Use of an anti-Claudin-1 antibody for treating cholangiocarcinoma

Abstract

The present disclosure relates to 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.

Description

Technical Field

[0001] Cross - reference to Related Applications This application claims the benefit of priority of U.S. Provisional Application No. 63 / 365,681, filed on September 15, 2014, which is hereby incorporated by reference in its entirety.

[0002] Reference to a Sequence Listing Submitted Electronically via EFS - Web The content 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 is hereby incorporated by reference in its entirety.

[0003] According to various aspects of the present disclosure, the present disclosure relates to methods of treating cholangiocarcinoma (CCA).

Background Art

[0004] Cholangiocarcinoma (CCA) is a progressive adenocarcinoma of the hepatobiliary tract (Brindley et al., 7 Nat Rev Disrimers 1-17 (2021)). CCA that occurs along the biliary tree and / or within the liver parenchyma is considered a heterogeneous malignancy that is mainly divided into three major anatomical subtypes, namely intrahepatic CCA (iCCA), perihilar region CCA (pCCA) or distal CCA (dCCA) (Banales et al., 17 Nat Rev Gastroenterol Hepatol 557-588 (2020)). Ranked as the second most common primary malignancy after hepatocellular carcinoma (HCC), CCA accounts 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 considered a rare cancer, the incidence of CCA has increased dramatically over the past decade (Bertuzzo et al., 71(1) J Hepatol. 104-114 (Jul 2019)), ranging from more than 1 to 4 cases per 100,000 in European countries (Al Mahjoub et al., 31(6) Eur J Gastroenterol Hepatol 678-684 (Jun 2019)). This can be partially explained by the fact that risk factors for CCA, such as diabetes, primary sclerosing cholangitis, intrahepatic lithiasis, cirrhosis, hepatitis B and C, are increasing globally (Clements et al., 72(1) J Hepatol. 95-103 (Jan 2020)). In particular, chronic cholangitis and / or biliary stasis have been identified as major factors in CCA initiation, and the prominent roles played by the tumor microenvironment (TME), especially cancer-associated fibroblasts (CAF), tumor-associated macrophages (TAM), inflammatory cytokines and growth factors, are emphasized (Leyva-Illades et al., 1(1) Transl Gastrointest Cancer 71-80 (2012)).These extracellular players intersect with dysregulation of several intracellular signaling pathways that are considered major drivers of cholangiocarcinoma carcinogenesis, 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)).

[0005] CCA is usually diagnosed very late, resulting in the progression of invasive disease, poor treatment response, and the worst prognosis with a median survival of less than two years (Vaquero et al., 13 Nat Rev Gastroenterol Hepatol 261-280 (2016)). Therapeutic hepatectomy is a treatment option for CCA management, but only 25% of patients are eligible due to metastatic or locally advanced tumors, and only 50% of the operated patients achieve a therapeutic or margin-free resection (Nagorney et al., 40 Adv Surg 159-171 (2006)). Standard first-line chemotherapy includes combinations of gemcitabine and cisplatin, in addition to recently approved targeted therapies such as pemigatinib, a fibroblast growth factor receptor inhibitor, andivosidenib, an isocitrate dehydrogenase 1 (IDH1) mutation inhibitor, which improve patient outcomes but respond only in a small subset of patients with advanced and / or metastatic CCA and are associated with significant side effects and potential treatment resistance (Sasaki et al., 10(14) J Clin Med 3108 (2021)).

[0006] There is a growing body of evidence indicating dysregulation of the expression of claudin family members in CCA and their contribution to carcinogenesis (Nemeth et al., 57(2) J Histochem Cytochem 113 - 121 (Feb 2009)). Claudin - 1 (CLDN1) is a transmembrane protein expressed at tight junctions (TJ), but is also expressed non - junctionally, for example, on the basolateral membrane of human hepatocytes, and functions as a cell entry factor for hepatitis C virus (Evans et al., 446 Nature 801 - 805 (2007)). CLDN1 has previously been identified as a mediator and therapeutic target in liver fibrosis and hepatocellular carcinoma (HCC) (WO20161 / 46809A1). Extensive studies in non - human primate and mouse models have not revealed significant toxicity even when high doses of mAb far exceeding therapeutic requirements were repeatedly applied.

[0007] Considering the lack of effective agents and the increasing incidence of this disease, there is an urgent unmet medical need for a new first - line therapeutic approach to treat CCA and improve patient outcomes.

Summary of the Invention

[0008] 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.

[0009] 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 to the human subject an effective amount of the anti - claudin - 1 antibody or a pharmaceutical composition thereof.

[0010] In some aspects, provided herein is a kit for treating a subject suffering from cholangiocarcinoma, the kit comprising a therapeutically effective amount of an anti-Claudin-1 antibody and a package insert comprising instructions for use of the kit.

[0011] In some aspects, provided herein is a pharmaceutical composition for the treatment of cholangiocarcinoma, the pharmaceutical composition comprising a therapeutically effective amount of an anti-Claudin-1 antibody.

[0012] In some aspects, Claudin-1 (CLDN1) is overexpressed in the human subject compared to the expression level in a normal subject.

[0013] In some embodiments, the human subject is further administered chemotherapy. In some aspects, the chemotherapy is gemcitabine. In some aspects, the chemotherapy is cisplatin.

[0014] In some aspects, 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 with DSMZ on July 29, 2008 under the accession number DSM ACC2938.

[0015] 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.

[0016] In some aspects, the anti-Claudin-1 antibody is humanized.

[0017] In some embodiments, the anti-claudin-1 antibody comprises a VH comprising the amino acid sequence set forth in SEQ ID NO: 3 or SEQ ID NO: 13.

[0018] In some embodiments, 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.

[0019] In some embodiments, the anti-claudin-1 antibody comprises a VH comprising the amino acid sequence set forth in SEQ ID NO: 3 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 4.

[0020] In some embodiments, the anti-claudin-1 antibody comprises a VH comprising the amino acid sequence set forth in SEQ ID NO: 13 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 14.

[0021] In some embodiments, the anti-claudin-1 antibody is administered intratumorally, intravenously, intraperitoneally, intramuscularly, intrathecally, or subcutaneously.

[0022] In some embodiments, the CCA is intrahepatic CCA.

[0023] In some embodiments, the CCA is hilar region CCA.

[0024] In some embodiments, the CCA is distal CCA.

[0025] In some embodiments, the CCA is combined or mixed hepatocellular cholangiocarcinoma (cHCC-CCA).

[0026] In some embodiments, the CCA is metastatic.

[0027] In some embodiments, the CCA is treated with chemotherapy such as gemcitabine and cisplatin.

[0028] In some embodiments, the CCA comprises 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), polybromo 1 (PBRM1), AT-rich interactive 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).

[0029] In some embodiments, the methods or uses provided herein further comprise administering a chemotherapeutic agent to a human subject in need thereof.

[0030] In some embodiments, the chemotherapeutic agent is gemcitabine.

[0031] In some embodiments, the chemotherapeutic agent is cisplatin. BRIEF DESCRIPTION OF THE DRAWINGS

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Mode for Carrying Out the Invention

[0053] I. Definitions Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. In case of conflict, this application, including definitions, will control. Unless the context otherwise requires, singular terms shall include pluralities and plural terms shall include singulars. All patents and references cited herein are hereby incorporated by reference in their entirety for all purposes as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

[0054] In practicing or testing the present disclosure, methods and materials similar or equivalent to those described herein can be used, but suitable methods and materials are described below. The materials, methods, and examples are illustrative only and not intended to be limiting. Other features and advantages of the present disclosure will become apparent from the mode for carrying out the invention and the claims.

[0055] To further define the present disclosure, the following terms and definitions are provided.

[0056] The singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. The terms "a" (or "an"), as well as "one or more" and "at least one", are used interchangeably herein. In certain embodiments, the term "a" or "an" means "single". In other embodiments, the term "a" or "an" includes "two or more" or "plural".

