Use of anti-tight connexin-1 antibodies to treat cholangiocarcinoma

Abstract

The present disclosure relates to a method of treating bile duct cancer in a human subject in need thereof, the method comprising administering to the human subject a therapeutically effective amount of an anti-tight junction protein-1 antibody.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of priority to U.S. Provisional Application No. 63 / 365,681, filed on June 1, 2022, which is hereby incorporated by reference in its entirety.

[0003] Reference to a sequence listing submitted electronically via EFS-WEB

[0004] The contents of the electronically submitted Sequence Listing (Name: 4872_020PC01_SequenceListing_ST26; Size: 20,121 bytes; and Creation Date: May 29, 2023) submitted with this application are incorporated herein by reference in its entirety. Technical Field

[0005] According to various aspects of the present disclosure, the present disclosure is directed to methods of treating cholangiocarcinoma (CCA). Background Art

[0006] Cholangiocarcinoma (CCA) is a highly aggressive adenocarcinoma of the hepatobiliary system (Brindley et al., 7 Nat Rev Dis Primers 1-17 (2021)). CCA originates from the biliary tree and / or liver parenchyma and is considered a heterogeneous malignancy that is divided into three major anatomical subtypes, namely intrahepatic CCA (iCCA), perihilar CCA (pCCA), or distal CCA (dCCA) (Banales et al., 17 Nat Rev Gastroenterol Hepatol 557-588 (2020)). CCA is the second most common primary liver malignancy after hepatocellular carcinoma (HCC), accounting for approximately 15% of all primary liver tumors and 2% of cancer-related deaths worldwide (Vaquero et al., 13 Nat Rev Gastroenterol Hepatol 261–280 (2016)). Although CCA is considered a rare cancer, the incidence of CCA has increased dramatically over the past decade (Bertuccio et al., 71(1) J Hepatol. 104-114 (July 2019)), with incidence ranging from 1 to more than 4 cases per 100,000 in European countries (Al Mahjoub et al., 31(6) Eur J Gastroenterol Hepatol 678-684 (June 2019)). This can be partially explained by the increasing trend of CCA risk factors worldwide, such as diabetes, primary sclerosing cholangitis, intrahepatic bile duct stones, cirrhosis, hepatitis B and hepatitis C (Clements et al., 72(1) J Hepatol. 95-103 (January 2020)). Specifically, chronic biliary inflammation and / or cholestasis have been identified as major drivers of CCA initiation, highlighting the important role played by the tumor microenvironment (TME), particularly cancer-associated fibroblasts (CAFs), tumor-associated macrophages (TAMs), proinflammatory cytokines and growth factors (Leyva-Illades et al., 1(1) Transl Gastrointest Cancer 71-80 (2012)). These extracellular players intersect with the dysregulation of several intracellular signaling pathways that are considered key drivers of cholangiocarcinogenesis, such as receptor tyrosine kinase (RTK) signaling, RAS–RAF–ERK, PI3K–AKT–mTOR, Notch, Hedgehog and Wnt signaling (Yang J et al., 21(5) Expert Opin Ther Targets 485-498 (May 2017)).

[0007] CCA is often diagnosed late, resulting in rapid disease progression, poor response to treatment, and a poor prognosis with a median survival of less than 2 years (Vaquero et al., 13 Nat Rev Gastroenterol Hepatol 261–280 (2016)). Although radical hepatectomy is a treatment option for the management of CCA, only 25% of patients are eligible due to metastatic or locally advanced tumors, and 50% of surgical patients achieve radical or margin-free resection (Nagorney et al., 40 Adv Surg 159-171 (2006)). First-line standard of care chemotherapy includes a combination of gemcitabine and cisplatin, in addition to recently approved targeted therapies such as the fibroblast growth factor receptor inhibitor pemigatinib and the isocitrate dehydrogenase 1 (IDH1) mutant inhibitor ivosidenib, which can improve patient outcomes but only respond in very few advanced and / or metastatic CCAs and have severe side effects and potential treatment resistance (Sasaki et al., 10(14) J Clin Med 3108(2021)).

[0008] There is increasing evidence that the expression of members of the tight junction protein family in CCA is dysregulated and contributes to carcinogenesis (Németh et al., 57 (2) J Histochem Cytochem 113-121 (February 2009)). Claudin-1 (CLDN1) is a transmembrane protein that is expressed in tight junctions (TJs), but is also expressed in a non-junctional form, such as at the basolateral membrane of human hepatocytes, where it acts as a cell entry factor for hepatitis C virus (Evans et al., 446 Nature 801-805 (2007)). CLDN1 was previously identified as a mediator and therapeutic target for liver fibrosis and hepatocellular carcinoma (HCC) (WO 20161 / 46809A1). Extensive studies in non-human primates and mouse models have shown that even repeated application of high doses of mAbs far exceeding those required for treatment did not produce any significant toxicity.

[0009] Due to the lack of highly effective drugs and the increasing incidence of the disease, there is a pressing unmet medical need for new first-line treatments to treat CCA and improve patient outcomes. Summary of the invention

[0010] The present disclosure provides a method of treating cholangiocarcinoma (CCA) in a human subject in need thereof, the method comprising administering to the human subject a therapeutically effective amount of an anti-Claudin-1 antibody.

[0011] In some aspects, provided herein is an anti-Claudin-1 antibody or a pharmaceutical composition thereof for use in a method of treating cholangiocarcinoma (CCA) in a human subject, the method comprising administering an effective amount of the anti-Claudin-1 antibody or a pharmaceutical composition thereof to the human subject.

[0012] In some aspects, provided herein is a kit for treating a subject having cholangiocarcinoma, the kit comprising a therapeutically effective amount of an anti-Claudin-1 antibody and instructions comprising instructions for use of the kit.

[0013] In some aspects, provided herein is a pharmaceutical composition for treating cholangiocarcinoma, the pharmaceutical composition comprising a therapeutically effective amount of an anti-Claudin-1 antibody.

[0014] In some aspects, claudin-1 (CLDN1) is overexpressed in a human subject compared to the expression level in a normal subject.

[0015] In some aspects, the human subject is also administered chemotherapy. In some aspects, the chemotherapy is gemcitabine. In some aspects, the chemotherapy is cisplatin.

[0016] In some aspects, the anti-Claudin-1 antibody comprises the six complementarity determining regions (CDRs) of the anti-Claudin-1 monoclonal antibody secreted by the hybridoma cell line deposited with the DSMZ on July 29, 2008 under accession number DSMACC2938.

[0017] In some aspects, the anti-Claudin-1 antibody comprises a heavy chain variable domain complementarity determining region (CDR) H1 comprising the amino acid sequence shown in SEQ ID NO:5, a CDR H2 comprising the amino acid sequence shown in SEQ ID NO:6, and a CDR H3 comprising the amino acid sequence shown in SEQ ID NO:7, and / or a light chain variable domain complementarity determining region (CDR) L1 comprising the amino acid sequence shown in SEQ ID NO:8, a CDR L2 comprising the amino acid sequence GAS, and a CDR L3 comprising the amino acid sequence shown in SEQ ID NO:10.

[0018] In some aspects, the anti-Claudin-1 antibody is humanized.

[0019] In some aspects, the anti-Claudin-1 antibody comprises a VH comprising the amino acid sequence shown in SEQ ID NO:3 or SEQ ID NO:13.

[0020] In some aspects, the anti-Claudin-1 antibody comprises a VL comprising the amino acid sequence set forth in SEQ ID NO:4 or SEQ ID NO:14.

[0021] In some aspects, the anti-Claudin-1 antibody comprises a VH comprising the amino acid sequence shown in SEQ ID NO:3; and a VL comprising the amino acid sequence shown in SEQ ID NO:4.

[0022] In some aspects, the anti-Claudin-1 antibody comprises a VH comprising the amino acid sequence shown in SEQ ID NO:13; and a VL comprising the amino acid sequence shown in SEQ ID NO:14.

[0023] In some aspects, the anti-Claudin-1 antibody is administered intratumorally, intravenously, intraperitoneally, intramuscularly, intrathecally, or subcutaneously.

[0024] In some aspects, the CCA is an intrahepatic CCA.

[0025] In some aspects, the CCA is a perihilar CCA.

[0026] In some aspects, the CCA is a distal CCA.

[0027] In some aspects, the CCA is mixed or mixed hepatocellular cholangiocarcinoma (cHCC-CCA).

[0028] In some aspects, CCA is metastatic.

[0029] In some aspects, CCA is treated with chemotherapy such as gemcitabine and cisplatin.

[0030] In some aspects, CCA contains mutations in the following genes: isocitrate dehydrogenase (NADP(+)) 1 (IDH1), isocitrate dehydrogenase (NADP(+)) 2 (IDH2), BRCA1-associated protein 1 (BAP1), fibroblast growth factor receptor 2 (FGFR2), Kirsten rat sarcoma viral oncogene homolog (KRAS), polybrominated 1 (PBRM1), AT-rich interaction domain 1A (ARID1A), phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA), Ephrin type A receptor 2 (EPHA2), cyclin-dependent kinase inhibitor 2A (CDKN2A), tumor protein P53 (TP53), SMAD family member 4 (SMAD4), transforming growth factor beta receptor 2 (TGFBR2).

[0031] In some aspects, the methods or uses provided herein further comprise administering a chemotherapeutic drug to a human subject in need thereof.

[0032] In some aspects, the chemotherapeutic agent is gemcitabine.

[0033] In some aspects, the chemotherapeutic agent is cisplatin. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figures 1A to 1C ( Figures 1A to 1C ) shows transcriptional analysis using a large number of RNA sequencing (RNA-seq) datasets deposited in the Genomic Data Sharing data portal, such as extrahepatic CLDN1 expression compared to non-tumor bile duct samples - GSE13205 ( Figure 1A ), CLDN1 expression in proliferative intrahepatic CCA compared with non-tumor bile duct samples - GSE32225 ( Figure 1B ), and CLDN1 expression in proliferative CCA compared with inflammatory CCA - GSE32225 ( Figure 1C ).

[0035] Figure 1D ( Figure 1D ) shows CLDN1 expression in CCA tumor tissues of patients with different genetic driver mutations based on a large number of RNA sequencing (RNA-seq) datasets deposited in the genomic data sharing data portal-GSE89747 and GSE89748. The corresponding CCA mutations are represented on the x-axis, and CLDN1 expression (signal intensity) is shown on the y-axis.

[0036] Figure 2A ( Figure 2A ) shows the experimental approach for an exemplary model of the cell line-derived xenograft (CDX) NRG mouse model.

[0037] FIG. 2B to FIG. 2D ( FIG. 2B to FIG. 2D ) shows tumor volume in EGI-1CDX model after treatment with anti-CLDN1 H3L3 antibody or control ( Figure 2B ) and skin ulcer grade ( Figure 2C to Figure 2D ).

[0038] FIG. 2E to FIG. 2F ( FIG. 2E to FIG. 2F ) shows tumor volume in HuCC-A1 CDX model after treatment with anti-CLDN1 H3L3 antibody or control ( FIG. 2E to FIG. 2F ).

[0039] Figure 2G ( Figure 2G ) shows tumor volume in HuCC-T1 CDX model after treatment with anti-CLDN1 H3L3 antibody or control.

