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

Abstract

The present disclosure relates to a method of treating cholangiopathy (e.g., primary sclerosing cholangitis or primary biliary cholangitis) 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. 62 / 050,745, filed on September 15, 2014, which is incorporated herein 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_019PC01_SequenceListing_ST26, Size: 20,119 bytes, and Creation Date: May 29, 2023) submitted with this application is incorporated herein by reference in its entirety.

[0003] According to various aspects of the present disclosure, the present disclosure relates to a method of treating cholangiopathy.

Background Art

[0004] Primary sclerosing cholangitis (PSC) is a chronic cholestatic disease characterized by periductal fibrosis, which causes biliary stricture, end - stage liver disease, and cholangiocarcinoma. There is no effective treatment strategy other than liver transplantation for the clinical care of PSC patients. Claudin - 1 (CLDN1) is a transmembrane protein involved in epithelial tight junctions, and is also expressed non - junctionally, mediating cell plasticity and signal transduction. In the art, there is a need for a treatment method targeting CLDN1 for the treatment of PSC.

Summary of the Invention

[0005] The present disclosure provides a method of treating cholangiopathy 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.

[0006] In some embodiments, provided herein is a method for effecting restoration of biliary epithelial integrity 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. In some embodiments, the administration results in modulation of hepatic progenitor cells and / or conversion of hepatocytes to ductal cells.

[0007] In some embodiments, provided herein is a method for reducing cholestasis and improving hepatitis and liver function.

[0008] In some embodiments, provided herein is a method for improving survival from cholangiopathy.

[0009] In some embodiments, provided herein is a method for preventing cholangiocarcinoma.

[0010] In some embodiments, provided herein is a method for reducing PSC-associated ulcerative colitis, the method comprising administering an anti-claudin-1 antibody to a human subject.

[0011] In some embodiments, provided herein is a method for reducing biliary fibrosis, the method comprising administering an anti-claudin-1 antibody to a human subject.

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

[0013] 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 deposit number DSM ACC2938.

[0014] In some embodiments, the anti-claudin-1 antibody is humanized.

[0015] 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.

[0016] 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.

[0017] 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.

[0018] 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.

[0019] In some embodiments, the anti-claudin-1 antibody comprises a heavy chain variable domain complementarity determining region (CDR) H1 comprising the amino acid sequence set forth in SEQ ID NO: 5, a CDR H2 comprising the amino acid sequence set forth in SEQ ID NO: 6, and a CDR H3 comprising the amino acid sequence set forth in SEQ ID NO: 7, and / or a light chain variable domain complementarity determining region (CDR) L1 comprising the amino acid sequence set forth in SEQ ID NO: 8, a CDR L2 comprising the amino acid sequence GAS, and a CDR L3 comprising the amino acid sequence set forth in SEQ ID NO: 10.

[0020] In some embodiments, the anti-claudin-1 antibody is administered intratumorally, intravenously, intraperitoneally, intramuscularly, intrathecally, or subcutaneously.

[0021] In some embodiments, provided herein is an anti-claudin-1 antibody or a pharmaceutical composition thereof for use in a method of treating PSC 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.

[0022] In some embodiments, the anti-claudin-1 antibody for use in a method of treating PSC in a human subject 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.

[0023] In some embodiments, the anti-claudin-1 antibody for use in a method of treating PSC in a human subject is humanized.

[0024] In some embodiments, the anti-claudin-1 antibody for use in a method of treating PSC in a human subject comprises a VH comprising the amino acid sequence set forth in SEQ ID NO: 3 or SEQ ID NO: 13.

[0025] In some embodiments, the anti-claudin-1 antibody for use in a method of treating PSC in a human subject comprises a VL comprising the amino acid sequence set forth in SEQ ID NO: 4 or SEQ ID NO: 14.

[0026] In some embodiments, the anti-claudin-1 antibody for use in a method of treating PSC in a human subject 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.