[0057] The term "about" is used herein to mean approximately, roughly, generally, or within the region of. When the term "about" is used in conjunction with a numerical range, it modifies the range by extending the boundaries above and below the stated numerical values. Generally, the term "about" is used herein to modify a value that is above and below the stated value by a variation of plus or minus 10%.

[0058] Throughout this disclosure, various aspects of the invention may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, a range of description should be considered to have specifically disclosed all the individual numerical values and all the possible sub-ranges within that range. For example, a range description such as 1-6 should be considered to specifically disclose sub-ranges such as 1-3, 1-4, 1-5, 2-4, 2-6, 3-6, etc., as well as the individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the width of the range. The recited numerical ranges include the boundary values defining the range and each integer within the defined range.

[0059] Units, prefixes, and symbols are shown in the form approved by the International System of Units (SI). Numerical ranges are to include the numbers defining the range. When a range of values is recited, it is to be understood that each integer value intervening between the recited upper and lower limits of that range, and each subrange therebetween, are also specifically disclosed, along with each part thereof. The upper and lower limits of any range may independently be included or excluded within the range, and each range that includes one or the other of these limiting values, or neither of these limiting values, or both of these limiting values, is included in this application. Accordingly, ranges provided herein are to be understood as a shorthand representation of all values within the range, including the recited endpoints. For example, the range of 1 to 10 is to be understood to include any number, combination of numbers, or subrange from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10.

[0060] When values are explicitly recited, it is understood that values that are substantially the same amount or quantity as the recited values are also within the scope of the present disclosure. When combinations are disclosed, each subcombination of the elements of that combination is also specifically disclosed and is within the scope of the present disclosure. Conversely, when different elements or groups of elements are disclosed individually, combinations thereof are also disclosed. When any element of the present disclosure is disclosed as having a plurality of alternatives, examples of that disclosure where each alternative is excluded, either alone or in any combination with other alternatives, are also disclosed herein, and a plurality of elements of the present disclosure may have such exclusions, and all combinations of elements having such exclusions are disclosed herein.

[0061] As used herein, the term "and / or" shall be construed as a specific disclosure of each of two designated features or components, regardless of the presence or absence of the others. Thus, the term "and / or" as used in phrases such as "A and / or B" herein is intended to include "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 include 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).

[0062] The term "treating" or "treatment" as used herein refers to administering a composition to a subject for therapeutic purposes.

[0063] The term "human claudin-1 (or CLDN1)" refers to a protein having the sequence shown in NCBI accession number NP_066924.1, or any naturally occurring variant commonly found in the HCV-permissive human population.

[0064] The term "antibody" as used herein refers to any immunoglobulin containing an antigen-binding site that immunospecifically binds to an antigen. Thus, the term antibody includes not only the entire antibody molecule, but also antibody fragments, as well as variants (including derivatives) of antibodies and antibody fragments insofar as the derivative and fragment maintain the specific binding ability. This term includes monoclonal and polyclonal antibodies. This term also includes any protein having a binding domain that is homologous or mostly homologous to an immunoglobulin binding domain. These proteins may be derived from natural sources or produced partially or wholly synthetically. The term "specifically binds" when used in reference to an antibody refers to an antibody that binds to a given antigen. Typically, an antibody has a binding affinity of at least 1×10 7 M 1It binds with an affinity that is at least two times greater than the affinity for binding to a non-specific antigen (e.g., BSA, casein) and binds to a predetermined antigen.

[0065] As used herein, a "humanized" antibody refers to a chimeric antibody that contains amino acid residues derived from non-human hypervariable regions and amino acid residues derived from human framework (FR) regions. In particular, a humanized antibody contains all or substantially all of at least one, typically two, variable domains, and all or substantially all of the complementarity determining regions (CDRs) are the CDRs of a human antibody. A humanized antibody may optionally also contain at least a portion of an antibody constant region derived from a human antibody. The "humanized form" of an antibody, e.g., a non-human antibody, refers to the antibody that has been humanized.

[0066] It is understood that when an embodiment is described herein using the language "comprising", similar embodiments are provided unless they are necessarily described using the terms "consisting of" and / or "consisting essentially of".

[0067] As used herein, the term "administering" refers to physically introducing a composition comprising a therapeutic agent (e.g., an anti-claudin-1 antibody) to a subject using any of a variety of methods and delivery systems known to those of skill in the art. Routes of administration include, for example, intravenous, intramuscular, subcutaneous, intraperitoneal, intraspinal, or other parenteral routes of administration by injection or infusion. The phrase "parenteral administration" as used herein means a route of administration other than enteral and topical administration, usually by injection, and includes intravenous injection and infusion, intramuscular injection and infusion, intraarterial injection and infusion, intrathecal injection and infusion, intralymphatic injection and infusion, intralesional injection and infusion, intracapsular injection and infusion, intraorbital injection and infusion, intracardiac injection and infusion, intradermal injection and infusion, intraperitoneal injection and infusion, intratracheal injection and infusion, subcutaneous injection and infusion, subepidermal injection and infusion, intraarticular injection and infusion, subcapsular injection and infusion, subdural injection and infusion, intrathecal injection and infusion, epidural injection and infusion, intrasternal injection and infusion, and in vivo electroporation, but is not limited thereto. Other parenteral routes of administration include topical, epithelial, or mucosal routes of administration, for example, intranasal, intravaginal, rectal, sublingual, or topical routes of administration. Administration can also be carried out, for example, once, multiple times, and / or over one or more extended periods of time.

[0068] The term "effective amount" refers to an amount of an agent that produces a desired biological, therapeutic, and / or prophylactic result. The result can be a reduction, improvement, alleviation, decrease, delay, and / or mitigation of one or more of the symptoms, signs, or causes of a disease, or any other desirable change in a biological system. With respect to solid tumors, an effective amount includes an amount sufficient to cause shrinkage of the tumor and / or to reduce the rate of tumor growth (e.g., to inhibit tumor growth), or to prevent or delay other undesirable cell proliferation. In some embodiments, the effective amount is an amount sufficient to delay the onset of a tumor. In some embodiments, the effective amount is an amount sufficient to prevent or delay the recurrence of a tumor. The effective amount can be administered in one or more administrations. An effective amount of a drug or composition can (i) decrease the number of cancer cells, (ii) decrease the tumor size, (iii) prevent, delay, slow to some extent, or stop cancer cell infiltration into peripheral organs, (iv) prevent tumor metastasis (i.e., delay or stop to some extent), (v) inhibit tumor growth, (vi) prevent or delay the occurrence and / or recurrence of a tumor, and / or (vii) reduce to some extent one or more of the symptoms associated with cancer. In one example, the "effective amount" is the amount of an anti-claudin-1 antibody that has been clinically proven to result in a significant decrease in cancer or a slowdown in cancer progression, such as in advanced solid tumors.

[0069] As used herein, "patient" includes any patient suffering from cancer (e.g., fibrosarcoma). The terms "subject" and "patient" are used interchangeably herein.

[0070] II. Anti-Claudin-1 Antibody The present invention relates to the use of an anti-claudin-1 antibody for the treatment of cholangiocarcinoma in a human subject in need thereof. In some embodiments, disclosed herein is a method for 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.

[0071] CLDN1 is a transmembrane protein with two major roles: (1) contributing to the barrier function by tight junctions together with other proteins, and (2) being expressed outside of the tight junctions on the basolateral membrane of epithelial cells, and CLDN1 has been shown to mediate oncogenic signaling, epithelial-mesenchymal transition (EMT), and cell fate. Furthermore, CLDN1 has been shown to be expressed also by non-epithelial cells such as myofibroblasts in the liver, lung, and kidney.

[0072] Antibodies against human Claudin-1 have been previously described to treat hepatitis C virus infection, hepatocellular carcinoma, and certain fibrotic diseases such as pulmonary fibrosis (see WO2010 / 034812, WO2016 / 146809, and WO2021 / 094469). Anti-Claudin-1 antibodies that can be used in the practice of the present invention include any antibodies produced against Claudin-1. Examples are disclosed in WO2010 / 034812 and WO2017 / 162678.