[0040] Figure 2H ( Figure 2H ) shows tumor volume in PDX mouse model after treatment with anti-CLDN1 H3L3 antibody or control.

[0041] FIG. 3A to FIG. 3C ( FIG. 3A to FIG. 3C ) shows the effect of anti-CLDN1 H3L3 antibody on cell migration in wound healing assays of intrahepatic CCA cell lines (HuCC-A1) and extrahepatic CCA cell lines (KKU100). Figure 3A Shown are representative micrographs of cell monolayers of HuCC-A1 or KKU100 cells co-cultured with LX2 cells 24 h after wounding. Figure 3B Shown are the effects of anti-CLDN1 H3L3 and control antibody treatment on the wound closure assay in HuCC-A1 assay cells after 24 hours. Figure 3C Shown are the effects of CLDN1-specific (H3L3) and control antibody treatment on the KKU100 cell wound closure assay after 24 hours (complete wound closure = 100%). The scale bar indicates 330 μm.

[0042] Figures 3D to 3G ( Figures 3D to 3G ) shows the effect of anti-CLDN1H3L3 antibody on cell invasion of extrahepatic (EGI-1) and intrahepatic (HuCC-T1) CCA cell lines studied using a transwell cancer invasion assay. Figure 3D The experimental method of the transwell Matrigel invasion assay is shown. Figure 3E Representative images of crystal violet staining to visualize invasive EGI-1 or HuCC-T1 cells in a transwell chamber assay are shown. Figure 3F Shown are the effects of anti-CLDN1 H3L3 and control antibody treatment on Matrigel invasion in EGI-1 cell transwell assays. Figure 3G Shown are the effects of anti-CLDN1 H3L3 and control antibody treatment on Matrigel invasion in HuCC-T1 cell transwell assays. Scale bar indicates 330 μm.

[0043] Figure 4A ( Figure 4A ) shows RNA-seq analysis comparing the effects of treatment with anti-CLDN1 H3L3 antibody versus control treatment on HuCC-A1 tumor tissues from CDX mice.

[0044] Figure 4B ( Figure 4B ) shows Western blot analysis of key signaling pathways in EGI-1CDX tumor samples after treatment with anti-CLDN1H3L3 antibody compared to animals treated with control.

[0045] Figure 4C ( Figure 4C ) shows Figure 4BQuantitative analysis of western blots.

[0046] FIG. 4D to FIG. 4F ( FIG. 4D to FIG. 4F ) shows the effect of anti-CLDN1 H3L3 and isotype control antibody treatment on Notch1 in EGI-1 / LX2 and EGI-1 cells studied using Western blot analysis ( Figure 4D )、SRC( Figure 4E ) and FAK signaling ( Figure 4F ). Protein and molecular weight are indicated.

[0047] Figure 4G ( Figure 4G ) shows Figure 2A RNA-Seq analysis of HuCC-A1 tumor tissue derived from the CDX model shown in Figure 1. Comparative analysis of tumor tissue from mice treated with anti-CLDN1 H3L3 antibody versus control treated mice is shown, demonstrating FIG. 4D to FIG. 4F Differential expression of downstream targets of Notch1, SRC and FAK signaling pathways as shown in .

[0048] Figure 5A ( Figure 5A ) shows the experimental approach for the CCA metastasis model using intravenous retro-orbital HuCC-T1 cell injection, followed by analysis of lung metastases in anti-CLDN1 H3L3 mAb versus control treated mice.

[0049] FIG. 5B to FIG. 5C ( FIG. 5B to FIG. 5C ) shows immunohistochemistry and quantification of CCA HuCC-T1 cell metastasis in lung sections visualized and detected by CK18 staining in mice treated with anti-CLDN1 H3L3 antibody or control.

[0050] Fig. 6A ( Fig. 6A ) shows flow cytometric analysis of CLDN1 expression using CLDN1-specific H3L3 mAb in EGI-1 cells after treatment with cisplatin and gemcitabine.

[0051] Figure 6B ( Figure 6B ) shows the corresponding Fig. 6A Delta mean fluorescence intensity (ΔMFI) fold change of flow cytometric analysis of CLDN1 expression detected by the CLDN1-specific mAb H3L3 is shown in FIG.

[0052] Figure 6C ( Figure 6C ) shows Western blot analysis of CLDN1 expression in EGI-1 cholangiocarcinoma cells after treatment with cisplatin and gemcitabine.

[0053] Fig.6D ( Fig.6D ) shows Figure 6C Quantification of proteins shown in Western blots.

[0054] FIG. 7A to FIG. 7B ( FIG. 7A to FIG. 7B ) shows the body weight of mice treated with anti-CLDN1 H1L1 mAb and control ( Fig. 7A ) and tumor volume ( Figure 7B ). DETAILED DESCRIPTION

[0055] I. Definitions

[0056] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those of ordinary skill in the art to which the present disclosure belongs. In the event of a conflict, the present application (including definitions) shall prevail. In addition, unless the context otherwise requires, singular terms shall include plural terms and plural terms shall include singular terms. All publications, patents and other references mentioned herein are incorporated by reference in their entirety for all purposes, as if each individual publication or patent application was clearly and individually indicated to be incorporated by reference.

[0057] Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described below. These materials, methods and examples are illustrative only and are not intended to be limiting. Other features and advantages of the present disclosure will be apparent from the detailed description and claims.

[0058] To further define the present disclosure, the following terms and definitions are provided.

[0059] Unless the context clearly indicates otherwise, the singular forms "a", "an", and "the" include plural referents. The term "a" (or "an") and the terms "one or more" and "at least one" are used interchangeably herein. In some aspects, the term "a" or "an" refers to "single". In other aspects, the term "a" or "an" includes "two or more" or "plurality".

[0060] The term "about" is used herein to mean approximately, roughly, around, or in the range of. When the term "about" is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values ​​set forth. Generally, the term "about" is used herein to modify numerical values ​​above and below the stated value by 10% up or down (higher or lower).

[0061] Throughout this disclosure, various aspects of the present invention are presented in range format.It should be understood that the description of range format is only for convenience and brevity, and should not be interpreted as an inflexible limitation to the scope of the invention.Therefore, the description of the scope should be considered to specifically disclose all possible sub-ranges and the single numerical values ​​in the scope.For example, the description of a scope such as 1 to 6 should be considered to specifically disclose sub-ranges, such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., and the single digits in the scope, such as 1, 2, 3, 4, 5 and 6.This is applicable regardless of the width of the scope.The numerical range listed includes the number defining the scope and includes each integer in the defined range.

[0062] Units, prefixes and symbols are expressed in a form acceptable to the Système International de Unites (SI). Numerical ranges include the number of limited ranges. In the case of enumerated numerical ranges, it is understood that each intermediate integer value and each fraction thereof between the upper and lower limits of the enumeration of the range, as well as each subrange between these values, is also specifically disclosed. The upper and lower limits of any range may be independently included in or excluded from the range, and each range including any one, zero or two limits is also encompassed in the present disclosure. Therefore, the ranges listed herein are understood to be a shorthand expression of all values ​​in the range including the enumerated endpoints. For example, a range of 1 to 10 is understood to include any number, combination of numbers or subranges in the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10.

[0063] In the case of explicitly enumerating values, it is understood that values ​​of the amount or quantity approximately the same as the enumerated values ​​are also within the scope of the present disclosure. In the case of a disclosed combination, each sub-combination of the elements of the combination is also specifically disclosed and within the scope of the present disclosure. Conversely, in the case of separately disclosing different elements or groups of elements, their combinations are also disclosed. In the case of any element of the present disclosure being disclosed as having multiple alternatives, examples of the disclosure are also disclosed herein, wherein each alternative is excluded individually or in any combination with other alternatives; more than one element of the present disclosure may have such exclusions, and all combinations of elements with such exclusions are disclosed herein.

[0064] The term "and / or" as used herein is considered to be a specific disclosure of each of the two specified features or components with or without the other. Thus, the term "and / or" as used herein in phrases such as "A and / or B" is meant to include: "both A and B"; "A or B"; "A" (alone); and "B" (alone). Similarly, the term "and / or" as used in phrases such as "A, B, and / or C" is intended to cover each of the following aspects: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0065] As used herein, the term "treating" or "treatment" refers to the administration of a composition to a subject for therapeutic purposes.

[0066] The term "human claudin-1 (or CLDN1)" refers to the protein having the sequence shown in NCBI Accession No. NP_066924.1, or any naturally occurring variant commonly found in HCV permissive populations.

[0067] As used herein, the term "antibody" refers to any immunoglobulin containing an antigen binding site that immunospecifically binds to an antigen. Thus, the term antibody encompasses not only whole antibody molecules, but also antibody fragments and variants (including derivatives) of antibodies and antibody fragments, as long as the derivatives and fragments retain specific binding ability. The term encompasses monoclonal antibodies and polyclonal antibodies. The term also encompasses any protein having a binding domain that is homologous or largely homologous to an immunoglobulin binding domain. These proteins may be derived from natural sources, or may be produced in part or in whole synthetically. When used with respect to antibodies, the term "specific binding" refers to the binding of an antibody to a predetermined antigen. Typically, antibodies bind to an antigen with at least 1 x 10 7 M 1 The antibody binds to the predetermined antigen with an affinity of at least twice that of the non-specific antigen (e.g., BSA, casein).

[0068] As used herein, the term "humanized antibody" refers to a chimeric antibody comprising amino acid residues from non-human hypervariable regions and amino acid residues from human framework regions (FRs). In particular, a humanized antibody comprises all or substantially all of at least one, usually two variable domains, wherein all or substantially all of the complementary determining regions (CDRs) are those of human antibodies. A humanized antibody optionally may comprise at least a portion of an antibody constant region derived from a human antibody. A "humanized form" of an antibody (e.g., a non-human antibody) refers to an antibody that has undergone humanization.

[0069] It should be understood that whenever an aspect is described herein with the term “comprising,” other similar aspects described with “consisting of” and / or “consisting essentially of” are also provided.

[0070] As used herein, the term "administering" refers to physically introducing a composition comprising a therapeutic agent (e.g., an anti-claudin-1 antibody) into a subject using any of a variety of methods and delivery systems known to those skilled in the art. Routes of administration include intravenous, intramuscular, subcutaneous, intraperitoneal, spinal or other parenteral routes of administration, such as by injection or infusion. As used herein, the phrase "parenteral administration" refers to modes of administration other than enteral and topical administration, typically by injection, and includes, but is not limited to, intravenous, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcutaneous, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion, and in vivo electroporation. Other non-parenteral routes include topical, epidermal or mucosal routes of administration, such as intranasal, vaginal, rectal, sublingual or topical administration. Administration may also be performed, for example, once, multiple times and / or over one or more extended time periods.

