Anti-CDH17 antibody-drug conjugate and method of making and using thereof

Abstract

The present application is related to a panel of antibody-drug conjugates (ADCs) in which cytotoxic tubulin inhibitor monomethyl auristatin E (MMAE) is conjugated onto monoclonal antibodies targeting cadherin-17 (CDH17), a tumor-associated antigen that is overexpressed in uterine cervical adenocarcinoma, mucinous carcinoma of the ovary, and gastrointestinal cancers including pancreatic, gastric, colorectal cancers, etc. These ADCs recognise different parts of CDH17, showing high-affinity binding to CDH17-expressing cells. All these properties allow the CDH17-targeting ADCs to demonstrate cytotoxicity specifically against CDH17-expressing cancer cells. The cytotoxic effect of MMAE would be highly localized in tumors, maximising the anti-tumor effect and simultaneously reducing the adverse effects of anti-cancer drug on non- cancerous normal tissues.

Description

ANTI-CDH17 ANTIBODY-DRUG CONJUGATE AND METHOD OF MAKING AND USING THEREOFCROSS REFERENCE TO RELATED APPLICATIONSThis application claims the benefit of the filing date of U.S. Provisional Application Ser. No. 63 / 538,238 filed September 13, 2023, under 35 U.S.C. 119(e), the entire disclosures of which are incorporated by reference herein.TECHNICAL FIELDThe present disclosure generally relates to the technical field of cancer immunotherapy, and more particularly to antibody-drug conjugates.BACKGROUNDGastrointestinal (GI) cancers such as the cancer of colorectal, gastric, pancreatic, esophageal, bile duct or liver are leading causes of morbidity and mortality worldwide. Colorectal carcinoma (CRC) alone represents approximately 10% of all cancer diagnosis and is the second leading cause of cancer deaths world-wide. In China, liver and stomach cancers are among the most lethal of malignancies and over half of the incidences diagnosed, causing >1.42 million deaths per year, which are believed attributable to the viral / bacterial endemic (Hepatitis B virus [HBV] and Helicobacter pylori infections), chemical intoxications, environmental pollutions and food contaminations. There are no effective therapies. New biomarkers and therapeutic targets are thus needed for potential drug development against these aggressive cancers. A proven molecular targeting agent that can eliminate or repress the growth of these cancers will have important clinical value and significant market impact. These tumors can be resected effectively by surgery if the diseases are diagnosed in early stages. Unfortunately, and very often, most of GI cancers are asymptomatic and detected at very advanced stages when presented in the clinic. Without effective treatment, these patients die shortly after the diagnosis or relapse after salvage therapies.Chemotherapeutics have been developed for cancer treatment, including GI cancer. Over the past few decades, despite their efficacy in some cancers, the clinical benefit of such therapeutics is limited by their unwanted adverse effects on normal tissues. Besides, many patients would acquire resistance to chemotherapeutics during treatment regimen. To overcome these challenges, antibody-drug conjugates (ADC) that can deliver chemotherapeutics to tumor are developed. This anti-body-guided delivery ensures the cytotoxic effect of chemotherapeutics be localized within the targeted tumor, reducing the adverse effects on normal tissues and simultaneously, minimizing the likelihood of drug resistance development.SUMMARYThe following summary is illustrative only and is not intended to be in any way limiting. In addition to the illustrative aspects, embodiments, and features described above, further aspects, embodiments, and features will become apparent by reference to the drawings and the following detailed description.Cadherin-17 (CDH17) is a prominent cancer biomarker characterized by its overexpression in both liver and stomach cancers but not normal tissues from healthy adults. Anti-CDH17 monoclonal antibody displays the growth inhibitory effect on liver and stomach tumour cells. CDH17 is highly expressed in gastrointestinal cancers, uterine cervical cancer, mucinous carcinoma of the ovary and metastatic cancers. The blockage of CDH17 expression and functions can markedly reduce lung metastasis of hepatocellular carcinoma (HCC).In one aspect, the application provides antibody-drug conjugates (ADCs). In one embodiment, this application discloses anti-CDH17 antibody-drug conjugates developed as target therapeutics for treating cancer patients with indication of CDH17 biomarker in tumour tissues and / or in serum samples.In one embodiment, the antibody-drug conjugate (ADC) includes a monoclonal antibody (mAb), and a cytotoxin conjugated to the monoclonal antibody through