[0073] Other examples of suitable anti-Claudin-1 antibodies can be found in European Patent EP1167389, US Patent 6,627,439, International Patent Application Publication WO2014 / 132307, International Patent Application Publications WO2015 / 014659 and WO2015 / 014357, and Yamashita et al., 353(1) J. Pharmacol. Exp. Ther. 112-118 (2015).

[0074] The anti-Claudin-1 antibodies suitable for use in the present invention may be polyclonal antibodies or monoclonal antibodies.

[0075] An anti-claudin-1 antibody suitable for use according to the present invention may also be "humanized". The sequence differences between rodent antibodies and human sequences can be minimized by site-directed mutagenesis of individual residues, transplantation of entire regions, or chemical synthesis to replace residues that differ from the human sequence. Humanized antibodies can also be produced using recombinant methods. In the humanized form of the 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. Minor additions, deletions, insertions, substitutions, or modifications of amino acids are tolerated as long as they do not significantly alter the biological activity of the resulting antibody. Suitable human "replacement" immunoglobulin molecules include IgG1, IgG2, IgG2a, IgG2b, IgG3, IgG4, IgA, IgM, IgD, or IgE molecules, and fragments thereof.

[0076] In some embodiments, the humanized anti-claudin-1 antibody for use according to the present invention is the one previously described in WO2017 / 162678. Exemplary sequences of the antibodies or antigen-binding fragments provided herein are listed in Table 1.

Table 1-1

Table 1-2

[0077] In some embodiments, the anti-claudin-1 antibody comprises complementarity-determining region (CDR) H1 comprising the amino acid sequence shown in SEQ ID NO: 5, CDR H2 comprising the amino acid sequence shown in SEQ ID NO: 6, and CDR H3 comprising the amino acid sequence shown in SEQ ID NO: 7.

[0078] In some embodiments, the anti-claudin-1 antibody comprises complementarity-determining region (CDR) L1 comprising the amino acid sequence shown in SEQ ID NO: 8, CDR L2 comprising the amino acid sequence shown as GAS, and CDR L3 comprising the amino acid sequence shown in SEQ ID NO: 10.

[0079] In some embodiments, the complementarity determining regions (CDRs) disclosed herein are defined according to IMGT®. However, it is understood that other methods of defining CDRs in the art may also be used.

[0080] In some embodiments, the six complementarity determining regions (CDRs) of the anti-claudin-1 antibody are the same as those of the anti-claudin-1 monoclonal antibody secreted by the hybridoma cell line deposited with DSMZ on July 29, 2008 under the accession number DSM ACC2938.

[0081]

[0082] In some embodiments, the anti-claudin-1 antibody comprises a VH comprising the amino acid sequence set forth in SEQ ID NO: 3 or SEQ ID NO: 13.

[0083] In some embodiments, 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.

[0084]

[0085] ​​In some embodiments, 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.

[0086] In some embodiments, 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.

[0087] In some embodiments, 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.

[0088] In some embodiments, 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.

[0089] In some embodiments, 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: 14.

[0090] In some embodiments, 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.

[0091] In some embodiments, 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.

[0092] In some embodiments, 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.

[0093] In some embodiments, 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.

[0094] In some embodiments, 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.

[0095] In some embodiments, 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.

[0096] In some embodiments, 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.

[0097] In some embodiments, 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.

[0098] In some embodiments, the heavy and light chains of the anti-claudin-1 antibody are the same as those of the anti-claudin-1 monoclonal antibody secreted by the hybridoma cell line deposited with DSMZ on July 29, 2008 under the accession number DSM ACC2938.

[0099] In some embodiments, 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.

[0100] In some embodiments, 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.

[0101] In some embodiments, 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.

[0102] In some embodiments, the anti-claudin-1 antibody comprises a light chain comprising the amino acid sequence set forth in SEQ ID NO: 2.

[0103] In some embodiments, 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.

[0104] In some embodiments, 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.

[0105] In some embodiments, 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, and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 2.

[0106] In some embodiments, 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.

[0107] In some embodiments, 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.

[0108] In some embodiments, the anti-claudin-1 antibody comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 11.

[0109] In some embodiments, 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.

[0110] In some embodiments, the anti-claudin-1 antibody comprises a heavy chain comprising an amino acid sequence having at least 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.

[0111] In some embodiments, the anti-claudin-1 antibody comprises a light chain comprising the amino acid sequence set forth in SEQ ID NO: 12.

[0112] In some embodiments, 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.

[0113] In some embodiments, 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.

[0114] In some embodiments, the anti-claudin-1 antibody comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 11, and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 12.

[0115] In some embodiments, 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.

[0116] In some embodiments, 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.

[0117] The humanized anti-claudin-1 antibody can be a full monoclonal antibody having an isotype selected from the group consisting of IgG1, IgG2, IgG3, and IgG4. Alternatively, the humanized anti-claudin-1 antibody can 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.

[0118] An anti-claudin-1 antibody (or a biologically active variant or fragment thereof) suitable for use according to the present invention may be functionally linked to one or more other molecular entities (e.g., by chemical bonding, gene fusion, non-covalent bonding or other means). 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 attached to a position on the antibody molecule that does not interfere with the binding properties of the resulting conjugate, e.g., a position that is not involved in the specific binding of the antibody to its target.

[0119] In some embodiments, the anti-claudin-1 antibodies described herein target extracellular loop 1 of claudin-1 that is exposed outside of the tight junction of the basolateral membrane of epithelial cells.

[0120] The antibody molecule and the molecular entity may be directly covalently bonded to each other. Alternatively, the antibody molecule and the molecular entity may be covalently bonded to each other via a linker group. This can be achieved by using any of a variety of well-known stable bifunctional agents in the art, including homofunctional and heterofunctional linkers.

[0121] In some embodiments, an anti-claudin-1 antibody (or a biologically active fragment thereof) for use according to the present invention is conjugated to a detectable agent. Any of a variety of detectable agents can be used, including but not limited to various ligands, radionuclides (e.g., 3 H, 125 I, 131I), Fluorescent dyes (e.g., fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthalaldehyde, and fluorescamine, etc.), chemiluminescent agents (e.g., luciferin, luciferase, aequorin), microparticles (e.g., quantum dots, nanocrystals, phosphors, etc.), enzymes (e.g., enzymes used in ELISA, i.e., horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase, etc.), colorimetric labels, magnetic labels, and biotin, digoxigenin, or other haptens and proteins such as antiserum or monoclonal antibodies that are available.

[0122] Other molecular entities that can be conjugated to the anti-claudin-1 antibody (or its biologically active fragment) of the present invention include, but are not limited to, linear or branched hydrophilic polymer groups, fatty acid groups, or fatty acid ester groups.

[0123] Therefore, in the practice of the present invention, the anti-claudin-1 antibody can be used in the form of a full-length antibody, its biologically active variant or its fragment, a chimeric antibody, a humanized antibody, and an antibody-derived molecule containing at least one complementarity-determining region (CDR) from either the heavy chain or the light chain variable region of the anti-claudin-1 antibody, which includes Fab fragments, F(ab’)2 fragments, Fd fragments, Fabc fragments, Sc antibodies (single-chain antibodies), diabodies, individual antibody light chains, individual antibody heavy chains, chimeric fusions between antibody chains and other molecules, and antibody conjugates such as antibodies conjugated to therapeutic agents or detectable drugs. Preferably, the anti-claudin-1 antibody-related molecule according to the present invention retains the ability of the antibody to bind to its antigen, particularly the extracellular domain of claudin-1.