[0071] The term "effective amount" refers to the amount of an agent that provides a desired biological, therapeutic and / or preventive result. The result can be a reduction, improvement, alleviation, mitigation, delay and / or alleviation of one or more signs, symptoms or causes of disease, or any other desired change in a biological system. With regard to solid tumors, an effective amount includes an amount sufficient to cause tumor reduction and / or reduce tumor growth rate (such as curbing tumor growth) or prevent or delay other unwanted cell proliferation. In some aspects, an effective amount is an amount sufficient to delay tumor development. In some aspects, an effective amount is an amount sufficient to prevent or delay tumor recurrence. The effective amount can be administered once or multiple times. An effective amount of a drug or composition can be one that: (i) reduces the number of cancer cells; (ii) reduces tumor size; (iii) inhibits, blocks, slows down, and possibly stops cancer cell infiltration into peripheral organs to some extent; (iv) inhibits (i.e., slows down and possibly stops) tumor metastasis to some extent; (v) inhibits tumor growth; (vi) prevents or delays the occurrence and / or recurrence of tumors; and / or (vii) alleviates to some extent one or more symptoms associated with cancer. In one example, an "effective amount" is an amount of an anti-claudin-1 antibody that has been clinically shown to affect a significant reduction in cancer or a slowing of the progression of cancer (such as advanced solid tumors).

[0072] As used herein, "patient" includes any patient suffering from cancer (eg, fibrotic cancer). The terms "subject" and "patient" are used interchangeably herein.

[0073] II. Anti-claudin-1 Antibodies

[0074] The present invention relates to the use of anti-claudin-1 antibodies for treating cholangiocarcinoma in a human subject in need thereof. In some aspects, disclosed herein is a method of treating cholangiocarcinoma in a human subject in need thereof, the method comprising administering to the human subject a therapeutically effective amount of an anti-claudin-1 antibody.

[0075] CLDN1 is a transmembrane protein with two major roles: (1) promoting barrier function through tight junctions together with other proteins; (2) CLDN1 is expressed outside the tight junctions of the basolateral membrane of epithelial cells, where it has been shown to mediate oncogenic signaling, epithelial-mesenchymal transition (EMT), and cell fate. In addition, CLDN1 has been shown to be expressed in non-epithelial cells such as myofibroblasts of the liver, lung, and kidney.

[0076] Antibodies against human claudin-1 have been previously described for the treatment of hepatitis C virus infection, hepatocellular carcinoma, and certain fibrotic diseases, such as pulmonary fibrosis (see WO 2010 / 034812, WO 2016 / 146809, and WO 2021 / 094469). Anti-claudin-1 antibodies useful in the practice of the present invention include any antibodies raised against claudin-1. Examples are disclosed in WO 2010 / 034812 and WO 2017 / 162678.

[0077] Other examples of suitable anti-claudin-1 antibodies include those disclosed in European Patent No. EP 1 167 389, U.S. Patent No. 6,627,439, International Patent Application Published as WO 2014 / 132307, International Patent Application Published as WO 2015 / 014659 and WO 2015 / 014357, and Yamashita et al., 353 (1) J. Pharmacol. Exp. Ther. 112-118 (2015).

[0078] The anti-Claudin-1 antibody suitable for use in the present invention may be a polyclonal antibody or a monoclonal antibody.

[0079] Anti-claudin-1 antibodies suitable for use according to the present invention may also be "humanized": sequence differences between rodent antibodies and human sequences may be minimized by replacing residues that differ from those in the human sequence with site-directed mutagenesis of single residues or by transplantation of entire regions or by chemical synthesis. Humanized antibodies may also be produced using recombinant methods. In a humanized form of an antibody, some, most or all of the amino acids outside the CDR regions are replaced with amino acids from a human immunoglobulin molecule, while some, most or all of the amino acids within one or more CDR regions remain unchanged. Small additions, deletions, insertions, substitutions or modifications of amino acids are permitted as long as they do not significantly modify the biological activity of the resulting antibody. Suitable human "alternative" immunoglobulin molecules include IgG1, IgG2, IgG2a, IgG2b, IgG3, IgG4, IgA, IgM, IgD or IgE molecules and fragments thereof.

[0080] In some aspects, the humanized anti-Claudin-1 antibody used in accordance with the present invention is an antibody previously described in WO 2017 / 162678. Table 1 describes exemplary sequences of antibodies or antigen-binding fragments provided herein.

[0081] Table 1 - Exemplary sequences

[0082]

[0083]

[0084]

[0085] In some aspects, the anti-Claudin-1 antibody comprises a complementarity determining region (CDR) H1 comprising the amino acid sequence shown in SEQ ID NO:5, a CDR H2 comprising the amino acid sequence shown in SEQ ID NO:6, and a CDR H3 comprising the amino acid sequence shown in SEQ ID NO:7.

[0086] In some aspects, the anti-Claudin-1 antibody comprises a complementarity determining region (CDR) L1 comprising the amino acid sequence shown in SEQ ID NO:8, a CDR L2 comprising the amino acid sequence shown as GAS, and a CDR L3 comprising the amino acid sequence shown in SEQ ID NO:10.

[0087] In some aspects, the complementarity determining regions (CDRs) disclosed herein are according to However, it will be appreciated that other methods of defining CDRs known in the art may also be used.

[0088] In some aspects, the six complementarity determining regions (CDRs) of the anti-Claudin-1 antibody are identical to those in the anti-Claudin-1 monoclonal antibody secreted by the hybridoma cell line deposited with the DSMZ on July 29, 2008 under accession number DSMACC2938.

[0089] In some aspects, the heavy chain variable region ("VH") and light chain variable region ("VL") of the anti-Claudin-1 antibody are identical to those in the anti-Claudin-1 monoclonal antibody secreted by the hybridoma cell line deposited with the DSMZ on July 29, 2008 under accession number DSM ACC2938.

[0090] In some aspects, the anti-Claudin-1 antibody comprises a VH comprising the amino acid sequence shown in SEQ ID NO:3 or SEQ ID NO:13.

[0091] In some aspects, the anti-Claudin-1 antibody comprises a VH comprising an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:3.

[0092] In some aspects, the anti-Claudin-1 antibody comprises a VH comprising an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:13.

[0093] In some aspects, the anti-Claudin-1 antibody comprises a VH comprising an amino acid sequence having about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% sequence identity to SEQ ID NO:3.

[0094] In some aspects, the anti-Claudin-1 antibody comprises a VH comprising an amino acid sequence having about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% sequence identity to SEQ ID NO:13.

[0095] In some aspects, the anti-Claudin-1 antibody comprises a VL comprising the amino acid sequence set forth in SEQ ID NO:4 or SEQ ID NO:14.

[0096] In some aspects, the anti-Claudin-1 antibody comprises a VL comprising an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:4.

[0097] In some aspects, the anti-Claudin-1 antibody comprises a VL comprising an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to SEQ ID NO:14.

[0098] In some aspects, the anti-Claudin-1 antibody comprises a VL comprising an amino acid sequence having about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% sequence identity to SEQ ID NO:4.

[0099] In some aspects, the anti-Claudin-1 antibody comprises a VL comprising an amino acid sequence having about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% sequence identity to SEQ ID NO:14.

[0100] In some aspects, the anti-Claudin-1 antibody comprises a VH comprising the amino acid sequence shown in SEQ ID NO:3; and a VL comprising the amino acid sequence shown in SEQ ID NO:4.

[0101] In some aspects, the anti-Claudin-1 antibody comprises a VH comprising an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:3, and a light chain variable region (VL) comprising an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:4.

[0102] In some aspects, the anti-Claudin-1 antibody comprises a VH comprising an amino acid sequence having about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% sequence identity to SEQ ID NO:3, and a light chain variable region (VL) comprising an amino acid sequence having about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% sequence identity to SEQ ID NO:4.

[0103] In some aspects, the anti-Claudin-1 antibody comprises a VH comprising the amino acid sequence shown in SEQ ID NO:13; and a VL comprising the amino acid sequence shown in SEQ ID NO:14.

[0104] In some aspects, the anti-Claudin-1 antibody comprises a VH comprising an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 13, and a light chain variable region (VL) comprising an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 14.

[0105] In some aspects, the anti-Claudin-1 antibody comprises a VH comprising an amino acid sequence having about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% sequence identity to SEQ ID NO: 13, and a light chain variable region (VL) comprising an amino acid sequence having about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% sequence identity to SEQ ID NO: 14.

[0106] In some aspects, the heavy and light chains of the anti-Claudin-1 antibody are identical to those in the anti-Claudin-1 monoclonal antibody secreted by the hybridoma cell line deposited with the DSMZ on July 29, 2008 under Accession No. DSMACC2938.

[0107] In some aspects, the anti-Claudin-1 antibody comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO:1 or SEQ ID NO:15.

[0108] In some aspects, the anti-Claudin-1 antibody comprises a heavy chain comprising an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 1 or SEQ ID NO: 15.

[0109] In some aspects, the anti-Claudin-1 antibody comprises a heavy chain comprising an amino acid sequence having about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% sequence identity to SEQ ID NO: 1 or SEQ ID NO: 15.

[0110] In some aspects, the anti-Claudin-1 antibody comprises a light chain comprising the amino acid sequence shown in SEQ ID NO:2.

[0111] In some aspects, the anti-Claudin-1 antibody comprises a light chain comprising an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:2.

[0112] In some aspects, the anti-Claudin-1 antibody comprises a light chain comprising an amino acid sequence having about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% sequence identity to SEQ ID NO:2.

[0113] In some aspects, the anti-Claudin-1 antibody comprises a heavy chain comprising the amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:15; and a light chain comprising the amino acid sequence shown in SEQ ID NO:2.

[0114] In some aspects, the anti-Claudin-1 antibody comprises a heavy chain comprising an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 1 or SEQ ID NO: 15, and a light chain comprising an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 2.

[0115] In some aspects, the anti-Claudin-1 antibody comprises a heavy chain comprising an amino acid sequence having about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% sequence identity to SEQ ID NO: 1 or SEQ ID NO: 15, and a light chain comprising an amino acid sequence having about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% sequence identity to SEQ ID NO: 2.

[0116] In some aspects, the anti-Claudin-1 antibody comprises a heavy chain comprising the amino acid sequence shown in SEQ ID NO:11.

[0117] In some aspects, the anti-Claudin-1 antibody comprises a heavy chain comprising an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:11.

[0118] In some aspects, the anti-Claudin-1 antibody comprises a heavy chain comprising an amino acid sequence having about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% sequence identity to SEQ ID NO:11.

[0119] In some aspects, the anti-Claudin-1 antibody comprises a light chain comprising the amino acid sequence shown in SEQ ID NO:12.

[0120] In some aspects, the anti-Claudin-1 antibody comprises a light chain comprising an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:12.

[0121] In some aspects, the anti-Claudin-1 antibody comprises a light chain comprising an amino acid sequence having about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% sequence identity to SEQ ID NO:12.

[0122] In some aspects, the anti-Claudin-1 antibody comprises a heavy chain comprising the amino acid sequence shown in SEQ ID NO:11; and a light chain comprising the amino acid sequence shown in SEQ ID NO:12.

[0123] In some aspects, the anti-Claudin-1 antibody comprises a heavy chain comprising an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 11, and a light chain comprising an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 12.

[0124] In some aspects, the anti-Claudin-1 antibody comprises a heavy chain comprising an amino acid sequence having about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% sequence identity to SEQ ID NO: 11, and a light chain comprising an amino acid sequence having about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% sequence identity to SEQ ID NO: 12.