a linker. In one embodiment, the mAb has a binding affinity to 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 VL domain form a Fab region having a binding affinity to human Cadherin-17 (CDH17).In one embodiment, the VH domain comprises an amino acid sequence having at least 60%, 70% 80%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 1, 3, 5, 7, or 9. In one embodiment, the VH domain comprises 3 complementary determining regions (CDRs) of SEQ ID NO: 1, 3, 5, 7, or 9. In one embodiment, the VH domain comprises an amino acid sequence of SEQ ID NO: 1.In one embodiment, the VL domain comprises an amino acid sequence having at least 60%, 70% 80%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 2, 4, 6, 8, or 10. In one embodiment, the VL domain comprises 3 CDRs of SEQ ID NO: 2, 4, 6, 8, or 10. In one embodiment, the VL domain comprises an amino acid sequence of SEQ ID NO: 2.In one embodiment, the mAb comprises amino acid sequence of SEQ ID NO: 1 and 2.In one embodiment, the cytotoxin comprises an anti-microtubule agent, a topoisomerase inhibitor, an RNA polymerase II inhibitor, a photosensitizing agent, a DNA alkylating agent, or a combination thereof. In one embodiment, the cytotoxin comprises an anti-microtubule agent selected from the group consisting of maytansinoid, auristatin, tubulysin, and the derivative thereof. In one embodiment, the auristatin comprises monomethyl auristatin E (MMAE) or a derivative thereof.The linker may include maleimidocaproyl (MC) group, valine-citruline p-aminobenzylcarbamate (VC-PAB) unit, or a combination thereof.In another aspect, the application provides pharmaceutical compositions. In one embodiment, the pharmaceutical composition comprises the antibody-drug conjugates as disclosed herein and a pharmaceutically acceptable carrier.In another aspect, the application provides method for treating or preventing cancer in a subject. In one embodiment, the method comprises administering to the subject a pharmaceutical composition comprising the 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 chemotherapy agent, an enzyme, or a combination thereof.In one embodiment, the cancer comprises a CDH17-expressing cancer. In one embodiment, the cancer comprises esophageal, lung, breast, gastric, pancreatic, liver, bile duct, colorectal, ovarian, cervical, uterine, renal, or prostate cancer.In a further aspect, the application comprises methods for making the antibody-drug conjugate as disclosed herein. In one embodiment, the method comprises conjugating the mAb with the cytotoxin.In a further aspect, the application provides isolated monoclonal antibodies (mAbs). In one embodiment, the mAb has a binding affinity to human Cadherin-17 (CDH17). In one embodiment, mAb comprises a heavy chain variable (VH) domain comprising a complementarity determining region 1 (HCDR1) amino acid sequence of SEQ ID NO: 1, 3, 5, 7, or 9, an HCDR2 amino acid sequence of SEQ ID NO: 1, 3, 5, 7, or 9, and an HCDR3 amino acid sequence of SEQ ID NO: 1, 3, 5, 7, or 9. In one embodiment, mAb comprises a light chain variable (VL) domain comprising a complementarity determining region 1 (LCDR1) amino acid sequence of SEQ ID NO: 2, 4, 6, 8, or 10, an LCDR2 amino acid sequence of SEQ ID NO: 2, 4, 6, 8, or 10, and an LCDR3 amino acid sequence of SEQ ID NO: 2, 4, 6, 8, or 10.In one embodiment, the isolated mAb comprises the VH domain having the amino acid sequence of SEQ ID NO: 1, 3, 5, 7, or 9. In one embodiment, the isolated mAb comprises the VL domain having the amino acid sequence of SEQ ID NO: 2, 4, 6, 8, or 10.In one embodiment, the isolated mAb comprises the VH domain having the amino acid sequence of SEQ ID NO: 1, 3, 5, 7, or 9, and the VL domain having the amino acid sequence of SEQ ID NO: 2, 4, 6, 8, or 10.In a further aspect, the application provides pharmaceutical compositions. In one embodiment, the pharmaceutical composition comprises the isolated mAb as disclosed thereof and a pharmaceutically acceptable carrier. In one embodiment, the composition includes the isolated mAb as disclosed herein or its derivative and a cytotoxic agent or its derivative thereof.In one embodiment, the cytotoxic agent comprises MMAE or its derivative thereof.BRIEF DESCRIPTION OF THE DRAWINGSThe foregoing and other features of this disclosure will become more fully apparent from the following description and appended claims, taken in conjunction with the accompanying drawings. Understanding that these drawings depict only several embodiments arranged in accordance with the disclosure and are, therefore, not to be considered limiting of its scope, the disclosure will be described with additional specificity and detail through use of the accompanying drawings, in which:Figure 1 shows the binding of ARB102-MMAE to CDH17 