[0124] III. Cholangiocarcinoma Cholangiocarcinoma (CCA) is a highly lethal epithelial malignancy that can occur anywhere along the biliary tree and / or within the hepatic parenchyma. CCA exhibits features of cholangiocyte differentiation and can arise from the epithelial cells that line the inside of the bile ducts, called cholangiocytes. However, these cancers can also originate from the peribiliary glands and hepatocytes, depending on the underlying liver disease and location. CCA is heterogeneous and is best classified as intrahepatic CCA (iCCA), perihilar region CCA (pCCA), or distal CCA (dCCA) according to the primary anatomical subtype. iCCA is located proximal to the secondary bile ducts within the hepatic parenchyma, pCCA is located between the secondary bile ducts and the insertion of the cystic duct into the common bile duct, and dCCA is limited to the portion of the common bile duct below the insertion of the cystic duct. The true incidence rates of pCCA and iCCA are unknown because in some national databases, pCCA is grossly misclassified as iCCA. Furthermore, improvements in diagnostic capabilities have made it possible to better clinically distinguish cancers of unknown origin from iCCA. Due to these factors, the incidence of iCCA has been reported to have increased over the past 20 or 30 years. Each of the anatomical subtypes is characterized by distinct genetic abnormalities, clinical symptoms, 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 carcinoma or clear cell carcinoma are rare. These cancers are highly fibrotic and are embedded within a dense network of inflammatory cells and matrix called the tumor immune microenvironment. The epidemiology of these cancers varies worldwide. Infections by certain trematodes (parasites, commonly called flukes) are the main cause of CCA in some regions. Fluke-related CCA may have specific etiologies, particularly genetic abnormalities, but the diagnosis and management are not different from non-fluke-related CCA. In the Western world, most CCA patients do not have identifiable risk factors, except for a subset with primary sclerosing cholangitis (PSC) (see Brindley et al., 7 Nat Rev Disrimers 1-17 (2021)).

[0125] In some embodiments, the CCA is composite or combined hepatocellular cholangiocarcinoma (cHCC-CCA). cHCC-CCA is a distinct type of primary liver cancer that shares the distinct phenotypic features of both hepatocellular carcinoma (HCC) and cholangiocarcinoma. cHCC-CCA is a rare and invasive primary malignant liver tumor with marked histological and biological heterogeneity. It exhibits a more aggressive behavior and poor survival rate compared to either HCC or cholangiocarcinoma alone. The World Health Organization classifies cHCC-CCA into two major types, namely, the classical type (characterized by the presence of mixed areas of typical HCC and CCA, as well as transitional areas with intermediate forms of both types) and the type with stem cell features (which is further divided into subtypes that are not common, typical, intermediate, and cholangiocytic). Specific histopathological criteria for the definitive diagnosis of cHCC-CCA have been established, which require the close admixture of fully differentiated hepatocellular carcinoma and CCA components, and the simultaneous identification of transitional areas containing cells with intermediate forms. This differentiates it from HCC and CCA seen in the same liver lobe representing collision tumors. Gene panels have revealed that mutations in the KRAS, ARID1A, TERT promoter, and TP53 genes are associated with different clinical phenotypes of cHCC-CCA (Stravrka et al., 6 J Hepatocell Carcinoma 11-21 (2018)).

[0126] In some embodiments, cholangiocarcinoma is treated with chemotherapy such as cisplatin and gemcitabine.

[0127] IV. Methods of Use The methods of the present invention can be achieved 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 involve administering an effective amount of an anti-claudin-1 antibody, or a biologically active fragment thereof, or its pharmaceutical composition to a subject in need thereof (i.e., a subject having a fibrotic tumor). Administration can be performed using any of the administration methods known to those of skill in the art (see below).

[0128] The present disclosure provides a method for 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.

[0129] In some embodiments, provided herein is an anti-claudin-1 antibody or a pharmaceutical composition thereof for use in a method for treating cholangiocarcinoma (CCA) in a human subject, the method comprising administering to the human subject an effective amount of the anti-claudin-1 antibody or a pharmaceutical composition thereof.

[0130] In some embodiments, the CCA is intrahepatic CCA.

[0131] In some embodiments, the CCA is hilar region CCA.

[0132] In some embodiments, the CCA is distal CCA.

[0133] In some embodiments, the CCA is combined or mixed hepatocellular cholangiocarcinoma (cHCC-CCA).

[0134] In some embodiments, 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), polybromo 1 (PBRM1), AT-rich interactive 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).

[0135] In some embodiments, Claudin-1 (CLDN1) is overexpressed in the human subject as compared to the expression level in a normal subject.

[0136] In some embodiments, Claudin-1 (CLDN1) is overexpressed in a human subject treated with chemotherapy.

[0137] In some embodiments, the anti-Claudin-1 antibody comprises the six complementarity-determining regions (CDRs) of an anti-Claudin-1 monoclonal antibody secreted by a hybridoma cell line deposited with DSMZ on July 29, 2008 under accession number DSM ACC2938.

[0138] In some embodiments, the anti-Claudin-1 antibody is humanized.

[0139] In some embodiments, the anti-Claudin-1 antibody comprises a VH comprising the amino acid sequence shown in SEQ ID NO: 3 or SEQ ID NO: 13.

[0140] In some embodiments, 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.

[0141] In some embodiments, 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.

[0142] In some embodiments, 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.

[0143] In some embodiments, 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.

[0144] In some embodiments, 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.

[0145] In some embodiments, 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.

[0146] In some embodiments, 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: 14.

[0147] In some embodiments, 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.

[0148] In some embodiments, 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.

[0149] In some embodiments, 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.

[0150] In some embodiments, 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.

[0151] In some embodiments, 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.

[0152] In some embodiments, 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.

[0153] In some embodiments, 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.

[0154] In some embodiments, 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.

[0155] In some embodiments, 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.

[0156] In some embodiments, 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.

[0157] In some embodiments, the anti-claudin-1 antibody is administered intratumorally, intravenously, intraperitoneally, intramuscularly, intrathecally, or subcutaneously.

[0158] In some embodiments, the method further comprises administering a chemotherapeutic agent.

[0159] In some embodiments, the chemotherapeutic agent is cisplatin.

[0160] In some embodiments, the chemotherapeutic agent is gemcitabine.

[0161] V. Administration The anti-claudin-1 antibody, or a biologically active fragment thereof (optionally, after formulation with one or more suitable pharmaceutically acceptable carriers or excipients), can be administered to a subject in need thereof at a desired dosage by any suitable route. Various delivery systems are known, including tablets, capsules, injection solutions, encapsulation in liposomes, microparticles, microcapsules, etc., and can be used to administer the antibody. Routes of administration include, but are not limited to, cutaneous, 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 absorption through an epithelial or mucosal layer (e.g., oral mucosa, bronchial mucosa, rectal and intestinal mucosa, etc.) by any convenient or other suitable route, such as infusion or bolus injection. Administration may be systemic or local.

[0162] In some embodiments, the anti-claudin-1 antibody is administered intratumorally, intravenously, intraperitoneally, intramuscularly, intrathecally, or subcutaneously.

[0163] An anti-claudin-1 antibody, or a biologically active fragment thereof (optionally, after formulation with one or more suitable pharmaceutically acceptable carriers or excipients), is administered in an amount effective for the intended purpose. The route of administration, formulation, and dosage are determined by the desired therapeutic effect, the severity of the condition to be treated if already present, the presence of any infectious disease, the age, sex, weight, and general condition of the patient, as well as the potency, bioavailability, and in vivo half-life of the antibody or composition used, the use (or non-use) of combination 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 from studies using animal models (e.g., non-human primates or rodents). Adjustment of the dosage to achieve maximum effectiveness based on these or other methods is well known in the art and within the capabilities of a trained physician. As studies are conducted using the anti-claudin-1 antibody, additional information regarding appropriate dosage levels and treatment durations will become apparent.

[0164] VI. Pharmaceutical Compositions As described above, the anti-claudin-1 antibody (and related molecules) can be administered by itself or as a pharmaceutical composition. Accordingly, the present invention provides a pharmaceutical composition comprising an effective amount of an anti-claudin-1 antibody described herein, or a biologically active fragment thereof, and at least one pharmaceutically acceptable carrier or excipient.

[0165] In some embodiments, provided herein is a pharmaceutical composition for the treatment of cholangiocarcinoma (CCA), comprising any of the anti-claudin-1 antibodies disclosed herein in a therapeutically effective amount.

[0166] In some embodiments, the CCA is intrahepatic CCA.

[0167] In some embodiments, the CCA is hilar region CCA.

