Anti-cdh17 antibody-drug conjugate and method of making and using thereof

Abstract

The present application relates to a set of antibody-drug conjugates (ADCs) in which the cytotoxic tubulin inhibitor monomethyl aurestatin E (MMAE) is conjugated to a monoclonal antibody targeting cadherin-17 (CDH17), which is a tumor-associated antigen that is overexpressed in gastrointestinal cancers such as cervical adenocarcinoma, ovarian mucinous cancer, and pancreatic cancer, gastric cancer, colorectal cancer. These ADCs recognize different parts of CDH17, exhibiting high affinity binding to cells expressing CDH17. All these characteristics allow CDH17-targeting ADCs to exhibit specific cytotoxicity against cancer cells that express CDH17. The cytotoxic effect of MMAE is highly positioned in tumors, so that the anti-tumor effect is maximized, and meanwhile, the adverse effects of anti-cancer drugs on non-cancerous normal tissues are reduced.

Description

(19) State Intellectual Property Office (12) Invention Patent Application (10) Application Publication Number (43) Application Publication Date (21) Application Number 202480061834.5 (22) Application Date 2024.09.13 (30) Priority Data 63 / 538,238 2023.09.13 US (85) PCT International Application Entering National Phase Date 2026.03.26 (86) PCT International Application Application Data PCT / US2024 / 046510 2024.09.13 (87) PCT International Application Publication Data WO2025 / 059391 EN 2025.03.20 (71) Applicant: Aibel Pharmaceutical Technology Co., Ltd. Address: Room 522, 5th Floor, Tower 2, Biotechnology Centre, 11 Science Avenue West, Sha Tin District, Hong Kong, China (72) Inventors: Lu Manqing, Huang Ziren, Huang Guanghui, Huang Baoyi (74) Patent Agency: Beijing Jingwantong Intellectual Property Agency Co., Ltd. 11440 Patent Attorney: Xu Tianyi (51) Int.Cl. C07K 16 / 28 (2006.01) A61K 47 / 68 (2006.01) A61P 35 / 00 (2006.01) (54) Invention Title: Anti-CDH17 Antibody-Drug Conjugate and its Preparation and Use Method (57) Abstract: This application relates to a group of antibody-drug conjugates (ADCs), among which the cytotoxic microtubule inhibitor monomethylolpropanetin E (MMAE) is conjugated to a monoclonal antibody targeting cadherin-17 (CDH17), a tumor-associated antigen overexpressed in cervical adenocarcinoma, ovarian mucinous carcinoma, and gastrointestinal cancers such as pancreatic, gastric, and colorectal cancers. These ADCs recognize different parts of CDH17 and exhibit high affinity binding to CDH17-expressing cells. All these properties enable CDH17-targeting ADCs to exhibit specific cytotoxicity against CDH17-expressing cancer cells. The cytotoxic effect of MMAE will be highly localized within the tumor, maximizing antitumor activity while reducing the adverse effects of anticancer drugs on non-cancerous normal tissues. Claims 2 pages, Description 9 pages, Sequence Listing (electronic publication), Drawings 14 pages, CN 121941709 A 2026.04.28 CN 1 21 94 17 09 A 1. An antibody-drug conjugate (ADC) comprising: a monoclonal antibody (mAb), and a cytotoxic agent conjugated to said mAb via a linker, wherein said mAb comprises a heavy chain variable (VH) domain and a light chain variable (VL) domain, and wherein said VH domain and VL domain form a Fab region having binding affinity for human cadherin-17 (CDH17).2. The antibody-drug conjugate of claim 1, wherein the VH domain comprises an amino acid sequence having at least 98% sequence identity with SEQ ID NO: 1, 3, 5, 7 or 9, or wherein the VL domain comprises an amino acid sequence having at least 98% sequence identity with SEQ ID NO: 2, 4, 6, 8 or 10. 3. The antibody-drug conjugate of claim 1, wherein the VH domain comprises three complementarity-determining regions (CDRs) of SEQ ID NO: 1, 3, 5, 7 or 9. 4. The antibody-drug conjugate of claim 1, wherein the VL domain comprises three CDRs of SEQ ID NO: 2, 4, 6, 8 or 10. 5. The antibody-drug conjugate of claim 1, wherein the cytotoxic agent is an anti-microtubule agent, a topoisomerase inhibitor, an RNA polymerase II inhibitor, a photosensitizer, or a DNA alkylating agent. 6. The antibody-drug conjugate of claim 5, wherein the cytotoxic agent comprises an antimicrotubule agent selected from maytansine, oliquistatin, and tubulolysin. 7. The antibody-drug conjugate of claim 6, wherein the oliquistatin comprises monomethyloliquistatin E (MMAE). 8. The antibody-drug conjugate of claim 1, wherein the linker comprises a maleimide hexanoyl (MC) group, a valine-citrulline p-aminobenzyl carbamate (VC-PAB) unit, or a combination thereof. 9. A pharmaceutical composition comprising the antibody-drug conjugate of claim 1 and a pharmaceutically acceptable carrier. 10. A method of treating or preventing cancer in a subject, the method comprising administering to the subject a pharmaceutical composition comprising an effective amount of the antibody-drug conjugate of claim 1. 11. The method of claim 10, further comprising co-administering an effective amount of a therapeutic agent, wherein the therapeutic agent comprises an antibody, a chemotherapeutic agent, an enzyme, or a combination thereof. 12. The method of claim 10, wherein the cancer comprises cancer expressing CDH17. 13. The method of claim 10, wherein the cancer includes esophageal cancer, lung cancer, breast cancer, gastric cancer, pancreatic cancer, liver cancer, bile duct cancer, colorectal cancer, ovarian cancer, cervical cancer, uterine cancer, kidney cancer, or prostate cancer. 14. A method for preparing the antibody-drug conjugate of claim 1, comprising conjugating the mAb to the cytotoxin. 15. An isolated monoclonal antibody (mAb) wherein the mAb has binding affinity for human cadherin-17 (CDH17), wherein the mAb comprises: a heavy chain variable (VH) domain, the heavy chain variable (VH) domain comprising the amino acid sequence of complementarity-determining region 1 (HCDR1) of SEQ ID NO: 1, 3, 5, 7, or 9, and SEQ ID NO: 9.The HCDR2 amino acid sequence of SEQ ID NO: 1, 3, 5, 7, or 9, and the HCDR3 amino acid sequence of SEQ ID NO: 1, 3, 5, 7, or 9, and the light chain variable (VL) domain comprising the complementarity-determining region 1 (LCDR1) amino acid sequence of SEQ ID NO: 2, 4, 6, 8, or 10, the LCDR2 amino acid sequence of SEQ ID NO: 2, 4, 6, 8, or 10, and the LCDR3 amino acid sequence of SEQ ID NO: 2, 4, 6, 8, or 10. Claims 1 / 2 page 2 CN 121941709 A 16. The isolated mAb according to claim 15, wherein the VH domain comprises the amino acid sequence of SEQ ID NO: 1, 3, 5, 7, or 9. 17. The isolated mAb according to claim 15, wherein the VL domain comprises the amino acid sequence of SEQ ID NO: 2, 4, 6, 8, or 10. 18. A pharmaceutical composition comprising the isolated mAb of claim 15 and a pharmaceutically acceptable carrier. 19. A composition comprising the isolated mAb or a derivative thereof of claim 15 and a cytotoxic agent or a derivative thereof. 20. The composition of claim 19, wherein the cytotoxic agent comprises MMAE or a derivative thereof. Claims 2 / 2 Page 3 CN 121941709 A Anti-CDH17 Antibody-Drug Conjugate and its Preparation and Use