protein using enzyme-linked immunosorbent assay;Figure 2 shows the binding of ARB102-MMAE to multiple cancer cells using flow cytometry, including AsPC1 cells (2A), MHCC97H cells (2B), NCI-N87-CDH17+ cells (2C), DLD1 cells (2D), T84 cells (2E), AGS-CDH17+ cells (2F), AGS-CDH17- cells (2G), SW480-CDH17+ cells (2H) and SW480-CDH17- (2I) cells, indicating ARB102-MMAE bound to cancer cells expressing CDH17 with affinity in nanomolar range;Figure 3 shows the internalization of ARB102-MMAE into multiple CDH17-expressing cancer cells using flow cytometry;Figure 4 depicts the cytotoxic effect of ARB102-MMAE towards 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 the receptor occupancy of AsPC1 cells (4F), NCI-N87-CDH17+ cells (G) and AGS-CDH17+ cells (H), showing ARB102-MMAE could kill 40% of cancer cells at less than 10% receptor occupancy on cancer cells (4I) Maximum killing and internalization of ARB102-MMAE were studied in 13 cancer cell lines;Figure 5 depicts the ex vivo stability of ARB102-MMAE in human plasma, showing the binding activity of ARB102-MMAE to CDH17 protein as measured by using ELISA, e.g., residual binding > 60% of ARB102-MMAE, after being incubated in human plasma at 37°C for 21 days (5A); and the level of intact ARB102-MMAE in human plasma over 21-day incubation (5B);Figure 6 depicts the anti-tumor efficacy, in vivo stability, biodistribution of ARB102-MMAE in a pancreatic xenograft model in mice, showing (6A) treatment with ARB102-MMAE resulted in reduction of tumor volume (mm³) in mice; (6B) ARB102-MMAE treatment was not associated with significant changes in body weight in mice; (6C) ARB102-MMAE but not unconjugated MMAE could be detected from plasma even 6 days after injection; and (6D and 6E) a significant amount of ARB102-MMAE was detected only in subcutaneous tumors but not in livers.DETAILED DESCRIPTIONIn the following detailed description, reference is made to the accompanying drawings, which form a part hereof. In the drawings, similar symbols typically identify similar components, unless context dictates otherwise. The illustrative embodiments described in the detailed description, drawings, and claims are not meant 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 will be readily understood that the aspects of the present disclosure, as generally described herein, and illustrated in the Figures, can be arranged, substituted, combined, separated, and designed in a wide variety of different configurations, all of which are explicitly contemplated herein.To enable immunotherapeutics for more effective cancer treatment, especially solid tumors, a combination therapeutic is paramount that incorporates multiple target specificities and / or mechanisms of action beyond that of typical bispecific antibodies. A therapeutic that is essentially a combination treatment such as that described here, is necessary to effectively treat cancer and to more frequently achieve a complete and durable response. Specifically, there is a need for a scaffold that possesses certain properties to create a combination therapeutic with advantageous mechanisms of action, manufacturing, pharmacokinetics, and low antigenic properties relative to approved bispecific antibodies. Many bispecific antibodies that are based on a whole antibody may have a greater mass relative to the tri-specific antibodies described here. While antibodies with a smaller mass, based on antibody fragments, may have greater tumor penetration, they generally possess relatively poor pharmacokinetic properties, such as FDA-approved bispecific antibody BLINCYTO® that does not possess an Fc region. In addition, many bispecific antibodies are based on knob-into-hole possess mutations within the constant domains of the Ig structure, which may contribute to an anti-drug antibody (ADA) response. In this context, a group of modified antibodies described here as Tri-axial or TriAx antibodies, do not require mutations in any constant domain yet have multiple antigen binding specificities.The terms "a", "an" and "the" as used herein are defined to mean "one or more" and include the plural unless the context is inappropriate.The term "antibody" is used in the broadest sense and specifically covers single monoclonal antibodies (including agonist and antagonist antibodies), antibody compositions with polyepitopic specificity, as well as antibody fragments, such as Fab, F(ab')2, and Fv, so long as they exhibit the desired biological activity. In some embodiments, the antibody may be monoclonal, chimeric, single chain, multi-specific, multi-effective, human and humanized antibodies. Examples of active antibody fragments that bind to known antigens include Fab, F(ab')2, scFv, and Fv fragments, as well as the products of a Fab immunoglobulin expression