[0168] In some embodiments, the CCA is a distal CCA.

[0169] In some embodiments, the CCA is a composite or mixed hepatocellular cholangiocarcinoma (cHCC-CCA).

[0170] The pharmaceutical composition can be administered in any amount effective to achieve the desired prophylactic and / or therapeutic effect and by any route of administration. 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.

[0171] The pharmaceutical composition of the present invention can be formulated in unit dosage forms for ease of administration and uniformity of dosage. It will be understood that the total daily dosage of the composition will be determined by the attending physician within the scope of sound medical judgment.

[0172] VII. Kit In another aspect, the present 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 components of the pharmaceutical composition of the present invention, enabling the administration of an anti-claudin-1 antibody, or a biologically active fragment thereof.

[0173] The various components of the pharmaceutical pack or kit can be supplied in solid (e.g., lyophilized) or liquid form. Each component is generally dispensed into its respective container or is preferably provided in concentrated form. The pharmaceutical pack or kit can include a medium for reconstituting the lyophilized components. The individual containers of the kit are preferably maintained in a sealed state for commercial sale.

[0174] A notice or attachment in a form specified by a government agency that regulates the manufacture, use, or sale of pharmaceuticals or biological products may be optionally attached to the container(s), and this notice represents the approval by the government agency for the manufacture, use, or sale for administration to humans. The notice of the attachment may include instructions for using the pharmaceutical composition according to the treatment methods disclosed herein.

[0175] Identifiers, such as barcodes, radio frequencies, ID tags, etc., may be present in or on the kit. The identifier may be used to uniquely identify the kit for purposes such as quality control, inventory management, tracking of movement between workstations, etc.

[0176] In some aspects, provided herein is a kit for treating a subject suffering from cholangiocarcinoma (CCA), the kit comprising a therapeutically effective amount of any of the anti-Claudin-1 antibodies disclosed herein and an attachment comprising instructions for use of the kit.

[0177] In some aspects, the CCA is intrahepatic CCA.

[0178] In some aspects, the CCA is hilar region CCA.

[0179] In some aspects, the CCA is distal CCA.

[0180] In some aspects, the CCA is combined or mixed hepatocellular cholangiocarcinoma (cHCC-CCA).

Examples

[0181] The following examples are illustrative and do not limit the scope of the claimed aspects.

[0182] Example 1. CLDN1 is highly expressed in intrahepatic and extrahepatic CCA tumors and correlates with tumor stemness 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 obtained from the Genomic Data Commons Data Portal (portal.gdc.cancer.gov) revealed that CLDN1 was highly expressed in extrahepatic CCA (GSE132305, p < 0.0001, t-test, Figure 1A) and proliferative intrahepatic CCA (GSE32225, p = 0.03, Mann-Whitney test, Figure 1B) compared to non-tumorous bile duct samples. Interestingly, CLDN1 expression was significantly higher in proliferative iCCA compared to inflammatory iCCA (GSE32225, p = 0.02, t-test, Figure 1C).

[0183] To examine the correlation between CLDN1 expression and common mutations in CCA patients, we used publicly available databases (Jusakul et al., 7(10)Cancer Discov 1116 - 1135 (Oct 2017)) to analyze CLDN1 expression at the transcriptional level across various common genetic mutations described in the tumors of CCA patients. These mutations included isocitrate dehydrogenase (NADP(+)) 1 (IDH1), isocitrate dehydrogenase (NADP(+)) 2 (IDH2), BRCA1-associated protein 1 (BAP1), fibroblast growth factor receptor 2 (FGFR2), Kirsten rat sarcoma virus oncogene homolog (KRAS), polybromo 1 (PBRM1), AT-rich interactive 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).

[0184] Comprehensive computational analysis revealed that CLDN1 was robustly expressed across various CCA gene mutations (Figure 1D). CLDN1 expression was highest in tumors with IDH1 and BAP1 gene mutations compared to other mutations (GSE89747 and GSE89748, **p<0.01, Mann-Whitney test, Figure 1D). CLDN1 expression was lower in tumors with TP53, SMAD4, and TGFBR2 mutations compared to other mutations (GSE89747 and GSE89748, **p<0.01, *p<0.05, Mann-Whitney test, Figure 1D). The frequency of driver gene mutations varies depending on the etiology of CCA, and mutations in BAP1 and IDH1 are highly abundant in non-fluke-related CCA, while TP53 and SMAD4 are highly abundant in fluke-related CCA (Brindley et al.,7(65)Nat Rev Dis Primes(Sep 2021)).

[0185] In summary, the upregulation of CLDN1 expression in both intrahepatic and extrahepatic CCA tumors highlights its potential involvement in the etiology and outcome of CCA disease, suggesting that CLDN1 is a therapeutic target for the treatment of CCA.

[0186] Example 2. CLDN1 mAb H3L3 inhibits tumor growth in both cell line-derived xenograft (CDX) mouse models and patient-derived xenograft (PDX) mouse models To evaluate the in vivo antitumor-forming activity of an anti-CLDN1 monoclonal (CLDN1 mAb H3L3) antibody in CCA, the effect of the anti-CLDN1 mAb on tumor growth was examined in cell line-derived xenograft (CDX) mouse models of both intrahepatic CCA and extrahepatic CCA.

[0187] Non-obese diabetic Rag1 - / - IL2Rgc - / - (NRG), 5×10 6 individual HuCC-A1 (intrahepatic CCA) or EGI-1 (extrahepatic CCA) cell lines were subcutaneously injected. Non-obese diabetic Prkdc scid L2Rg - / - (NSG), 5×106 Individual HuCC-T1 (intrahepatic CCA) cell lines were subcutaneously injected. When the average tumor volume reached 40 - 50 mm 3 , the 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 digital calipers, and tumor volume was calculated using the formula of 1 / 2 (length × width 2 ) (Figure 2A).

[0188] CLDN1 mAb H3L3 significantly reduced tumor growth in three CDX models (Figure 2B - 2G). In HuCC-A1 CDX mice, a strong and robust inhibition of tumor growth over time was observed (p < 0.001, Mann - Whitney test), which was highly reflected by the macroscopic appearance of the explanted tumors (Figure 2E - 2F). Furthermore, this antibody delayed tumor growth in EGI-1 CDX (p < 0.05, Mann - Whitney test, Figure 2B) and HuCC-T1 CDX mice (p < 0.05, Mann - Whitney test, Figure 2G). Additionally, anti-CLDN1 mAb H3L3 showed a significant decrease in skin ulcers induced by tumors in the EGI-1 model (p < 0.01, Mann - Whitney test, Figure 2C - 2D), suggesting an effect on tumor cell infiltration.

[0189] On the other hand, molecular drivers and treatment responses vary greatly among different CCA subtypes and patients (Banales et al., 17 Nat Rev Gastroenterol Hepatol 557 - 588 (2020)). In this regard, PDX mouse models have been shown to partially recapitulate tumor heterogeneity and are currently one of the most widely used in vivo systems for testing cancer therapeutics and predicting clinical outcomes (Xu et al., 17(1) Oncol Lett 3 - 10 (Jan 2019)). Therefore, a PDX mouse model was established to evaluate the anti-tumor effect and efficacy of anti-CLDN1 mAb H3L3.

[0190] Non-obese diabetic Rag1- / - IL2Rgc - / - (NRG) was subcutaneously injected with fresh tumor tissue cut into small pieces (approximately 2 - 3 mm in diameter). When the average tumor volume reached 40 mm 3 , the 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 vehicle control (PBS, intraperitoneally, once a week, n = 11 mice). Tumor growth was monitored using digital calipers, and tumor volume was calculated using the formula 1 / 2 (length × width 2 ).

[0191] Consistent with the results obtained in CDX mice, treatment with anti-CLDN1 mAb H3L3 significantly and markedly retarded tumor growth in the PDX model (p < 0.05, Mann - Whitney test, Figure 2H). Overall, these data demonstrated the antitumor effect of non - conjugated CLDN1 - targeting mAb in both cell - line - derived and patient - derived tumor models, providing a robust pre - clinical proof - of - concept for CLDN1 mAb for CCA treatment.