[0001] Cross-Reference to Related Applications This application claims the benefit of U.S. Provisional Application Serial No. 63 / 538,238, filed September 13, 2023, under 35 USC 119(e), the entire disclosure of which is incorporated herein by reference. Technical Field

[0002] This disclosure relates generally to the field of cancer immunotherapy technology, and more particularly to antibody-drug conjugates. Background Art

[0003] Gastrointestinal (GI) cancers, such as colorectal cancer, stomach cancer, pancreatic cancer, esophageal cancer, bile duct cancer, or liver cancer, are leading causes of morbidity and mortality worldwide. Colorectal cancer (CRC) alone accounts for approximately 10% of all diagnosed cancers and is the second leading cause of cancer death globally. In China, liver and stomach cancers are the deadliest malignancies, accounting for more than half of all diagnosed cases and causing more than 1.42 million deaths annually. This is believed to be caused by endemic viral / bacterial diseases (hepatitis B virus [HBV] and Helicobacter pylori infection), chemical poisoning, environmental pollution, and food contamination. Currently, there are no effective treatments. Therefore, there is an urgent need for novel biomarkers and therapeutic targets to develop potential drugs for these aggressive cancers. Molecularly targeted agents that have been proven to eliminate or inhibit the growth of these cancers will have significant clinical value and market impact. If the disease is diagnosed early,These tumors can then be effectively removed surgically. Unfortunately, most GI cancers are usually asymptomatic and are only detected at a very late stage when they appear clinically. Without effective treatment, these patients die shortly after diagnosis or relapse after salvage therapy.

[0004] Chemotherapy drugs for the treatment of cancer, including GI cancers, have been developed. In the past few decades, although such therapeutic drugs have been effective in some cancers, undesirable adverse effects on normal tissues have limited their clinical benefits. In addition, many patients develop resistance to chemotherapy drugs during treatment regimens. To overcome these challenges, antibody-drug conjugates (ADCs) that can deliver chemotherapy drugs to tumors have been developed. This antibody-guided delivery ensures that the cytotoxic effects of chemotherapy drugs are localized within the target tumor, thereby reducing adverse effects on normal tissues and minimizing the possibility of resistance development. Summary of the Invention

[0005] The following summary of the invention is illustrative only and is not intended to be limiting in any way. Other aspects, embodiments, and features will become apparent from the accompanying drawings and the following detailed description, in addition to the illustrative aspects, embodiments, and features described above.

[0006] Cadmin-17 (CDH17) is an important cancer biomarker, characterized by overexpression in both liver and gastric cancers, but not in normal tissues of healthy adults. Anti-CDH17 monoclonal antibodies have shown growth inhibitory effects on liver and gastric tumor cells. CDH17 is highly expressed in gastrointestinal cancers, cervical cancer, ovarian mucinous carcinoma, and metastatic cancers. Blocking the expression and function of CDH17 can significantly reduce lung metastases of hepatocellular carcinoma (HCC).

[0007] In one aspect, this application provides antibody-drug conjugates (ADCs). In one embodiment, this application discloses an anti-CDH17 antibody-drug conjugate developed as a targeted therapeutic agent for treating cancer patients with CDH17 biomarker indicators in tumor tissues and / or serum samples. Specification 1 / 9 pages 4 CN 121941709 A

[0008] In one embodiment, the antibody-drug conjugate (ADC) comprises a monoclonal antibody (mAb) and a cytotoxic agent conjugated to the monoclonal antibody via a linker. In one embodiment, the mAb has binding affinity for human cadherin-17 (CDH17). In one embodiment, the mAb comprises a heavy chain variable (VH) domain and a light chain variable (VL) domain, and the VH domain and the VL domain form a Fab region having binding affinity for human cadherin-17 (CDH17).

[0009] In one embodiment, the VH domain comprises having a binding affinity of at least 60%, 70%, or 80% with SEQ ID NO: 1, 3, 5, 7, or 9.The amino acid sequence has 80%, 90%, 95%, 98%, or 99% sequence identity. In one embodiment, the VH domain comprises three complementarity-determining regions (CDRs) of SEQ ID NO: 1, 3, 5, 7, or 9. In one embodiment, the VH domain comprises the amino acid sequence of SEQ ID NO: 1.

[0010] In one embodiment, the VL domain comprises an amino acid sequence having at least 60%, 70%, 80%, 90%, 95%, 98%, or 99% sequence identity with SEQ ID NO: 2, 4, 6, 8, or 10. In one embodiment, the VL domain comprises three CDRs of SEQ ID NO: 2, 4, 6, 8, or 10. In one embodiment, the VL domain comprises the amino acid sequence of SEQ ID NO: 2.