[0125] The humanized anti-claudin-1 antibody may be a fully monoclonal antibody having an isotope selected from the group consisting of IgG1, IgG2, IgG3 and IgG4. Alternatively, the humanized anti-claudin-1 antibody may be a fragment of a monoclonal antibody selected from the group consisting of Fv, Fab, F(ab')2, Fab', dsFv, scFv, sc(Fv)2 and diabody.

[0126] Anti-claudin-1 antibodies (or biologically active variants or fragments thereof) suitable for use according to the present invention may be functionally linked (e.g., by chemical coupling, genetic fusion, non-covalent binding or other means) to one or more other molecular entities. Methods for preparing such modified antibodies (or conjugated antibodies) are known in the art (see, e.g., "Affinity Techniques. Enzyme Purification: Part B", Methods in Enzymol., 1974, Vol. 34, Jakoby and Wilneck (eds.), Academic Press: New York, NY; and Wilchek and Bayer, Anal. Biochem., 1988, 171: 1-32). Preferably, the molecular entity is linked to a position on the antibody molecule that does not interfere with the binding properties of the resulting conjugate, such as a position that does not participate in the specific binding of the antibody to its target.

[0127] In some aspects, the anti-Claudin-1 antibodies described herein target the extracellular loop 1 of Claudin-1 that is exposed outside of tight junctions in the basolateral membrane of epithelial cells.

[0128] The antibody molecule and the molecular entity can be covalently and directly linked to each other. Alternatively, the antibody molecule and the molecular entity can be covalently linked to each other via a linker group. This can be achieved by using any of a variety of stable bifunctional agents well known in the art, including homofunctional and heterofunctional linkers.

[0129] In some aspects, the anti-Claudin-1 antibodies (or biologically active fragments thereof) used in accordance with the present invention are conjugated to a detectable agent. Any of a variety of detectable agents may be used, including but not limited to various ligands, radionuclides (e.g., 3 H. 125 I. 131I, etc.), fluorescent dyes (e.g., fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthalaldehyde and fluorescamine), chemiluminescent agents (e.g., luciferin, luciferase and aequorin), microparticles (e.g., quantum dots, nanocrystals, phosphors, etc.), enzymes (e.g., those used in ELISA, i.e., horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase), colorimetric labels, magnetic labels and biotin, digoxigenin or other haptens and proteins for which antisera or monoclonal antibodies are available.

[0130] Other molecular entities that can be conjugated to the anti-Claudin-1 antibodies (or biologically active fragments thereof) of the present invention include, but are not limited to, linear or branched hydrophilic polymer groups, fatty acid groups, or fatty ester groups.

[0131] Therefore, in the practice of the present invention, the anti-claudin-1 antibody can be used in the form of a full-length antibody, a biologically active variant or fragment thereof, a chimeric antibody, a humanized antibody, and an antibody-derived molecule comprising at least one complementarity determining region (CDR) from the heavy chain or light chain variable region of the anti-claudin-1 antibody, including molecules such as: Fab fragments, F(ab')2 fragments, Fd fragments, Fabc fragments, Sc antibodies (single-chain antibodies), diabodies, single antibody light chain single chains, single antibody heavy chains, chimeric fusions between antibody chains and other molecules, and antibody conjugates, such as antibodies conjugated to therapeutic agents or detectable agents. Preferably, the anti-claudin-1 antibody-related molecules according to the present invention retain the antibody ability to bind to its antigen, particularly the extracellular domain of claudin-1.

[0132] III. Cholangiocarcinoma

[0133] Cholangiocarcinoma (CCA) is a highly lethal epithelial cell malignancy that arises anywhere within the biliary system and / or liver parenchyma. CCA exhibits features of cholangiocytic differentiation and may arise from epithelial cells lining the bile ducts, called cholangiocytes. However, the cancer may also develop from peribiliary glands and hepatocytes, depending on the underlying liver disease and location. CCA is heterogeneous and can be classified according to the predominant anatomical subtype as intrahepatic CCA (iCCA), perihilar CCA (pCCA), or distal CCA (dCCA). iCCA is located proximal to the secondary bile ducts within the liver parenchyma, pCCA is located between the secondary bile ducts and the insertion of the cystic duct into the common bile duct, and dCCA is confined to the common bile duct below the insertion of the cystic duct. The true incidence of pCCA and iCCA is unknown, as some national databases misclassify pCCA as iCCA in large numbers. In addition, enhanced diagnostic capabilities have made the clinical distinction between bile duct carcinoma of unknown primary and iCCA more apparent. These factors have contributed to the increase in the reported incidence of iCCA over the past two or three decades. Each anatomical subtype is characterized by unique genetic aberrations, clinical manifestations and management options. However, many databases classify both pCCA and dCCA as extrahepatic CCA. Most CCAs are adenocarcinomas and other histological subtypes, such as adenosquamous carcinomas or clear cell carcinomas, are rarely encountered. These cancers have a high degree of desmoplastic tissue proliferation and are wrapped in a dense network of inflammatory cells and matrix known as the tumor immune microenvironment. The epidemiology of these cancers varies around the world. In some regions, infection with specific trematodes (flatworm parasites, commonly referred to as trematodes) is the main cause of CCA. Trematode-related CCA may have a specific pathogenesis, especially genetic aberrations, but diagnosis and management are no different from non-trematode-related CCA. In the Western world, most CCA patients have no identifiable risk factors, except for some patients with primary sclerosing cholangitis (PSC) (see Brindley et al., 7Nat Rev Dis Primers 1-17 (2021)).

[0134] In some aspects, CCA is a mixed or hybrid hepatocellular cholangiocarcinoma (cHCC-CCA). cHCC-CCA is a unique primary liver cancer that has clear phenotypic features of both hepatocellular carcinoma (HCC) and cholangiocarcinoma. cHCC-CCA is a rare and aggressive primary liver malignancy with significant histological and biological heterogeneity. It exhibits more aggressive behavior and worse survival outcomes than hepatocellular carcinoma or cholangiocarcinoma. The World Health Organization divides cHCC-CCA into two main types: the classic type, which is characterized by intermixed areas of typical HCC and CCA and the presence of a transition zone with an intermediate morphology between the two types; and the type with stem cell characteristics, which is less common and is further subdivided into typical, intermediate, and cholangiocellular subtypes. Certain histopathological criteria have been established to confirm the diagnosis of cHCC-CCA, which require the presence of fully differentiated hepatocyte and CCA components, which are closely intermixed, and the presence of evidence of a transition zone containing cells with intermediate morphology. This distinguishes it from HCC and CCA found in the same liver lobe, which represent collision tumors. A gene panel showed that mutations in the genes KRAS, ARID1A, TERT promoter, and TP53 were associated with different clinical phenotypes of cHCC-CCA (see Stravraka et al., 6 J Hepatocell Carcinoma 11-21 (2018)).

[0135] In some aspects, cholangiocarcinoma is treated with chemotherapy such as cisplatin and gemcitabine.

[0136] IV. How to use

[0137] The methods of the present invention can be accomplished using an anti-claudin-1 antibody or a biologically active fragment thereof, or a pharmaceutical composition comprising such an antibody or fragment (see below). These methods generally comprise administering an effective amount of an anti-claudin-1 antibody or a biologically active fragment thereof, or a pharmaceutical composition thereof, to a subject in need thereof (i.e., a subject with a fibrotic tumor). Any administration method known to those skilled in the art can be used for administration (see below).

[0138] The present disclosure provides a method of treating cholangiocarcinoma (CCA) in a human subject in need thereof, the method comprising administering to the human subject a therapeutically effective amount of an anti-Claudin-1 antibody.

[0139] In some aspects, provided herein is an anti-Claudin-1 antibody or a pharmaceutical composition thereof for use in a method of treating cholangiocarcinoma (CCA) in a human subject, the method comprising administering an effective amount of the anti-Claudin-1 antibody or a pharmaceutical composition thereof to the human subject.

[0140] In some aspects, the CCA is an intrahepatic CCA.

[0141] In some aspects, the CCA is a perihilar CCA.

[0142] In some aspects, the CCA is a distal CCA.

[0143] In some aspects, the CCA is mixed or mixed hepatocellular cholangiocarcinoma (cHCC-CCA).

[0144] In some aspects, the CCA has mutations in isocitrate dehydrogenase (NADP(+)) 1 (IDH1), isocitrate dehydrogenase (NADP(+)) 2 (IDH2), BRCA1-associated protein 1 (BAP1), fibroblast growth factor receptor 2 (FGFR2), Kirsten rat sarcoma viral oncogene homolog (KRAS), polybrominated 1 (PBRM1), AT-rich interaction domain 1A (ARID1A), phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA), Ephrin type A receptor 2 (EPHA2), cyclin-dependent kinase inhibitor 2A (CDKN2A), tumor protein P53 (TP53), SMAD family member 4 (SMAD4), transforming growth factor beta receptor 2 (TGFBR2).

[0145] In some aspects, claudin-1 (CLDN1) is overexpressed in a human subject compared to the expression level in a normal subject.

[0146] In some aspects, claudin-1 (CLDN1) is overexpressed in a human subject treated with chemotherapy.

[0147] In some aspects, the anti-Claudin-1 antibody comprises the six complementarity determining regions (CDRs) of the anti-Claudin-1 monoclonal antibody secreted by the hybridoma cell line deposited with the DSMZ on July 29, 2008 under accession number DSMACC2938.

[0148] In some aspects, the anti-Claudin-1 antibody is humanized.

[0149] In some aspects, the anti-Claudin-1 antibody comprises a VH comprising the amino acid sequence shown in SEQ ID NO:3 or SEQ ID NO:13.

[0150] In some aspects, the anti-Claudin-1 antibody comprises a VH comprising an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:3.

[0151] In some aspects, the anti-Claudin-1 antibody comprises a VH comprising an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:13.

[0152] In some aspects, the anti-Claudin-1 antibody comprises a VH comprising an amino acid sequence having about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% sequence identity to SEQ ID NO:3.

[0153] In some aspects, the anti-Claudin-1 antibody comprises a VH comprising an amino acid sequence having about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% sequence identity to SEQ ID NO:13.

[0154] In some aspects, the anti-Claudin-1 antibody comprises a VL comprising the amino acid sequence set forth in SEQ ID NO:4 or SEQ ID NO:14.

[0155] In some aspects, the anti-Claudin-1 antibody comprises a VL comprising an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:4.

[0156] In some aspects, the anti-Claudin-1 antibody comprises a VL comprising an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to SEQ ID NO:14.

[0157] In some aspects, the anti-Claudin-1 antibody comprises a VL comprising an amino acid sequence having about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% sequence identity to SEQ ID NO:4.

[0158] In some aspects, the anti-Claudin-1 antibody comprises a VL comprising an amino acid sequence having about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% sequence identity to SEQ ID NO:14.

[0159] In some aspects, the anti-Claudin-1 antibody comprises a VH comprising the amino acid sequence shown in SEQ ID NO:3; and a VL comprising the amino acid sequence shown in SEQ ID NO:4.

[0160] In some aspects, the anti-Claudin-1 antibody comprises a VH comprising an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:3, and a light chain variable region (VL) comprising an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:4.