[0192] Example 3. Anti - CLDN1 antibody inhibits migration and invasion of CCA cell lines In a cell - based wound - healing assay using an extrahepatic CCA cell line (KKU100) and an intrahepatic CCA cell line (HuCC - A1), the effect of anti - CLDN1 mAb H3L3 on cell migration was evaluated.

[0193] In the wound - healing assay, 1.5×10 5Individual CCA cells were co-cultured with LX2 at a 1:1 ratio in 12-well plates (Corning) to recapitulate the stromal structures seen in CCA tumors. The co-cultures were treated with 20 μg / mL of isotype control mAb or 20 μg / mL of anti-CLDN1 mAb H3L3 for 3 days. On day 3, a wound was created 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 re-treated with 20 μg / mL of isotype control mAb or 20 μg / mL of anti-CLDN1 mAb H3L3. The plates were incubated at 37 °C, and the wounds were imaged at 0, 24, and 48 hours. The relative percentage of wound closure was measured using ImageJ. Each experiment was independently repeated at least 3 times.

[0194] Treatment with anti-CLDN1 mAb H3L3 significantly and specifically decreased the migration of CCA cells in the wound healing assay compared to treatment with the isotype antibody (p = 0.02, Mann-Whitney test, Figures 3A - 3C).

[0195] In additional studies, 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 of isotype control mAb or 20 μg / mL of anti-CLDN1 mAb H3L3 for 3 days (Colpitts et al., 67(4) Gut 736 - 745 (Mar 2017)). Then, 0.5×10 5Individual cells were seeded into a Transwell (8-μm porous polycarbonate membrane insert) in a 24-well dish containing 75 μl of Matrigel (Corning) and serum-free medium (1:20). After solidifying for 10 minutes in an incubator (37 °C, 5% CO2), 600 μL of 1% serum-supplemented medium was added to the bottom well. Non-invasive cells were removed by rubbing with a cotton swab, and invasive cells were fixed using 70% ethanol and stained with crystal violet (0.2%) after 72 hours. Next, the cells on the lower side of the membrane were visualized in five randomly selected fields and quantified using ImageJ. Each experiment was repeated at least three times independently.

[0196] Treatment with anti-CLDN1 mAb H3L3 significantly and notably decreased 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 - 3G).

[0197] Collectively, these data indicate that treatment with anti-CLDN1 mAb inhibited the migration and invasion abilities of CCA cell lines, further confirming the antitumor effect of anti-CLDN1 antibody.

[0198] Example 4. CLDN1 mAb mediates antitumor effects by interfering with cancer cell differentiation, metabolism, and oncogenic pathways To elucidate the molecular mechanism underlying the antitumor effect provided by anti-CLDN1 mAb (H3L3), RNA sequencing (RNA-seq) was performed on HuCC-A1 tumor tissues derived from CDX mice.

[0199] Unbiased pathway analysis using the Molecular Signatures Database (MSigDB v.7.4) was performed using gene set enrichment analysis (GSEA). To evaluate the effect of anti-CLDN1 mAb H3L3 treatment in the HuCC-A1 CDX mouse model, samples from animals treated with anti-CLDN1 H3L3 mAb (n = 5 mice) were compared to respective control samples (n = 5 mice) of the same model. The results of GSEA were adjusted considering the false discovery rate (FDR). An FDR < 0.05 was considered statistically significant.

[0200] Gene sets related to cell survival, metabolism, and differentiation (including EMT) were significantly downregulated in mice treated with CLDN1 mAb H3L3 (Figure 4A, left panel). In addition to the downregulation of these pathways, targeting non-junctional CLDN1 showed strong suppression of several major oncogenic signaling pathways, with the strongest effects observed in TNF-α / NF-κB, TGF-β, IL-6 / JAK / STAT3, and PI3K / Akt / mTOR signaling (Figure 4A, right panel), highlighting the important role of non-junctional CLDN1 in driving oncogenic signaling.

[0201] To further investigate the antitumor effect of CLDN1-targeting mAbs, the major signaling pathways involved in the development and progression of CCA were analyzed in EGI-1 CDX tumor samples (Figure 4B - 4C).

[0202] For protein extraction from CDX tumors, tumor pieces were lysed in lysis buffer (1% Triton, 50 mM NaCl, 50 mM Tris, pH 7.6, 2 mM MgCl2 in ddH2O) supplemented with protease inhibitor (Roche) and phosphatase inhibitors (Sigma Phosphatase Cocktail numbers 2 and 3) at 4°C for 30 minutes. Next, the samples were centrifuged at 10,000×g for 10 minutes and the supernatants were collected. Protein quantification was evaluated using a Thermo Scientific BCA Kit. Gels were prepared according to the Biorad TGX gel protocol (BioRad). Gels were transferred to PVDF membranes using the Biorad Trans-Blot Turbo protocol (BioRad). Membrane blocking was performed in TBS-T containing 5% BSA for 1 hour. The membranes were incubated overnight at 4°C with YAP / TAZ (D24E4) rabbit mAb (number 8418, Cell Signaling Technology), phosphorylated YAP (Ser127) (D9W2I) rabbit mAb (number 13008, Cell Signaling Technology), SRC rabbit mAb (number 21085, Cell Signaling Technology), phosphorylated SRC (Tyr416) rabbit mAb (number 21015, Cell Signaling Technology), Notch1 (D1E11) XP® rabbit mAb (number 3608, Cell Signaling Technology), cleaved Notch1 (Val1744) (D3B8) rabbit mAb (number 4147, Cell Signaling Technology), and monoclonal anti-β-actin antibody produced in mouse (Sigma) in TBS-T containing 2.5% BSA at a dilution of 1:1000. The membranes were incubated with horseradish peroxidase-conjugated secondary antibody in TBS-T containing 2.5% BSA at a dilution of 1:10,000 for 1 hour at room temperature. Protein immunodetection of the membranes was performed using Clarity ECL Western blot substrate (BioRad) in a ChemiDoc MP imaging system (BioRad).The immunoblot images were analyzed using Image Lab Software v6.1 (Biorad).

[0203] By Western blot quantitative analysis, it was revealed that in tumors treated with anti-CLDN1 mAb H3L3, the cleavage form of Notch1, which is known to promote the proliferation and survival of CCA cells compared to control tumors (Singrang et al., 13(2) J Cell Commun Signal 245 - 254 (Jun 2019)), was decreased (p < 0.001, Mann-Whitney test, Figure 4C). Furthermore, the activation of the non-receptor tyrosine kinase protein SRC was strongly reduced in tumors treated with 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. Indeed, a strong increase in YAP S127 phosphorylation was observed upon treatment with anti-CLDN1 mAb H3L3 (p < 0.01, Mann-Whitney test, Figure 4C), which leads to the cytoplasmic sequestration and inactivation of YAP (Sugihara et al., 54(6) J Gastroenterol 485 - 491 (Jun 2019)).

[0204] In summary, based on the results of Examples 1 - 4, non-junctional CLDN1 was identified as an oncogenic driver and therapeutic target in CCA. Targeting CLDN1 by perturbation studies using highly specific antibodies 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 state-of-the-art human CCA cell-based models. Mechanistic studies based on RNA-Seq and proteome analysis of signaling revealed that this antitumor effect is likely mediated through interference with cancer cell differentiation, metabolism, and major oncogenic pathways involved in the pathogenesis of CCA.

[0205] Lack of response to pharmacological treatment is a major limitation in the management of advanced CCA. The mechanisms of chemotherapy resistance (MOC) are diverse and depend on the expression and / or function of MOC gene molecular targets (Marin et al., 1864(4 Pt B) Biochim Biophys Act Mol Basis Dis 1444 - 1453 (Apr 2018)). For example, downregulation of human passive diffusion nucleoside transporter 1 and copper transporter CTR1 results in decreased sensitivity to gemcitabine and cisplatin, respectively, the first - line standard chemotherapy for CCA (Borbath et al., 48(7) Eur J Cancer 990 - 996 (May 2012)). Advantageously, the unique mechanism of action of CLDN1 mAb constitutes an important therapeutic option for avoiding multidrug intrinsic or acquired resistance phenotypes in CCA.