[0011] In one embodiment, the mAb comprises the amino acid sequences of SEQ ID NO: 1 and 2.

[0012] In one embodiment, the cytotoxic agent comprises an antimicrotubule agent, a topoisomerase inhibitor, an RNA polymerase II inhibitor, a photosensitizer, a DNA alkylating agent, or a combination thereof. In one embodiment, the cytotoxic agent comprises an antimicrotubule agent selected from maytansines, oliquistatins, microtubule-lysins, and derivatives thereof. In one embodiment, oliquistatins comprise monomethyl oliquistatin E (MMAE) or a derivative thereof.

[0013] The linker may comprise a maleimide hexanoyl (MC) group, a valine-citrulline p-aminobenzyl carbamate (VC-PAB) unit, or a combination thereof.

[0014] In another aspect, this application provides pharmaceutical compositions. In one embodiment, the pharmaceutical composition comprises an antibody-drug conjugate as disclosed herein and a pharmaceutically acceptable carrier.

[0015] In another aspect, this application provides a method of treating or preventing cancer in a subject. In one embodiment, the method comprises administering to a subject a pharmaceutical composition comprising an antibody-drug conjugate as disclosed herein. In one embodiment, the method further comprises co-administering an effective amount of a therapeutic agent. In one embodiment, the co-administered therapeutic agent comprises an antibody, a chemotherapeutic agent, an enzyme, or a combination thereof.

[0016] In one embodiment, the cancer comprises cancer expressing CDH17. In one embodiment, the cancer includes esophageal cancer, lung cancer, breast cancer, gastric cancer, pancreatic cancer, liver cancer, bile duct cancer, colorectal cancer, ovarian cancer, cervical cancer, uterine cancer, kidney cancer, or prostate cancer.

[0017] In another aspect, this application includes methods for preparing antibody-drug conjugates as disclosed herein. In one embodiment, the method includes conjugating the mAb with a cytotoxin.

[0018] In another aspect, this application provides isolated monoclonal antibodies (mAbs). In one embodiment, the mAb is effective against human...cadherin-17 (CDH17) has binding affinity. In one embodiment, the mAb comprises a heavy chain variable (VH) domain containing the amino acid sequence of complementarity-determining region 1 (HCDR1) of SEQ ID NO: 1, 3, 5, 7, or 9, the amino acid sequence of HCDR2 of SEQ ID NO: 1, 3, 5, 7, or 9, and the amino acid sequence of HCDR3 of SEQ ID NO: 1, 3, 5, 7, or 9. In one embodiment, the mAb comprises a light chain variable (VL) domain containing the amino acid sequence of complementarity-determining region 1 (LCDR1) of SEQ ID NO: 2, 4, 6, 8, or 10, the amino acid sequence of LCDR2 of SEQ ID NO: 2, 4, 6, 8, or 10, and the amino acid sequence of LCDR3 of SEQ ID NO: 2, 4, 6, 8, or 10.

[0019] In one embodiment, the isolated mAb comprises a VH domain having an amino acid sequence of SEQ ID NO: 1, 3, 5, 7, or 9. In one embodiment, the isolated mAb comprises a VL domain having an amino acid sequence of SEQ ID NO: 2, 4, 6, 8, or 10.

[0020] In one embodiment, the isolated mAb comprises a VH domain having an amino acid sequence of SEQ ID NO: 1, 3, 5, 7, or 9, and a VL domain having an amino acid sequence of SEQ ID NO: 2, 4, 6, 8, or 10.

[0021] In another aspect, this application provides pharmaceutical compositions. In one embodiment, the pharmaceutical composition comprises an isolated mAb as disclosed herein and a pharmaceutically acceptable carrier. In one embodiment, the composition comprises an isolated mAb or a derivative thereof as disclosed herein and a cytotoxic agent or a derivative thereof.

[0022] In one embodiment, the cytotoxic agent comprises MMAE or a derivative thereof.

[0023] The foregoing and other features of this disclosure will become more apparent from the accompanying drawings, the following description, and the appended claims. It should be understood that these drawings depict only a few embodiments arranged according to this disclosure and should not be considered as limiting its scope. Further specific and detailed description of the disclosure will be given below using the drawings, in which: Figure 1 illustrates the binding of ARB102-MMAE to CDH17 protein as detected by enzyme-linked immunosorbent assay (ELISA); Figure 2 illustrates the binding of ARB102-MMAE to various cancer cells, including AsPC1 cells (2A), MHCC97H cells (2B), NCI-N87-CDH17+ cells (2C), DLD1 cells (2D), T84 cells (2E), and AGS-...CDH17+ cells (2F), AGS-CDH17- cells (2G), SW480-CDH17+ cells (2H), and SW480-CDH17- cells (2I) demonstrate that ARB102-MMAE binds to CDH17-expressing cancer cells with nanomolar affinity; Figure 3 shows the internalization of ARB102-MMAE in various CDH17-expressing cancer cells as detected by flow cytometry; Figure 4 depicts the cytotoxic effects of ARB102-MMAE on AsPC1 cells (4A), NCI-N87-CDH17+ cells (4B), NCI-N87-CDH17- cells (4C), AGS-CDH17+ cells (4D), and AGS-CDH17- cells (4E), as well as AsPC1 cells. Receptor occupancy rates of ARB102-MMAE in cells (4F), NCI-N87-CDH17+ cells (G), and AGS-CDH17+ cells (H) were shown, indicating that ARB102-MMAE could kill 40% of cancer cells when the receptor occupancy rate was less than 10% (4I). The maximum killing and internalization of ARB102-MMAE were investigated in 13 cancer cell lines. Figure 5 depicts the in vitro stability of ARB102-MMAE in human plasma, showing the binding activity of ARB102-MMAE to CDH17 protein as measured by ELISA, for example, after incubation at 37°C for 21 days in human plasma, residual binding >60% (5A); and the level of intact ARB102-MMAE in human plasma during the 21-day incubation period (5B). Figure 6 depicts the antitumor efficacy and in vivo stability of ARB102-MMAE in a mouse pancreas xenograft model. Biodistribution showed that (6A) ARB102-MMAE treatment resulted in a reduction in tumor volume (mm3) in mice; (6B) ARB102-MMAE treatment was not associated with significant changes in mouse body weight; (6C) ARB102-MMAE was still detectable in plasma 6 days after injection, while unconjugated MMAE was undetectable; and (6D and 6E) significant amounts of ARB102-MMAE were detected only in subcutaneous tumors, but not in the liver. Detailed Description

