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3.

[0009] The HCDR1 contains the amino acid sequence shown in SEQ ID NO:1;

[0010] The HCDR2 contains the amino acid sequence shown in SEQ ID NO:2;

[0011] The HCDR3 contains the amino acid sequence shown in SEQ ID NO:3;

[0012] The LCDR1 contains the amino acid sequence shown in SEQ ID NO:5;

[0013] The LCDR2 contains the amino acid sequence shown in SEQ ID NO:6; and,

[0014] The LCDR3 contains an amino acid sequence as shown in SEQ ID NO:7.

[0015] In some specific embodiments, the heavy chain variable region comprises an amino acid sequence having at least 90%, at least 95%, or at least 99% sequence identity with, for example, SEQ ID NO:4; and / or, the light chain variable region comprises an amino acid sequence having at least 90%, at least 95%, or at least 99% sequence identity with, for example, SEQ ID NO:8.

[0016] The amino acid sequence described in this application having at least 90%, at least 95%, or at least 99% sequence identity maintains at least the same antigen-binding function as the original sequence; this identity is based on the aforementioned CDR combination, i.e., the site of sequence change is the FR region.

[0017] In some specific embodiments, the amino acid sequence of the heavy chain variable region VH is as shown in SEQ ID NO:4; and / or, the amino acid sequence of the light chain variable region VL is as shown in SEQ ID NO:8.

[0018] In some specific embodiments, the anti-CDH17 antibody or its antigen-binding fragment includes one or more of the following: (1) fully human antibody, humanized antibody, chimeric antibody, probody, bispecific antibody, multispecific antibody, monoclonal antibody and polyclonal antibody; (2) Fab, Fab', F(ab')2, Fv, ScFv, biantibody, Fd, sdAb, VHH and complementarity-determining region (CDR); and (3) the CDH17 is human CDH17.

[0019] In some implementations, the antibody molecule is humanized. Different methods for humanizing antibodies are known to those skilled in the art, as reviewed by Almagro & Fransson, the contents of which are incorporated herein by reference in their entirety (Almagro JC and Fransson J (2008) Frontiers in Bioscience 13:1619-1633).

[0020] In some specific embodiments, the anti-CDH17 antibody or its antigen-binding fragment is a full-length antibody, which includes a heavy chain constant region of the heavy chain of the human antibody and a light chain constant region of the light chain of the human antibody; the heavy chain constant region is preferably the heavy chain constant region of the human antibody IgG1, and the light chain constant region is preferably the light chain constant region of the human antibody κ chain.

[0021] In some specific embodiments, the amino acid sequence of the heavy chain constant region of the human antibody IgG1 is as shown in SEQ ID NO:9 or 11, or has at least 90%, at least 95%, or at least 99% sequence identity with SEQ ID NO:9 or 11; and / or, the amino acid sequence of the light chain constant region of the human antibody κ chain is as shown in SEQ ID NO:10, or has at least 90%, at least 95%, or at least 99% sequence identity with SEQ ID NO:10; the amino acid sequence having at least 90%, at least 95%, or at least 99% sequence identity maintains at least the same antigen-binding function as the original sequence.

[0022] In some specific embodiments, the amino acid sequence of the heavy chain constant region of the human antibody IgG1 is as shown in SEQ ID NO:9 or 11; and / or, the amino acid sequence of the light chain constant region of the human antibody κ chain is as shown in SEQ ID NO:10.

[0023] In some specific embodiments, the amino acid sequence of the heavy chain of the anti-CDH17 antibody or its antigen-binding fragment is as shown in SEQ ID NO:12 or 14; and / or, the amino acid sequence of the light chain is as shown in SEQ ID NO:13.

[0024] In some specific embodiments, the cytotoxic drug M has the structure shown in formula (A-1), its stereoisomer, pharmaceutically acceptable salt, solvate, or solvate of a salt thereof.

[0025] in,

[0026] M is -L 2 -L 1 -C(O)-;

[0027] L 2 It is -O-, and L 2 Connected to the connector unit L;

[0028] L 1 -(C(R) 1a (R) 1b )) m -CH2-;

[0029] m is selected from 1, 2, 3, or 4;

[0030] Each R 1a and R 1b Independently hydrogen, halogen, hydroxyl, amino, or C1-C6 alkyl, wherein the C1-C6 alkyl is optionally substituted with one or more R;

[0031] Each R is independently either hydrogen or halogen.

[0032] In some specific implementation schemes, each R 1a Independently hydrogen, halogen, or C1-C6 alkyl; and / or, each R 1b It can be hydrogen, halogen, or C1-C6 alkyl independently.

[0033] In some specific implementation schemes, L 1 for

[0034] In some specific embodiments, the cytotoxic drug M has any of the following structures:

[0035] In some specific implementations, the connector unit L is -L a -L b -L c -; and the L c Connected to the cytotoxic drug M;

[0036] -L a -for Preferred The a-terminus is linked to the anti-CDH17 antibody or its antigen-binding fragment, and the b-terminus is linked to L... b Connected;

[0037] -L b - For any of the following structures: Preferred More Among them, C-end and L a Connected, d end and L c Connected;

[0038] -L c-for

[0039] In some specific implementations, the connector unit L is Preferred

[0040] In some specific embodiments, the antibody-drug conjugate or its pharmaceutically acceptable salt structure is as shown in formula (A-2):

[0041] Wherein, p represents the average number of connections, and p is any integer or decimal from 1 to 10; preferably any integer or decimal from 3 to 9; more preferably an integer or decimal from 7 to 8, such as 7.77 or 7.79;

[0042] Ab is the anti-CDH17 antibody or its antigen-binding fragment as described above;

[0043] M is the cytotoxic drug M as described above;

[0044] L refers to the connector unit L as described above.

[0045] In some specific embodiments, the antibody-drug conjugate or a pharmaceutically acceptable salt thereof is selected from the following structural formulas:

[0046] in,

[0047] p represents the average number of connections, and p is any integer or decimal from 1 to 10, preferably any integer or decimal from 3 to 9; more preferably an integer or decimal from 7 to 8, such as 7.77 or 7.79;

[0048] Ab is the anti-CDH17 antibody or its antigen-binding fragment as described above.

[0049] In some specific embodiments, the antibody-drug conjugate or its pharmaceutically acceptable salt is the following conjugate or its pharmaceutically acceptable salt:

[0050] Wherein, p is any integer or decimal from 1 to 10, preferably any integer or decimal from 3 to 9; more preferably any integer or decimal from 6 to 8; for example, p is 7.77 or 7.79;

[0051] DB1001 and DB1002 are anti-CDH17 antibodies. The heavy chain amino acid sequence of DB1001 is shown in SEQ ID NO:12 and the light chain amino acid sequence is shown in SEQ ID NO:13. The heavy chain amino acid sequence of DB1002 is shown in SEQ ID NO:14 and the light chain amino acid sequence is shown in SEQ ID NO:13.

[0052] In some specific embodiments, the antibody-drug conjugate or its pharmaceutically acceptable salt is the following conjugate or its pharmaceutically acceptable salt:

[0053] Where p' represents the number of connections, and p' is any integer from 1 to 10, preferably any integer from 3 to 9; more preferably any integer from 4 to 8; for example, p' is 4, 5, 6, 7 or 8;

[0054] DB1001 and DB1002 are anti-CDH17 antibodies. The heavy chain amino acid sequence of DB1001 is shown in SEQ ID NO:12 and the light chain amino acid sequence is shown in SEQ ID NO:13. The heavy chain amino acid sequence of DB1002 is shown in SEQ ID NO:14 and the light chain amino acid sequence is shown in SEQ ID NO:13.

[0055] In some specific embodiments, the antibody-drug conjugate or its pharmaceutically acceptable salt described in this application comprises at least two antibody-drug conjugates or their pharmaceutically acceptable salts as described above, wherein the number of links p' is the same or different, and the average value of the number of links p' is any integer or decimal from 1 to 10, preferably any integer or decimal from 3 to 9; more preferably any integer or decimal from 6 to 8; for example, the average value of the number of links p' is 7.77 or 7.79.

[0056] To address the aforementioned technical problems, the second technical solution provided in this application is: a method for preparing an antibody-drug conjugate or a pharmaceutically acceptable salt thereof as described in one of the technical solutions of this application, wherein the preparation method comprises reacting the anti-CDH17 antibody or its antigen-binding fragment with a compound as shown in Formula II to obtain the antibody-drug conjugate or a pharmaceutically acceptable salt thereof.

[0057] L'-cytotoxic drugs

[0058] II;

[0059] L' is a linker unit L formed with the anti-CDH17 antibody or its antigen-binding fragment as described in one of the technical solutions of this application;

[0060] The cytotoxic drug M is as described in one of the technical solutions of this application.

[0061] In some specific embodiments, the antibody-drug conjugate or its pharmaceutically acceptable salt satisfies one or more of the following conditions:

[0062] (1) The compound shown in Formula II is:

[0063] (2) The anti-CDH17 antibody or its antigen-binding fragment is DB1001 or DB1002;

[0064] The heavy chain amino acid sequence of DB1001 is preferably as shown in SEQ ID NO:12, and the light chain amino acid sequence is preferably as shown in SEQ ID NO:13; the heavy chain amino acid sequence of DB1002 is as shown in SEQ ID NO:14, and the light chain amino acid sequence is as shown in SEQ ID NO:13.

[0065] To solve the above-mentioned technical problems, the third technical solution provided in this application is: a pharmaceutical composition comprising an antibody-drug conjugate or a pharmaceutically acceptable salt thereof as described in one of the technical solutions of this application, and a pharmaceutically acceptable carrier.

[0066] To solve the above-mentioned technical problems, the fourth technical solution provided in this application is: the use of antibody-drug conjugates or their pharmaceutically acceptable salts as described in the first technical solution of this application and / or pharmaceutical compositions as described in the third technical solution of this application in the preparation of drugs for diagnosing, preventing and / or treating cancer.

[0067] In some specific implementations, the cancer is a CDH17-expressing cancer, such as colorectal cancer, pancreatic cancer, and gastric cancer.

[0068] To address the aforementioned technical problems, the fifth technical solution provided in this application is: a method for diagnosing, preventing, and / or treating cancer, the method comprising administering to a subject in need an effective amount of an antibody-drug conjugate as described in one of the technical solutions of this application or a pharmaceutically acceptable salt thereof and / or a pharmaceutical composition as described in another technical solution of this application.

[0069] In some specific implementations, the cancer is a CDH17-expressing cancer, such as colorectal cancer, pancreatic cancer, and gastric cancer.

[0070] To solve the above-mentioned technical problems, the sixth technical solution provided in this application is: an antibody-drug conjugate as described in the first technical solution of this application or a pharmaceutically acceptable salt thereof and / or a pharmaceutical composition as described in the third technical solution of this application, for the diagnosis, prevention and / or treatment of cancer.

[0071] In some specific implementations, the cancer is a CDH17-expressing cancer, such as colorectal cancer, pancreatic cancer, and gastric cancer.

[0072] To solve the above-mentioned technical problems, the seventh technical solution provided in this application is: a combination therapy, which includes administering to a subject in need an antibody-drug conjugate as described in one of the technical solutions of this application or a pharmaceutically acceptable salt thereof and / or a pharmaceutical composition as described in three of the technical solutions of this application, and a second therapeutic agent.

[0073] In some specific embodiments, the second therapeutic agent comprises other anti-CDH17 antibodies or antigen-binding fragments thereof, or antibody-drug conjugates or pharmaceutical compositions comprising said other anti-CDH17 antibodies or antigen-binding fragments thereof, and / or other drugs for treating cancer.

[0074] In some specific implementations, the cancer is a CDH17-expressing cancer, such as colorectal cancer, pancreatic cancer, and gastric cancer.

[0075] Based on common knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of this application.

[0076] All reagents and raw materials used in this application are commercially available.

[0077] The positive and progressive effects of this application are as follows:

[0078] The antibody of this application has the following technical effects: (1) it specifically binds to human CDH17 and does not bind to its family proteins or other non-target-related proteins; (2) it has excellent affinity for CDH17 protein and tumor cells expressing CDH17; (3) it has species cross-binding activity for human, rat and monkey CDH17 protein; (4) it can mediate CDH17 endocytosis; (5) it has good thermal stability, low pH stability and freeze-thaw stability.

[0079] The antibody-drug conjugate of this application exhibits strong inhibitory activity against tumor cell proliferation both in vivo and in vitro. The antibody-drug conjugate retains the affinity of the parent monoclonal antibody for CDH17 in humans, cynomolgus monkeys, and rats; it shows high affinity for CDH17 from different tumors and at different expression levels; it specifically binds to human CDH17 protein and shows no cross-reactivity with other CDH family proteins; it exhibits significant, dose-dependent antitumor activity after administration and has no effect on animal weight gain. Attached Figure Description

[0080] Figure 1 shows the expression detection of HEK293T-hCDH17 overexpressing cell line.

[0081] Figure 2A shows the protein affinity detection between the anti-human CDH17 humanized antibody and human CDH17-mFc.

[0082] Figure 2B shows the protein affinity detection between the anti-human CDH17 humanized antibody and human CDH17-his.

[0083] Figure 3 shows the protein affinity detection between the anti-human CDH17 humanized antibody and monkey CDH17-His.

[0084] Figure 4 shows the protein affinity test between the anti-human CDH17 humanized antibody and rat CDH17-His.

[0085] Figure 5 shows the affinity test between the anti-human CDH17 humanized antibody and HEK293T-h.CDH17 overexpressing cells.

[0086] Figure 6 shows the affinity test between the anti-human CDH17 humanized antibody and NCI-H716 tumor cells.

[0087] Figure 7 shows the affinity test between the anti-human CDH17 humanized antibody and LS1034 tumor cells.

[0088] Figure 8 shows the affinity test between the anti-human CDH17 humanized antibody and DLD-1 tumor cells.

[0089] Figure 9 shows the affinity test between the anti-human CDH17 humanized antibody and MKN45 tumor cells.

[0090] Figure 10 shows the ADCC activity detection of the anti-human CDH17 humanized antibody.

[0091] Figure 11 shows the detection of ADCP activity of the anti-human CDH17 humanized antibody.

[0092] Figure 12 shows the affinity of antibody-drug conjugates and their parent antibodies for human, monkey, rat, and mouse CDH17.

[0093] Figure 13 shows the affinity of the anti-CDH17 antibody-drug conjugate, its parent monoclonal antibody, and different tumor cell lines.

[0094] Figure 14 shows the affinity of the anti-CDH17 antibody-drug conjugate and its parent antibody for human CDH family proteins.

[0095] Figure 15 shows the efficacy evaluation of the antibody-drug conjugate in human colorectal cancer NCI-H716 tumor-bearing mice.

[0096] Figure 16 shows the efficacy evaluation of the antibody-drug conjugate in human colorectal cancer HT-55 tumor-bearing mice.

[0097] Figure 17 shows the efficacy evaluation of the antibody-drug conjugate in AsPC-1 tumor-bearing mice with human pancreatic cancer.

[0098] Figure 18 shows the growth inhibition of colorectal organoids by the drug.

[0099] Figure 19 shows the in vivo efficacy and weight changes of antibody-drug conjugates with different DAR values ​​in NCI-H716 and HT-55 tumor-bearing mice.

[0100] Figure 20 shows the changes in efficacy and body weight of the antibody-drug conjugate in human HT-29 tumor-bearing mice.

[0101] Figure 21 shows the in vivo efficacy and weight changes of the antibody-drug conjugate in a human colorectal cancer LD1-2012-200671 model.

[0102] Figure 22 shows the changes in efficacy and body weight of the antibody-drug conjugate and the positive control antibody-drug conjugate in human NCI-H716 tumor-bearing mice.

[0103] Figure 23 shows the changes in efficacy and body weight of the antibody-drug conjugate and the positive control antibody-drug conjugate in human HT-55 tumor-bearing mice.