[0161] In some aspects, the anti-Claudin-1 antibody comprises a VH comprising an amino acid sequence having about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% sequence identity to SEQ ID NO:3, and a light chain variable region (VL) comprising an amino acid sequence having about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% sequence identity to SEQ ID NO:4.

[0162] In some aspects, the anti-Claudin-1 antibody comprises a VH comprising the amino acid sequence shown in SEQ ID NO:13; and a VL comprising the amino acid sequence shown in SEQ ID NO:14.

[0163] In some aspects, the anti-Claudin-1 antibody comprises a VH comprising an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 13, and a light chain variable region (VL) comprising an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 14.

[0164] In some aspects, the anti-Claudin-1 antibody comprises a VH comprising an amino acid sequence having about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% sequence identity to SEQ ID NO: 13, and a light chain variable region (VL) comprising an amino acid sequence having about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% sequence identity to SEQ ID NO: 14.

[0165] In some aspects, the anti-Claudin-1 antibody comprises a complementarity determining region (CDR) H1 comprising the amino acid sequence shown in SEQ ID NO:5, a CDR H2 comprising the amino acid sequence shown in SEQ ID NO:6, and a CDR H3 comprising the amino acid sequence shown in SEQ ID NO:7.

[0166] In some aspects, the anti-Claudin-1 antibody comprises a complementarity determining region (CDR) L1 comprising the amino acid sequence shown in SEQ ID NO:8, a CDR L2 comprising the amino acid sequence shown as GAS, and a CDR L3 comprising the amino acid sequence shown in SEQ ID NO:10.

[0167] In some aspects, the anti-Claudin-1 antibody is administered intratumorally, intravenously, intraperitoneally, intramuscularly, intrathecally, or subcutaneously.

[0168] In some aspects, the method further comprises administering a chemotherapeutic agent.

[0169] In some aspects, the chemotherapeutic agent is cisplatin.

[0170] In some aspects, the chemotherapeutic agent is gemcitabine.

[0171] V. Application

[0172] The desired dose of the anti-claudin-1 antibody, or a biologically active fragment thereof (optionally after formulation with one or more suitable pharmaceutically acceptable carriers or excipients) may be administered to a subject in need thereof by any suitable route. Various delivery systems are known and may be used to administer the antibody, including tablets, capsules, injectable solutions, encapsulation in liposomes, microparticles, microcapsules, and the like. Methods of administration include, but are not limited to, dermal, intradermal, intramuscular, intraperitoneal, intralesional, intravenous, subcutaneous, intranasal, pulmonary, epidural, and oral routes. The anti-claudin-1 antibody, or a biologically active fragment thereof, or a pharmaceutical composition thereof may be administered by any convenient or other appropriate route, such as by infusion or bolus injection, absorption through epithelial or mucosal linings (e.g., oral mucosa, bronchial mucosa, rectal and intestinal mucosa, etc.). It may be administered systemically or topically.

[0173] In some aspects, the anti-Claudin-1 antibody is administered intratumorally, intravenously, intraperitoneally, intramuscularly, intrathecally, or subcutaneously.

[0174] The anti-claudin-1 antibody or biologically active fragment thereof (optionally after formulation with one or more appropriate pharmaceutically acceptable carriers or excipients) will be administered at a dosage such that the amount delivered is effective for the intended purpose. The route of administration, formulation, and dosage administered will depend on the desired therapeutic effect, the severity of the condition to be treated (if already present), the presence of any infection, the age, sex, weight, and general health of the patient, as well as on the potency, bioavailability, and in vivo half-life of the antibody or composition used, the use (or non-use) of concomitant therapy, and other clinical factors. These factors can be readily determined by the attending physician during the course of treatment. Alternatively or additionally, the dosage to be administered can be determined based on studies using animal models (e.g., non-human primates or rodents). Adjusting the dosage based on these or other methods to achieve maximum efficacy is well known in the art and is within the capabilities of a trained physician. Further information on appropriate dosage levels and duration of treatment will emerge as studies are conducted using anti-claudin-1 antibodies.

[0175] VI. Pharmaceutical Compositions

[0176] As described above, anti-Claudin-1 antibodies (and related molecules) can be administered alone or as a pharmaceutical composition. Therefore, the present invention provides a pharmaceutical composition comprising an effective amount of an anti-Claudin-1 antibody or a biologically active fragment thereof as described herein and at least one pharmaceutically acceptable carrier or excipient.

[0177] In some aspects, provided herein is a pharmaceutical composition for treating cholangiocarcinoma (CCA), the pharmaceutical composition comprising a therapeutically effective amount of any anti-Claudin-1 antibody disclosed herein.

[0178] In some aspects, the CCA is an intrahepatic CCA.

[0179] In some aspects, the CCA is a perihilar CCA.

[0180] In some aspects, the CCA is a distal CCA.

[0181] In some aspects, the CCA is mixed or mixed hepatocellular cholangiocarcinoma (cHCC-CCA).

[0182] The pharmaceutical composition can be administered in any amount and using any administration route that is effective to achieve the desired preventive and / or therapeutic effect. The optimal pharmaceutical formulation can vary depending on the route of administration and the desired dosage. Such formulations can affect the physical state, stability, in vivo release rate, and in vivo clearance rate of the administered active ingredient.

[0183] The pharmaceutical compositions of the present invention may be formulated in dosage unit form for ease of administration and uniformity of dosage. It will be understood, however, that the total daily dosage of the composition will be decided by the attending physician within the scope of sound medical judgment.

[0184] VII.Medicine Box

[0185] In another aspect, the invention provides a pharmaceutical pack or kit comprising one or more containers (e.g., vials, ampoules, test tubes, flasks or bottles) containing one or more ingredients of the pharmaceutical compositions of the invention, thereby allowing administration of the anti-claudin-1 antibody or biologically active fragment thereof.

[0186] The different components of a drug package or medicine box can be supplied in solid (e.g., lyophilized) or liquid form. Each component is generally suitable for being equally divided in its respective container or provided in a concentrated form. The drug package or medicine box may include a medium for reconstructing the lyophilized components. The individual containers of the medicine box will preferably remain tightly closed for commercial sale.

[0187] Optionally, associated with the container may be a notice or package insert in the form prescribed by a governmental agency regulating the manufacture, use or sale of drugs or biological products, which notice reflects the agency's approval of manufacture, use or sale for human administration. The notice or package insert may contain instructions for use of the pharmaceutical composition according to the methods of treatment disclosed herein.

[0188] An identifier (e.g., barcode, radio frequency, ID tag, etc.) may be present in or on the kit. For example, the identifier may be used to uniquely identify the kit for purposes such as quality control, inventory control, tracking movement between workstations, etc.

[0189] In some aspects, provided herein is a kit for treating a subject having cholangiocarcinoma (CCA), the kit comprising a therapeutically effective amount of any anti-Claudin-1 antibody disclosed herein and instructions comprising instructions for use of the kit.

[0190] In some aspects, the CCA is an intrahepatic CCA.

[0191] In some aspects, the CCA is a perihilar CCA.

[0192] In some aspects, the CCA is a distal CCA.

[0193] In some aspects, the CCA is mixed or mixed hepatocellular cholangiocarcinoma (cHCC-CCA).

[0194] Example

[0195] The following embodiments are illustrative and do not limit the scope of the claimed aspects.

[0196] Example 1. CLDN1 is highly expressed in intrahepatic and extrahepatic CCA tumors and is associated with tumor stemness

[0197] To investigate the role of CLDN1 in cholangiocarcinoma, we first analyzed CLDN1 expression at the transcriptional level in CCA patients. Comprehensive computational analysis of data retrieved from the Genomic Data Commons data portal (portal.gdc.cancer.gov) showed that CLDN1 was upregulated in extrahepatic CCA (GSE132305, p<0.0001, t-test, Figure 1A ) and proliferative intrahepatic CCA (GSE32225, p = 0.03, Mann-Whitney test, Figure 1B Interestingly, CLDN1 expression in proliferative iCCA was significantly higher than that in inflammatory iCCA (GSE32225, p=0.02, t-test, Figure 1C ).

[0198] To investigate the correlation between CLDN1 expression and common mutations in CCA patients, CLDN1 expression was analyzed at the transcriptional level for different common gene mutations described in CCA patient tumors using a public database (Jusakul et al., 7(10) Cancer Discov 1116-1135 (October 2017)). These mutations include isocitrate dehydrogenase (NADP(+)) 1 (IDH1), isocitrate dehydrogenase (NADP(+)) 2 (IDH2), BRCA1-associated protein 1 (BAP1), fibroblast growth factor receptor 2 (FGFR2), Kirsten rat sarcoma viral oncogene homolog (KRAS), polybrominated 1 (PBRM1), AT-rich interaction domain 1A (ARID1A), phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA), Ephrin type A receptor 2 (EPHA2), cyclin-dependent kinase inhibitor 2A (CDKN2A), tumor protein P53 (TP53), SMAD family member 4 (SMAD4), and transforming growth factor beta receptor 2 (TGFBR2).

[0199] Comprehensive computational analysis showed that CLDN1 was robustly expressed in different CCA gene mutations ( Figure 1D ). CLDN1 expression was highest in tumors with IDH1 and BAP1 gene mutations compared with other mutations (GSE89747 and GSE89748, **p<0.01, Mann-Whitney test, Figure 1D ). Compared with other mutations, CLDN1 expression was the lowest in TP53, SMAD4, and TGFBR2 mutations (GSE89747 and GSE89748, **p<0.01, *p<0.05, Mann-Whitney test, Figure 1D ). The mutation frequencies of driver genes vary between CCA etiologies, with BAP1 and IDH1 mutations being highly enriched in non-trematode-related CCA, while TP53 and SMAD4 are highly enriched in trematode-related CCA (Brindley et al., 7(65) Nat Rev Dis Primers (September 2021)).

[0200] In summary, upregulation of CLDN1 expression in both intrahepatic and extrahepatic CCA tumors emphasizes its potential involvement in CCA disease pathogenesis and outcome and suggests that CLDN1 is a therapeutic target for the treatment of CCA.

[0201] Example 2. CLDN1 mAb H3L3 suppresses tumor growth in cell line-derived xenograft (CDX) and patient-derived xenograft (PDX) mouse models

[0202] To evaluate the in vivo anti-tumorigenic activity of anti-CLDN1 monoclonal (CLDN1 mAb H3L3) antibody in CCA, the effect of anti-CLDN1 mAb on tumor growth was investigated in both intrahepatic CCA and extrahepatic CCA cell line-derived xenograft (CDX) mouse models.

[0203] Non-obese diabetes Rag1 - / - IL2Rgc - / - (NRG) subcutaneous injection 5×10 6 Cells of HuCC-A1 (intrahepatic CCA) or EGI-1 (extrahepatic CCA) cell lines. Non-obese diabetic Prkdc scid IL2Rg - / - (NSG) subcutaneous injection 5×10 6 When the average tumor volume reached 40-50mm 3 At 4 hr, mice were randomized into two different groups and treated with anti-CLDN1 mAb H3L3 (25 mg / kg intraperitoneally, once a week) or vehicle control (PBS intraperitoneally, once a week) for 6 weeks. Tumor growth was monitored using a digital caliper and measured using 1 / 2 (length x width) 2 ) formula to calculate tumor volume. Figure 2A ).