[0024] In the following detailed description, reference is made to the accompanying drawings, which form a part thereof. In the drawings, similar symbols generally identify similar components unless the context otherwise requires. The illustrative embodiments described in the detailed description, drawings, and claims are not intended to be limiting. Other embodiments may be utilized, and other changes may be made, without departing from the spirit or scope of the subject matter presented herein. It is readily understood that aspects of this disclosure (as shown in the general description and drawings herein) can be arranged, substituted, combined, separated, and designed in a variety of different configurations, all of which are described on pages 3 / 9 of this specification, 6 CN.121941709 A This is explicitly anticipated.

[0025] In order for immunotherapies to treat cancer, especially solid tumors, more effectively, combination therapies are essential, which, in addition to typical bispecific antibodies, also possess multiple target specificities and / or mechanisms of action. Therapies that are essentially combination therapies (such as those described herein) are necessary for the effective treatment of cancer and for achieving complete and durable responses more frequently. Specifically, there is a need for a scaffold with certain properties to construct combination therapies that have a favorable mechanism of action, preparation, pharmacokinetics, and low antigenicity compared to approved bispecific antibodies. Many bispecific antibodies based on whole antibodies can have a greater mass than the trispecific antibodies described herein. Although low-mass antibodies based on antibody fragments can have stronger tumor penetration, they generally have relatively poor pharmacokinetic properties, such as the FDA-approved bispecific antibody BLINCYTO®, which does not have an Fc region. Furthermore, many knob-into-hole-based bispecific antibodies have mutations in constant domains of the Ig structure that can trigger anti-drug antibody (ADA) responses. In this context, a group of modified antibodies described herein as triaxial or TriAx antibodies can possess multiple antigen-binding specificities without mutations in any constant domain.

[0026] As used herein, the terms “an,” “a,” and “the” are defined to mean “one or more” and include the plural unless the context is inappropriate.

[0027] The term “antibody” is used in the broadest sense and specifically covers single monoclonal antibodies (including agonist and antagonist antibodies), antibody compositions with multi-epitope specificity, and antibody fragments such as Fab, F(ab')2, and Fv, provided they exhibit the desired biological activity. In some embodiments, the antibody may be a monoclonal antibody, chimeric antibody, single-chain antibody, multispecific antibody, pleiotropic antibody, human antibody, and humanized antibody. Examples of active antibody fragments that bind to known antigens include Fab, F(ab')2, scFv, and Fv fragments, as well as products of Fab immunoglobulin expression libraries and epitope-binding fragments of any of the aforementioned antibodies and fragments. In some embodiments, the antibody may include an immunoglobulin molecule and the immunoactive portion of an immunoglobulin molecule, i.e., a molecule containing a binding site that specifically binds to an antigen. Immunoglobulins can be any type (IgG, IgM, IgD, IgE, IgA, and IgY) or class (IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2) or subclass of immunoglobulin molecules. In one embodiment, the antibody may be a complete antibody and any antigen-binding fragment derived from a complete antibody. A typical antibody is a heterotetramer that typically comprises two heavy (H) chains and two light (L) chains.Proteins. Each heavy chain consists of a heavy chain variable domain (abbreviated as VH) and a heavy chain constant domain. Each light chain portion consists of a light chain portion variable domain (abbreviated as VL) and a light chain portion constant domain. The VH and VL regions can be further subdivided into highly variable complementarity-determining regions (CDRs) and more conserved regions called framework regions (FRs). Each variable domain (VH or VL) typically consists of three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. Binding regions that interact with antigens exist within the variable regions of the heavy and light chains.

[0028] The term "multispecific" antibody, as used herein, refers to an antibody having at least two binding sites, each binding site having binding affinity for an epitope of an antigen. The terms "bispecific, trispecific, tetraspecific, or pentaspecific" antibodies, as used herein, refer to antibodies having two, three, four, five, or six antigen binding sites.

[0029] The term “humanized antibody” refers to a class of engineered antibodies whose CDR is derived from a non-human donor immunoglobulin, and the remaining immunoglobulin-derived portions of the molecule are derived from one (or more) human immunoglobulins. Furthermore, the framework support residues can be modified to maintain binding affinity. Methods for obtaining “humanized antibodies” are well known to those skilled in the art (see Queen et al., Proc. Natl Acad Sci USA, 1989; Hodgson et al., Bio / Technology, 1991). In one embodiment, “humanized antibodies” can be obtained by genetic engineering methods that enable the production of affinity-matured human-like polyclonal antibodies in large animals (e.g., rabbits) (see U.S. Patent No. 7,129,084).

[0030] The term “antigen” refers to an entity or fragment thereof that can induce an immune response in an organism, particularly an animal, and more particularly in mammals including humans. This term includes immunogens and the regions responsible for antigenicity or antigenic determinants.

[0031] The term "epitope," also known as "antigenic determinant," is a portion of an antigen that is recognized by the immune system, particularly by antibodies, B cells, or T cells, and is a specific fragment of the antigen to which antibodies bind.

[0032] The term "immunogenicity" refers to a substance that induces or enhances the production of antibodies, T cells, or other reactive immune cells against an immunogenic agent and contributes to an immune response in a human or animal. An immune response occurs when an individual produces sufficient amounts of antibodies, T cells, and other reactive immune cells against the administered immunogenic composition of this application to alleviate or reduce the condition to be treated.