[0104] Figure 24 shows the changes in efficacy and body weight of the antibody-drug conjugate and the positive control antibody-drug conjugate in human AsPC-1 tumor-bearing mice. Detailed Implementation

[0105] In this application, unless otherwise stated, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the operational procedures used herein, such as molecular genetics, nucleic acid chemistry, chemistry, molecular biology, biochemistry, cell culture, microbiology, cell biology, genomics, and recombinant DNA, are all routine procedures widely used in their respective fields. To better understand this application, definitions and explanations of relevant terms are provided below:

[0106] In this application, the letters in the amino acid sequence represent single-letter abbreviations of amino acids known in the art, such as those described in J. Biol. Chem, 243, p3558 (1968): alanine: Ala-A, arginine: Arg-R, aspartic acid: Asp-D, cysteine: Cys-C, glutamine: Gln-Q, glutamic acid: Glu-E, histidine: His-H, glycine: Gly-G, asparagine: Asn-N, tyrosine: Tyr-Y, proline: Pro-P, serine: Ser-S, methionine: Met-M, lysine: Lys-K, valine: Val-V, isoleucine: Ile-I, phenylalanine: Phe-F, leucine: Leu-L, tryptophan: Trp-W, threonine: Thr-T.

[0107] In this application, the term "and / or" should be understood to mean any one of the options or any combination of two or more of the options.

[0108] In this application, the term "or" should be understood to have the same meaning as "and / or" as defined above. For example, when separating items in a list, "or" or "and / or" should be interpreted inclusively, that is, including at least one in the quantity or element list, but also including more than one, and optionally, additional unlisted items. Only when explicitly indicated by the opposite terms, such as "only one" or "exactly one" or when used in the claims as "consisting of...", will it refer to only one number or one element of the list.

[0109] In this application, the term "antibody-drug conjugate" generally refers to an antibody linked to a biologically active cytotoxic drug via a stable linker unit. In this application, "antibody-drug conjugate" can also refer to an antibody or its antigen-binding fragment linked to a biologically active cytotoxic drug fragment via a stable linker unit.

[0110] In this application, the term "cytotoxic drug" generally refers to a toxic drug that possesses a strong chemical molecule within tumor cells that disrupts their normal growth. Cytotoxic drugs can kill tumor cells at sufficiently high concentrations. The "cytotoxic drug" may include toxins, such as small molecule toxins or enzyme-active toxins derived from bacteria, fungi, plants, or animals, and radioactive isotopes (e.g., At). 211 I 131 I 125 Y 90 Re 186 Re 188 、Sm 153 Bi 212 P 32 (or radioactive isotopes of Lu), toxic drugs, chemotherapeutic drugs, antibiotics or ribolysins, or their derivatives, for example, can be toxic drugs, including but not limited to camptothecin derivatives, such as camptothecin derivative essanotecan (chemical name: (1S,9S)-1-amino-9-ethyl-5-fluoro-2,3-dihydro-9-hydroxy-4-methyl-1H,12H-benzo[de]pyrano[3',4':6,7]imidazo[1,2-b]quinoline-10,13(9H,15H)-dione).

[0111] In this application, the term "antibody" generally refers to an immunoglobulin that reacts to a specified protein or peptide or fragment thereof. Antibodies can be from any class, including but not limited to IgG, IgA, IgM, IgD, and IgE, and antibodies from any subclass (e.g., IgG1, IgG2, IgG3, and IgG4). Antibodies may have a heavy chain constant region selected from, for example, IgG1, IgG2, IgG3, or IgG4. Antibodies may also have a light chain selected from, for example, kappa (κ) or lambda (λ). The antibodies of this application may be derived from any species. The term "antibody" may include complete polyclonal antibodies, complete monoclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), chimeric antibodies, humanized antibodies, human antibodies, fusion proteins containing antibodies, and any other modified immunoglobulin molecules, provided that these antibodies exhibit the desired biological activity.

[0112] In this application, the term "antigen-binding fragment" generally refers to a portion of an antibody molecule containing amino acids responsible for the specific binding between the antibody and the antigen. The portion of the antigen that is specifically recognized and bound by the antibody is called an "epitope." As described above, an antigen-binding domain typically includes a variable region (VL) and a variable region (VH) of the antibody light chain; however, it is not necessary to include both. Fd fragments, for example, have two VH regions and typically retain some of the antigen-binding function of the intact antigen-binding domain. Examples of antigen-binding fragments of antibodies include (1) Fab fragments, monovalent fragments having VL, VH, constant light chain (CL) and CH1 domains; (2) F(ab′)2 fragments, bivalent fragments having two Fab fragments connected by disulfide bridges of hinge regions; (3) Fd fragments having two VH and CH1 domains; (4) Fv fragments having VL and VH domains of antibody single arms; (5) dAb fragments (Ward et al., “Binding Activities of a Repertoire of Single Immunoglobulin Variable Domains Secreted From Escherichia coli”, Nature 341:544-546 (1989), which are incorporated herein by reference in their entirety) having a VH domain; (6) separate complementarity-determining regions (CDRs); and (7) single-chain Fv (scFv), for example derived from scFV libraries.Although the two domains VL and VH of the Fv fragment are encoded by independent genes, they can be joined together using a recombination method via a synthetic linker. The synthetic linker allows it to be prepared as a single protein chain in which the VL and VH regions pair to form a monovalent molecule (called single-chain Fv (scFv)) (see, for example, Huston et al., “Protein Engineering of Antibody Binding Sites: Recovery of Specific Activity in an Anti-Digoxin Single-ChainFv Analogue Produced in Escherichia coli,” Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988)); (8) “VHH” refers to the variable antigen-binding domain of heavy chain antibodies from camelids (camels, dromedaries, llamas, alpacas, etc.) (see Nguyen VK et al., 2000, The EMBO Journal, 19, 921-930; Muyldermans S., 2001, J Biotechnol., 74, 277-302 and a review by Vanlandschoot P. et al., 2011, Antiviral Research 92, 389-407). VHH can also be called nanobody (Nb).

[0113] In this application, the term "variable region" or "variable domain" generally refers to the domain of the antibody heavy or light chain involved in antibody-antigen binding. In this application, the term "variable" generally means that certain portions of the sequence of the variable domain of an antibody vary significantly, resulting in various specific antibody binding and specificity to its specific antigen. This variability is not uniformly distributed throughout the entire variable region of the antibody. It is concentrated in three segments within the light and heavy chain variable regions, referred to as complementarity-determining regions (CDRs) or hypervariable regions (HVRs), namely LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, and HCDR3. The more highly conserved portions of the variable domain are called framework regions (FRs). The variable domains of the natural heavy and light chains each contain four FR regions (H-FR1, H-FR2, H-FR3, H-FR4, L-FR1, L-FR2, L-FR3, L-FR4), mostly in a β-sheet configuration, connected by three CDR loop regions. The CDRs in each chain are closely packed together through the FR region and together with the CDRs from the other chain, they form the antigen-binding site of the antibody.

[0114] In one respect, "CDR" is defined as the amino acid sequence of the complementarity-determining region of an antigen-binding protein. These are hypervariable regions of the heavy and light chains of immunoglobulins. The variable portion of an immunoglobulin has three heavy chain CDRs and three light chain CDRs (or CDR regions). Therefore, as used herein, "CDR" refers to all three heavy chain CDRs, all three light chain CDRs, all heavy chain CDRs and light chain CDRs, or at least two CDRs.

[0115] In this specification, amino acid residues in the variable domain sequence and variable domain regions in the full-length antigen-binding sequence (e.g., antibody heavy chain or antibody light chain sequences) are numbered according to the Chothia, AbM, and / or Kabat numbering scheme. Similarly, the terms “CDR,” “CDRL1,” “CDRL2,” “CDRL3,” “CDRH1,” “CDRH2,” and “CDRH3” used in the examples follow the Chothia, AbM, and / or Kabat numbering scheme. For more information, see Kabat et al., Sequences of Proteins of Immunological Interest, 4th edition, USDapartment of Health and Human Services, National Institutes of Health (1987) and Chothia et al. (1989) Nature 342:877-883. Other numbering schemes for CDR sequences available to those skilled in the art include the “AbM” (University of Bath) and “contact” (University College London) methods.

[0116] The structure and protein folding of antigen-binding proteins may imply that other residues are considered to be part of the CDR sequence, as those skilled in the art would understand.

[0117] In this application, the amino acid sequences of the listed CDRs are all given according to the Kabat definition rules. However, it is well known to those skilled in the art that antibody CDRs can be defined in various ways, such as Chothia et al. (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)), based on antibody sequence variability (Kabat et al., Sequences of Proteins of Immunological Interest, 4th edition, USDepartment of Health and Human Services, National Institutes of Health (1987)), AbM (University of Bath), Contact (University College London), the international ImMunoGeneTics database (IMGT) (imgt.cines.fr / ), and the North CDR definition based on affinity propagation clustering using a large number of crystal structures. Those skilled in the art will understand that, unless otherwise specified, the terms "CDR" and "complementarity-determining region" for a given antibody or its region (e.g., variable region) should be understood to encompass complementarity-determining regions defined by any of the known schemes described herein. While the scope of protection claimed in this application is based on the sequence shown in the Kabat definition rules, amino acid sequences corresponding to other CDR definition rules should also fall within the scope of protection of this application. Therefore, when referring to antibodies defined by a specific CDR sequence as defined in this application, the scope of said antibody also includes antibodies whose variable region sequence contains the specific CDR sequence, but whose claimed CDR boundaries differ from those defined in this application due to the application of different schemes (e.g., different assignment system rules or combinations).

[0118] The sequence identity between sequences is calculated as follows. To determine the percentage of identity between two amino acid sequences, the sequences are aligned for optimal comparison purposes (e.g., vacancies may be introduced in the first and second amino acid sequences for optimal alignment, or non-homologous sequences may be discarded for comparison purposes). In a preferred embodiment, for comparison purposes, the length of the reference sequence being compared is at least 30%, preferably at least 40%, more preferably at least 50%, 60%, and even more preferably at least 70%, 80%, 90%, or 100% of the reference sequence length. The amino acid residues at corresponding amino acid positions are then compared. When a position in the first sequence is occupied by the same amino acid residue at the corresponding position in the second sequence, the molecules are identical at that position. Sequence comparison and the calculation of the percentage of identity between two sequences can be performed using mathematical algorithms. In a preferred embodiment, the Needlema and Wunsch ((1970) J. Mol. Biol. 48: 444-453) algorithm (available at http: / / www.gcg.com) is used in the GAP program, which is integrated into the GCG software package. The Blossum 62 matrix or PAM250 matrix and vacancy weights of 16, 14, 12, 10, 8, 6, or 4, and length weights of 1, 2, 3, 4, 5, or 6 are used to determine the percentage of identity between two amino acid sequences. A particularly preferred set of parameters (and one set of parameters that should be used unless otherwise specified) is a Blossum 62 scoring matrix with a vacancy penalty of 12, a vacancy extension penalty of 4, and a frameshift vacancy penalty of 5. Alternatively, the PAM120 weighted remainder table, gap length penalty of 12, and gap penalty of 4 can be used to determine the percentage of identity between two amino acid sequences using the E. Meyers and W. Miller algorithm ((1989) CABIOS, 4:11-17), which has been incorporated into the ALIGN program (version 2.0). Additionally or alternatively, the protein sequence described in this application can be further used as a "query sequence" to perform a search against public databases to, for example, identify other family member sequences or related sequences.

[0119] In this application, the term "full-length antibody" is used interchangeably to refer to a glycoprotein comprising at least two heavy chains (HC) and two light chains (LC) linked together by disulfide bonds. Each heavy chain consists of a heavy chain variable region (abbreviated as VH in this application) and a heavy chain constant region. The heavy chain constant region consists of three domains: CH1, CH2, and CH3. Each light chain consists of a light chain variable region (abbreviated as VL in this application) and a light chain constant region (abbreviated as CL in this application). The light chain constant region consists of one domain: CL. Mammalian heavy chains are classified as α, δ, ε, γ, and μ. Mammalian light chains are classified as λ or κ. Immunoglobulins containing α, δ, ε, γ, and μ heavy chains are classified as immunoglobulin (Ig) A, IgD, IgE, IgG, and IgM. Complete antibodies form a "Y" shape. The stem of Y consists of the second and third constant regions (and a fourth constant region for IgE and IgM) of two heavy chains linked together, with disulfide bonds (interchain) forming hinges. Heavy chains γ, α, and δ have constant regions consisting of three tandem (in a row) Ig domains, and hinge regions for increased flexibility; heavy chains μ and ε have constant regions consisting of four immunoglobulin domains. The second and third constant regions are referred to as the "CH2 domain" and the "CH3 domain," respectively. Each arm of Y includes a variable region of a single heavy chain and a first constant region that binds to a variable and constant region of a single light chain. The variable regions of the light and heavy chains are responsible for antigen binding.

[0120] In this application, "Fab" consists of a light chain and a heavy chain, CH1, and a variable region. The heavy chain of the Fab molecule cannot form disulfide bonds with another heavy chain molecule. The "Fc" region contains two heavy chain segments containing the CH2 and CH3 domains of the antibody. The two heavy chain segments are held together by two or more disulfide bonds and through the hydrophobic interaction of the CH3 domain. "Fab'" contains a portion of a light chain and a heavy chain containing the VH domain, the CH1 domain, and the region between the CH1 and CH2 domains, thereby allowing interchain disulfide bonds to form between the two heavy chains of the two Fab's to form the F(ab')2 molecule. "F(ab')2" contains two light chains and two heavy chains containing portions of the constant region between the CH1 and CH2 domains, thereby allowing interchain disulfide bonds to form between the two heavy chains. Therefore, the F(ab')2 fragment consists of two Fab' fragments held together by disulfide bonds between the two heavy chains. The term "Fv" refers to an antibody fragment consisting of the VL and VH domains of a single arm of the antibody, but lacking the constant region.

[0121] In this application, the term "monoclonal antibody" refers to an antibody derived from a basic homogeneous group of antibodies, meaning that the individual antibodies comprising this group are identical except for the possibility of naturally occurring mutations that may be present in small amounts. Monoclonal antibodies are highly specific, targeting a single antigenic epitope. In contrast, conventional (polyclonal) antibody preparations typically comprise a large number of antibodies targeting different epitopes (or specific to different epitopes). The modifier "monoclonal" indicates the characteristic of antibodies derived from a basic homogeneous group of antibodies and should not be construed as requiring the production of antibodies by any particular method.

[0122] In this application, the term "humanized antibody" refers to an antibody form containing sequences derived from human and non-human (e.g., mouse, rat) antibodies. Generally, humanized antibodies contain substantially all of at least one, typically two, variable domains, wherein all or substantially all of the hypervariable loops correspond to the hypervariable loops of non-human immunoglobulins, and all or substantially all of the framework (FR) regions are framework regions of human immunoglobulin sequences. Humanized antibodies may optionally contain at least a portion of the constant region (Fc) of human immunoglobulins.

[0123] "Isotype" antibodies refer to antibody classes provided by heavy chain constant region genes (e.g., IgM, IgE, IgG such as IgG1, IgG2, or IgG4). Isotypes also include modified forms of one of these classes, where modifications have been generated to alter Fc function, such as to enhance or weaken effector function or binding to the Fc receptor.

[0124] In this application, "affinity" or "binding affinity" refers to the intrinsic binding affinity that reflects the interaction between members of a binding pair. The affinity of molecule X for its partner Y can generally be determined by the equilibrium dissociation constant (K0). D The equilibrium dissociation constant represents the dissociation rate constant and the binding rate constant (k, k, k) respectively. dis and k on The ratio of affinity to antigen. Affinity can be measured by common methods known in the art. In some embodiments of this application, surface plasmon resonance (SPR) technology is used to measure affinity, such as the affinity between the antibody and the antigen in this application. In some preferred embodiments of this application, a specific method for measuring affinity is the BIAcore method described herein.