[0204] CLDN1 mAb H3L3 significantly reduced tumor growth in three CDX models ( Figures 2B to 2G A strong and robust inhibition of tumor growth over time was observed in HuCC-A1CDX mice (p<0.001, Mann-Whitney test), which was highly reflected in the macroscopic appearance of transplanted tumors ( FIG. 2E to FIG. 2F In addition, the antibody delayed the EGI-1CDX mice (p<0.05, Mann-Whitney test, Figure 2B ) and HuCC-T1 CDX mice (p < 0.05, Mann-Whitney test, Figure 2G In addition, anti-CLDN1 mAb H3L3 showed a significant reduction in tumor-induced skin ulcers in the EGI-1 model (p<0.01, Mann-Whitney test, Figure 2C to Figure 2D ), indicating that it has an effect on tumor cell invasion.

[0205] On the other hand, there are great differences in molecular drivers and treatment responses between different CCA subclasses and patients (Banales et al., 17 Nat Rev Gastroenterol Hepatol 557-588 (2020)). In this context, PDX mouse models have been shown to partially reproduce tumor heterogeneity and are currently one of the most widely used in vivo systems for studying cancer treatment and predicting clinical outcomes (Xu et al., 17 (1) Oncol Lett 3-10 (January 2019)). Therefore, a PDX mouse model was established to evaluate the anti-tumor efficacy and response rate of anti-CLDN1 mAb H3L3.

[0206] Non-obese diabetes Rag1 - / - IL2Rgc - / - Fresh tumor tissue cut into small pieces (about 2-3 mm in diameter) was injected subcutaneously (NRG). When the average tumor volume reached 40 mm 3 At 4 hr, mice were randomized into two different groups and treated with anti-CLDN1 mAb H3L3 (25 mg / kg intraperitoneally, once a week, n=10 mice) or with vehicle control (PBS intraperitoneally, once a week, n=11 mice). Tumor growth was monitored using a digital caliper and 1 / 2 (length x width) was used. 2 ) formula to calculate tumor volume.

[0207] Consistent with the results obtained in CDX mice, treatment with the anti-CLDN1 mAb H3L3 significantly and significantly slowed tumor growth in the PDX model (p<0.05, Mann-Whitney test, Figure 2H Together, these data demonstrate that non-ligating CLDN1-targeting mAbs have antitumor effects in both cell line-derived and patient-derived tumor models, providing robust preclinical proof-of-concept for the use of CLDN1 mAbs for CCA therapy.

[0208] Example 3. Anti-CLDN1 antibodies inhibit migration and invasion of CCA cell lines

[0209] Next, the effect of anti-CLDN1 mAb H3L3 on cell migration was evaluated in a cell-based wound healing assay using an extrahepatic CCA cell line (KKU100) and an intrahepatic CCA cell line (HuCC-A1).

[0210] In the wound healing assay, 1.5 x 10 5CCA cells were co-cultured with LX2 in a 1:1 ratio in 12-well plates (Corning) to reproduce the matrix structure seen in CCA tumors. Co-cultures were treated with 20 μg / mL isotype control mAb or 20 μg / mL anti-CLDN1 mAb H3L3 for 3 days. On the 3rd day, wounds were made in the confluent cell monolayer using a pipette tip. The cells were then washed once with PBS to remove debris, covered with 1% FBS medium, and treated again with 20 μg / mL isotype control mAb or 20 μg / mL anti-CLDN1 mAb H3L3. The plates were incubated at 37°C and the wounds were imaged at 0, 24, and 48 h. The relative percentage of wound closure was measured using ImageJ. Each experiment was repeated at least three times independently.

[0211] Treatment with anti-CLDN1 mAb H3L3 significantly and specifically reduced CCA cell migration in the wound healing assay compared to treatment with isotype antibody (p=0.02, Mann-Whitney test, FIG. 3A to FIG. 3C ).

[0212] In another study, a transwell invasion assay was used to evaluate the effect of anti-CLDN1 mAb H3L3 treatment on cancer invasion. In this assay, EGI-1 or HuCC-T1 cells were seeded in 12-well plates and treated with 20 μg / mL isotype control mAb or 20 μg / mL anti-CLDN1 mAb H3L3 (Colpitts et al., 67(4) Gut 736-745 (March 2017)) for 3 days. 0.5 x 10 5 Cells were seeded in a transwell (8 μm pore size polycarbonate membrane insert) of a 24-well culture dish containing 75 μl of Matrigel (Corning) and serum-free medium (1:20). After solidification in an incubator (37°C, 5% CO2) for 10 minutes, 600 μL of 1% serum-supplemented medium was added to the bottom wells. Non-invasive cells were removed by scrubbing with a cotton swab, and invasive cells were fixed with 70% ethanol and stained with crystal violet (0.2%) after 72 hours. The cells on the underside of the membrane were then visualized in 5 randomly selected areas and quantified using ImageJ. Each experiment was repeated at least three times independently.

[0213] Treatment with the anti-CLDN1 mAb H3L3 significantly and significantly reduced the number of invasive EGI-1 and HuCC-T1 cells compared to cells treated with an isotype control antibody (p=0.02, Mann-Whitney test, Figures 3D to 3G ).

[0214] Taken together, these data indicate that treatment with anti-CLDN1 mAb inhibited the migration and invasion capabilities of CCA cell lines, further confirming the antitumor effect of anti-CLDN1 antibodies.

[0215] Example 4. CLDN1 mAb mediates anti-tumorigenic effects by interfering with cancer cell differentiation, metabolism and oncogenic pathways

[0216] To elucidate the molecular mechanisms behind the antitumor effects provided by anti-CLDN1 mAb (H3L3), RNA sequencing (RNA-seq) of HuCC-A1 tumor tissues from CDX mice was performed.

[0217] Unbiased pathway analysis was performed using gene set enrichment analysis (GSEA) using the Molecular Signature Database (MSigDB v.7.4). For the evaluation of the therapeutic effect of anti-CLDN1 mAb H3L3 in the HuCC-A1 CDX mouse model, samples derived from anti-CLDN1H3L3 mAb-treated animals (n=5 mice) were compared with corresponding control samples (n=5 mice) from the same model. The results of GSEA were adjusted for the false discovery rate (FDR). FDR<0.05 was considered statistically significant.

[0218] Gene sets associated with cell survival, metabolism and differentiation, including EMT, were significantly downregulated in mice treated with CLDN1 mAb H3L3 ( Figure 4A , left panel). In addition to downregulation of these pathways, targeting nonjunctive CLDN1 also showed strong suppression of several key oncogenic signaling pathways, with the strongest effects observed on TNF-α / NF-κB, TGF-β, IL-6 / JAK / STAT3, and PI3K / Akt / mTOR signaling ( Figure 4A , right), highlighting the major role of nonjunctional CLDN1 in driving oncogenic signaling.

[0219] To further investigate the antitumor effects of CLDN1-targeted mAbs, key signaling pathways involved in the development and progression of CCA were analyzed in EGI-1CDX tumor samples. FIG. 4B to FIG. 4C ).

[0220] For protein extraction from CDX tumors, tumor fragments were lysed with lysis buffer (Triton 1%; NaCl 50mM; Tris 50mM pH 7.6; MgCl2 2mM ddH2O) supplemented with protease inhibitors (Roche) and phosphatase inhibitors (Sigma phosphatase cocktail No. 2 and 3) at 4°C for 30 min. The samples were then centrifuged at 10000xg for 10 min and the supernatant was collected. Protein quantification was assessed using the Thermo Scientific BCA kit. Gels were prepared following the BioradTGX gel protocol (BioRad). Gels were transferred to PVDF membranes using the Biorad trans blot turbo protocol (BioRad). The membranes were blocked with 5% BSA in TBS-T for 1 h. The membrane was incubated with YAP / TAZ (D24E4) rabbit mAb (#8418, Cell Signaling Technology), Phospho-YAP (Ser127) (D9W2I) rabbit mAb (#13008, Cell Signaling Technology), SRC rabbit mAb (#21085, Cell Signaling Technology), phosphorylated SRC (Tyr416) ​​rabbit mAb (#21015, Cell Signaling Technology), Notch1 (D1E11) rabbit mAb (#21085, Cell Signaling Technology), and phospho-SRC (Tyr416) ​​rabbit mAb (#21015, Cell Signaling Technology). Rabbit mAb (#3608, Cell Signaling Technology), cleaved Notch1 (Val1744) (D3B8) rabbit mAb (#4147, Cell Signaling Technology), and monoclonal anti-β-actin antibody (Sigma) produced in mouse were incubated at a dilution of 1:1000 at 4°C overnight in TBS-T containing 2.5% BSA. The membrane was incubated with horseradish peroxidase-conjugated secondary antibody at a dilution of 1:10000 in TBS-T containing 2.5% BSA for 1 h at room temperature. The membrane was immunodetected for proteins using Clarity ECL Western Blotting Substrate (Biorad) in a ChemiDoc MP imaging system (Biorad). Immunoblot images were analyzed using ImageLab software v6.1 (Biorad).

[0221] Western blot quantification analysis showed that cleaved Notch1 was reduced in tumors treated with the anti-CLDN1 mAb H3L3 compared with control-treated tumors (p < 0.001, Mann-Whitney test, Figure 4C), which is known to promote CCA cell proliferation and survival (Singrang et al., 13(2) J Cell Commun Signal 245-254 (June 2019)). In addition, activation of the non-receptor tyrosine kinase protein SRC was strongly reduced in tumors treated with the anti-CLDN1 mAb H3L3, as indicated by a significant decrease in SRC phosphorylation (p<0.0001, Mann-Whitney test, Figure 4C The Yes-associated protein (YAP) / Hippo pathway was also affected by anti-CLDN1 antibody treatment. In fact, a strong increase in YAP S127 phosphorylation was observed after anti-CLDN1 mAb H3L3 treatment (p < 0.01, Mann-Whitney test, Figure 4C ), which leads to YAP cytoplasmic sequestration and inactivation (Sugihara et al., 54(6) J Gastroenterol 485-491 (June 2019)).

[0222] Collectively, the results in Examples 1-4 identify non-junctional CLDN1 as an oncogenic driver and therapeutic target in CCA. In perturbation studies using highly specific antibodies, targeting CLDN1 demonstrated significant and robust suppression of tumor growth in all human CDX and PDX mouse models tested, as well as inhibition of invasion and migration in the most advanced human CCA cell-based models. Mechanistic studies based on RNA-Seq and proteomic analysis of signaling revealed that this anti-tumorigenic effect may be mediated by interference with cancer cell differentiation, metabolism, and key oncogenic pathways involved in CCA pathogenesis.

[0223] Lack of response to drug therapy is a major limiting factor in the management of advanced CCA. The mechanisms of chemoresistance (MOC) are diverse and depend on the expression and / or function of the molecular targets of the MOC genes (Marin et al., 1864 (4Pt B) Biochim Biophys Act Mol Basis Dis 1444-1453 (April 2018)). For example, downregulation of human equilibrative nucleoside transporter 1 and copper transporter CTR1 leads to reduced sensitivity to gemcitabine and cisplatin (first-line standard of care chemotherapy for CCA), respectively (Borbath et al., 48 (7) Eur J Cancer 990-996 (May 2012)). Advantageously, the unique mechanism of action of CLDN1 mAb will become an important therapeutic option to circumvent the multidrug intrinsic or acquired resistance phenotype in CCA.