[0033] As used herein, the term "tumor antigen" refers to an antigenic molecule produced in tumor cells. Tumor antigens can...Inducing an immune response in the host. In one embodiment, tumor cells express tumor antigens, including but not limited to tumor-specific antigens (TSA), neoantigens, and tumor-associated antigens (TAA).

[0034] As used herein, the terms “specifically bind to,” “specifically bind to,” or “specifically target” a particular antigen or epitope mean a binding that is measurably different from a nonspecific interaction. Specific binding can be measured by identifying the binding of a molecule and comparing it to the binding of a control molecule (typically a molecule with a similar structure that does not have binding activity). Specific binding can be determined by competition with a control molecule similar to the target. Specific binding to a particular antigen or epitope can be represented by an antibody with a KD of at least about 10⁻⁴ M, at least about 10⁻⁵ M, at least about 10⁻⁶ M, at least about 10⁻⁷ M, at least about 10⁻⁸ M, at least about 10⁻⁹ M, optionally at least about 10⁻¹⁰ M, at least about 10⁻¹¹ M, at least about 10⁻¹² M, or greater, where KD refers to the dissociation rate of a particular antibody-antigen interaction. In some embodiments, the KD of the multispecific antibody that specifically binds to the antigen is 20, 50, 100, 500, 1000, 5000, 10000, or higher than that of the control molecule relative to the antigen or epitope. Furthermore, specific binding to a particular antigen or epitope can be represented by an antibody whose KA or Ka is at least 20, 50, 100, 500, 1000, 5000, 10000, or higher than that of the control, where KA or Ka refers to the association rate of the specific antibody-antigen interaction.

[0035] As used herein, the term “antibody-drug conjugate” refers to the connection of an antibody or its antigen-binding fragment to another pharmaceutical agent such as a chemotherapeutic agent, toxin, immunotherapeutic agent, imaging probe, etc. This connection can be a covalent bond or a non-covalent interaction, such as by electrostatic force. Various connectors known in the art can be used to form immunoconjugates.

[0036] The term “subject” includes humans and non-human animals. Non-human animals include all vertebrates, such as mammals and non-mammals, such as non-human primates, sheep, dogs, cattle, chickens, amphibians, and reptiles. Unless otherwise stated, the terms "patient" or "subject" are used interchangeably herein.

[0037] As used herein, the term "cytotoxic agent" means any agent that is harmful to cell growth and proliferation and can reduce, inhibit, or destroy cells or malignant tumors.

[0038] As used herein, the term "anticancer agent" means any agent that can be used to treat cell-proliferating diseases such as cancer, including but not limited to cytotoxic agents, chemotherapy agents, radiotherapy and radiotherapy agents, targeted anticancer agents, and immunotherapy agents.

[0039] As used herein, the terms "pharmaceutical portion" or "payload" refer to the chemical portion conjugated to the antibody or antigen-binding fragment of this application and may include any therapeutic or diagnostic agent, such as an anticancer agent, anti-inflammatory agent, anti-infective agent (e.g., antifungal agent, antibacterial agent, antiparasitic agent, antiviral agent), or anesthetic agent. For example, the pharmaceutical portion may be an anticancer agent, such as a cytotoxin. In some embodiments, the pharmaceutical component is selected from V-ATPase inhibitors, HSP90 inhibitors, IAP inhibitors, mTor inhibitors, microtubule stabilizers, microtubule destabilizers, olistatin, dolalastatin, maytanyl derivatives, MetAP (methionine aminopeptidase), CRM1 inhibitors, DPPIV inhibitors, mitochondrial phosphoryl transfer reaction inhibitors, protein synthesis inhibitors, kinase inhibitors, CDK2 inhibitors, CDK9 inhibitors, proteasome inhibitors, kinase inhibitors, HDAC inhibitors, DNA damaging agents, DNA alkylating agents, DNA intercalating agents, DNA minor groove binding agents, and DHFR inhibitors. Methods for linking each of these to a connector compatible with the antibodies and methods of this application are known in the art. In addition, the payload can be a biophysical probe, fluorophore, spin label, infrared probe, affinity probe, chelating agent, spectral probe, radioactive probe, lipid molecule, polyethylene glycol, polymer, spin label, DNA, RNA, protein, peptide, surface, antibody, antibody fragment, nanoparticle, quantum dot, liposome, PLGA particle, sugar or polysaccharide.

[0040] Several short peptide compounds have been isolated from the marine mollusc Dolabella auricularia and found to be bioactive. Analogs of these compounds have also been prepared and some of them have been found to be bioactive (see Pettit et al., Anti-Cancer Drug Design 13:243-277, 1998). For example, olistatin E is a synthetic analog of the marine natural product doralastatin 10, which is an agent that inhibits tubulin polymerization by binding to the same domain of tubulin as the anticancer drug vincristine (US 5,635,483). Dolatatine 10, olistatin PE, and olistatin E are linear peptides containing four amino acids and a C-terminal amide, three of which are characteristic of doralastatin compounds. Olistatin peptides (olistatin E (AE) and monomethylolistatin (MMAE)) are synthetic analogues of doralastatin. MMAE is an antimitotic agent that inhibits cell division by blocking the polymerization of tubulin. The linkers of monoclonal antibodies are stable in the extracellular fluid, but once the conjugates enter tumor cells, they are blocked by tissue proteins.Enzyme cleavage, thereby activating the anti-mitotic mechanism. MMAE is one of the most commonly used payloads for preparing ADCs, including anti-CD20 ADCs such as RITUXAN® (WO 2004 / 032828), ADCETRIS® (brentuximab velituximab), PADCEV® (enrofloxacin / velituximab), and POLIVY® (velituximab for injection) for the treatment of cancers and immune diseases expressing CD20.

[0041] Examples The present disclosure is further described with reference to the following examples. These examples are provided for illustrative purposes only and are not intended to be limiting unless otherwise stated. Those skilled in the art will readily recognize the various non-critical parameters that can be changed or modified to produce substantially the same or similar results.