[0125] In this application, the term "halogen" generally refers to fluorine, chlorine, bromine, or iodine, for example, it can be fluorine or chlorine.

[0126] In this application, the term "alkyl" generally refers to a residue derived from an alkane by removing a hydrogen atom. Alkyl groups can be substituted or unsubstituted, substituted or non-substituted. The term "alkyl" generally refers to a saturated straight-chain or branched aliphatic hydrocarbon group having residues derived from the removal of hydrogen atoms from the same carbon atom or two different carbon atoms of the parent alkane, and can be a straight-chain or branched group containing 1 to 20 carbon atoms, for example, containing 1 to 12 carbon atoms, such as a chain alkyl containing 1 to 6 carbon atoms. Non-limiting examples of alkyl groups include, but are not limited to, methyl, ethyl, propyl, propyl, butyl, etc.

[0127] In this application, the term "independently" generally means that the variable applies to any situation, regardless of whether there are variables with the same or different definitions in the same compound. For example, the variable may refer to the type or number of substituents in the compound, or the type of atoms in the compound. For example, when R appears twice in a compound and R is defined as "independently carbon or nitrogen", both Rs can be carbon, both Rs can be nitrogen, or one R can be carbon and the other R can be nitrogen.

[0128] In this application, the terms “optional” or “optionally” generally mean that the event or environment described below may but does not have to occur, and the description includes the occasion in which the event or environment occurs or does not occur. For example, “C1-C6 alkyl group optionally substituted with one or more R” means that R may but does not have to be present, and the description can include the case where the C1-C6 alkyl group is substituted with R and the case where the C1-C6 alkyl group is not substituted with R.

[0129] In this application, the term "substituted" generally refers to one or more hydrogen atoms in a group, for example, up to five, or for example, one to three hydrogen atoms, independently substituted by the corresponding number of substituents. Substituents are only in their possible chemical positions, and those skilled in the art can determine (by experiment or theory) possible or impossible substitutions without much effort. For example, an amino or hydroxyl group with free hydrogen may be unstable when bonded to a carbon atom with an unsaturated bond (such as an alkene).

[0130] In this application, as those skilled in the art will know, terms such as "alkyl," "alkenyl," and "cycloalkyl" may be preceded by an identifier indicating the number of atoms present in the group under specific conditions, for example, C1-C4 alkyl, C3-C7 cycloalkoxy, C1-C4 alkylcarbonylamino, etc., and the subscript number following "C" indicates the number of carbon atoms present in the group. For example, C3 alkyl refers to an alkyl group having three carbon atoms (e.g., n-propyl, isopropyl); C 1-10 In this context, the members of the group can have any number of carbon atoms falling within the range of 1-10.

[0131] In this application, the compounds or antibody-drug conjugates of this application include their tautomers, meso compounds, racemates, enantiomers, and / or diastereomers. In this application, the term "diastereomer" generally refers to a stereoisomer having two or more chiral centers and whose molecules are not mirror images of each other. Diastereomers can have different physical properties, such as melting point, boiling point, spectral properties, and reactivity. In this application, the terms "tautomer" or "tautomer form" are used interchangeably and generally refer to structural isomers with different energies that can be interconverted through a low energy barrier. For example, proton tautomers (also known as prototropic tautomers) include interconversions via proton migration, such as keto-enol isomerization and imine-enamine isomerization. Valence tautomers include interconversions via the rearrangement of some bonding electrons. In this application, the term "meta-polymorph" generally refers to a molecule containing asymmetric atoms but possessing symmetry factors that result in zero total optical rotation within the molecule. The term "racemic mixture" or "racemic mixture" refers to a composition consisting of two enantiomers in equimolar amounts.

[0132] In this application, the terms "connector unit" or "connector structure" generally refer to a chemical structural fragment or bond that is connected to a ligand at one end and to a cytotoxic drug at the other end. It may also refer to connecting other connectors before being connected to the cytotoxic drug. The direct or indirect connection to the ligand can refer to the group directly connecting to the ligand via a covalent bond, or it can refer to the connection of the ligand via a connector structure. For example, chemical structural fragments or bonds containing acid-labile connector structures (e.g., hydrazones), protease-sensitive (e.g., peptidase-sensitive) connector structures, light-labile connector structures, dimethyl connector structures, or disulfide-containing connector structures can be used as connector structures.

[0133] In some embodiments of this application, antibody-drug conjugates refer to compositions comprising different DAR (drug-antibody ratio) distributions. In some embodiments of this application, compositions comprising antibodies conjugated with bioactive cytotoxic drugs at different DARs are provided.

[0134] In some embodiments of this application, antibody-drug conjugates refer to compositions containing the same DAR distribution. In some embodiments of this application, compositions comprising an antibody conjugated with a bioactive cytotoxic drug using the same DAR are provided.

[0135] In some embodiments of this application, the terms "drug loading" or DAR are used interchangeably, referring to the average amount of cytotoxic drug loaded on each ligand, which may be called the average number of linkages p, or expressed as the ratio of cytotoxic drug to antibody. The range of cytotoxic drug loading can be 0-12 linkages per ligand (Ab), for example, 1-10 cytotoxic drugs. The drug loading of each ADC molecule after the coupling reaction can be identified using conventional methods such as UV / Vis spectroscopy, mass spectrometry, ELISA assays, and HPLC characterization. The average number of linkages p can be an integer or decimal from 1 to 10. For example, the average number of linkages p can be an integer or decimal from 2 to 8. For example, the average number of linkages p can be an integer or decimal from 3 to 8. For example, the average number of linkages p can be an integer or decimal from 1 to 2, 2 to 3, 3 to 4, 4 to 5, 5 to 6, 6 to 7, 7 to 8, 8 to 9, or 9 to 10.

[0136] In some embodiments of this application, an antibody-drug conjugate refers to a single compound containing the same DAR distribution. The term "drug loading" refers to the number of cytotoxic drugs loaded onto each ligand, which may be called the linker number p', or expressed as the ratio of cytotoxic drug to antibody amount. The cytotoxic drug loading can range from 0 to 12 per ligand (Ab), for example, 1 to 10 cytotoxic drugs. The linker number p' can be any integer from 1 to 10. For example, the linker number p' can be any integer from 3 to 9. For example, the linker number p' can be any integer from 6 to 8. For example, the linker number p' can be 4, 5, 6, 7, or 8.

[0137] In this application, certain atoms of the compound or antibody-drug conjugate may appear in more than one isotopic form. For example, hydrogen may appear as protium (…). 1 H), deuterium ( 2 H) and tritium ( 3 Carbon exists in the form of H, and it may exist in three different isotopes (H). 12 C 13 C and 14 C) Naturally occurring. Examples of isotopes that may be incorporated into the compounds of this application include, but are not limited to, those that exist naturally. 15 N、 18 O、 17 O、 18 F, 32 P, 33 P, 129 I, 131 I, 123 I, 124 I, 125I, or similar isotopes. Therefore, the compounds or antibody-drug conjugates of this application can be enriched in one or more of these isotopes relative to their natural abundance. As those skilled in the art will know, such isotope-enriched compounds can be used for a variety of purposes. For example, with heavy isotopes such as deuterium (I, or similar isotopes). 2 H) substitution may offer certain therapeutic advantages, possibly due to greater metabolic stability. For example, deuterium (H) 2 The natural abundance of deuterium (H) is approximately 0.015%. Therefore, there is approximately one deuterium atom for every 6500 hydrogen atoms in nature. Consequently, the deuterium abundance of the deuterium-containing compounds or antibody-drug conjugates of this application is greater than 0.015% at one or more positions (as the case may be). Unless otherwise specified, the structures described in this application may also include compounds or antibody-drug conjugates that differ only in the presence or absence of one or more isotopically enriched atoms. For example, compounds or antibody-drug conjugates whose structures are identical to those of this application except that hydrogen atoms are replaced by deuterium or tritium, or carbon atoms are replaced by carbon-13 or carbon-14, are within the scope of this application.

[0138] In this application, the term "pharmaceutical composition" generally refers to a mixture containing one or more of the compounds or antibody-drug conjugates described in this application, or their physiologically / pharmaceutical acceptable salts or prodrugs, along with other chemical components, such as physiologically / pharmaceutical acceptable carriers and excipients. Pharmaceutical compositions can facilitate administration to an organism, promote the absorption of the active ingredient, and thus exert its biological activity. Conventional preparation methods for pharmaceutical compositions can be found in the Chinese Pharmacopoeia. Pharmaceutical compositions can be in the form of sterile injectable aqueous or oil suspensions for intramuscular and subcutaneous administration. Such suspensions can be formulated using suitable dispersants or wetting agents and suspending agents as described above, according to known techniques. Sterile injectable formulations can also be sterile injectable solutions or suspensions prepared in non-toxic, parenteral-acceptable diluents or solvents, such as solutions prepared in 1,3-butanediol. Furthermore, sterile fixative oils can conveniently be used as solvents or suspension media. For example, any blended fixative oil, including synthetic mono- or diglycerides, can be used. Additionally, fatty acids such as oleic acid can also be used to prepare injectable formulations.

[0139] In this application, the terms "pharmaceutically acceptable salt" or "pharmaceutically usable salt" generally refer to salts of compounds or antibody-drug conjugates of this application, or salts of compounds or antibody-drug conjugates described in this application. Such salts may be safe and / or effective when used in mammals and may have the desired biological activity. The compounds or antibody-drug conjugates of this application may form salts with acids. Non-limiting examples of pharmaceutically acceptable salts include: hydrochloride, hydrobromide, hydroiodide, sulfate, hydrogen sulfate, citrate, acetate, succinate, ascorbate, oxalate, nitrate, sorbate, hydrogen phosphate, dihydrogen phosphate, salicylate, hydrogen citrate, tartrate, maleate, fumarate, formate, benzoate, methanesulfonate, ethanesulfonate, benzenesulfonate, and p-toluenesulfonate.

[0140] In this application, a pharmaceutically acceptable carrier is any of those conventionally used carriers, limited only by physicochemical considerations (such as solubility and lack of reactivity with antibodies targeting CDH17) and by route of administration. Pharmaceutically acceptable carriers described herein, such as mediators, adjuvants, excipients, and diluents, are well known to those skilled in the art and are readily available to the public. In one aspect, a pharmaceutically acceptable carrier is a carrier that is chemically inert to the active ingredient of a pharmaceutical composition and does not have adverse side effects or toxicity under the conditions of use. In some embodiments, the carrier does not produce adverse, allergic, or other inappropriate reactions when administered to animals or humans. In some aspects, the pharmaceutical composition is free of pyrogens and other impurities that would be harmful to humans or animals. Pharmaceutically acceptable carriers include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonics, and absorption delay agents, etc.; their uses are well known in the art.

[0141] As used in this application, the term "effective amount" means the amount of a drug or agent that elicits a biological or pharmaceutical response in a tissue, system, animal, or human, as sought by, for example, an investigator or clinician. Furthermore, the term "effective amount" means the amount that causes improved treatment, cure, prevention, or reduction of disease, symptom, or side effects, or reduces the rate of progression of a disease or condition, compared to a corresponding subject who did not receive that amount. Within its scope, the term also includes amounts that effectively enhance normal physiological function.

[0142] As used herein, “cancer disease” or “cancer” includes diseases characterized by abnormally regulated cell growth, proliferation, differentiation, adhesion, and / or migration. Cancer cells are abnormal cells that grow through rapid, uncontrolled cell proliferation and continue to grow even after the stimuli that initiated new growth have ceased.

[0143] As used herein, the term "CDH17-expressing cancer" refers to cancer involving cancer cells expressing CDH17, preferably on the surface of said cancer cells.

[0144] The present application is further illustrated below by way of embodiments, but these embodiments are not intended to limit the scope of the present application. Experimental methods not specifically described in the following embodiments are performed according to conventional methods and conditions, or as selected in accordance with the product instructions.

[0145] Example 1: Preparation of anti-CDH17 antibody

[0146] Humanized monoclonal antibodies against human CDH17, DB1001 and DB1002, reference antibody 1 (BM-1, sequence from patent application CN 110582513 A, SEQ ID NO:116 and SEQ ID NO:117), and reference antibody 2 (BM-2, sequence from the heavy chain variable region and light chain variable region amino acid sequences of SEQ ID NO:47 and SEQ ID NO:48 in patent application WO2023107558A1), were modified by adding the heavy chain constant region (SEQ ID NO:9) and light chain constant region (SEQ ID NO:10) sequences, respectively. These were then submitted to Universal Biotech for codon optimization and gene synthesis. The modified antibodies were cloned into mammalian cell expression vectors and expressed in 293E cells. The cell expression supernatant was centrifuged at high speed to remove impurities. The expression supernatant was purified using a Protein A column. The supernatant was loaded onto the column at a specific flow rate, and the column was washed with PBS until the A280 reading returned to baseline. The target protein was eluted with 100 mM sodium acetate at pH 3.5. The samples were neutralized with 2M Tris HCl at 8.0 g / L, concentrated by elution, and then aliquoted with PBS to prepare DB1001, DB1002, BM-1, and BM-2.

[0147] The sequence numbers of the CDRs and variable regions of the heavy and light chains of the DB1001 and DB1002 antibodies are shown in Table 1.

[0148] Table 1. Heavy and light chain CDRs and variable region sequences of the antibody (Kabat)

[0149] The underlines below the amino acid sequences in Table 1 indicate CDR regions defined according to the Kabat numbering system.

[0150] The heavy chain and light chain variable region sequences are linked to the heavy chain constant region and light chain constant region sequences of the human antibody, respectively. For example, the antibody heavy chain constant region is selected from the human IgG1 heavy chain constant region as shown in SEQ ID NO:9 or 11; the antibody light chain constant region is selected from the human κ chain constant region as shown in SEQ ID NO:10, and the antibody constant region sequences are shown in Table 2.

[0151] Table 2. Sequence Numbering of Constant Regions

[0152] The heavy chain amino acid sequence of DB1001 is (SEQ ID NO:12):

[0153] The light chain amino acid sequences of DB1001 & DB1002 are (SEQ ID NO:13):

[0154] The heavy chain amino acid sequence of DB1002 is (SEQ ID NO:14):

[0155] Bold text with underline indicates the CDR region defined according to the Kabat numbering system, and bold text with italics indicates the LALA mutation site at the Fc end.

[0156] Example 2: Synthesis of Connector-Cytoxin

[0157] Connector - Cytotoxin X2:

[0158] Synthesis route:

[0159] first step

[0160] 34a (5 g, 48.0 mmol) and K₂CO₃ (19.9 g, 144.0 mmol) were dissolved in DMF (20 mL), and benzyl bromide (12.3 g, 72.0 mmol) was added dropwise. The reaction was carried out at 25 °C for 17 hours. TLC (PE / EA = 3 / 1) was used to determine if the reaction was complete. The reaction solution was added to water (200 mL), extracted with EA (250 mL), washed with saturated NaCl, dried over anhydrous Na₂SO₄, and concentrated and column filtered (PE:EA = 2:1) to give 8.7 g of colorless liquid 34b, yield 93%. MS-ESI: m / z 195.1 [M+H]⁺.

[0161] Step 2

[0162] Dissolve 7.3 g (19.8 mmol) of 43c and 1.46 g (8.5 mmol) of TsOH in 20 mL of THF. Under nitrogen protection and cooling to 0 °C, add 10 mL of THF solution containing 7.7 g (39.6 mmol) of 34b. After the addition is complete, react at 0 °C for 2 hours. TLC (PE / EA = 2 / 1) shows that most of the starting material has reacted. Pour the reaction solution into 100 mL of water, extract with 100 mL of DCM, separate the layers, wash with saturated NaCl, dry with anhydrous Na₂SO₄, and pass through a column (PE / EA = 1 / 1) to obtain 3.9 g of colorless viscous 34d, yield: 39%. MS-ESI: m / z 503.3 [M+H]+.