[0206] Hepatocellular carcinoma - cholangiocarcinoma complex (HCC - CC) is an invasive dual - phenotype primary liver cancer with unmet needs and unsatisfactory outcomes (Azizi et al., 10 Front Oncol 570958 (Sep 2020)). Despite recent advances, the options for standard first - line systemic therapy are not well - established, and surgical approaches are the only available curative treatments (Leoni et al., 12(4) Cancers (Basel) 794 (Mar 2020)). Claudin - 1, advantageously, may also provide a dual - targeting therapeutic target for HCC - CC.

[0207] The intricate interplay between various signaling pathways that regulate cholangiocarcinoma formation and mediate tumorigenesis has been outlined over the past few decades (Banales et al., 17 Nat Rev Gastroenterol Hepatol 557 - 588 (2020)). In particular, the Hippo pathway effector protein YAP, and highly conserved pathways such as Notch, are significantly active in CCA and are upregulated by 67.2% and 79.5% respectively in CCA tumors (Wu et al., 37(10) Tumor Biol. 13499 - 13508 (Oct 2016), Wu et al., 31(6) Oncol Rep 2515 - 2524 (Jun 2014)). These data showed strong downregulation of Notch1 and YAP signaling upon treatment with anti - CLDN1 mAb H3L3 (Figure 4B - 4C). By targeting two independent but interconnected signaling pathways, anti - CLDN1 mAb may avoid the acquired treatment resistance experienced by previous targeted therapies for CCA (Simile et al., 55(2) Medicina(Kaunas) 42 (Feb 2019)). Furthermore, targeting SRC, a central component upstream of multiple transduction pathways (Ishizawar et al., 6(3) Cancer Cell 209 - 214 (Sep 2004)), may reduce the activation of compensatory signaling in single - target approaches due to functional crosstalk and redundancy in the maintenance of major pathways of this disease. In summary, this unique mechanism of action reveals a novel therapeutic approach to address inter - and intra - tumor heterogeneity that dysregulates distinct signaling cascades in different subsets of CCA.

[0208] To confirm the effect of anti - CLDN1 mAb H3L3 on CCA signaling observed in vivo (Figure 4A - 4C), further signaling assays were performed in a CCA cell - based model. The CCA model was 1.5×10 5Consisted of individual EGI-1 CCA cells, or co-cultures (ratio 1:5) of CCA EGI-1 and LX2 stellate cells treated with anti-CLDN1 mAb H3L3 (Colpitts et al., 67(4) Gut 736 - 745 (Mar 2017)) (10 μg / mL) or isotype control mAb (10 μg / mL) for 2 days. On the second day after treatment, cells were stimulated with TNF-α (10 ng / mL) for 24 hours to evaluate SRC and FAK signaling, or with Jagged-1 (50 ng / mL, SRP8012, Sigma-Aldrich) for 24 hours to evaluate Notch1 signaling. For protein extraction from cell cultures, cells were lysed at 4 °C for 20 minutes using Glo lysis buffer (E2661, Promega) supplemented with protease and phosphatase inhibitors (78442, Halt™ Protease and Phosphatase Inhibitor Single-Use Cocktail, Thermo Fisher). Samples were then centrifuged at 15,000 rpm for 10 minutes, 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 Trans-Blot Turbo protocol (BioRad). Membrane blocking was performed for 1 hour in TBS-T containing 5% BSA.The membrane was incubated with anti-SRC rabbit mAb (number 21085, Cell Signaling Technology), phospho-SRC (Tyr416) rabbit mAb (number 21015, Cell Signaling Technology), anti-Notch1 (D1E11) XP® rabbit mAb (number 3608, Cell Signaling Technology), anti-cleaved Notch1 (Val1744) (D3B8) rabbit mAb (number 4147, Cell Signaling Technology), anti-FAK rabbit mAb (number 3285, Cell Signaling Technology), anti-phospho-FAK (Tyr576 / 577) rabbit mAb (#3281, Cell Signaling Technology) and anti-β-actin antibody (Sigma) in TBS-T containing 5% milk at room temperature for 2 hours. The secondary antibodies used were Peroxidase AffiniPure goat anti-rabbit IgG (H+L) mAb (Jackson ImmunoResearch) or ECL mouse IgG, HRP-conjugated whole Ab (Amersham). Protein immunodetection of the membrane was performed using Clarity ECL Western blot substrate (Biorad) in a ChemiDoc MP imaging system (Biorad). Each experiment was repeated at least 3 times independently.

[0209] Western blot analysis of the CCA cell line EGI-1 treated with anti-CLDN1 mAb H3L3 or isotype control mAb showed a robust decrease in cleaved Notch1 and its downstream target Hes1 (Figure 4D), confirming that treatment with anti-CLDN1 mAb inhibits Notch1 signaling in CCA cells. Furthermore, phosphorylation of SRC and FAK decreased upon treatment with anti-CLDN1 mAb compared to cells treated with the control (Figures 4E-4F), confirming the effect of anti-CLDN1 mAb on these signaling pathways in CCA cells. Since the Notch1, SRC, and FAK pathways have been identified as major signaling pathways driving the development and progression of CCA (Banales et al., 17(9) Nat Rev Gastroenterol Hepatol 557-588 (Sep 2020)), these data indicate that inhibition of Notch1, SRC, and FAK signaling by anti-CLDN1 mAb contributes to the antitumor effect of the antibody.

[0210] To further understand and confirm the effect of anti-CLDN1 mAb treatment on the identified oncogenic signaling pathways, gene set enrichment analysis (GSEA) was applied to unbiased pathway analysis using the Molecular Signatures Database (MSigDB v.7.4) in HuCC-A1 CDX mouse tumors. Samples from animals treated with anti-CLDN1 H3L3 mAb (n = 5 mice) were compared to respective control samples (n = 5 mice) of the same model. The results of GSEA were adjusted considering the false discovery rate (FDR). An FDR < 0.05 was considered statistically significant.

[0211] The expression of downstream targets and effector genes of the YAP / TAZ, FAK, SRC, and Notch1 signaling pathways was consistently downregulated in the group treated with anti-CLDN1 mAb H3L3 compared to the control (Figure 4G), confirming the functional relevance of the identified pathways in CCA treated with anti-CLDN1 mAb.

[0212] In summary, these results provide a robust preclinical proof-of-concept of the therapeutic efficacy of CLDN1-specific monoclonal antibodies, revealing that unmet medical needs are rapidly increasing worldwide and that anti-CLDN1 mAb represents an innovative treatment modality for CCA treatment where there are no satisfactory treatment options.

[0213] Example 5. In vivo inhibition of lung metastasis by treatment with CLDN1 mAb in a mouse model of CCA metastasis. To evaluate the in vivo activity of anti-CLDN1 mAb (H3L3) against invasion and metastasis, treatment with the anti-CLDN1 antibody was examined in a state-of-the-art mouse model of CCA metastasis based on intravenous injection of metastatic HuCC-T1 CCA cells.

[0214] One day before intravenous injection of 0.5×10 6 HuCC-T1 cells, non-obese diabetic Rag1 - / - IL2Rgc - / - (NRG) mice were treated with anti-CLDN1 mAb H3L3 (Colpitts et al., 67(4) Gut 736 - 745 (Mar 2017)) (25 mg / kg, intraperitoneally) or vehicle control (PBS, intraperitoneally). The mice were randomized into two groups and treated with anti-CLDN1 mAb H3L3 (Colpitts et al., 67(4) Gut 736 - 745 (Mar 2017)) (25 mg / kg, intraperitoneally, twice a week) or vehicle control (PBS, intraperitoneally, twice a week) for 6 weeks. After 6 weeks, the mice were sacrificed, the lungs were harvested, and cytokeratin 18 (CK18) staining was used to identify and quantify CK18-positive cholangiocarcinoma metastases in the lungs (Figure 5A).