library and epitope-binding fragments of any of the antibodies and fragments mentioned above. In some embodiments, antibody may include immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e. molecules that contain a binding site that immunospecifically bind to an antigen. The immunoglobulin can be of any type (IgG, IgM, IgD, IgE, IgA and IgY) or class (IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclasses of immunoglobulin molecule. In one embodiment, the antibody may be a whole antibody and any antigen-binding fragment derived from the whole antibody. A typical antibody refers to heterotetrameric protein comprising typically of two heavy (H) chains and two light (L) chains. Each heavy chain is comprised of a heavy chain variable domain (abbreviated as VH) and a heavy chain constant domain. Each light chain moiety is comprised of a light chain moiety variable domain (abbreviated as VL) and a light chain moiety constant domain. The VH and VL regions can be further subdivided into domains of hypervariable complementarity determining regions (CDR), and more conserved regions called framework regions (FR). Each variable domain (either VH or VL) is typically composed of three CDRs and four FRs, arranged in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 from amino-terminus to carboxy-terminus. Within the variable regions of the heavy and light chain there are binding regions that interacts with the antigen.The term "multi-specific" antibody as used herein denotes an antibody that has at least two binding sites each having a binding affinity to an epitope of an antigen. The term "bi-specific, tri-specific, tetra-specific, or penta-specific" antibody as used herein denotes an antibody that has two, three, four, five, or six antigen-binding sites.The term "humanized antibody" antibody refers to a type of engineered antibody having its CDRs derived from a non-human donor immunoglobulin, the remaining immunoglobulin-derived parts of the molecule being derived from one (or more) human immunoglobulin(s). In addition, framework support residues may be altered to preserve binding affinity. Methods to obtain "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, the "humanized antibody" may be obtained by genetic engineering approach that enables production of affinity-matured humanlike polyclonal antibodies in large animals such as, for example, rabbits (see U.S. Pat. No. 7,129,084).The term "antigen" refers to an entity or fragment thereof which can induce an immune response in an organism, particularly an animal, more particularly a mammal including a human. The term includes immunogens and regions thereof responsible for antigenicity or antigenic determinants.The term "epitope", also known as "antigenic determinant", is the part of an antigen that is recognized by the immune system, specifically by antibodies, B cells, or T cells, and is the specific piece of the antigen to which an antibody binds.The term "immunogenic" refers to substances which elicit or enhance the production of antibodies, T-cells, or other reactive immune cells directed against an immunogenic agent and contribute to an immune response in humans or animals. An immune response occurs when an individual produces sufficient antibodies, T-cells, and other reactive immune cells against administered immunogenic compositions of the present application to moderate or alleviate the disorder to be treated.The term "tumor antigen" as used herein means an antigenic molecule produced in tumor cells. A tumor antigen may trigger an immune response in the host. In one embodiment, the tumor cells express tumor antigens, including without limitation, tumor-specific antigens (TSA), neoantigens, and tumor-associated antigens (TAA).The term "specific binding to" or "specifically binds to" or "specific for" a particular antigen or an epitope as used herein means the binding that is measurably different from a non-specific interaction. Specific binding can be measured by determining binding of a molecule compared to binding of a control molecule, which generally is a molecule of similar structure that does not have binding activity. Specific binding can be determined by competition with a control molecule that is similar to the target. Specific binding for a particular antigen or an epitope can be exhibited by an antibody having a KD for an antigen or epitope 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, alternatively at least about 10^-10 M, at least about 10^-11 M, at least about 10^-12 M, or greater, where KD refers to a dissociation rate of a particular antibody-antigen interaction. In some embodiments, a multi-specific antibody that specifically binds to an antigen will have a KD that is 20-, 50-, 100-, 500-, 1000-, 5,000-, 10,000- or