[0163] Step 3

[0164] Under hydrogen atmosphere and at 0°C, Pd / C (1 g, 10 wt.%) was added to a mixed solution of EtOH (100 mL) and EA (100 mL) containing 34d (1.9 g, 3.78 mmol) and reacted at 0°C for 3 hours. TLC (PE / EA = 2 / 1) showed that the reaction was complete. The reaction solution was filtered through diatomaceous earth, and the filter cake was washed with EA / EtOH (1:1, 100 mL × 3). The filtrate was concentrated, dissolved in THF (50 mL × 3), and evaporated to dryness. This process was repeated three times to obtain 1 g of gray solid 34e, yield: 64%. MS-ESI: m / z 435.2 [M+Na]+.

[0165] Step 4

[0166] Under nitrogen protection, DIEA (303 mg, 2.35 mmol) was added dropwise to a DMF (20 mL) solution of 34e (426 mg, 1.03 mmol), KI4 (500 mg, 0.94 mmol), and HATU (429 mg, 1.13 mmol) at 0 °C. After the addition was complete, the reaction was allowed to proceed at 0 °C for 2 hours. LC-MS showed the reaction was complete. The reaction solution was added dropwise to 300 mL of water, stirred, and allowed to stand for 5 minutes. After filtration, the filter cake was dissolved in a DCM / MeOH (10:1, 100 mL) solution, dried, and stirred. Column chromatography (EA:MeOH = 30:1) yielded 600 mg of yellow solid 34f, yield: 77%. MS-ESI: m / z 830.3 [M+H]+.

[0167] Step 5

[0168] Under nitrogen protection, diethylamine (5 mL) was added dropwise to a 34f (150 mg, 0.18 mmol) DCM (5 mL) solution at 0 °C, and the reaction was carried out at 0 °C for 2 hours. LCMS showed that the reaction was complete. Petroleum ether solution (100 mL × 6) was added to the reaction solution, and a solid precipitated. After standing to allow the solid to settle, the solution was poured off and then dried using an oil pump to obtain 34 g of 120 mg white powder. LCMS showed that the product content was 70%, yield: 76%. MS-ESI: m / z 608.3 [M+H]+.

[0169] Step 6

[0170] Under nitrogen protection, HATU (45 mg, 0.118 mmol) in DMF (1 mL) was added to 34 g (60 mg, 0.099 mmol), 43 h (51 mg, 0.108 mmol), and DIEA (32 mg, 0.25 mmol) solutions at 0 °C, and the reaction was carried out at 0 °C for 2 h. LC-MS showed that the starting material reacted completely. The reaction solution was directly passed through a reversed-phase column with eluent (MeCN / MeOH = 1 / 1):H2O = 60%:40%) to purify 14.8 mg of yellow solid x 2, yield 14%.

[0171] MS-ESI: m / z 1062.4[M+H]+.

[0172] 1H NMR(400MHz, Methanol-d4)δ7.69–7.61(m,2H),7.22–7.16(m,2H),7.16–7.09(m,3H),6.76(s,2H),5.70–5.64(m,1H),5.6 0(d,J=16.4Hz,1H),5.40–5.31(m,2H),5.26(d,J=19.0Hz,1H),4.65–4.50(m,7H),4.25–4.16(m,1H),3.87(d,J=16.7Hz,1 H),3.83–3.76(m,3H),3.72(d,J=17.0Hz,2H),3.44(t,J=7.1Hz,2H),3.25–3.17(m,2H),3.10–3.02(m,1H),2.92–2.83(m, 1H),2.45–2.39(m,5H),2.32–2.20(m,5H),1.97–1.89(m,2H),1.63–1.50(m,4H),1.34–1.20(m,6H),0.99(t,J=7.3Hz,3H).

[0173] Example 3: Preparation of anti-CDH17 antibody drug conjugate

[0174] ADC-1(DB1001-X2_DAR8)

[0175] The reducing agent and the protective agent were prepared separately using ultrapure water as follows: 5 mmol / L TCEP (Tris-2-carboxyethyl-phosphine, manufacturer: Thermo) aqueous solution and 100 mmol / L EDTA (disodium ethylenediaminetetraacetate, manufacturer: Sigma) aqueous solution.

[0176] Take 200 mg of DB1001 monoclonal antibody (25.00 mg / ml) and place it in a 50 ml centrifuge tube. Dilute the antibody concentration to 10 mg / ml with 20 mM His-HCl, pH 6.0 buffer. Add 100 mM EDTA aqueous solution at 5% of the total reaction volume. After vortexing and mixing, add 5 mmol / L TCEP aqueous solution for antibody reduction. The molar ratio of TCEP to antibody is 10:1. After vortexing and mixing, react in a refrigerated thermostat at 37°C for 2 h. Add the aforementioned adapter-cytotoxic agent X2 DMSO solution at a final drug-to-antibody molar ratio of 12:1. Add DMSO at 10% of the total reaction volume. After vortexing and mixing, react in a refrigerated thermostat at 4°C for 1 h. The sample preservation buffer was replaced using an ultrafiltration tube (MWCO 30KD, manufacturer: Millipore). The sample was first ultrafiltered three times with 20mM His-HCl buffer (pH 6.0) containing 10% DMSO, and then six times with 20mM His-HCl buffer (pH 6.0) without DMSO. This yielded 166.5 mg of antibody-drug conjugate ADC-1 (DB1001-X2_DAR8), with a concentration of 17.6 mg / mL and a yield of 83.2%.

[0177] The drug loading (DAR / p) of antibody-drug conjugate ADC-1 (DB1001-X2_DAR8) was 7.77 and the SEC purity was 99.19%, as determined by RP-HPLC and SEC-HPLC.

[0178] ADC-1-2(DB1002-X2_DAR8)

[0179] The reducing agent and the protective agent were prepared separately using ultrapure water as follows: 5 mmol / L TCEP (Tris-2-carboxyethyl-phosphine, manufacturer: Thermo) aqueous solution and 100 mmol / L EDTA (disodium ethylenediaminetetraacetate, manufacturer: Sigma) aqueous solution.

[0180] 23 mg of DB1002 monoclonal antibody (6.00 mg / ml) was placed in a 50 ml centrifuge tube. The antibody concentration was diluted to 4 mg / ml with 20 mM His-HCl, pH 6.0 buffer. 100 mM EDTA aqueous solution was added at 5% of the total reaction volume. After vortexing and mixing, 5 mmol / L TCEP aqueous solution was added for antibody reduction (TCEP to antibody molar ratio 10:1). After vortexing and mixing, the mixture was incubated at 37°C for 2 h using a refrigerated incubator. Then, DMSO solution of the aforementioned adapter-cytotoxic agent X2 was added at a final drug to antibody molar ratio of 12:1. DMSO was added at 10% of the total reaction volume. After vortexing and mixing, the mixture was incubated at 4°C for 1 h using a refrigerated incubator. The sample preservation buffer was replaced using an ultrafiltration tube (MWCO 30KD, manufacturer: Millipore). The sample was first ultrafiltered three times with 20mM His-HCl buffer containing 10% DMSO at pH 6.0, and then ultrafiltered six times with 20mM His-HCl buffer without DMSO at pH 6.0 to obtain 20mg of antibody-drug conjugate DB1002-X2 at a concentration of 5mg / mL, with a yield of 86.9%.

[0181] The drug loading (DAR / p) of the antibody-drug conjugate DB1002-X2 was 7.79, and the SEC purity was 99.3%, as determined by RP-HPLC and SEC-HPLC.

[0182] ADC-2 (DB1001-X2 DAR6)

[0183] The reducing agent and the protective agent were prepared separately using ultrapure water as follows: 5 mmol / L TCEP (Tris-2-carboxyethyl-phosphine, manufacturer: Thermo) aqueous solution and 100 mmol / L EDTA (disodium ethylenediaminetetraacetate, manufacturer: Sigma) aqueous solution.

[0184] Take 23 mg of DB1001 monoclonal antibody (6.00 mg / ml) and place it in a 50 ml centrifuge tube. Dilute the antibody concentration to 4 mg / ml with 20 mM His-HCl and pH 6.0 buffer. Add 100 mM EDTA aqueous solution at 5% of the total reaction volume. After vortexing and mixing, add 5 mmol / L TCEP aqueous solution for antibody reduction. The molar ratio of TCEP to antibody is 3.6:1. After vortexing and mixing, place the mixture on a refrigerated constant temperature mixer at 37°C for 2 h.

[0185] Add the pre-prepared DMSO solution of adapter-cytotoxic X2 at a drug-to-antibody molar ratio of 8:1, and supplement with DMSO at 10% of the total volume of the reaction solution. After shaking and mixing, place the mixture on a refrigerated constant temperature mixer and react at 4°C for 1 hour.

[0186] The sample preservation buffer was replaced using an ultrafiltration tube (MWCO 30KD, manufacturer: Millipore). The sample was first ultrafiltered three times with 20mM His-HCl buffer (pH 6.0) containing 10% DMSO, and then ultrafiltered six times with 20mM His-HCl buffer (pH 6.0) without DMSO to obtain 20mg of antibody-drug conjugate ADC-2 at a concentration of 5mg / mL, with a yield of 88.5%.

[0187] The drug loading (DAR / p) of antibody-drug conjugate ADC-2 was 6.1 as determined by RP-HPLC and SEC-HPLC, and its purity by size exclusion chromatography (SEC) was 99.2%.

[0188] ADC-3 (Isotype IgG1-X2_DAR8)

[0189] The preparation of ADC-3 was similar to that of ADC-2 in this example, except that DB1001 was replaced with isotype IgG1. The drug loading (DAR / p) of ADC-3 was 8.0.

[0190] ADC-4(BM3-X2_DAR8)

[0191] The preparation of ADC-4 was similar to that of ADC-2 in this embodiment, except that DB1001 was replaced with the CDH17-targeting antibody BM3. The drug loading (DAR / p) of ADC-4 was 8.0. Antibody BM3 was prepared according to patent CN110582513A (SEQ ID NO:118, SEQ ID NO:119).

[0192] ADC-5 (BM4-X2_DAR8)

[0193] The preparation of ADC-5 was similar to that of ADC-2 in this embodiment, except that DB1001 was replaced with the CDH17-targeting antibody BM4. The drug loading (DAR / p) of ADC-5 was 8.0. Antibody BM4 was prepared according to patent WO2023107558A1 (SEQ ID NO:49, SEQ ID NO:50).

[0194] Example 4: Biological activity of anti-CDH17 antibody

[0195] 4.1 Quality Detection of Anti-human CDH17 Humanized Antibodies

[0196] 4.1.1 Humanized antibody purity detection

[0197] The purity of humanized antibodies was determined by SEC, and the detection method is as follows:

[0198] Instrument: Waters Alliance e2695 HPLC;

[0199] Column: Thermo MabPac SEC-1, 5µm, 7.8*300mm;

[0200] Mobile phase: 61 mmol / L Na2HPO4, 39 mmol / L NaH2PO4, 200 mmol / L NaCl, 5% IPA;

[0201] Instrument parameters: Sample chamber temperature: 8℃; Column temperature: 30℃; Flow rate: 0.5ml / min; Injection volume: 20μg; Detection wavelength: 280nm; Isocratic run: 30min.

[0202] 4.1.2 Detection of hydrophobicity of humanized antibodies

[0203] The hydrophilicity / hydrophobicity of humanized antibodies was detected by HIC assay as follows: A hydrophobic column (TOSOH Tskgel Buty-NPR(2.5), 4.6*100) from Tosoh Corporation was used on an Agilent HPLC system. Mobile phase A was 1.5M (NH4)2SO4, and mobile phase B was 25mM Na2HPO4 (pH=7.0) + 25% IPA. Instrument parameters were set as follows: sample chamber temperature: 8℃, column temperature: 30℃, flow rate: 0.5mL / min, detection wavelength: 280nm. The sample was diluted with mobile phase A to a final concentration of 1mg / mL, and 20μL was injected for gradient elution. The elution gradient is shown in Table 3 below.

[0204] Table 3 Elution gradient

[0205] The test results are shown in Table 4. DB1001 has an SEC of >95%, indicating its good purity properties. DB1001 has a weak binding to the hydrophobic chromatography column, a short HIC retention time, and good hydrophilicity.

[0206] Table 4. Quality test results of anti-human CDH17 humanized antibodies

[0207] 4.2 Determination of Tm value of anti-human CDH17 humanized antibody

[0208] The thermostability (Tm) of humanized antibodies was determined using DSF. The experimental procedure was as follows: the antibody sample was diluted to 1 mg / mL with PBS; the SYPRO Orange dye (Thermo#56651) was diluted to 40 μL with ddH2O; the reaction mixture consisted of 12.5 μL sample + 2.5 μL 40 μL dye + 5 μL ddH2O; the membrane was sealed and briefly centrifuged; and the results were detected by Q-PCR. The Q-PCR parameters were set as follows: Target (ROX), program (25℃, 3 min; 1% rate, 95℃; 95℃, 2 min). The results showed that the Tm of the DB1001 humanized antibody was 69.2℃, indicating good thermostability.

[0209] 4.3 Affinity evaluation of anti-human CDH17 humanized antibody

[0210] 4.3.1 Construction of overexpression of CDH17 protein on the surface of HEK293T cells

[0211] The amino acid sequence of human CDH17 was obtained by querying databases. Referring to the Uniprot protein database, the full-length protein encoding Q12864 and its extracellular domains EC1-EC7 were deleted. The corresponding genes were constructed into lentiviral vectors, and HEK293T cells were infected with the lentivirus to obtain HEK293T-hCDH17 and stable cell lines with deletions of each domain of HEK293T-hCDH17 (HEK293T-h.CDH17-△EC1-7). The positive rate of target expression was determined by FACS detection for subsequent screening and identification. The results are shown in Figure 1. HEK293T-hCDH17 showed high expression of hCDH17, with a positive rate of 99%, which can be used for subsequent mouse immunization and antibody evaluation. As shown in Table 5, the positive rates of each cell line of HEK293T-h.CDH17-△EC1-7 ranged from 40% to 100%, which can be used for subsequent domain evaluation.

[0212] Table 5. Results of positive rate detection of HEK293-hCDH17 domain-deficient cell lines

[0213] 4.3.2 Protein Expression

[0214] The extracellular domain genes of proteins, including the extracellular domain of human CDH17 protein (23-787AA), the extracellular domain of cynomolgus monkey CDH17 protein (XP_005563762.1) (23-787AA), and the extracellular domain of hCDH6 protein (P55285) (54-615AA), were synthesized. The extracellular domains of hCDH7 gene (Sinochem, HG22474-UT) (28-607AA) and hCDH20 gene (Sinochem, HG25413-UT) (60-619AA) were cloned into mammalian cell expression vectors, expressed and purified in 293E cells, and used for subsequent experiments.

[0215] 4.3.3 Protein purification

[0216] mFC and hFc protein purification: Cell expression supernatant samples were centrifuged at high speed to remove impurities. Recombinant proteins containing Fc, chimeric antibody expression supernatants, and hybridoma expression supernatants were purified using a Protein A column. The supernatant was loaded onto the column at a certain flow rate, and the column was washed with PBS until the A280 reading dropped to baseline. The target protein was eluted with 100mM sodium acetate at pH 3.5, neutralized with 2M Tris-HCl at pH 8.0, concentrated, replaced with PBS, and aliquoted for use. HIS-tagged protein purification: The same method was used for autonomous expression. The protein was eluted using a nickel column (brand: Borglon, catalog number: AA0051) with a 20-500mM imidazole gradient, replaced with PBS ultrafiltration, and aliquoted for use.