[0042] Example 1. Generation and production of CDH17-targeting ADCs Recombinant humanized anti-CDH17 monoclonal antibody ARB102 (US20210115152) was produced using the cytotoxic microtubule inhibitor monomethylolpropionate E (MMAE) as the payload for ADC production. Then, mc-vc-PAB-MMAE was conjugated to the cysteine ​​residues on ARB102 via a valine-citrulline dipeptide linker to generate the ADC ARB102-MMAE. In short, the antibody was first dialyzed at 4°C in conjugation buffer (25 mM Na2B4O7, 25 mM NaCl, 1 mM DPTA, pH 7.4). The dialyzed antibody was then mixed with mc-vc-PAB-MMAE solution (MedChemExpress) at the desired molar ratio and incubated at 25°C. The conjugation product was dialyzed at 4°C in phosphate buffer (pH 7.4) for at least 4 hours. The resulting ADC was analyzed using SEC-HPLC and LC-MS. The drug conjugate prepared via maleimide-thiol chemistry does not affect the targeting properties of the cysteine-containing monoclonal antibody. This linker has been used in many FDA-approved ADCs, and the mechanism of action is expected to be the same, i.e., the linker is recognized by cathepsin B, which cleaves the citrulline-PAB amide bond. MMAE is then released to exert its anticancer effect. This conjugation achieves an antibody-drug ratio (DAR) of approximately 4.

[0043] Example 2. Characterization of CDH17-targeting ADCs The binding of ARB102-MMAE to CDH17 was examined using a CDH17 enzyme-linked immunosorbent assay (ELISA) (Figure 1). Recombinant CDH17 protein (5 μg / ml) was coated onto an immunoassay plate and incubated with ARB102 (i.e., the parental antibody) and ARB102-MMAE. The bound antibody was detected using anti-human IgG-HRP. The signal was generated by a chemiluminescent substrate and detected at 450 nm.The absorbance was measured at the optical density. The results showed that ARB102-MMAE exhibited CDH17 binding comparable to that of ARB102, indicating that conjugation does not affect the binding properties of ARB102. Similar to the parent antibody ARB102, ARB102-MMAE showed high affinity for CDH17, with a titer in the sub-nanomolar range. The results indicate that conjugation of MMAE does not affect the binding of the antibody to CDH17.

[0044] The binding of ARB102-MMAE to cancer cells was demonstrated using flow cytometry (Figure 2). The study investigated CDH17-expressing cancer cells, including AsPC1 pancreatic adenocarcinoma cells (Fig. 2A), hepatocellular carcinoma MHCC97H cells (Fig. 2B), gastric adenocarcinoma NCI-N87-CDH17 cells (Fig. 2C), colorectal adenocarcinoma DLD1 cells (Fig. 2D), and colonic adenocarcinoma T84 cells (Fig. 2E). ARB102-MMAE showed binding to all CDH17-positive cell lines, all in the single-digit nanomolar range. The CDH17 binding specificity of ARB102-MMAE was also tested in CDH17-expressing gastric AGS cell syngeneic lines (Fig. 2F and 2G) and colorectal cancer SW480 cell syngeneic lines (Fig. 2H and 2I).

[0045] Internalization of ARB102-MMAE in CDH17-positive cancer cells: Effective internalization in cells is essential for the cytotoxic function of ADCs. Flow cytometry was used to investigate the internalization of ARB102-MMAE in various cancer cells (Fig. 3). ARB102-MMAE was labeled with a dye, which showed enhanced fluorescence when internalized into cells. The development of fluorescence was tracked by flow cytometry. ARB102-MMAE was shown to be internalized into pancreatic AsPC1 cells, colorectal SW480-CDH17+ cells, gastric AGS-CDH17+ cells, liver MHCC97H cells, gastric SNU1-CDH17+ cells, gastric NCI-N87-CDH17+ cells, and colonic T84 cells. No significant internalization was observed in lung A549 cells, colorectal DLD1 cells, and Colo205 cells.

[0046] Cytotoxicity of ARB102-MMAE against CDH17-positive cancer cells The cytotoxicity of ARB102-MMAE was examined in vitro (Figure 4). Pancreatic AsPC1 cells express CDH17. Treatment of this cell line with ARB102-MMAE resulted in dose-dependent cytotoxicity (Figure 4A). ARB102 alone did not induce any cytotoxicity, indicating that MMAE internalization is essential for the observed cytotoxicity. In gastric NCI- with and without CDH17 overexpression...The selective killing effect of ARB102-MMAE on CDH17-positive cancer cells was examined in N87 cell syngeneic lines (Fig. 4B and 4C) and AGS cell syngeneic lines (Fig. 4D and 4E). ARB102-MMAE killed CDH17-positive NCI-N87 cells but not CDH17-negative NCI-N87 cells, indicating that the ADC is selective for CDH17-positive tumors. Calculations showed that the receptor occupancy rates of 40% killed AsPC1 cells (Fig. 4F), NCI-N87-CDH17+ cells (Fig. 4G), and AGS-CDH17+ cells (Fig. 4H) were 0.4%, 1.8%, and 8.3%, respectively, indicating that ARB102-MMAE only requires a low receptor occupancy rate to exert its cytotoxic effect on cancer cells. In addition to AsPC1, NCI-N87, and AGS cells, the killing and internalization of ARB102-MMAE were also investigated in a group of gastrointestinal cancer cells (Fig. 4I). These results indicate that the maximum killing effect of ARB102-MMAE on cancer cells is positively correlated with the percentage of internalization.

[0047] In vitro stability of ARB102-MMAE in human plasma The stability of ADCs is important for their cytotoxic function in humans. ARB102-MMAE was added to human plasma and incubated at 37°C for up to 3 weeks (Figure 5). In vitro stability was determined by measuring binding activity (Figure 5A) and the concentration of intact ARB102-MMAE (Figure 5B). ARB102-MMAE showed good stability with an active half-life of about 21 days and a concentration half-life of about 60 days.