more times greater for a control molecule relative to the antigen or epitope. Also, specific binding for a particular antigen or an epitope can be exhibited by an antibody having a KA or Ka for an antigen or epitope of at least 20-, 50-, 100-, 500-, 1000-, 5,000-, 10,000- or more times greater for the epitope relative to a control, where KA or Ka refers to an association rate of a particular antibody-antigen interaction.The term "antibody drug conjugate" as used herein refers to the linkage of an antibody or an antigen binding fragment thereof with another agent, such as a chemotherapeutic agent, a toxin, an immunotherapeutic agent, an imaging probe, and the like. The linkage can be covalent bonds, or non-covalent interactions such as through electrostatic forces. Various linkers, known in the art, can be employed to form the immunoconjugate.The term "subject" includes human and non-human animals. Non-human animals include all vertebrates, e.g., mammals and non-mammals, such as non-human primates, sheep, dog, cow, chickens, amphibians, and reptiles. Except when noted, the terms "patient" or "subject" are used herein interchangeably.The term "cytotoxin", or "cytotoxic agent" as used herein, refers to any agent that is detrimental to the growth and proliferation of cells and may act to reduce, inhibit, or destroy a cell or malignancy.The term "anti-cancer agent" as used herein refers to any agent that can be used to treat a cell proliferative disorder such as cancer, including but not limited to, cytotoxic agents, chemotherapeutic agents, radiotherapy and radiotherapeutic agents, targeted anti-cancer agents, and immunotherapeutic agents.The term "drug moiety" or "payload" as used herein refers to a chemical moiety that is conjugated to an antibody or antigen binding fragment of the application, and can include any therapeutic or diagnostic agent, for example, an anti-cancer, anti-inflammatory, anti-infective (e.g., anti-fungal, antibacterial, anti-parasitic, anti-viral), or an anesthetic agent. For example, the drug moiety can be an anti-cancer agent, such as a cytotoxin. In certain embodiments, a drug moiety is selected from a V-ATPase inhibitor, a HSP90 inhibitor, an IAP inhibitor, an mTor inhibitor, a microtubule stabilizer, a microtubule destabilizer, an auristatin, a dolastatin, a maytansinoid, a MetAP (methionine aminopeptidase), an inhibitor of nuclear export of proteins CRM1, a DPPIV inhibitor, an inhibitor of phosphoryl transfer reactions in mitochondria, a protein synthesis inhibitor, a kinase inhibitor, a CDK2 inhibitor, a CDK9 inhibitor, a proteasome inhibitor, a kinesin inhibitor, an HDAC inhibitor, a DNA damaging agent, a DNA alkylating agent, a DNA intercalator, a DNA minor groove binder and a DHFR inhibitor. Methods for attaching each of these to a linker compatible with the antibodies and method of the application are known in the art. In addition, a payload can be a biophysical probe, a fluorophore, a spin label, an infrared probe, an affinity probe, a chelator, a spectroscopic probe, a radioactive probe, a lipid molecule, a polyethylene glycol, a polymer, a spin label, DNA, RNA, a protein, a peptide, a surface, an antibody, an antibody fragment, a nanoparticle, a quantum dot, a liposome, a PLGA particle, a saccharide or a polysaccharide.Several short peptidic compounds have been isolated from the marine mollusc Dolabella auricularia and found to have biological activity. Analogs of these compounds have also been prepared, and some were found to have biological activity (see Pettit et al. Anti-Cancer Drug Design 13:243-277, 1998). For example, auristatin E is a synthetic analogue of the marine natural product Dolastatin 10, an agent that inhibits tubulin polymerization by binding to the same domain on tubulin as the anticancer drug vincristine (US 5,635,483). Dolastatin 10, auristatin PE, and auristatin E are linear peptides having four amino acids, three of which are unique to the dolastatin class of compounds, and a C-terminal amide. The auristatin peptides, auristain E (AE) and monomethylauristatin (MMAE) are synthetic analogs of dolastatin. MMAE is an antimitotic agent which inhibits cell division by blocking the polymerisation of tubulin. The linker to the monoclonal antibody is stable in extracellular fluid but is cleaved by cathepsin once the conjugate has entered a tumor cell, thus activating the antimitotic mechanism. MMAE is one of the most commonly used payloads to make ADCs, including anti-CD20 ADC, RITUXAN® (WO 2004 / 032828) for the treatment of CD20-expressing cancers and immune disorders, ADCETRIS® (brentuximab vedotin), PADCEV® (enfortumab vedotin-ejfv), and POLIVY® (polatuzumab vedotin-piiq).EXAMPLESThe present disclosure is further described with reference to the following