[0217] 4.3.4 Evaluate the affinity of the anti-human CDH17 human antibody

[0218] Detected by ELISA with hCDH17-His or hCDH17-mFc protein. Dilute hCDH17-His or hCDH17-mFc to 100 ng / well using CBS coating buffer and incubate overnight at 4°C. Remove the coating buffer, add 100 μL / well blocking buffer, and incubate at 37°C for 2 hours. Dilute the antibody to be tested with PBS (containing 2% BSA), starting at 50 μg / ml, 3-fold dilution, 12 concentration spots, and add 100 μL / well to the microplate, incubating at 37°C for 2 hours. Remove the supernatant, wash three times with 300 μL / well PBST, add 100 μL / well 1:10000-1:20000 anti-human HRP secondary antibody (Jackson Immuno Research, 109-035-088), and incubate at 37°C for 1 hour. Remove the secondary antibody, wash five times with 300 μL / well PBST, then add 100 μL TMB substrate to each well and develop for 5-20 minutes. Add 50 μL 2N to each well. The reaction was terminated with HCl, and the absorbance at OD450 nM was read using an MD microplate reader. The EC50 value was calculated using the four-parameter method.

[0219] The experimental results are shown in Figures 2A and 2B and Table 6. The anti-human CDH17 humanized antibody DB1001 has a better affinity for hCDH17-his or hCDH17-mFc than the control antibody BM-2.

[0220] Table 6. Results of protein affinity assay for anti-CDH17 humanized antibody

[0221] 4.4 Dynamic affinity evaluation of anti-human CDH17 humanized antibody

[0222] To detect the dynamic affinity of the anti-human CDH17 humanized antibody, the binding and dissociation rates of the antibody were detected using a Fortebio device. DB1001 and the control antibody BM-2 were diluted to 5 μg / mL with PBST, and the antibody molecules were captured using a ProA biosensor chip. The hCDH17-his antigen (described in 4.3) was diluted with PBST, starting at 1800 nM, with 2-fold dilutions, five concentration points, and a 0 concentration point, as the analyte. The program was set, the sensor plate and sample plate were placed, and the reaction signal was detected in real time to obtain binding and dissociation curves. After dissociation in each experimental cycle, the biosensor chip was washed and regenerated with 20 mM glycine solution (pH 1.7). The data were fitted using a 1:1 model to obtain the affinity values.

[0223] The experimental results are shown in Table 7. DB1001 and the control antibody BM-2 showed comparable dynamic affinity to the hCDH17-his antigen, at the E-08 level.

[0224] Table 7. Results of dynamic affinity assay between anti-CDH17 humanized antibody and hCDH17-his

[0225] 4.5 Evaluation of species affinity of anti-human CDH17 humanized antibody

[0226] The cross-species activity of the anti-human CDH17 monoclonal antibody was evaluated by ELISA with monkey CDH17-His or rat CDH17-his protein. Specific experimental methods are described in Example 4.3.

[0227] The results are shown in Figure 3 and Table 8. The anti-human CDH17 humanized antibody DB1001 has good cross-activity with monkeys, while the control antibody BM-2 has slightly weaker cross-activity with monkeys. The results are shown in Figure 4 and Table 8. Both the anti-human CDH17 humanized antibody DB1001 and the control antibody BM-2 have good cross-activity with rats. According to Table 8, the control antibody BM-1 has no cross-activity with rats.

[0228] Table 8. Results of protein affinity assay for anti-CDH17 humanized antibody

[0229] 4.6 Specificity evaluation of anti-human CDH17 humanized antibody

[0230] The non-specific binding of the anti-human CDH17 humanized monoclonal antibody to the following family proteins was evaluated by ELISA: hCDH6-His (see Example 4.3), hCDH7-His (see Example 4.3), hCDH9-His (ACRO, CA9-H52H6), hCDH10-His (ACRO, CA0-H52H5), hCDH12-His (Sinochem, 10317-H08H), hCDH16-His (Sinochem, 10915-H08H), and hCDH20-His (see Example 4.3). The specific experimental method is as follows: Dilute the syngeneic protein antigen to 1 μg / mL using CBS coating buffer, add 100 μL / well of the ELISA plate, and incubate overnight at 4°C; wash once with 300 μL / well of PBS, add 100 μL / well of blocking buffer, and incubate at 37°C for 2 hours; dilute the humanized monoclonal antibody with PBS (2% BSA) to concentrations of 20 μg / ml, 5 μg / ml, and 1 μg / ml, and add 100 μL of each diluted solution to the plate, incubate at 37°C for 2 hours; remove the supernatant, wash three times with 300 μL of PBST in each well, add 100 μL of 1:10000-1:20000 anti-human HRP secondary antibody in each well, and incubate at 37°C for 1 hour; remove the secondary antibody, wash five times with 300 μL of PBST in each well, then add 100 μL of TMB substrate in each well, and develop at room temperature for 5-20 minutes; add 50 μL of 2N to each well. The reaction was terminated with HCl, and the ELISA reader was used to read the OD450nM value.

[0231] Experimental results showed that the anti-human CDH17 humanized antibody DB1001 did not bind to any of the family proteins hCDH6, hCDH7, hCDH9, hCDH10, hCDH12, hCDH16 and hCDH20, demonstrating excellent specificity.

[0232] 4.7 Evaluation of tumor affinity of anti-human CDH17 humanized antibody

[0233] To detect the affinity of the humanized anti-human CDH17 antibody DB1001 for cells with different expression levels, FACS was used to detect its binding to HEK293T-h.CDH17 cells, NCI-H716, LS1034, DLD-1, and MKN45 tumor cells. NCI-H716 tumor cells with high CDH17 expression were collected by centrifugation at 500g for 3 minutes. After EDTA digestion, HEK293T-h.CDH17 cells and LS1034, DLD-1, and MKN45 tumor cells expressing CDH17 were collected by centrifugation at 500g for 3 minutes. Cells were resuspended in FACS buffer (PBS containing 1% BSA), and the test antibody was added in serially diluted FACS buffer. The cells were incubated at 4°C for 1 hour, washed five times with pre-chilled PBS, and then 1 μL of anti-human fluorescent secondary antibody (BioLegend, catalog number 410712) was added to each well. The cells were incubated at 4°C for 1 hour, washed three times with pre-chilled PBS, and resuspended. Flow cytometry analysis was performed. EC50 values ​​were calculated based on fluorescence signal values.

[0234] The experimental results are shown in Figures 5 to 9 and Table 9: DB1001 has comparable affinity to BM2 on HEK293T-h.CDH17 overexpressing cells; DB1001 has stronger affinity than BM2 on MKN45 tumor cells; overall, DB1001 has strong cell affinity.

[0235] Table 9. Results of cell affinity assay for anti-CDH17 humanized antibody

[0236] 4.8 Detection of the binding domain of anti-human CDH17 humanized antibody

[0237] The binding domain of the anti-human CDH17 human monoclonal antibody was evaluated using FACS. HEK293T-h.CDH17-△EC1-7 cells were collected by centrifugation, resuspended in PBS (containing 1% BSA), and cultured at 3 × 10⁻⁶ cells per well. 5 Cells were seeded into 96-well conical plates, diluted test antibody was added, and incubated at 4°C for 1 hour. After washing three times with pre-chilled PBS, 1 μL of anti-human fluorescent secondary antibody was added to each well, and the plates were incubated at 4°C for 0.5 hours. After washing three times with pre-chilled PBS, the cells were resuspended and analyzed by flow cytometry. The binding domain of the human monoclonal antibody was determined.

[0238] Experimental results show that DB1001 binds to the EC1 domain of human CDH17 protein.

[0239] 4.9 Evaluation of the endocytic activity of the humanized monoclonal antibody against human CDH17

[0240] The endocytic activity of the humanized monoclonal antibody against human CDH17 was detected by FACS. The experimental procedure was as follows: HEK293T-h.CDH17 cells were collected and incubated with the antibody at 4°C for 60 minutes. After washing with PBS, a portion of the cells were incubated with secondary antibody. After incubation, the fluorescence intensity was measured as "F0". The remaining cells, after incubation with the antibody, were incubated at 37°C for 2 hours. After incubation, the cells were washed with PBS, then incubated with secondary antibody again before fluorescence detection, where the fluorescence intensity was measured as "FX".

[0241] The internalization rate at a certain time point is calculated as follows: [1-(FX sample to be tested - FX isotype control) / (F0 sample to be tested - F0 isotype control)]×100.

[0242] Experimental results showed that the endocytosis rates of the anti-human CDH17 humanized antibody DB1001, control antibody BM-1, and control antibody BM-2 at 2 hours were 32.80%, 14.51%, and 17.05%, respectively. DB1001 exhibited superior endocytosis activity.

[0243] 4.10 Evaluation of the thermal stability, low pH stability, and freeze-thaw stability of anti-human CDH17 humanized antibody

[0244] To evaluate the thermal stability, low pH stability, and freeze-thaw stability of the anti-human CDH17 humanized antibody: 1) Thermal stability: The anti-human CDH17 humanized antibody DB1001 sample was ultrafiltered and replaced with PBS (pH=7.4) at a concentration of 10 mg / mL. After filtration and sterilization, 0.5 mL / vial was stored at 37°C for 0, 7, and 14 days, respectively. The SEC and CE purity of the sample were then determined by HPLC. 2) Freeze-thaw stability: The sample was repeatedly frozen and thawed 4 and 8 times at -80°C. The SEC purity of the sample was then determined by HPLC. 3) Low pH stability: The DB1001 sample to be tested was ultrafiltered and replaced with NaAc-Ac (pH=3.3), stored for 4 hours, and then neutralized with 2M Tris to approximately pH 7.0. The SEC and CE purity of the sample were then determined by HPLC.

[0245] The experimental results are shown in Table 10 below. DB1001 exhibits good stability after high-temperature acceleration, repeated freeze-thaw cycles, and low-pH treatment.

[0246] Table 10. Stability study of anti-CDH17 humanized antibodies

[0247] 4.11 Evaluation of antibody-dependent cell-mediated cytotoxicity (ADCC) activity of anti-human CDH17 humanized antibody

[0248] The ADCC activity of the humanized monoclonal antibody against human CDH17 was detected using a luciferin reporter system. The experimental procedure was as follows: HEK293T-h.CDH17 cells were collected by digestion and centrifugation, resuspended in DMEM + 1% FBS, and seeded at 50,000 cells per well; Jurkat-NFAT-CD16a cells were collected by centrifugation, resuspended in RPMI 1640 + 1% FBS, and seeded at 100,000 cells per well; serially diluted antibody was added, starting at a final concentration of 50 μg / ml, and diluted 4-fold, incubated at 37°C for 5 hours; 50 μL of Bio-Lite-Luciferase Assay Buffer (Nanjing Novizan, DD1201-02) was added to each well, and the readings were taken using a microplate reader (MD, i3x). Humanized CCR8 antibody and HEK293T-CCR8 cells were used as positive controls (refer to patent application: WO2024008110A1).

[0249] The experimental results are shown in Figure 10. DB1001 did not show obvious ADCC activity.

[0250] 4.12 Evaluation of antibody-dependent cell-mediated phagocytosis (ADCP) activity of anti-human CDH17 humanized antibody

[0251] HEK293T-h.CDH17 cells were collected by trypsin digestion and centrifugation, and 50,000 cells were seeded per well after resuspending the cells in DEME + 1% FBS. Jurkat-NFAT-H131 cells were collected by centrifugation, and 100,000 cells were seeded per well after resuspending the cells in RPMI 1640 + 1% FBS. Serially diluted test antibodies were added, starting at a final concentration of 50 μg / ml, and then diluted 3-fold. The cells were incubated at 37°C for 5 hours. 50 μL of Bio-Lite-Luciferase Assay Buffer was added to each well, and the readings were taken using a microplate reader (MD, i3x).

[0252] The experimental results are shown in Figure 11. DB1001 did not show significant ADCP activity.

[0253] Example 5: Binding of anti-CDH17 antibody-drug conjugate and its parent antibody to human, mouse, and monkey antigens (enzyme-linked immunosorbent assay).

[0254] Test objective

[0255] The cross-species reaction of antibody-drug conjugate ADC-1 (DB1001-X2) and its parent antibody DB1001 with CDH17 proteins from humans, cynomolgus monkeys, rats and mice was detected.

[0256] Experimental methods

[0257] 1. Coating: Add the stock solutions of human CDH17-his (Kai Ka, CDH-HM117) / cynomolgus monkey CDH17-his (Kai Ka, CDH-CM127) / rat CDH17-his (Yi Qiao Shen Zhou, 80283-R08H) / mouse CDH17-his (Kai Ka, CDH-MM117) to 1×PBS to a final concentration of 2 μg / mL. Add 30 μL to each well and incubate overnight at 4°C.

[0258] 2. Blocking: Wash three times with PBST at room temperature, and block with 5% PBSM for 2 hours.

[0259] 3. Incubation: Wash the plate three times with PBST. Add 30 μL of antibody-drug conjugate or maternal antibody diluted with 1% PBSM to each well of the ELISA plate. The initial concentration of the first well is 33.33 μg / mL. Dilute 3 times and make a total of 7 concentration points. Incubate at room temperature for 60 minutes.

[0260] 4. Secondary antibody: Wash the plate three times with PBST, add 1:8000 Anti-Human-IgG-Fc-HRP (abcam,ab97225) to 1% PBSM and incubate at room temperature for 50 minutes.

[0261] 5. Color development: Wash the plate 6 times with PBST, add TMB, stop the reaction with 2M H2SO4 stop solution, and read OD450.

[0262] Experimental results are shown in Table 11 and Figure 12.

[0263] Experimental conclusions: ADC-1 (DB1001-X2) and the parent antibody DB1001 have similar affinity for CDH17 in humans, cynomolgus monkeys, and rats, but do not bind to CDH17 in mice. ADC-1 (DB1001-X2) maintains the affinity of the parent monoclonal antibody DB1001 for CDH17 in humans, cynomolgus monkeys, and rats.

[0264] Isotype: IgG1 isotype control;

[0265] Isotype ADC: The antibody was an IgG1 isotype control, the adaptor-cytotoxin was X2, and it was prepared with reference to ADC-1 (DB1001-X2).

[0266] Table 11 Affinity with CDH17 in humans, monkeys, rats, and mice

[0267] Example 6: Affinity (SPR) of the anti-CDH17 antibody-drug conjugate and its parent antibody to human monkey antigen.

[0268] Objective: To detect the affinity kinetics of antibody-drug conjugate ADC-1 (DB1001-X2) and its parent antibody DB1001 with human and cynomolgus monkey CDH17 protein using a Biacore T200 (Cytiva) analyzer.

[0269] Experimental methods:

[0270] 1. The ProA chip captured the maternal monoclonal antibody DB1001 (1 μg / mL). The analytes were human CDH17 (human CDH17, C-His Tag, Kactus Biosystem, CDH-HM117), serially diluted in the range of 6.25-100 nM, for a total of 5 concentrations; and cynomolgus CDH17 (Cynomolgus CDH17, C-His Tag, Kactus Biosystem, CDH-CM127), serially diluted in the range of 4.69-75 nM, for a total of 5 concentrations.

[0271] 2. ProA chip captured antibody-drug conjugate ADC-1 (DB1001-X2) (1 μg / mL). The analytes were human CDH17 (human CDH17, C-His Tag, Kactus Biosystem, CDH-HM117), serially diluted in the range of 12.5-200 nM, for a total of 5 concentrations; and cynomolgus CDH17 (Cynomolgus CDH17, C-His Tag, Kactus Biosystem, CDH-CM127), serially diluted in the range of 4.69-75 nM, for a total of 5 concentrations.

[0272] 3. Affinity data were analyzed and fitted using a 1:1 Langmuir binding model, where affinity (KD) is the ratio of dissociation constant (Kd) to binding constant (Ka). The results are shown in Table 12.

[0273] Table 12 Affinity of Anti-CDH17 Antibody Drug Conjugates and Their Parent Antibodies to Human Monkey Antigens

[0274] Experimental conclusion: ADC-1 (DB1001-X2) and the maternal antibody have similar affinity dynamics for human and cynomolgus monkey CDH17, and ADC-1 (DB1001-X2) maintains the affinity of the maternal monoclonal antibody for human and cynomolgus monkey CDH17.