[0048] Antitumor efficacy, in vivo stability and biodistribution of ARB102-MMAE in a mouse pancreatic xenograft model The antitumor efficacy of ARB102-MMAE was examined in mice carrying pancreatic cancer xenografts. Pancreatic AsPC1 cancer cells were subcutaneously inoculated into NOD-SCID mice. When the tumors became palpable, mice were intravenously injected with ARB102-MMAE (3 mg / kg), equimolar amounts of MMAE, or sterile PBS once a week for 4 weeks. ARB102-MMAE treatment resulted in complete inhibition of tumor growth (Fig. 6A). No significant changes in body changes associated with ARB102-MMAE were observed (Fig. 6B). Plasma, tumor tissue, and liver tissue were collected from mice one day before the fourth dose, two days after the fourth dose, and six days after the fourth dose to monitor the level and distribution of ARB102-MMAE (Fig. 6C-6E). The level of ARB102-MMAE in plasma (Fig. 6C) increased after the fourth dose, began to decrease after two days, and remained in circulation at detectable levels after six days. A similar pattern was observed in tumor tissue (Fig. 6C).6D), but not observed in liver tissue, as only trace amounts of ARB102-MMAE were detected in the liver (Figure 6E), indicating that ARB102-MMAE has target specificity, can penetrate to the target tumor tissue and bind only to the target tumor tissue to exert cytotoxic effects, without infiltrating other organs and causing damage to other organs.

[0049] Example 3. Screening and optimization of anti-CDH17 antibodies against CDH17 ADC Internalization of CDH17 monoclonal antibodies in pancreatic AsPC1 cells In addition to ARB102 and ARB102-MMAE, the internalization of monoclonal antibodies targeting different extracellular regions of CDH17 in AsPC1 cells was also tested. As described in Example 2, antibodies were labeled with dyes, which showed enhanced fluorescence when they were internalized into cells. The development of fluorescence was tracked by flow cytometry. The results in Figure 7 show that monoclonal antibodies with different epitopes were internalized to different degrees, indicating that the internalization of CDH17 monoclonal antibodies may be affected by their epitopes on CDH17. These findings will provide a reference for the subsequent development of other ADCs targeting CDH17. References: 1. Cadherin-17 as diagnostic marker and therapeutic target for liver cancer. US9207242B2. 2. Composition of bispecific antibodies and method of use thereof. US20210115152A1. 3. Jendryczko K, Rzeszotko J, Szlachcic A. Drug conjugate via maleimide-thiol chemistry does not affect targeting properties of cysteine‑ containing anti‑FGFR1 peptibodies.Mol Pharmaceutics, 2022, 19, 1422‑33. 4. Lum YL, Luk JM, Staunton DE, Ng DK, Fong WP. Cadherin‑17 targeted near‑infrared photoimmunotherapy for treatment of gastrointestinal cancer. Mol Pharmaceutics, 2020, 17, 3941-51. 5.Wang Y, Shek FH, Wong KF, et al.al. Anti-cadherin-17 antibody modulates beta-catenin signaling and tumorigenicity of hepatocellular carcinoma. PLoS One. 2013, 8(9):e72386. 6. Lee NP, Poon RT, Shek FH, Ng IO, Luk JM. Role of cadherin-17 in oncogenesis and potential therapeutic implications in hepatocellular carcinoma. Biochim Biophys Acta. 2010, 1806(2):138-45. 7. Liu LX, Lee NP, Chan VW, et al. Targeting cadherin-17 inactivates Wnt signaling and inhibits tumor growth in liver carcinoma. Hepatology. 2009, 50(5):1453-63. Sequence Listing >Seq ID 1: ARB102 (h10C12) VH EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQTPGKGLEWVAVIDSNGGSTYYPDTVKDRFT ISRDNSKNTLYLQMNSLRAEDTAVYYCSSYTNLGAYWGQGTLVTVSAAST >Seq ID 2: ARB102 (h10C12) VL DIQMTQSPSSLSASVGDRVTITCRASQDISGYLNWLQQKPGGAIKRLIYTTSTLDSGVPKRFSGSGSGT DFTLTISSLQSEDFATYYCLQYASSPFTFGGGTKVEIK >Seq ID 3: mLic3 VH Description of the Manual Page 8 / 9 11 CN 121941709 A EVQLVESGGGLVKPGGSLKLSCAASGFSFSDYYMYWVRQAPEKRLEWVASISFDGTYTYYTDRVKGRFT ISRDNAKNNLYLQMSSLKSEDTAMYYCARDRPAWFPYWGQGTLVTVSA >Seq ID 4: mLic3 VLDVLMTQIPLSLTVSLGDQASISCRSSQSIVHSNGNTYLEWYLQRPGQSPKLLIYKVSNRFSGVPDRFSG SGSGTDFTLKISRVEAEDLGVYYCFQGSHVPLTFGAGTKLELK >Seq ID 5: m5F6 VH QIQLQQSGPELVKPGASVKISCKASGYTFTDYYLSWVKQRPGQGLEWIGWIYPGTGNTKYNEKLKDKAT LTVDTYSSTAYMHLRSLTSEDSAVYFCARSLGLRDCWGQGTTLTVSS >Seq ID 6: m5F6 VL DIVLTQSHKFMSTSVGDRVPITCKASQDVGTAVAWYQQKPGQSPKLLIYWASTRHTGVPDRFTGSGSGS DFTLTISNVQSDDLADYFCQQYSHYPYTFGGGTKLEIK >Seq ID 7: m7C5 VH QVQLQQSGAELARPGASVKLSCKASGYTFTSYGLSWVKQRTGQGLEWIGEIFPRSGNSYYNEKFKGKAA LTADKSSSTAYMQLSSLTSEDSAVYFCARHYYSSLYYAMDYWGQGTSVTVS >Seq ID 8: m7C5 VL DIQVTQSPASLSASVGESVSITCGTNENLYGALNWYQRKQGKSPQLLIYGATNLADGMSSRFSGSGSGR QYSLKISSLHPDDVATYYCQNVLSTPRTFGGGTKLEIK >Seq ID 9: m9A6 VH EVKLQESGPELVKPGASVTISCKASGYTFTDYYINWVKQRPGQGLEWIGWLFPGSGTTYYNEKFKGKAT LTVAKSSSTAYMLLSSLTSEDSAVYFCARWGFGNYAFAYWGQGTLVTVSA >Seq ID 10: m9A6 VL DIVLTQSQKFMSATVGDRVSITCKASQNVGTAVAWYQQKPGQSPKLLIYSPSSRNTGVPDRFTGSGSGT DFTLTISSVQSEDLADYFCQQYSTYPRTFGGGTKLEIK Specification 9 / 9 pages 12 CN 121941709 A Figure 1 Figure 2A Specification attached drawing 1 / 14 pages 13 CN 121941709 A Figure 2B Figure 2C DescriptionFigure 2 / 14, page 14, CN 121941709 A, Figure 2D, Figure 2E; Figure 3 / 14, page 15, CN 121941709 A, Figure 2F, Figure 2G; Figure 4 / 14, page 16, CN 121941709 A, Figure 2H, Figure 2I; Figure 5 / 14, page 17, CN 121941709 A, Figure 3, Figure 4A; Figure 6 / 14, page 18, CN 121941709 A, Figure 4B, Figure 4C; Figure 7 / 14, page 19, CN 121941709 A, Figure 4D, Figure 4E; Figure 8 / 14, page 20, CN 121941709 A, Figure 4F, Figure 4G; Figure 9 / 14, page 21, CN 121941709 A, Figure 4H, Figure 4I; Figure 10 / 14, page 22, CN 121941709 A Figure 5A Figure 5B Appendix to the Instruction Manual, Page 11 / 14, 23 CN 121941709 A Figure 6A Figure 6B Appendix to the Instruction Manual, Page 12 / 14, 24 CN 121941709 A Figure 6C Figure 6D Appendix to the Instruction Manual, Page 13 / 14, 25 CN 121941709 A Figure 6E Figure 7 Appendix to the Instruction Manual, Page 14 / 14, 26 CN 121941709 A