examples. These examples are provided for purposes of illustration only and are not intended to be limiting unless otherwise specified. Those of skill in the art will readily recognize a variety of non-critical parameters that could be changed or modified to yield essentially the same or similar results.Example 1. Generation and production of CDH17-targeting ADCHumanized anti-CDH17 monoclonal antibody ARB102 (US20210115152) was recombinantly expressed and the cytotoxic tubulin inhibitor monomethyl auristatin E (MMAE) was used as the payload for producing ADC. The resulting ADC ARB102-MMAE was then produced by conjugating mc-vc-PAB-MMAE to cysteine residues on ARB102 via dipeptide valine-citrulline linker. In brief, the antibody was first dialysed at 4°C against conjugation buffer (25 mM Na2B4O7, 25 mM NaCl, 1 mM DPTA, pH 7.4). The dialysed antibody was then mixed with mc-vc-PAB-MMAE solution (MedChemExpress) at desired molar ratio and was allowed to incubate at 25°C. The conjugation product was dialyzed against phosphate-buffered saline (pH 7.4) at 4°C for more than 4 hours. The resulting ADC was analysed using SEC-HPLC and LC-MS. Drug conjugate via maleimide-thiol chemistry does not affect targeting properties of cysteine-containing monoclonal antibodies. This linker has been utilized in many FDA-approved ADCs and the mechanism of action is expected to be the same, i.e., the linker would be recognized by cathepsin B, which cleaves the citrulline-PAB amide bond. MMAE is then released for its anti-cancer effects. This conjugation achieved an antibody-drug ratio of about 4 (DAR).Example 2. Characterization of CDH17-targeting ADCBinding of ARB102-MMAE to CDH17CDH17 enzyme-linked immunosorbent assay (ELISA) was used to examine the binding of ARB102-MMAE to CDH17 (Figure 1). Recombinant CDH17 protein (5 ug / ml) was coated on immunoassay plates, and was allowed to incubate with ARB102 (i.e., the parental antibody) and ARB102-MMAE. The bound antibodies were detected using anti-human-IgG-HRP. The signal was developed from chemiluminescent substrate and was measured as absorbance at optical density of 450 nm. Results showed that ARB102-MMAE displayed CDH17 binding that was comparable to ARB102, suggesting that the conjugation would not affect the binding properties of ARB102. Like the parental antibody ARB102, ARB102-MMAE showed high-affinity to CDH17 with potency in sub-nanomolar range. Result suggested that the conjugation of MMAE did not affect the binding of antibody to CDH17.Binding of ARB102-MMAE to cancer cellsThe binding of ARB102-MMAE to cancer cells was illustrated using flow cytometry (Figure 2). The cancer cells expressing CDH17 including AsPC1 pancreas adenocarcinoma cells (Figure 2A), hepatocellular carcinoma MHCC97H cells (Figure 2B), gastric adenocarcinoma NCI-N87-CDH17 cells (Figure 2C), colorectal adenocarcinoma DLD1 cells (Figure 2D) and colonic adenocarcinoma T84 cells (Figure 2E) were studied. ARB102-MMAE showed binding to the all CDH17-positive cell lines in single digit nanomolar range. Isogenic pair of gastric AGS cells (Figure 2F and 2G) and colorectal carcinoma SW480 cells (Figure 2H and 2I) with and without CDH17 expression were also tested to demonstrate the CDH17-binding specificity of ARB102-MMAE.Internalization of ARB102-MMAE into CDH17-positive cancer cellsEfficient internalization into the cells is essential to the cytotoxic function of ADC. The internalization of ARB102-MMAE into multiple cancer cells was investigated using flow cytometry (Figure 3). ARB102-MMAE was labelled with a dye that would show an increase in fluorescence as they are internalized into cells. The development of fluorescence was followed by flow cytometry. ARB102-MMAE was shown to be internalized into pancreatic AsPC1 cells, colorectal SW480-CDH17+ cells, gastric AGS-CDH17+ cells, hepatic MHCC97H cells, stomach SNU1-CDH17+ cells, gastric NCI-N87-CDH17+ cells and colonic T84 cells. No significant internalizations into lung A549 cells, colorectal DLD1 cells and Colo205 cells were seen.Cytotoxic effect of ARB102-MMAE on CDH17-positive cancer cellsThe cytotoxic effect of ARB102-MMAE was examined in vitro (Figure 4). Pancreatic AsPC1 cell expresses CDH17. Treatment of this cell line with ARB102-MMAE resulted in dose-dependent cytotoxicity (Figure 4A). ARB102 alone did not cause any cytotoxicity, suggesting the internalization of MMAE would be essential to the observed cytotoxicity. The selective killing of ARB102-MMAE on CDH17-positive cancer cell was examined in an isogenic pair of gastric NCI-N87 cells (Figure 4B and 4C) and AGS cells (Figure 4D and 4E) with and without CDH17 overexpression. ARB102-MMAE killed CDH17-positive but not CDH17-negative NCI-N87 cells, implicating the ADC would offer selectivity against