[0275] Example 7: Affinity of anti-CDH17 antibody-drug conjugates, their parent monoclonal antibodies, and different tumor cell lines expressing CDH17

[0276] Experimental Objective: To detect the affinity of antibody-drug conjugate ADC-1 (DB1001-X2) and its parent antibody DB1001 for CDH17 tumor cell lines from different sources by flow cytometry.

[0277] Experimental methods:

[0278] 1. Gastric cancer cell line SNU-5, colorectal cancer cell line NCI-H716, and pancreatic cancer cell line AsPC-1 were cultured in complete medium at 37°C and 5% CO2.

[0279] 2. Harvest cells in the logarithmic growth phase and assess cell viability using the trypan blue rejection method, ensuring cell viability is above 90%. Centrifuge at 1000 rpm for 5 min and discard the supernatant. Wash cells once with PBS, resuspend in FACS Buffer to prepare a single-cell suspension, and adjust the cell density to 5 × 10⁶ cells / mL. 6 cells / mL;

[0280] 3. Add 50 μL of cell suspension to each well of a 96-well plate to make the initial concentration of the test sample working solution 5 μg / mL, dilute 4 times to make a total of 8 concentrations; mix well and incubate at 4℃ for 40 min.

[0281] 4. Wash the cells three times with 400 μL of FACS Buffer each time, centrifuge at 1000 r / min for 5 min, and finally resuspend the cells with 100 μL of FACS Buffer.

[0282] 5. Add 2 μL of PE-labeled secondary antibody (PE anti-human IgG Fc Antibody), mix well, and incubate at 4°C in the dark for 40 min;

[0283] 6. Wash the cells three times with 400 μL of FACS Buffer each time, centrifuge at 1000 r / min for 5 min, and finally resuspend the cells with 250 μL of FACS Buffer.

[0284] 7. Fluorescence values ​​were detected by flow cytometry.

[0285] The experimental results are shown in Figure 13 (Isotype IgG1 in the figure is the IgG1 isotype control) and Table 13.

[0286] Experimental conclusions

[0287] ADC-1 (DB1001-X2) and the parent antibody both have high affinity for CDH17 from different tumors and at different expression levels. Furthermore, ADC-1 (DB1001-X2) retains the same affinity for cellular CDH17 as the parent antibody.

[0288] Table 13 Affinity of anti-CDH17 antibody-drug conjugates, their parent monoclonal antibodies, and different tumor cell lines

[0289] Example 8: Anti-CDH17 antibody drug conjugate and its parent antibody targeting specificity

[0290] Test objective

[0291] The binding specificity of antibody-drug conjugate ADC-1 (DB1001-X2) and parental monoclonal antibody DB1001 to human CDH family proteins was detected by enzyme-linked immunosorbent assay.

[0292] Experimental methods

[0293] 1. Coating: Add 1×PBS to the stock solutions of seven human CDH family proteins to a final concentration of 2 μg / mL, 30 μL per well, and incubate overnight at 4°C.

[0294] 2. Blocking: Wash three times with PBST at room temperature, and block with 5% PBSM for 2 hours.

[0295] 3. Incubation: Wash the plate three times with PBST, add 30 μL of antibody-drug conjugate or maternal antibody diluted with 1% PBSM to the ELISA plate, with a final concentration of 100 μg / mL for the first well, 10-fold dilution, for a total of 4 concentration points, and incubate at room temperature for 60 minutes.

[0296] 4. Secondary antibody: Wash the plate three times with PBST, add 1:8000 Anti-Human-IgG-Fc-HRP (abcam,ab97225) to 1% PBSM and incubate at room temperature for 50 minutes.

[0297] 5. Color development: Wash the plate 6 times with PBST, add TMB, stop the reaction with 2M H2SO4 stop solution, and read OD450.

[0298] Experimental results

[0299] As shown in Figure 14, in this embodiment, the antibody-drug conjugate ADC-1 (DB1001-X2) and the parent monoclonal antibody DB1001 bind to human CDH17, but no binding to other human CDH family proteins CDH16, CDH1, CDH2, CDH3, CDH4 and CDH6 is detected.

[0300] Experimental conclusions

[0301] ADC-1 (DB1001-X2) and the parental monoclonal antibody DB1001 can specifically bind to human CDH17 protein and have no cross-reactivity with other CDH family proteins.

[0302] Example 9: Evaluation of the efficacy of the antibody-drug conjugate in CDH17-expressing human colorectal cancer cell line NCI-H716 tumor-bearing mice.

[0303] To investigate the inhibitory effect of ADC-1 (DB1001-X2) on tumor formation in vivo, xenografts of human colorectal cancer cells NCI-H716 expressed in CDH17 were formed in mice, and the anti-tumor effect of ADC-1 (DB1001-X2) was studied.

[0304] 1. Test drug and materials

[0305] Blank control group (control group): physiological saline

[0306] ADC-1 (DB1001-X2) (treatment group): 3 mg / kg

[0307] 2. Preparation method: All samples were prepared by diluting with physiological saline.

[0308] 3. Experimental animals: 6-8 week old female BALB / c Nude mice, purchased from Jicui Pharmaceutical Technology Co., Ltd.

[0309] 4. Test methods:

[0310] 5 × 10⁵ cells were subcutaneously injected into the right nape of the neck of each mouse. 6 NCI-H716 cells were seeded at a volume of 0.2 mL, with the cell suspension consisting of PBS and matrix gel (volume ratio 1:1). The experimental animals were simultaneously tagged with 5-digit ear tags as the sole confirmation marker for subsequent experiments. In vivo efficacy experiments were conducted on day 17 post-cell seeding, when the average tumor volume reached 150 mmHg. 3 Mice were randomly assigned to groups of five mice each, based on tumor volume. The test drug was administered intravenously (iv) starting on day 0, once a week for a total of two weeks, at a dose of 3 mg / kg. The endpoint was day 28 after grouping. Tumor volume and body weight were measured twice weekly, and data were recorded.

[0311] The relative tumor-suppressive efficacy of the compounds was evaluated using TGI (%) or relative tumor proliferation rate T / C (%).

[0312] The relative tumor volume (RTV) is calculated based on the tumor measurement results. The formula is: RTV = V t / V0, where V0 is the tumor volume of the animals in the group when the drug is administered, V t The tumor volume of the animals in this group is t days after treatment.

[0313] Relative tumor proliferation rate T / C (%): The calculation formula is T / C (%) = TRTV / C RTV ×100, where T RTV For the treatment group RTV, C RTV For the control group, RTV, T RTV With C RTV Take data from the same day.

[0314] Tumor growth inhibition rate is calculated using the following formula: TGI (%) = [1 - (T i -T0) / (V i -V0)]×100, where T i V represents the average tumor volume of a specific treatment group on a given day; T0 represents the average tumor volume of this treatment group on the day of administration. i For a certain day (with T) i The mean tumor volume of the control group on the same day (V0) was used. Independent samples t-test (T-Test) was used to compare the two groups, with P < 0.05 considered statistically significant.

[0315] Table 14 Evaluation of the antitumor efficacy of the test substances against the NCI-H716 cell subcutaneous xenograft tumor model (calculated based on tumor volume 28 days after administration). a. Tumor volume is expressed as mean ± standard error; b. Tumor growth inhibition is calculated using T / C (T / C(%) = T²⁸ / V²⁸ × 10⁰) and TGI (TGI(%) = [1 - (T²⁸ / V²⁸ × 10⁰)]²⁰. 28 -T0) / (V 28 - V0)]×100) reflects; cp value is calculated based on tumor volume (p<0.05 indicates statistical difference, p<0.01 indicates significant difference).

[0316] The experimental results are shown in Figure 15 and Table 14. The antibody-drug conjugate ADC-1 (DB1001-X2) showed significant antitumor activity after administration and had no effect on the weight gain of the animals.

[0317] Example 10: Evaluation of the efficacy of antibody-drug conjugates against CDH17-overexpressing human colorectal cancer cell line HT-55 tumor-bearing mice.

[0318] To investigate the inhibitory effect of ADC-1 (DB1001-X2) on tumor formation in vivo, the anti-tumor effect of ADC-1 (DB1001-X2) was detected after xenografts were formed in mice using human colorectal cancer cells HT-55 with high CDH17 expression.

[0319] 1. Test drug and materials

[0320] Blank control group (control group): physiological saline

[0321] ADC-1 (DB1001-X2) (treatment group): 5 mg / kg

[0322] 2. Preparation method: All samples were prepared by diluting with physiological saline.

[0323] 3. Experimental animals: 6-8 week old female CB-17SCID mice, purchased from Vital River.

[0324] 4. Test methods:

[0325] 3 × 10⁻⁶ cells were subcutaneously injected into the right nape of the neck of each mouse. 6 HT-55 cells were seeded at a volume of 0.2 mL, with the cell suspension consisting of PBS and matrix gel (volume ratio 1:1). The experimental animals were simultaneously tagged with 5-digit ear tags, which served as the sole confirmation marker for subsequent experiments. In vivo efficacy studies were conducted on day 7 post-cell seeding, when the average tumor volume reached approximately 200 mmHg. 3 Mice were randomly assigned to groups of five mice each, based on tumor volume. The test drug was administered intravenously (iv) starting on day 0, once a week for a total of two weeks, at a dose of 5 mg / kg. The endpoint was day 22 after grouping. Tumor volume and body weight were measured twice weekly, and data were recorded.

[0326] The relative tumor volume (RTV) is calculated based on the tumor measurement results. The formula is: RTV = V t / V0, where V0 is the tumor volume of the animals in the group when the drug is administered, V t The tumor volume of the animals in this group is t days after treatment.

[0327] Relative tumor proliferation rate T / C (%): The calculation formula is T / C (%) = T RTV / C RTV ×100, where T RTV For the treatment group RTV, C RTV For the control group, RTV, T RTV With C RTV Take data from the same day.

[0328] Tumor growth inhibition rate is calculated using the following formula: TGI (%) = [1 - (T i -T0) / (V i -V0)]×100, where T i V represents the average tumor volume of a specific treatment group on a given day; T0 represents the average tumor volume of this treatment group on the day of administration. i For a certain day (with T) iThe mean tumor volume of the control group on the same day (V0) was used. Independent samples t-test (T-Test) was used to compare the two groups, with P < 0.05 considered statistically significant.

[0329] Table 15 Evaluation of the antitumor efficacy of the test substances in a subcutaneous xenograft tumor model of HT-55 cells (calculated based on tumor volume 22 days after administration). a. Tumor volume is expressed as mean ± standard error; b. Tumor growth inhibition is reflected by T / C (T / C(%) = T22 / V22 × 100) and TGI (TGI(%) = [1 - (T22 - T0) / (V22 - V0)] × 100); cp value is calculated based on tumor volume (p < 0.05 indicates statistical difference, p < 0.01 indicates significant difference).

[0330] The experimental results are shown in Figure 16 and Table 15. The antibody-drug conjugate ADC-1 (DB1001-X2) showed significant antitumor activity after administration, and had no statistically significant effect on the weight gain of the animals.

[0331] Example 11: Evaluation of the efficacy of antibody-drug conjugates against CDH17-overexpressing human pancreatic cancer cell line AsPC-1 tumor-bearing mice.

[0332] To investigate the inhibitory effect of ADC-1 (DB1001-X2) on tumor formation in vivo, xenografts of human pancreatic cancer AsPC-1 with high CDH17 expression were formed in mice, and the antitumor effect of ADC-1 (DB1001-X2) and its dose-dependent relationship were examined.

[0333] 1. Test drug and materials

[0334] Blank control group (control group): physiological saline

[0335] ADC-1 (DB1001-X2) (treatment group): 5 mg / kg

[0336] ADC-1 (DB1001-X2) (treatment group): 10 mg / kg

[0337] 2. Preparation method: All samples were prepared by diluting with physiological saline.

[0338] 3. Experimental animals: 6-8 week old female CB-17SCID mice, purchased from Vital River.

[0339] 4. Test methods:

[0340] 5 × 10⁵ cells were subcutaneously injected into the right nape of the neck of each mouse. 6AsPC-1 cells were seeded at a volume of 0.2 mL, with the cell suspension consisting of PBS and matrix gel (volume ratio 1:1). The experimental animals were simultaneously tagged with 5-digit ear tags as the sole confirmation marker for subsequent experiments. In vivo efficacy experiments were conducted on day 10 post-cell seeding, when the average tumor volume reached approximately 124 mm³. Mice were randomly assigned to groups of 6 mice each, based on tumor volume. The test drug was administered intravenously (iv) once on day 0, at a dose of either 5 mg / kg or 10 mg / kg. The endpoint was day 27 post-grouping. Tumor volume and body weight were measured twice weekly, and data were recorded.

[0341] The relative tumor-suppressive efficacy of the compounds was evaluated using TGI (%) or relative tumor proliferation rate T / C (%).

[0342] The relative tumor volume (RTV) is calculated based on the tumor measurement results. The formula is: RTV = V t / V0, where V0 is the tumor volume of the animals in the group when the drug is administered, V t The tumor volume of the animals in this group is t days after treatment.

[0343] Relative tumor proliferation rate T / C (%): The calculation formula is T / C (%) = T RTV / C RTV ×100, where T RTV For the treatment group RTV, C RTV For the control group, RTV, T RTV With C RTV Take data from the same day.

[0344] Tumor growth inhibition rate is calculated using the following formula: TGI (%) = [1 - (T i -T0) / (V i -V0)]×100, where T i V represents the average tumor volume of a specific treatment group on a given day; T0 represents the average tumor volume of this treatment group on the day of administration. i For a certain day (with T) i The mean tumor volume of the control group on the same day (V0) is the mean tumor volume of the control group on the day of administration. One-way ANOVA was used to analyze the differences between multiple groups, and P < 0.05 was considered statistically significant.

[0345] Table 16 Evaluation of the antitumor efficacy of the test substances in a subcutaneous xenograft tumor model of AsPC-1 cells (calculated based on tumor volume 27 days after administration). a. Tumor volume is expressed as mean ± standard error; b. Tumor growth inhibition is reflected by T / C (T / C(%) = T27 / V27 × 100) and TGI (TGI(%) = [1 - (T27 - T0) / (V27 - V0)] × 100); cp value is calculated based on tumor volume (p < 0.05 indicates statistical difference, p < 0.01 indicates significant difference).

[0346] The experimental results are shown in Figure 17 and Table 16. The antibody-drug conjugate ADC-1 (DB1001-X2) exhibited significant antitumor activity after administration, and this activity was dose-dependent. The test product had no significant effect on the weight gain of the animals.

[0347] Example 12: In vitro organoid killing of colorectal cancer and adjacent tissues

[0348] Objective: To detect the in vitro killing ability of ADC-1 on colorectal cancer organoids and paired normal colorectal organoids from adjacent tissues.

[0349] method:

[0350] 1. Two pairs of colorectal cancer tissues expressing CDH17 and their paired adjacent normal tissues were selected: 2300094 (adjacent normal tissue, CDH17 IHC H-score 177) and 2300095 (colorectal cancer, CDH17 IHC H-score 152); 2300093 (adjacent normal tissue, CDH17 IHC H-score 131) and 2300092 (colorectal cancer, CDH17 IHC H-score 124).

[0351] 2. When the revived and expanded organoids grow to 50-100 μm and are in good condition (no contamination, intact organoid morphology, and good light transmittance), add Calcein-AM (Invitrogen, C34852, 2 μM) to the culture medium and stain for 30 min.

[0352] 3. Collect the organoids stained with Calcein-AM, resuspend them in 10% Matrigel (diluted with organoid culture medium), and adjust the organoid density to approximately 2.0 × 10⁻⁶. 4 per mL.

[0353] 4. Add 10 μL of organoid suspension to each well of a 96-well plate with a base layer of 50% matrix gel, and incubate in a CO2 cell culture incubator for 15 min.