[0215] Anti-CLDN1 mAb H3L3 significantly reduced metastasis formation in the lung compared to the control group (p<0.05, Mann-Whitney test) (Figures 5B - 5C), as shown by the decrease in CK18 positive area upon treatment with anti-CLDN1 mAb H3L3 in lung sections. These data indicate that anti-CLDN1 mAb H3L3 treatment results in inhibition of CCA metastasis formation in vivo.

[0216] Example 6. CLDN1 expression targeted by CLDN1 mAb H3L3 is upregulated in CCA upon treatment with cisplatin and gemcitabine The first-line chemotherapy treatment for advanced CCA is cisplatin and gemcitabine (Borbath et al., 48(7) Eur J Cancer 990 - 996 (May 2012)). To examine the expression of CLDN1 upon treatment with cisplatin and gemcitabine, 1.5×10 5 CCA EGI-1 cells were treated with cisplatin (100 nM) and gemcitabine (10 nM) for 24 hours. Cells were then harvested and CLDN1 expression was evaluated by flow cytometry and Western blot using a CLDN1-specific Ab.

[0217] For flow cytometry, cells were incubated with anti-CLDN1 mAb H3L3 or isotype control mAb at a concentration of 10 μg / ml (repeated 3 times for each condition). After incubation with the primary antibody for 1 hour, cells were washed and incubated with a human Alexa647-conjugated secondary antibody at 4°C for 45 minutes. 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 mean fluorescence intensity between cells stained with anti-CLDN1 mAb H3L3 and cells stained with isotype control mAb. The experiment was repeated independently 3 times.

[0218] For protein extraction for Western blot analysis, cells were lysed at 4 °C for 20 minutes using Glo lysis buffer (E2661, Promega) supplemented with protease and phosphatase inhibitors (78442, Halt™ Protease and Phosphatase Inhibitor Single-Use Cocktail, Thermo Fisher). Samples were then centrifuged at 15,000 rpm for 10 minutes and the supernatants were collected. Protein quantification was performed using a 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 Trans-Blot Turbo protocol (BioRad). Membrane blocking was performed in TBS-T containing 5% BSA for 1 hour. Membranes were incubated with anti-CLDN1 rabbit mAb (number E-AB-15674, ElabScience) and anti-β-actin antibody (Sigma) in TBS-T containing 5% milk at room temperature for 2 hours. The secondary antibodies used were peroxidase AffiniPure goat anti-rabbit IgG (H+L) mAb (Jackson ImmunoResearch) or ECL mouse IgG, HRP-conjugated whole Ab (Amersham). Protein immunodetection of membranes was performed using Clarity ECL Western blot substrate (BioRad) in a ChemiDoc MP imaging system (BioRad). Experiments were repeated independently at least 3 times.

[0219] Flow cytometry analysis of the CCA cell line EGI-1 treated with cisplatin and gemcitabine using anti-CLDN1 mAb H3L3 or control Ab showed a robust increase in CLDN1 expression upon treatment with cisplatin and gemcitabine (Figs. 6A–6B). This upregulation was further confirmed by Western blot analysis using CLDN1-specific Ab (Figs. 6C–6D).

[0220] These data indicate that chemotherapy with cisplatin and gemcitabine results in upregulation of exposed and targetable CLDN1 on the CCA cell surface that is detected and accessible by anti-CLDN1 mAb H3L3. Since CLDN1 expression was thus increased in CCA treated with chemotherapy, patients are provided with the opportunity to be treated with anti-CLDN1 mAb for CCA either in combination with, or after, treatment with chemotherapy including cisplatin and gemcitabine to improve patient outcomes.

[0221] Example 7. Anti-CLDN1 mAb H1L1 inhibits tumor growth in a cholangiocarcinoma PDX model The objective of this experiment was to preclinically evaluate the in vivo therapeutic effect of anti-CLDN1 mAb H1L1 in the treatment of a subcutaneous PDX model (CC6702 model) of cholangiocarcinoma in female BALB / c nude mice. The number of animals was set to n = 2 for the placebo and n = 3 for the treatment group.

[0222] Fresh tumor tissue was collected from mice bearing an established PDX model and cut into small fragments (approximately 2 - 3 mm in diameter). To generate tumors, PDX tumor fragments collected from donor mice were subcutaneously inoculated into the upper right dorsal side of the test mice. Randomization was initiated when the average tumor size reached approximately 100 - 150 mm 3 At this point. A total of 5 mice per model were enrolled in the study and assigned to 2 groups. Randomization was performed based on the "stratification" method (StudyDirector™ software, version 3.1.399.19). The date of grouping was designated as day 0.

[0223] For the control group, 0.2 mL of placebo was administered by intraperitoneal injection (QW×4). For the treatment group, 0.2 mL of anti-CLDN1 mAb H1L1 (2.5 mg / mL) was administered by intraperitoneal injection (QW×4).

[0224] After tumor inoculation, the animals were checked daily for morbidity and mortality. During regular monitoring, the animals were checked for any effects of tumor growth and treatment on behavior, such as locomotor ability, food and water intake, weight gain or loss (weight was measured twice a week after randomization), wetness of eyes / hair, and any other abnormalities. For each individual animal, the number of deaths and the observed clinical signs were recorded in detail.

[0225] After randomization, tumor volume was measured twice a week in two dimensions using calipers and expressed in mm3 using the formula: V = (L × W × W) / 2 (where V is the tumor volume, L is the tumor length (the longest dimension of the tumor), and W is the tumor width (the longest dimension of the tumor perpendicular to L)). Administration as well as tumor and body weight measurements were performed inside a laminar flow cabinet. Body weight and tumor volume were measured using StudyDirector™ software (version 3.1.399.19).

[0226] Figure 7A shows the body weight curves at various time points for the CC6702 PDX model after treatment with vehicle or anti-CLDN1 mAb H1L1. Figure 7B shows the tumor volume growth curves at various time points for the CC6702 PDX model after treatment with vehicle or anti-CLDN1 mAb H1L1. The control group was terminated after day 14 due to humane considerations arising from overly strong tumor growth. Figure 7B demonstrates that anti-CLDN1 mAb H1L1 inhibited tumor growth in the cholangiocarcinoma PDX model compared to the group treated with vehicle. ***

[0227] In practicing the present disclosure, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art, will be used. Such techniques are well explained in the literature.

[0228] All cited references are hereby incorporated by reference in their entirety.

[0229] The examples provided in this specification are provided by way of example and not by way of limitation.

Claims

Claim 1 A method for 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. Claim 2 The method of claim 1, wherein the CCA is intrahepatic CCA. Claim 3 The method of claim 1, wherein the CCA is hilar region CCA. Claim 4 The method of claim 1, wherein the CCA is distal CCA. Claim 5 The method of claim 1, wherein the CCA is combined or mixed hepatocellular cholangiocarcinoma (cHCC-CCA). Claim 6 The method according to 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. Claim 7 The method according to 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 with DSMZ on July 29, 2008 under the accession number DSM ACC2938. Claim 8 The method according to 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. Claim 9 The method according to any one of claims 1 to 8, wherein the anti-claudin-1 antibody is humanized. Claim 10 The method according to 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. Claim 11 The method according to 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. Claim 12 The method according to 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. Claim 13 The method according to 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 according to 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 according to 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, the anti-claudin-1 antibody or a pharmaceutical composition thereof.

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

18. Use according to claim 16, wherein the CCA is hilar region CCA.

19. Use according to claim 16, wherein the CCA is distal CCA.

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

21. Use according to any one of claims 16 to 20, 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 with DSMZ on July 29, 2008 under the accession number DSM ACC2938.

22. 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. Use according to any one of claims 16 to 22, wherein the anti-claudin-1 antibody is humanized.

24. 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. 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. 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. 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. 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. 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. Use according to claim 16, further comprising administering a chemotherapeutic agent to the human subject.

31. Use according to claim 16, wherein the CCA comprises 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 virus oncogene homolog (KRAS), polybromo 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 according to claim 1, further comprising administering a chemotherapeutic agent to the human subject in need thereof.

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

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

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

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

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

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