CDH17-positive tumors. The receptor occupancy of AsPC1 cells (Figure 4F), NCI-N87-CDH17+ cells (Figure 4G) and AGS-CDH17+ cells (Figure 4H) for 40% killing were calculated to be 0.4%, 1.8% and 8.3% respectively, suggesting a low receptor occupancy was required for ARB102-MMAE to perform cytotoxic effect towards cancer cells. In addition to AsPC1, NCI-N87 and AGS cells, the killing and internalization of ARB102-MMAE were studied in a panel of gastrointestinal cancer cells (Figure 4I). These results indicate that the maximal killing of ARB102-MMAE on cancer cells positively correlated with the percentage of internalization.Ex-vivo stability of ARB102-MMAE in human blood plasmaStability of ADC is important for its cytotoxic function inside human body. ARB102-MMAE was spiked into human blood plasma and incubated at 37°C for up to 3 weeks (Figure 5). The Ex-vivo stability was determined by measuring the binding activity (Figure 5A) and the concentration of the intact ARB102-MMAE concentration (Figure 5B). ARB102-MMAE demonstrated favourable stability with half-life of its activity around 21 days and that of its concentration around 60 days.Anti-tumor efficacy, in vivo stability and biodistribution of ARB102-MMAE in a pancreatic xenograft model in mice.The anti-tumor efficacy of ARB102-MMAE was examined in mice bearing pancreatic cancer xenograft. NOD-SCID mice were inoculated with pancreatic AsPC1 cancer cells subcutaneously, and when tumors became palpable, mice were intravenously injected with ARB102-MMAE (3 mg / kg), equimolar of MMAE or sterile PBS once a week for 4 weeks. Treatment with ARB102-MMAE resulted in a complete tumor growth inhibition (Figure 6A). Significant changes in body changes associated with ARB102-MMAE were not seen (Figure 6B). Blood plasma, tumor tissue and liver tissue were collected from the mice at the day before the 4th dose, 2 days and 6 days after the 4th dose to monitor the level and distribution of ARB102-MMAE (Figure 6C - 6E). The ARB102-MMAE level in plasma (Figure 6C) accelerated after the 4th dose and started to drop after 2 days and remained in blood circulation in a detectable level after 6 days. Similar pattern was observed in tumor tissue (Figure 6D), but not in liver tissue as only trace amount of ARB102-MMAE was detected in liver (Figure 6E), showing ARB102-MMAE was target specific and was capable to penetrate to and engaged only with target tumor tissue to exert cytotoxic effect without infiltrating into and causing harm to other organs.Example 3. Selecting and optimizing anti-CDH17 antibodies for CDH17-targeting ADCInternalization of CDH17 monoclonal antibodies into pancreatic AsPC1In addition to ARB102 and ARB102-MMAE, monoclonal antibodies targeting different extracellular regions of CDH17 were also tested for their internalization into AsPC1. As described in Example 2, antibodies were labelled with a dye that would show an increase in fluorescence as they are internalized into cells. The development of fluorescence was followed by flow cytometry. The results in Figure 7 showed that monoclonal antibodies with different epitopes would internalize to different extents, indicating that the internalization of CDH17 monoclonal antibodies may be affected by their epitopes on CDH17. These findings would shed light into the future development of other CDH17-targeting ADCs.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. 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) VHEVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQTPGKGLEWVAVIDSNGGSTYYPDTVKDRFTISRDNSKNTLYLQMNSLRAEDTAVYYCSSYTNLGAYWGQGTLVTVSAAST>Seq ID 2: ARB102 (h10C12) VLDIQMTQSPSSLSASVGDRVTITCRASQDISGYLNWLQQKPGGAIKRLIYTTSTLDSGVPKRFSGSGSGTDFTLTISSLQSEDFATYYCLQYASSPFTFGGGTKVEIK>Seq ID 3: mLic3 VHEVQLVESGGGLVKPGGSLKLSCAASGFSFSDYYMYWVRQAPEKRLEWVASISFDGTYTYYTDRVKGRFTISRDNAKNNLYLQMSSLKSEDTAMYYCARDRPAWFPYWGQGTLVTVSA>Seq ID 4: mLic3 VLDVLMTQIPLSLTVSLGDQASISCRSSQSIVHSNGNTYLEWYLQRPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHVPLTFGAGTKLELK>Seq ID 5: m5F6 VHQIQLQQSGPELVKPGASVKISCKASGYTFTDYYLSWVKQRPGQGLEWIGWIYPGTGNTKYNEKLKDKATLTVDTYSSTAYMHLRSLTSEDSAVYFCARSLGLRDCWGQGTTLTVSS>Seq ID 6: m5F6 VLDIVLTQSHKFMSTSVGDRVPITCKASQDVGTAVAWYQQKPGQSPKLLIYWASTRHTGVPDRFTGSGSGSDFTLTISNVQSDDLADYFCQQYSHYPYTFGGGTKLEIK>Seq ID 7: m7C5 VHQVQLQQSGAELARPGASVKLSCKASGYTFTSYGLSWVKQRTGQGLEWIGEIFPRSGNSYYNEKFKGKAALTADKSSSTAYMQLSSLTSEDSAVYFCARHYYSSLYYAMDYWGQGTSVTVS>Seq ID 8: m7C5 VLDIQVTQSPASLSASVGESVSITCGTNENLYGALNWYQRKQGKSPQLLIYGATNLADGMSSRFSGSGSGRQYSLKISSLHPDDVATYYCQNVLSTPRTFGGGTKLEIK>Seq ID 9: m9A6 VHEVKLQESGPELVKPGASVTISCKASGYTFTDYYINWVKQRPGQGLEWIGWLFPGSGTTYYNEKFKGKATLTVAKSSSTAYMLLSSLTSEDSAVYFCARWGFGNYAFAYWGQGTLVTVSA>Seq ID 10: m9A6 VLDIVLTQSQKFMSATVGDRVSITCKASQNVGTAVAWYQQKPGQSPKLLIYSPSSRNTGVPDRFTGSGSGTDFTLTISSVQSEDLADYFCQQYSTYPRTFGGGTKLEIK