[0224] Hybrid hepatocellular carcinoma and cholangiocarcinoma (HCC-CC) is an aggressive biphenotypic primary liver cancer with unmet needs and unsatisfactory outcomes (Azizi et al., 10 Front Oncol 570958 (September 2020)). Despite recent advances, standard first-line systemic therapy options have not been refined, and surgical approaches are the only available radical treatment (Leoni et al., 12 (4) Cancers (Basel) 794 (March 2020)). Claudin-1 may be beneficial in providing a dual-targeted therapeutic target for HCC-CC.

[0225] Over the past few decades, the complex interactions between various signal transduction pathways that regulate bile duct carcinogenesis and mediate tumor development have been elucidated (Banales et al., 17 Nat Rev Gastroenterol Hepatol 557-588 (2020)). Among them, highly conserved pathways such as YAP, Hippo pathway effector protein and Notch are significantly active in CCA, upregulated by 67.2% and 79.5% in CCA tumors, respectively (Wu et al., 37 (10) Tumour Biol. 13499-13508 (October 2016); Wu et al., 31 (6) Oncol Rep 2515-2524 (June 2014)). The data showed that Notch1 and YAP signaling were strongly downregulated after anti-CLDN1 mAb H3L3 treatment ( FIG. 4B to FIG. 4C ). By targeting two independent but interconnected signaling pathways, anti-CLDN1 mAbs may circumvent the acquired therapeutic resistance encountered by previous targeted therapies for CCA (Simile et al., 55(2) Medicina (Kaunas) 42 (February 2019)). In addition, targeting SRC, a central component upstream of multiple transduction pathways (Ishizawar et al., 6(3) Cancer Cell 209-214 (September 2004)), may reduce the activation of compensatory signaling in single-target approaches due to functional crosstalk and redundancy in the maintenance of key pathways in this disease. In summary, this unique mechanism of action reveals a novel therapeutic approach that can address the heterogeneous nature of inter- and intratumoral deregulation of different signaling cascades in different CCA subsets.

[0226] To confirm the effect of anti-CLDN1 mAb H3L3 on CCA signaling observed in vivo ( FIG. 4A to FIG. 4C ), additional signaling studies were performed in a CCA cell-based model. The CCA model consists of 1.5 x 10 5EGI-1CCA cells or CCA EGI-1 and LX2 stellate cells (ratio 1:5) were co-cultured and treated for 2 days with anti-CLDN1 mAb H3L3 (Colpitts et al., 67(4) Gut 736-745 (March 2017)) (10 μg / mL) or isotype control mAb (10 μg / mL). On day 2 after treatment, cells were stimulated for 24 hours with TNF-α (10 ng / mL) to evaluate SRC and FAK signaling, or with Jagged-1 (50 ng / mL, SRP8012, Sigma-Aldrich) to evaluate Notch1 signaling. For protein extraction from cell cultures, 1% TNF-α (10 ng / mL, SRP8012, Sigma-Aldrich) was used supplemented with protease and phosphatase inhibitors (78442, Halt TM Cells were lysed at 4°C for 20 min with Glo lysis buffer (E2661, Promega) with protease and phosphatase inhibitor single-use cocktail, Thermo Fisher). The samples were then centrifuged at 15,000 rpm for 10 min and the supernatant was collected. Protein quantification was performed using the Bio-Rad DC assay kit. Gels were prepared according to the Biorad TGX gel protocol (BioRad). Gels were transferred to PVDF membranes using the Biorad transblot turbo protocol (BioRad). The membranes were blocked with 5% BSA in TBS-T for 1 h. The membranes were incubated with anti-SRC rabbit mAb (#21085, Cell Signaling Technology), phospho-SRC (Tyr416) ​​rabbit mAb (#21015, Cell Signaling Technology), anti-Notch1 (D1E11) Rabbit mAb (#3608, Cell Signaling Technology), anti-cleaved Notch1 (Val1744) (D3B8) rabbit mAb (#4147, Cell Signaling Technology), anti-FAK rabbit mAb (#3285, Cell Signaling Technology), anti-phospho-FAK (Tyr576 / 577) rabbit mAb (#3281, Cell Signaling Technology) and anti-β-actin antibody (Sigma) were incubated in TBS-T containing 5% milk for 2 hours at room temperature. The secondary antibodies used were peroxidase AffiniPure goat anti-rabbit IgG (H+L) mAb (Jackson ImmunoResearch) or ECL mouse IgG, HRP-linked whole antibody (Amersham). The membrane was immunodetected for proteins using Clarity ECL Western Blotting Substrate (Biorad) in the ChemiDoc MP imaging system (Biorad). Each experiment was repeated at least three times independently.

[0227] Western blot analysis of the CCA cell line EGI-1 treated with the anti-CLDN1 mAb H3L3 or an isotype control mAb showed a robust reduction in cleaved Notch1 and its downstream target Hes1 ( Figure 4D ), confirming that treatment with anti-CLDN1 mAb inhibited Notch1 signaling in CCA cells. In addition, SRC and FAK phosphorylation were reduced after treatment with anti-CLDN1 mAb compared with control-treated cells ( FIG. 4E to FIG. 4F ), confirming the effects of anti-CLDN1 mAbs on those signaling pathways in CCA cells. Since the Notch1, SRC, and FAK pathways have been identified as key signaling pathways driving the development and progression of CCA (Banales et al., 17(9) Nat Rev Gastroenterol Hepatol 557-588 (September 2020)), these data suggest that inhibition of Notch1, SRC, and FAK signaling by anti-CLDN1 mAbs contributes to the anti-tumor effects of the antibodies.

[0228] To further understand and confirm the effects of anti-CLDN1 mAb treatment on the identified oncogenic signaling pathways, unbiased pathway analysis was performed using gene set enrichment analysis (GSEA) in HuCC-A1CDX mouse tumors using the Molecular Signature Database (MSigDB v.7.4). Samples derived from anti-CLDN1 mAb H3L3-treated animals (n=5 mice) were compared to corresponding control samples (n=5 mice) from the same model. Results from GSEA were adjusted for the false discovery rate (FDR). FDR<0.05 was considered statistically significant.

[0229] Compared with the control group, the expression of downstream target genes and effector genes of YAP / TAZ, FAK, SRC and Notch1 signaling pathways was significantly downregulated in the anti-CLDN1 mAb H3L3 treatment group ( Figure 4G ), confirming the functional relevance of the identified pathway in anti-CLDN1 mAb-treated CCA.

[0230] Collectively, these results provide robust preclinical proof-of-concept for the therapeutic efficacy of CLDN1-specific monoclonal antibodies and reveal anti-CLDN1 mAbs as an innovative therapeutic modality for the treatment of CCA, a rapidly growing unmet medical need worldwide with unsatisfactory treatment options.

[0231] Example 5. In vivo inhibition of lung metastasis by treatment with CLDN1 mAb in a CCA metastasis mouse model.

[0232] To evaluate the in vivo activity of anti-CLDN1 mAb (H3L3) on invasion and metastasis, treatment with anti-CLDN1 antibody was investigated in a state-of-the-art CCA metastasis mouse model based on intravenous injection of metastatic HuCC-T1CCA cells.

[0233] Non-obese diabetic Rag1 mice were treated with anti-CLDN1 mAb H3L3 (Colpitts et al., 67(4) Gut 736-745 (March 2017)) (25 mg / kg, ip) or vehicle control (PBS, ip) - / - IL2Rgc - / - (NRG) mice were injected intravenously via the retro-orbital vein 1 day later with 0.5 x 10 6h HuCC-T1 cells. Mice were randomly divided into two groups and treated with anti-CLDN1 mAb H3L3 (Colpitts et al., 67(4) Gut 736-745 (March 2017)) (25 mg / kg, intraperitoneal, twice a week) or vehicle control (PBS, intraperitoneal, twice a week) for 6 weeks. After 6 weeks, mice were sacrificed, lungs were harvested, and cytokeratin 18 (CK18) staining was used to identify and quantify CK18-positive cholangiocarcinoma metastases in the lungs ( Figure 5A ).

[0234] Compared with the control group, anti-CLDN1 mAb H3L3 significantly reduced metastasis formation in the lungs, as evidenced by a decrease in CK18-positive area in lung sections after treatment with anti-CLDN1 mAb H3L3 (p<0.05, Mann-Whitney test) ( FIG. 5B to FIG. 5C ). These data suggest that anti-CLDN1 mAb H3L3 treatment can induce inhibition of CCA metastasis formation in vivo.

[0235] Example 6. CLDN1 expression targeted by CLDN1 mAb H3L3 is upregulated in CCA following treatment with cisplatin and gemcitabine

[0236] First-line chemotherapy for advanced CCA is cisplatin plus gemcitabine (Borbath et al., 48(7) Eur J Cancer 990-996 (May 2012)). To investigate the expression of CLDN1 after cisplatin and gemcitabine treatment, 1.5 x 10 5 The cells were then harvested and CLDN1 expression was assessed by flow cytometry and western blotting using a CLDN1-specific antibody.

[0237] For flow cytometry, cells were incubated with anti-CLDN1 mAb H3L3 or isotype control mAb at a concentration of 10 μg / ml in triplicate under each condition. After incubation with the primary antibody for 1 hour, the cells were washed and incubated with a secondary antibody conjugated to human Alexa647 at 4 ° C for 45 minutes. The cells were then washed and fixed with 2% paraformaldehyde (PFA). Data were acquired using Cytoflex B2R2V0 (Beckman Coulter) and analyzed using CytExpert 2.1 and FlowJo v10 (Beckman Coulter). CLDN1 expression was calculated as the difference in the mean fluorescence intensity of cells stained with anti-CLDN1 mAb H3L3 and isotype control mAb. The experiment was repeated three times independently.

[0238] For protein extraction for Western blotting, ELISA was used supplemented with protease and phosphatase inhibitors (78442, Halt TM Cells were lysed at 4°C for 20 minutes with Glo lysis buffer (E2661, Promega) with protease and phosphatase inhibitor disposable cocktail, Thermo Fisher). The samples were then centrifuged at 15000 rpm for 10 minutes and the supernatant was collected. Protein quantification was performed using the Bio-Rad DC assay kit. The gel was prepared according to the Biorad TGX gel protocol (BioRad). The gel was transferred to a PVDF membrane using the Biorad trans blot turbo protocol (BioRad). The membrane was blocked for 1 hour using 5% BSA in TBS-T. The membrane was incubated with anti-CLDN1 rabbit mAb (#E-AB-15674, ElabScience) and anti-β-actin antibody (Sigma) at room temperature in TBS-T containing 5% milk for 2 hours. The secondary antibodies used were peroxidase AffiniPure goat anti-rabbit IgG (H+L) mAb (Jackson ImmunoResearch) or ECL mouse IgG, HRP-linked whole antibody (Amersham). The membranes were subjected to protein immunodetection using Clarity ECL Western Blotting Substrate (Biorad) in a ChemiDoc MP imaging system (Biorad). The experiments were repeated at least three times independently.