[0354] 5. Dilute the test sample with organoid culture medium. The antibody-drug conjugate should start at a concentration of 100 nM, be diluted 4 times, for a total of 8 concentrations, with 3 replicates for each concentration; the toxin (P-III-30, a cytotoxic drug, see page 138 of the instruction manual WO2022068878A1) should start at a concentration of 800 nM, be diluted 4 times, for a total of 8 concentrations, with 3 replicates for each concentration.

[0355] 6. Add 200 μL of organoid culture medium containing the test sample to each well (the control group is the culture medium without the drug), and acquire bright field and fluorescence images (image of day 0, the starting point of the drug efficacy test) using the Z-stack image acquisition technology of a fluorescence microscope (Nikon, Ti2-E).

[0356] 7. On day 6 of incubation, add 5 μL of AO / PI staining solution (ViaStain™ AOPI Staining Solution, Nexcelom, CS2-0106-5 mL) to each well. After staining for 1 hour, acquire bright-field and fluorescence images (images of day 6, the endpoint of the drug efficacy test) using the Z-stack image acquisition technology of a fluorescence microscope.

[0357] 8. Use Nikon's built-in software to statistically analyze the cell cluster size of the live organoids on day 0 and day 6 of the drug efficacy test.

[0358] 9. Divide the data from day 6 by the data from day 0 for all groups to obtain the growth data of organoids after drug treatment within each group.

[0359] 10. Using the growth data of the control group (no drug-treated group) as 100% standardized growth data of the drug-treated group, the relative organoid growth rate of the drug-treated group was obtained.

[0360] Results: See Table 17 and Figure 18

[0361] Conclusion: ADC-1 has specific killing effect on colorectal tumors in vitro, but only at high concentrations does it have weak killing effect on normal colorectal tissue.

[0362] Table 17. Growth inhibition of colorectal organoids by drugs

[0363] Example 13: Efficacy evaluation of antibody-drug conjugates with different DAR values ​​in NCI-H716 and HT-55 tumor-bearing mice

[0364] Objective: To investigate the in vivo efficacy of DB-1001 conjugated with a toxin-cytotoxic agent X2 at different DAR values ​​in colorectal models with varying CDH17 expression levels, adult female mice with subcutaneous NCI-H716 colorectal cancer cell lines exhibiting moderate CDH17 expression and adult female mice with subcutaneous HT-55 colorectal cancer cell lines exhibiting high CDH17 expression were treated with different ADCs. After ectopic inoculation of NCI-H716 or HT-55 tumors into the right dorsal region of mice to form xenografts, the antitumor effect of DB-1001 conjugated with toxins of different DAR values ​​was evaluated.

[0365] method:

[0366] 1. The test drugs and dosing regimens are shown in Tables 18 and 19.

[0367] Table 18: Administration routes, dosages, and regimens in the NCI-H716 subcutaneous xenograft model of colorectal cancer in 18 patients.

[0368] Table 19: Administration routes, dosages, and regimens in subcutaneous xenograft models of HT-55 cell lines in colorectal cancer.

[0369] 2. Preparation method: All samples were prepared by diluting with DPBS.

[0370] 3. Experimental animals: 6-8 week old female CB-17SCID mice, purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.

[0371] 4. Experimental Methods: Cells in the exponential growth phase were collected and resuspended in a 1:1 mixture of DPBS and matrix gel. 5 × 10⁶ cells were subcutaneously inoculated into the right back of experimental mice. 6 NCI-H716 or HT-55 cells were used to regularly monitor tumor growth. When the tumor grew to approximately 200 mm... 3 (NCI-H716) or 194mm 3 (HT-55) Tumor-bearing mice were randomly assigned to groups, with the grouping day designated as Day 1. Specific dosing regimens are shown in Tables 18 and 19. The endpoint was either day 27 (NCI-H716) or day 28 (HT-55) after grouping. Tumor volume and body weight were measured twice weekly, and data were recorded.

[0372] 5. Six mice were used in each of the control and treatment groups. The tumor inhibition rate was calculated by measuring tumor volume. The formula for calculating tumor volume is: V = 0.5a × b², where a and b represent the long and short diameters of the tumor, respectively. The tumor growth inhibition rate was calculated using the following formula: TGI (%) = [1 - (Ti - T0) / (Vi - V0)] × 100, where Ti is the average tumor volume of a given treatment group on a certain day, T0 is the average tumor volume of this treatment group at the start of administration, Vi is the average tumor volume of the solvent control group on a certain day (the same day as Ti), and V0 is the average tumor volume of the solvent control group at the start of administration. One-way ANOVA was used for comparisons between multiple groups. P < 0.05 was considered statistically significant, and P < 0.01 was considered statistically significant.

[0373] Results: As shown in Tables 20 and 21 and Figure 19.

[0374] Conclusion: In colorectal cancer models with intermediate and high levels of CDH17 expression, DB1001 conjugates with DAR6 and DAR8 of the cytotoxin X2 showed significant antitumor effects, and the antitumor effect was significantly enhanced with increasing DAR value. The antitumor effects of low-dose conjugates with DAR8 and high-dose conjugates with DAR6 were comparable. Meanwhile, with increasing DAR value and dosage, there was no significant difference in mouse body weight gain compared with the blank control group.

[0375] Table 20 Evaluation of the antitumor efficacy of the test substances in the NCI-H716 cell subcutaneous xenograft tumor model (based on 20 days after administration) 1 (Tumor volume calculated)

[0376] Table 21 Evaluation of the antitumor efficacy of the test substance on a subcutaneous xenograft tumor model of HT-55 cells (calculated based on tumor volume 28 days after administration).

[0377] 1. Because some mice in the NCI-H716 blank control group met the euthanasia criteria after 20 days, the statistical analysis data was incomplete. Therefore, the statistical analysis was conducted on the 20th day when the number of animals was complete.

[0378] 2. Tumor volume is expressed as mean ± standard error;

[0379] 3. The p-value is calculated based on the tumor volume.

[0380] Example 14: Evaluation of the efficacy of antibody-drug conjugate in CDH17-negative HT-29 tumor-bearing mice

[0381] Objective: To investigate the target specificity of the antitumor efficacy of the antibody-drug conjugate of this application, the in vivo antitumor effect of the antibody-drug conjugate was evaluated after ectopic inoculation of CDH17-negative HT-29 into the right back of mice to form xenografts.

[0382] method:

[0383] 1. The test drugs and dosing regimens are shown in Table 22.

[0384] Table 22: Administration routes, dosages, and regimens in the HT-29 subcutaneous xenograft model of colorectal cancer.

[0385] 2. Experimental animals: 6-8 week old female CB-17SCID mice, purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.

[0386] 3. Experimental Methods: Cells in the exponential growth phase were collected and resuspended in a 1:1 mixture of DPBS and matrix gel. 2 × 10⁶ cells were subcutaneously inoculated into the right back of experimental mice. 6 HT-29 cells were used to regularly monitor tumor growth. When the tumor grew to approximately 200 mm... 3 Tumor-bearing mice were randomly assigned to groups as shown in Table 22. The day of group assignment was designated Day 1. The experimental endpoint was day 27 after group assignment. Tumor volume and body weight were measured twice weekly, and the data were recorded.

[0387] 4. Six mice were used in each of the control and treatment groups. The tumor inhibition rate was calculated by measuring tumor volume. The formula for calculating tumor volume is: V = 0.5a × b², where a and b represent the long and short diameters of the tumor, respectively. The tumor growth inhibition rate was calculated using the following formula: TGI (%) = [1 - (Ti - T0) / (Vi - V0)] × 100, where Ti is the average tumor volume of a given treatment group on a certain day, T0 is the average tumor volume of this treatment group at the start of treatment, Vi is the average tumor volume of the solvent control group on a certain day (the same day as Ti), and V0 is the average tumor volume of the solvent control group at the start of treatment. Independent samples t-test (T-Test) was used to compare the two groups. P < 0.05 was considered statistically significant, and P < 0.01 was considered statistically significant.

[0388] Results: As shown in Table 23 and Figure 20.

[0389] Conclusion: In a CDH17-negative colorectal cancer model, the TGI of ADC1 at high doses was only 18.52%, showing only a slight statistical difference compared to the blank control group. Mouse weight gain was not significantly different from the blank control group. Therefore, the significant antitumor efficacy of ADC1 depends on CDH17 expression.

[0390] Table 23 Evaluation of the antitumor efficacy of the test substance on the HT-29 cell subcutaneous xenograft tumor model (calculated based on tumor volume 27 days after administration).

[0391] 1. Tumor volume is expressed as mean ± standard error;

[0392] 2. The p-value is calculated based on the tumor volume.

[0393] Example 15: In vivo efficacy of antibody-drug conjugates in patient-derived xenograft (PDX) models

[0394] Objective: To investigate the in vivo efficacy of the drug of this application in a more clinically relevant tumor model, the in vivo antitumor effect of the antibody-drug conjugate was evaluated after ectopic inoculation of tumor tissue derived from colorectal cancer patients into the right back of mice to form xenografts.

[0395] method:

[0396] 1. NU / NU mice, female, 6-8 weeks old, weighing 18-22g, purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.

[0397] 2. Successfully resuscitated FP1+5 generation LD1-2012-200671 human colorectal cancer xenografts were cut into small pieces approximately 3mm × 3mm × 3mm in size. These pieces were then subcutaneously injected into the right back of NU / NU mice (efficacy experiment generation: FP1+6) using a tumor inoculation needle. Each animal received approximately 50–90 mg of tumor tissue mixed with 20 μL of Matrigel. Mice were observed after inoculation, and tumor growth was monitored. Grouping and administration of the drug occurred when the average tumor volume of the tumor-bearing mice reached 164 mm³. The day of group administration was defined as day 0. Tumor volume and body weight were measured twice weekly, and data were recorded.

[0398] 3. The test drugs and dosing regimens are shown in Table 24.

[0399] Table 24: Administration routes, dosages, and regimens for LD1 in a subcutaneous xenograft model of colorectal cancer (2012-200671).

[0400] 4. Five mice were used in each of the control and treatment groups. The tumor inhibition rate was calculated by measuring tumor volume. The formula for calculating tumor volume was: V = 0.5a × b², where a and b represent the long and short diameters of the tumor, respectively. The tumor growth inhibition rate was calculated using the following formula: TGI (%) = [1 - (Ti - T0) / (Vi - V0)] × 100, where Ti is the average tumor volume of a given treatment group on a certain day, T0 is the average tumor volume of this treatment group at the start of administration, Vi is the average tumor volume of the solvent control group on a certain day (the same day as Ti), and V0 is the average tumor volume of the solvent control group at the start of administration. One-way ANOVA was used for comparisons between multiple groups. P < 0.05 was considered statistically significant, and P < 0.01 was considered statistically significant.

[0401] Results: As shown in Table 25 and Figure 21.

[0402] Conclusion: Compared with the blank control group, the antibody-drug conjugate of this application showed no antitumor effect at low doses (1 mg / kg), but significant in vivo tumor inhibition was detected at 3 mg / kg and 10 mg / kg. The conjugate of the isotype control showed no antitumor effect at high doses (10 mg / kg), indicating the target specificity of this application. No significant decrease in body weight was observed in any group of mice.

[0403] Table 25 Evaluation of the antitumor efficacy of the test substance against the LD1-2012-200671 subcutaneous xenograft tumor model (calculated based on tumor volume 25 days after administration).

[0404] 1. Tumor volume is expressed as mean ± standard error;

[0405] 2. The p-value is calculated based on the tumor volume.

[0406] Example 16: In vivo efficacy comparison of antibody-drug conjugate ADC1 and control antibody-drug conjugate in NCI-H716 xenograft model

[0407] Objective: To compare the in vivo antitumor efficacy of the antibody-drug conjugate of this application with that of other control antibodies targeting CDH17, conjugated with the same linker toxin X2, in a CDH17-expressing colorectal cancer NCI-H716 model. The other control antibodies targeting CDH17 are the baseline antibody BM3.

[0408] method:

[0409] 1. Preparation of antibody-drug conjugates is described above.

[0410] 2. The test drugs and dosing regimens are shown in Table 26.

[0411] Table 26: Administration routes, dosages, and regimens for NCI-H716 subcutaneous xenograft models of colorectal cancer.

[0412] 3. Experimental animals: 6-8 week old female BALB / c nude mice, purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd.

[0413] 4. Experimental Methods: Cells in the exponential growth phase were collected and resuspended in a 1:1 mixture of PBS and matrix gel. 5 × 10⁶ cells were subcutaneously inoculated into the right nape of the neck of experimental mice. 6 NCI-H716 cells were used to regularly monitor tumor growth. When the tumor grew to approximately 150 mm... 3 Tumor-bearing mice were randomly assigned to groups as shown in Table 26. The day of group assignment was designated Day 0. The experimental endpoint was day 28 after group assignment. Tumor volume and body weight were measured twice weekly, and the data were recorded.

[0414] 5. Five mice were used in each of the control and treatment groups. The tumor inhibition rate was calculated by measuring tumor volume. The formula for calculating tumor volume is: V = 0.5a × b², where a and b represent the long and short diameters of the tumor, respectively. The tumor growth inhibition rate was calculated using the following formula: TGI (%) = [1 - (Ti - T0) / (Vi - V0)] × 100, where Ti is the average tumor volume of a given treatment group on a certain day, T0 is the average tumor volume of this treatment group at the start of administration, Vi is the average tumor volume of the solvent control group on a certain day (the same day as Ti), and V0 is the average tumor volume of the solvent control group at the start of administration. One-way ANOVA was used for comparisons between multiple groups. P < 0.05 was considered statistically significant, and P < 0.01 was considered statistically significant.

[0415] Results: As shown in Table 27 and Figure 22.

[0416] Conclusion: Both the antibody-drug conjugate (ADC-1) and the control antibody-drug conjugate of this application showed significant in vivo tumor-suppressing effects in the NCI-H716 colorectal cancer model with moderate CDH17 expression. The antibody-drug conjugate ADC-1 showed superior tumor-suppressing effects compared to the control antibody-drug conjugate ADC-4. No significant decrease in body weight was observed in any group of mice.

[0417] Table 27 Evaluation of the antitumor efficacy of the test substance against the NCI-H716 cell subcutaneous xenograft tumor model (calculated based on tumor volume 28 days after administration).

[0418] 1. Tumor volume is expressed as mean ± standard error;

[0419] 2. The p-value is calculated based on the tumor volume.

[0420] Example 17: In vivo efficacy comparison of antibody-drug conjugate ADC1 and control antibody-drug conjugate in HT-55 xenograft model

[0421] Objective: To compare the in vivo antitumor efficacy of the antibody-drug conjugate of this application with that of other control antibodies targeting CDH17, conjugated with the same linker toxin X2, in a CDH17-overexpressing colorectal cancer HT-55 model. The other control antibodies targeting CDH17 are the baseline antibody BM3.

[0422] method:

[0423] 1. Preparation of antibody-drug conjugates is described above.

[0424] 2. The test drugs and dosing regimens are shown in Table 28.

[0425] Table 28: Administration routes, dosages, and regimens for HT-55 subcutaneous xenograft models of colorectal cancer in 28 patients.

[0426] 3. Experimental animals: 6-8 week old female CB-17SCID mice, purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.

[0427] 4. Experimental Methods: Cells in the exponential growth phase were collected and resuspended in a 1:1 mixture of DPBS and matrix gel. 3 × 10⁶ cells were subcutaneously inoculated into the right back of experimental mice. 6 HT-55 cells were used to regularly monitor tumor growth. When the tumor grew to approximately 198 mm... 3 Tumor-bearing mice were randomly assigned to groups as shown in Table 28. The day of group assignment was designated as Day 0. The experimental endpoint was day 22 after group assignment. Tumor volume and body weight were measured twice weekly, and the data were recorded.