Claims

1. An antibody-drug conjugate (ADC) comprisinga monoclonal antibody (mAb), anda cytotoxin conjugated to the mAb through a linker,wherein the mAb comprises a heavy chain variable (VH) domain and a light chain variable (VL) domain, and wherein the VH domain and VL domain form a Fab region having a binding affinity to 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% of sequence identity to SEQ ID NO: 1, 3, 5, 7, or 9, or wherein the VL domain comprises the amino acid sequence having at least 98% of sequence identity to SEQ ID NO: 2, 4, 6, 8, or 10.

3. The antibody-drug conjugate of claim 1, wherein the VH domain comprises 3 complementary 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 3 CDRs of SEQ ID NO: 2, 4, 6, 8, or 10.

5. The antibody-drug conjugate of claim 1, wherein the cytotoxin is an anti-microtubule agent, a topoisomerase inhibitor, an RNA polymerase II inhibitor, a photosensitizing agent, or a DNA alkylating agent.

6. The antibody-drug conjugate of claim 5, wherein the cytotoxin comprises an anti-microtubule agent selected from the group consisting of maytansinoid, auristatin, and tubulysin.

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

8. The antibody-drug conjugate of claim 1, wherein the linker comprises maleimidocaproyl (MC) group, valine-citruline p-aminobenzylcarbamate (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 for treating or preventing cancer in a subject, said 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 chemotherapy agent, an enzyme, or a combination thereof.

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

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

14. A method for making the antibody-drug conjugate of claim 1, comprising conjugating the mAb with the cytotoxin.

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

16. The isolated mAb of 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 of 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 thereof and a pharmaceutically acceptable carrier.

19. A composition, comprising the isolated mAb of claim 15 or its derivative and a cytotoxic agent or its derivative thereof.

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

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