Claims

1. An antibody-drug conjugate (ADC) comprising: Monoclonal antibodies (mAbs), and Cytotoxins coupled to the mAb via a linker The mAb comprises a heavy chain variable (VH) domain and a light chain variable (VL) domain, wherein the VH domain and the VL domain form a Fab region having a binding affinity for human cadherin-17 (CDH17).

2. The antibody-drug conjugate according to claim 1, wherein the VH domain comprises an amino acid sequence having at least 98% sequence identity with SEQ ID NO: 1, 3, 5, 7 or 9, or wherein the VL domain comprises an amino acid sequence having at least 98% sequence identity with SEQ ID NO: 2, 4, 6, 8 or 10.

3. The antibody-drug conjugate according to claim 1, wherein the VH domain comprises three complementarity-determining regions (CDRs) of SEQ ID NO: 1, 3, 5, 7 or 9.

4. The antibody-drug conjugate according to claim 1, wherein the VL domain comprises three CDRs of SEQ ID NO: 2, 4, 6, 8 or 10.

5. The antibody-drug conjugate according to claim 1, wherein the cytotoxic agent is an antimicrotubule agent, a topoisomerase inhibitor, an RNA polymerase II inhibitor, a photosensitizer, or a DNA alkylating agent.

6. The antibody-drug conjugate according to claim 5, wherein the cytotoxic agent comprises an antimicrotubule agent selected from maytansine, olrestatin and tubulolysin.

7. The antibody-drug conjugate of claim 6, wherein the oprestatin class comprises monomethyl oprestatin E (MMAE).

8. The antibody-drug conjugate of claim 1, wherein the linker comprises a maleimide hexanoyl (MC) group, a valine-citrulline p-aminobenzyl carbamate (VC-PAB) unit, or a combination thereof.

9. A pharmaceutical composition comprising the antibody-drug conjugate according to claim 1 and a pharmaceutically acceptable carrier.

10. A method of treating or preventing cancer in a subject, the method comprising administering to the subject a pharmaceutical composition comprising an effective amount of the antibody-drug conjugate according to claim 1.

11. The method of claim 10, further comprising co-administering an effective amount of a therapeutic agent, wherein the therapeutic agent comprises an antibody, a chemotherapeutic agent, an enzyme, or a combination thereof.

12. The method of claim 10, wherein the cancer comprises cancer expressing CDH17.

13. The method of claim 10, wherein the cancer includes esophageal cancer, lung cancer, breast cancer, gastric cancer, pancreatic cancer, liver cancer, bile duct cancer, colorectal cancer, ovarian cancer, cervical cancer, uterine cancer, kidney cancer, or prostate cancer.

14. A method for preparing the antibody-drug conjugate according to claim 1, comprising conjugating the mAb with the cytotoxin.

15. An isolated monoclonal antibody (mAb), wherein the mAb has binding affinity for human cadherin-17 (CDH17), wherein the mAb comprises: The heavy chain variable (VH) domain comprises the complementarity-determining region 1 (HCDR1) amino acid sequence of SEQ ID NO: 1, 3, 5, 7 or 9, the HCDR2 amino acid sequence of SEQ ID NO: 1, 3, 5, 7 or 9, and the HCDR3 amino acid sequence of SEQ ID NO: 1, 3, 5, 7 or 9. The light chain variable (VL) domain comprises the complementarity-determining region 1 (LCDR1) amino acid sequence of SEQ ID NO: 2, 4, 6, 8 or 10, the LCDR2 amino acid sequence of SEQ ID NO: 2, 4, 6, 8 or 10, and the LCDR3 amino acid sequence of SEQ ID NO: 2, 4, 6, 8 or 10.

16. The isolated mAb according to claim 15, wherein the VH domain comprises an amino acid sequence of SEQ ID NO: 1, 3, 5, 7 or 9.

17. The isolated mAb according to claim 15, wherein the VL domain comprises the amino acid sequence of SEQ ID NO: 2, 4, 6, 8 or 10.

18. A pharmaceutical composition comprising the isolated mAb according to claim 15 and a pharmaceutically acceptable carrier.

19. A composition comprising the isolated mAb or a derivative thereof as claimed in claim 15 and a cytotoxic agent or a derivative thereof.

20. The composition of claim 19, wherein the cytotoxic agent comprises MMAE or a derivative thereof.

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