[0239] Flow cytometric analysis of the CCA cell line EGI-1 treated with cisplatin and gemcitabine using the anti-CLDN1 mAb H3L3 or a control antibody showed a robust increase in CLDN1 expression after treatment with cisplatin and gemcitabine ( FIG. 6A to FIG. 6B This upregulation was further confirmed by Western blot analysis using a CLDN1-specific antibody ( FIG. 6C to FIG. 6D ).

[0240] These data suggest that chemotherapy with cisplatin and gemcitabine results in upregulation of exposed and targetable CLDN1 on the surface of CCA cells, which can be detected and reached by the anti-CLDN1 mAb H3L3. Increased CLDN1 expression in chemotherapy-treated CCA provides an opportunity to treat CCA in patients using anti-CLDN1 mAbs in combination with chemotherapy including cisplatin and gemcitabine or after treatment with chemotherapy including cisplatin and gemcitabine to improve patient outcomes.

[0241] Example 7. Anti-CLDN1 mAb H1L1 inhibits tumor growth in a cholangiocarcinoma PDX model

[0242] The purpose of this experiment is to preclinically evaluate the in vivo therapeutic effect of anti-CLDN1 mAb H1L1 in the treatment of female BALB / c nude mice cholangiocarcinoma subcutaneous injection PDX model (CC6702 model). The number of animals in the placebo group was set to n=2, and the number of animals in the treatment group was set to n=3.

[0243] Fresh tumor tissue was harvested from mice bearing established PDX models and cut into small pieces (approximately 2-3 mm in diameter). PDX tumor fragments harvested from donor mice were subcutaneously inoculated in the upper right back of study mice for tumor development. When the average tumor size reached approximately 100-150 mm 3 Randomization was initiated at 1:00 p.m. Each model was enrolled in 5 mice and divided into 2 groups. Randomization was based on the “stratified” method (Study Director TM Software, version 3.1.399.19). The grouping date is represented as day 0.

[0244] For the control group, 0.2 mL of placebo was administered via intraperitoneal injection (QW x 4). For the treatment group, 0.2 mL of anti-CLDN1 mAb H1L1 (2.5 mg / mL) was administered via intraperitoneal injection (QW x 4).

[0245] After tumor inoculation, animals were checked daily for morbidity and mortality. During routine monitoring, animals were checked for tumor growth and the effects of treatment on behavior, such as activity, food and water consumption, weight gain / loss (weight was measured twice a week after randomization), eye / hair tangles, and any other abnormalities. Mortality and observed clinical signs of individual animals were recorded in detail.

[0246] After randomization, tumor volume was measured in two dimensions using a caliper twice a week, and the volume was expressed in mm3 using the following formula: V = (L x W x W) / 2, where V is the tumor volume, L is the tumor length (the longest tumor dimension), and W is the tumor width (the longest tumor dimension perpendicular to L). Dosing and tumor and body weight measurements were performed in a laminar flow cabinet. Body weight and tumor volume were measured using Study Director™ software (version 3.1.399.19).

[0247] Fig. 7A Shown are body weight curves of the CC6702 PDX model at different time points after treatment with vehicle or anti-CLDN1 mAb H1L1. Figure 7B Tumor volume growth curves of CC6702 PDX model at different time points after treatment with vehicle or anti-CLDN1 mAb H1L1 are shown. Due to vigorous tumor growth, the control group was terminated after day 14 for humanitarian considerations. Figure 7Bshowed that the anti-CLDN1 mAb H1L1 suppressed tumor growth in a cholangiocarcinoma PDX model compared with the control treatment group.

[0248] ***

[0249] Unless otherwise indicated, the practice of the present disclosure will employ conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art. Such techniques are fully explained in the literature.

[0250] All references cited above and all references cited herein are incorporated by reference in their entirety.

[0251] Any examples provided herein are provided by way of illustration and not limitation.

Claims

1. A method of treating cholangiocarcinoma (CCA) in a human subject in need thereof, the method comprising administering to the human subject a therapeutically effective amount of an anti-Claudin-1 antibody.

2. The method of claim 1, wherein the CCA is an intrahepatic CCA. The method of claim 1 , wherein the CCA is a perihilar CCA. The method of claim 1 , wherein the CCA is a distal CCA.

5. The method of claim 1, wherein the CCA is mixed or mixed hepatocellular cholangiocarcinoma (cHCC-CCA). 6 . The method of any one of claims 1 to 5 , wherein Claudin-1 (CLDN1) is overexpressed in the human subject compared to the expression level in a normal subject.

7. The method of any one of claims 1 to 6, wherein the anti-Claudin-1 antibody comprises six complementarity determining regions (CDRs) of an anti-Claudin-1 monoclonal antibody secreted by a hybridoma cell line deposited at DSMZ on July 29, 2008 under accession number DSMACC2938.

8. The method of any one of claims 1 to 7, wherein the anti-Claudin-1 antibody comprises a heavy chain variable domain complementarity determining region (CDR) H1 comprising the amino acid sequence shown in SEQ ID NO: 5, a CDR H2 comprising the amino acid sequence shown in SEQ ID NO: 6, and a CDR H3 comprising the amino acid sequence shown in SEQ ID NO: 7, and / or a light chain variable domain complementarity determining region (CDR) L1 comprising the amino acid sequence shown in SEQ ID NO: 8, a CDR L2 comprising the amino acid sequence GAS, and a CDR L3 comprising the amino acid sequence shown in SEQ ID NO:

10.

9. The method of any one of claims 1 to 8, wherein the anti-Claudin-1 antibody is humanized. 10 . The method of any one of claims 1 to 9 , wherein the anti-Claudin-1 antibody comprises a VH comprising the amino acid sequence shown in SEQ ID NO: 3 or SEQ ID NO:

13. 11 . The method of any one of claims 1 to 10 , wherein the anti-Claudin-1 antibody comprises a VL comprising the amino acid sequence shown in SEQ ID NO: 4 or SEQ ID NO:

14. 12 . The method of any one of claims 1 to 11 , wherein the anti-Claudin-1 antibody comprises a VH comprising the amino acid sequence shown in SEQ ID NO: 3; and a VL comprising the amino acid sequence shown in SEQ ID NO:

4. 13 . The method of any one of claims 1 to 12 , wherein the anti-Claudin-1 antibody comprises a VH comprising the amino acid sequence shown in SEQ ID NO: 13; and a VL comprising the amino acid sequence shown in SEQ ID NO:

14. 14 . The method of any one of claims 1 to 13 , wherein the anti-Claudin-1 antibody comprises a heavy chain comprising the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 15; and a light chain comprising the amino acid sequence shown in SEQ ID NO:

2.

15. The method of any one of claims 1 to 14, wherein the anti-Claudin-1 antibody is administered intratumorally, intravenously, intraperitoneally, intramuscularly, intrathecally, or subcutaneously.

16. An anti-Claudin-1 antibody or a pharmaceutical composition thereof for use in a method of treating cholangiocarcinoma (CCA) in a human subject, the method comprising administering an effective amount of the anti-Claudin-1 antibody or a pharmaceutical composition thereof to the human subject.

17. The use according to claim 16, wherein the CCA is intrahepatic CCA.

18. The use according to claim 16, wherein the CCA is a perihilar CCA.

19. The use of claim 16, wherein the CCA is a distal CCA.

20. The use according to claim 16, wherein the CCA is mixed or mixed hepatocellular cholangiocarcinoma (cHCC-CCA).

21. The use according to any one of claims 16 to 20, wherein the anti-Claudin-1 antibody comprises six complementary determining regions (CDRs) of an anti-Claudin-1 monoclonal antibody secreted by a hybridoma cell line deposited with DSMZ on July 29, 2008 under the accession number DSMACC2938.

22. The use according to any one of claims 16 to 21, wherein the anti-Claudin-1 antibody comprises a heavy chain variable domain complementarity determining region (CDR) H1 comprising the amino acid sequence shown in SEQ ID NO: 5, a CDR H2 comprising the amino acid sequence shown in SEQ ID NO: 6, and a CDR H3 comprising the amino acid sequence shown in SEQ ID NO: 7, and / or a light chain variable domain complementarity determining region (CDR) L1 comprising the amino acid sequence shown in SEQ ID NO: 8, a CDR L2 comprising the amino acid sequence GAS, and a CDR L3 comprising the amino acid sequence shown in SEQ ID NO:

10.

23. The use according to any one of claims 16 to 22, wherein the anti-Claudin-1 antibody is humanized. 24 . The use according to any one of claims 16 to 23, wherein the anti-Claudin-1 antibody comprises a VH comprising the amino acid sequence shown in SEQ ID NO: 3 or SEQ ID NO:

13.

25. The use according to any one of claims 16 to 24, wherein the anti-Claudin-1 antibody comprises a VL comprising the amino acid sequence shown in SEQ ID NO:4 or SEQ ID NO:

14. 26 . The use according to any one of claims 16 to 25 , wherein the anti-Claudin-1 antibody comprises a VH comprising the amino acid sequence shown in SEQ ID NO: 3; and a VL comprising the amino acid sequence shown in SEQ ID NO:

4. 27 . The use according to any one of claims 16 to 26, wherein the anti-Claudin-1 antibody comprises a VH comprising the amino acid sequence shown in SEQ ID NO: 13; and a VL comprising the amino acid sequence shown in SEQ ID NO:

14.

28. The use according to any one of claims 16 to 27, wherein the anti-Claudin-1 antibody comprises a heavy chain comprising the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 15; and a light chain comprising the amino acid sequence shown in SEQ ID NO:

2.

29. The use according to any one of claims 16 to 28, wherein the anti-Claudin-1 antibody is administered intratumorally, intravenously, intraperitoneally, intramuscularly, intrathecally or subcutaneously.

30. The use of claim 16, further comprising administering a chemotherapeutic agent to the human subject.

31. The use as claimed in claim 16, wherein the CCA contains the following gene mutations: isocitrate dehydrogenase (NADP(+)) 1 (IDH1), isocitrate dehydrogenase (NADP(+)) 2 (IDH2), BRCA1 associated protein 1 (BAP1), fibroblast growth factor receptor 2 (FGFR2), Kirsten rat sarcoma viral oncogene homolog (KRAS), polybrominated 1 (PBRM1), AT-rich interaction domain 1A (ARID1A), phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA), Ephrin type A receptor 2 (EPHA2), cyclin-dependent kinase inhibitor 2A (CDKN2A), tumor protein P53 (TP53), SMAD family member 4 (SMAD4), transforming growth factor beta receptor 2 (TGFBR2).

32. The method of claim 1, further comprising administering a chemotherapeutic drug to the human subject in need thereof.

33. The method of claim 30, wherein the chemotherapeutic agent is gemcitabine.

34. The method of claim 30, wherein the chemotherapeutic agent is cisplatin.

35. The use of claim 32, wherein the chemotherapeutic agent is gemcitabine.

36. The use of claim 32, wherein the chemotherapeutic agent is cisplatin.

37. The method of claim 1, wherein the CCA is metastatic.

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