[0428] 5. Five mice were used in each of the control and treatment groups. The tumor inhibition rate was calculated by measuring tumor volume. The formula for calculating tumor volume is: V = 0.5a × b², where a and b represent the long and short diameters of the tumor, respectively. The tumor growth inhibition rate was calculated using the following formula: TGI (%) = [1 - (Ti - T0) / (Vi - V0)] × 100, where Ti is the average tumor volume of a given treatment group on a certain day, T0 is the average tumor volume of this treatment group at the start of administration, Vi is the average tumor volume of the solvent control group on a certain day (the same day as Ti), and V0 is the average tumor volume of the solvent control group at the start of administration. One-way ANOVA was used for comparisons between multiple groups. P < 0.05 was considered statistically significant, and P < 0.01 was considered statistically significant.

[0429] Results: As shown in Table 29 and Figure 23.

[0430] Conclusion: Both the antibody-drug conjugate and the control antibody-drug conjugate of this application showed significant in vivo tumor-suppressing effects. The antibody-drug conjugate (ADC-1) of this application showed superior tumor-suppressing effects compared to the control antibody-drug conjugate (ADC-4). There was no significant decrease in body weight in mice in the treatment group compared to the control group.

[0431] Table 29 Evaluation of the antitumor efficacy of the test substance on a subcutaneous xenograft tumor model of HT-55 cells (calculated based on tumor volume 22 days after administration).

[0432] 1. Tumor volume is expressed as mean ± standard error;

[0433] 2. The p-value is calculated based on the tumor volume.

[0434] Example 18: In vivo efficacy comparison of antibody-drug conjugate ADC1 and control antibody-drug conjugate in AsPC-1 xenograft model

[0435] Objective: To compare the in vivo antitumor efficacy of the antibody-drug conjugate of this application with that of other control antibodies targeting CDH17, conjugated with the same linker toxin X2, in a CDH17-overexpressing pancreatic cancer AsPC-1 model. The other control antibodies targeting CDH17 are the baseline antibody BM4.

[0436] method:

[0437] 1. Preparation of antibody-drug conjugates is described above.

[0438] 2. The test drugs and dosing regimens are shown in Table 30.

[0439] Table 30: Administration routes, dosages, and regimens for AsPC-1 subcutaneous xenograft models of pancreatic cancer in 30 patients.

[0440] 3. Experimental animals: 6-7 week old female CB-17SCID mice, purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.

[0441] 4. Experimental Methods: Cells in the exponential growth phase were collected and resuspended in a 1:1 mixture of DPBS and matrix gel. 5 × 10⁶ cells were subcutaneously inoculated into the right back of experimental mice. 6 AsPC-1 cells were used to regularly monitor tumor growth. When the tumor grew to approximately 124 mm... 3 Tumor-bearing mice were randomly assigned to groups as shown in Table 30. The day of group assignment was designated as Day 0. The experimental endpoint was day 27 after group assignment. Tumor volume and body weight were measured twice weekly, and the data were recorded.

[0442] 5. Six mice were used in each of the control and treatment groups. The tumor inhibition rate was calculated by measuring tumor volume. The formula for calculating tumor volume is: V = 0.5a × b², where a and b represent the long and short diameters of the tumor, respectively. The tumor growth inhibition rate was calculated using the following formula: TGI (%) = [1 - (Ti - T0) / (Vi - V0)] × 100, where Ti is the average tumor volume of a given treatment group on a certain day, T0 is the average tumor volume of this treatment group at the start of administration, Vi is the average tumor volume of the solvent control group on a certain day (the same day as Ti), and V0 is the average tumor volume of the solvent control group at the start of administration. One-way ANOVA was used for comparisons between multiple groups. P < 0.05 was considered statistically significant, and P < 0.01 was considered statistically significant.

[0443] Results: As shown in Table 31 and Figure 24.

[0444] Conclusion: Both the antibody-drug conjugate (ADC-1) and the control antibody-drug conjugate (ADC-5) of this application showed significant in vivo tumor-suppressing effects, with the antibody-drug conjugate of this application exhibiting superior tumor-suppressing effects compared to the control antibody-drug conjugate. There was no significant decrease in body weight in mice in the treatment group compared to the control group.

[0445] Table 31 Evaluation of the antitumor efficacy of the test substance on the AsPC-1 cell subcutaneous xenograft tumor model (calculated based on tumor volume 27 days after administration).

[0446] 1. Tumor volume is expressed as mean ± standard error;

[0447] 2. The p-value is calculated based on the tumor volume.

Claims

1. An antibody drug conjugate or a pharmaceutically acceptable salt thereof, characterized in that, The antibody drug conjugate or the pharmaceutically acceptable salt thereof comprises an anti-CDH17 antibody or an antigen binding fragment thereof, a linker unit L, and a cytotoxic drug M; The anti-CDH17 antibody or the antigen binding fragment thereof comprises a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3, and a light chain variable region comprising LCDR1, LCDR2, and LCDR3, The HCDR1 comprises an amino acid sequence as set forth in SEQ ID NO: 1; The HCDR2 comprises an amino acid sequence as set forth in SEQ ID NO: 2; The HCDR3 comprises an amino acid sequence as set forth in SEQ ID NO: 3; The LCDR1 comprises an amino acid sequence as set forth in SEQ ID NO: 5; The LCDR2 comprises an amino acid sequence as set forth in SEQ ID NO: 6; and The LCDR3 comprises an amino acid sequence as set forth in SEQ ID NO:

7.

2. The antibody drug conjugate or a pharmaceutically acceptable salt thereof according to claim 1, wherein The heavy chain variable region comprises an amino acid sequence having at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO: 4; and / or, the light chain variable region comprises an amino acid sequence having at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO: 8; The amino acid sequence having at least 90%, at least 95%, or at least 99% sequence identity maintains at least equivalent antigen binding function to the original sequence; Preferably, the amino acid sequence of the heavy chain variable region VH is as set forth in SEQ ID NO: 4; and / or, the amino acid sequence of the light chain variable region VL is as set forth in SEQ ID NO:

8.

3. The antibody drug conjugate or a pharmaceutically acceptable salt thereof according to claim 1 or 2, wherein The anti-CDH17 antibody or the antigen binding fragment thereof comprises one or more of the following: (1) a fully human antibody, a humanized antibody, a chimeric antibody, a pro-antibody, a bispecific antibody, a multispecific antibody, a monoclonal antibody, or a polyclonal antibody; (2) a Fab, a Fab’, a F(ab’)2, a Fv, a ScFv, a diabody, a Fd, a sdAb, a VHH, or a complementarity determining region; and (3) the CDH17 is a human CDH17.

4. The antibody drug conjugate or a pharmaceutically acceptable salt thereof according to claim 3, wherein The anti-CDH17 antibody or the antigen binding fragment thereof is a full-length antibody comprising a heavy chain constant region of a heavy chain of a human antibody and a light chain constant region of a light chain of a human antibody; the heavy chain constant region is preferably a heavy chain constant region of a human antibody IgG1, and the light chain constant region is preferably a light chain constant region of a human antibody kappa chain; The anti-CDH17 antibody or the antigen binding fragment thereof is a full-length antibody comprising a heavy chain constant region of a heavy chain of a human antibody and a light chain constant region of a light chain of a human antibody; the heavy chain constant region is preferably a heavy chain constant region of a human antibody IgG1, and the light chain constant region is preferably a light chain constant region of a human antibody kappa chain; Preferably, the amino acid sequence of the heavy chain constant region of the humanized antibody IgG1 is as set forth in SEQ ID NO: 9 or 11, or has at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO: 9 or 11; and / or, the amino acid sequence of the light chain constant region of the humanized antibody kappa chain is as set forth in SEQ ID NO: 10, or has at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO: 10; the amino acid sequence having at least 90%, at least 95%, or at least 99% sequence identity maintains at least equivalent antigen binding function to the original sequence; More preferably, the amino acid sequence of the heavy chain constant region of the humanized antibody IgG1 is as set forth in SEQ ID NO: 9 or 11; and / or, the amino acid sequence of the light chain constant region of the humanized antibody kappa chain is as set forth in SEQ ID NO: 10; Further more preferably, the amino acid sequence of the heavy chain of the anti-CDH17 antibody or antigen binding fragment thereof is as set forth in SEQ ID NO: 12 or 14; and / or, the amino acid sequence of the light chain is as set forth in SEQ ID NO:

13.

5. The antibody drug conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 4, wherein The cytotoxic drug M is of the structure shown in formula (A-1), a stereoisomer, a pharmaceutically acceptable salt, a solvate, or a solvate of a salt thereof, wherein, M is -L 2 -L 1 -C(O)-; L 2 is -O-, and L 2 is connected to the linker unit L; L 1 -(C(R) 1a (R) 1b )) m -CH2-; m is selected from 1, 2, 3, or 4; each R 1a and R 1b is independently hydrogen, halogen, hydroxyl, amino, or C1-C6alkyl optionally substituted with one or more R; each R is independently hydrogen or halogen.

6. The antibody drug conjugate or a pharmaceutically acceptable salt thereof according to claim 5, wherein each R 1a independently hydrogen, halogen, or Ci-C6alkyl; and / or, each R 1b independently hydrogen, halogen, or Ci-C6alkyl.

7. The antibody drug conjugate or a pharmaceutically acceptable salt thereof according to claim 6, wherein wherein L 1 is 8. The antibody drug conjugate or a pharmaceutically acceptable salt thereof according to claim 5, wherein wherein the cytotoxic drug M is any one of the following structures:

9. The antibody drug conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 5 to 8, wherein, wherein the linker unit L is -L a -L b -L c -; and the L c is linked to the cytotoxic drug M; - L a - is preferably wherein a is attached to the anti-CDH17 antibody or antigen binding fragment thereof, and b is attached to L b ; - L b - is any one of the following structures: Preferably more preferably wherein the c-terminus and L a are connected, and the d-terminus and L c are connected; - L c - is 10. The antibody drug conjugate or a pharmaceutically acceptable salt thereof according to claim 9, wherein wherein the linker unit L is preferably 11. The antibody drug conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 10, wherein, wherein, The antibody drug conjugate or pharmaceutically acceptable salt thereof has a structure as shown in formula (A-2): wherein, p represents the average number of connections, and p is any integer or decimal number from 1 to 10; preferably any integer or decimal number from 3 to 9; more preferably an integer or decimal number from 7 to 8, for example 7.77 or 7.79; Ab is the anti-CDH17 antibody or antigen binding fragment thereof as set forth in any one of claims 1 to 4; M is the cytotoxic drug M as set forth in any one of claims 5 to 10; L is the linker unit L as set forth in any one of claims 1 to 10.

12. The antibody drug conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 11, wherein, wherein the antibody drug conjugate or a pharmaceutically acceptable salt thereof is selected from the following structural formulae: wherein, p represents the average number of connections, and p is any integer or decimal number from 1 to 10; preferably any integer or decimal number from 3 to 9; more preferably an integer or decimal number from 7 to 8, for example 7.77 or 7.79; Ab is the anti-CDH17 antibody or antigen binding fragment thereof as set forth in any one of claims 1 to 4.

13. The antibody drug conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 12, wherein, The antibody drug conjugate or pharmaceutically acceptable salt thereof is a conjugate as follows or a pharmaceutically acceptable salt thereof: wherein, p is any integer or decimal number from 1 to 10; preferably any integer or decimal number from 3 to 9; more preferably any integer or decimal number from 6 to 8; for example, p is 7.77 or 7.79; DB1001 and DB1002 are anti-CDH17 antibodies, the heavy chain amino acid sequence of DB1001 is as set forth in SEQ ID NO: 12 and the light chain amino acid sequence is as set forth in SEQ ID NO: 13; the heavy chain amino acid sequence of DB1002 is as set forth in SEQ ID NO: 14 and the light chain amino acid sequence is as set forth in SEQ ID NO:

13.

14. The antibody drug conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 13, wherein, The antibody drug conjugate or pharmaceutically acceptable salt thereof is a conjugate as follows or a pharmaceutically acceptable salt thereof: wherein, p’ represents the number of connections, and p’ is any integer from 1 to 10; preferably any integer from 3 to 9; more preferably any integer from 4 to 8; for example, p’ is 4, 5, 6, 7, or 8; DB1001 and DB1002 are anti-CDH17 antibodies, the heavy chain amino acid sequence of DB1001 is shown as SEQ ID NO: 12 and the light chain amino acid sequence is shown as SEQ ID NO: 13; the heavy chain amino acid sequence of DB1002 is shown as SEQ ID NO: 14 and the light chain amino acid sequence is shown as SEQ ID NO:

13.

15. An antibody drug conjugate or a pharmaceutically acceptable salt thereof, characterized in that, It comprises the antibody drug conjugate or its pharmaceutically acceptable salt as claimed in claim 14, wherein the number of linkages p' is the same or different, and the average value of the number of linkages p' is any integer or decimal number between 1 and 10, preferably any integer or decimal number between 3 and 9; more preferably any integer or decimal number between 6 and 8; for example, the average value of the number of linkages p' is 7.77 or 7.

79.

16. A method of preparing an antibody drug conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 15, characterized by, The preparation method comprises reacting the anti-CDH17 antibody or its antigen binding fragment with a compound as shown in formula II to obtain the antibody drug conjugate or its pharmaceutically acceptable salt, L'-cytotoxic drug II; L' is a linker unit L as claimed in any one of claims 1-15, which forms with the anti-CDH17 antibody or its antigen binding fragment; The cytotoxic drug M is as claimed in any one of claims 1-15; Preferably, the antibody drug conjugate or its pharmaceutically acceptable salt satisfies one or more of the following conditions: (1) the compound is of Formula II: (2) the anti-CDH17 antibody or its antigen binding fragment is DB1001 or DB1002; The heavy chain amino acid sequence of DB1001 is preferably shown as SEQ ID NO: 12, and the light chain amino acid sequence is preferably shown as SEQ ID NO: 13; the heavy chain amino acid sequence of DB1002 is shown as SEQ ID NO: 14 and the light chain amino acid sequence is shown as SEQ ID NO:

13.

17. A pharmaceutical composition comprising, in combination, a compound of any one of claims 1-16 and a pharmaceutically acceptable carrier. It comprises the antibody drug conjugate or its pharmaceutically acceptable salt as claimed in any one of claims 1-15, and a pharmaceutically acceptable carrier.

18. Use of the antibody drug conjugate or its pharmaceutically acceptable salt as claimed in any one of claims 1-15 and / or the pharmaceutical composition as claimed in claim 17 in the preparation of a medicament for the diagnosis, prevention and / or treatment of cancer. Preferably, the cancer is a CDH17-expressing cancer, such as colorectal cancer, pancreatic cancer and gastric cancer.

19. A method of diagnosing, preventing and / or treating cancer, characterized in that, The method comprises administering to a subject in need an effective amount of the antibody drug conjugate or its pharmaceutically acceptable salt as claimed in any one of claims 1-15 and / or the pharmaceutical composition as claimed in claim 17; Preferably, the cancer is a CDH17-expressing cancer, such as colorectal cancer, pancreatic cancer and gastric cancer.

20. The antibody drug conjugate or its pharmaceutically acceptable salt as claimed in any one of claims 1-15 and / or the pharmaceutical composition as claimed in claim 17 for use in the diagnosis, prevention and / or treatment of cancer. Preferably, the cancer is a CDH17-expressing cancer, such as colorectal cancer, pancreatic cancer and gastric cancer.

21. A combination therapy, characterized in that, It comprises administering to a subject in need thereof the antibody drug conjugate or a pharmaceutically acceptable salt thereof of any one of claims 1-15 and / or the pharmaceutical composition of claim 17, and a second therapeutic agent; Preferably, the second therapeutic agent comprises another anti-CDH17 antibody or antigen binding fragment thereof or an antibody drug conjugate or a pharmaceutical composition comprising the other anti-CDH17 antibody or antigen binding fragment thereof, and / or another cancer therapeutic agent; More preferably, the cancer is a CDH17-expressing cancer, such as colorectal cancer, pancreatic cancer, and gastric cancer.

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