[0027] In some embodiments, the anti-claudin-1 antibody for use in a method of treating PSC in a human subject 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.

[0028] In some embodiments, the anti-claudin-1 antibody for use in a method of treating PSC in a human subject comprises a heavy chain variable domain complementarity determining region (CDR) H1 comprising the amino acid sequence set forth in SEQ ID NO:5, a CDR H2 comprising the amino acid sequence set forth in SEQ ID NO:6, and a CDR H3 comprising the amino acid sequence set forth in SEQ ID NO:7, and / or a light chain variable domain complementarity determining region (CDR) L1 comprising the amino acid sequence set forth in SEQ ID NO:8, a CDR L2 comprising the amino acid sequence GAS, and a CDR L3 comprising the amino acid sequence set forth in SEQ ID NO:10.

[0029] In some embodiments, the anti-claudin-1 antibody for use in a method of treating PSC in a human subject is administered intratumorally, intravenously, intraperitoneally, intramuscularly, intrathecally or subcutaneously.

[0030] In some embodiments, provided herein is a kit for treating a subject suffering from cholangiopathy, the kit comprising a therapeutically effective amount of an anti-claudin-1 antibody and a package insert comprising instructions for use of the kit.

[0031] In some embodiments, provided herein is a pharmaceutical composition for the treatment of cholangiopathy, the pharmaceutical composition comprising a therapeutically effective amount of an anti-claudin-1 antibody.

[0032] In some embodiments, the cholangiopathy treated by the methods, uses, kits, or pharmaceutical compositions disclosed herein is primary sclerosing cholangitis (PSC).

[0033] In some embodiments, the cholangiopathy treated by the methods, uses, kits, or pharmaceutical compositions disclosed herein is primary biliary cirrhosis (PBC).

[0034] In some embodiments, the cholangiopathy treated by the methods, uses, kits, or pharmaceutical compositions disclosed herein is biliary fibrosis. In some embodiments, biliary fibrosis is caused by cystic fibrosis. In some embodiments, biliary fibrosis is caused by IgG4-related disease.

[0035] In some embodiments, cholangiopathy is biliary atresia.

[0036] In some embodiments, cholangiopathy is Alagille syndrome. BRIEF DESCRIPTION OF THE DRAWINGS

[0037]

Figure 1

[0038]

Figure 2

[0039]

Figure 3

[0040]

Figure 4

[0041]

Figure 5

[0042]

Figure 6A

Figure 6B

Figure 6C

[0043]

Figure 7

[0044]

Figure 8

[0045]

Figure 9

[0046]

Figure 10

[0047]

Figure 11

[0048]

Figure 12

[0049]

Figure 13

[0050]

Figure 14

[0051]

Figure 15A

Figure 15B

Figure 15C

Figure 15D

Figure 15E

Figure 15F

Figure 15G

[0052]

Figure 16A

Figure 16B

Figure 16C

[0053]

Figure 17

[0054]

Figure 18A

[0055]

Figure 18B

Figure 18C

Mode for Carrying Out the Invention

[0056] 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, the present application, including definitions, will control. Unless the context requires otherwise, the singular terms herein shall include the plural, and the plural terms shall include the singular. All patents and references cited herein are 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.

[0057] 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 detailed description and the claims.

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

[0059] 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.”

[0060] The term “about” as used herein is used to mean approximately, roughly, around, or in the region of. When the term “about” is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the stated numerical value. Generally, the term “about” is used herein to modify a value that exceeds and is less than the recited value by a variation of plus or minus 10%.

[0061] 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 immutable limitation on the scope of the invention. Thus, a range description should be regarded as specifically disclosing not only the individual numerical values within that range but also all possible sub-ranges. For example, a range description such as 1 to 6 is to be regarded as specifically disclosing sub-ranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, as well as the individual numbers within that range, e.g., 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.

[0062] Units, prefixes, and symbols are shown in the form recognized in the International System of Units (SI). Numerical ranges are to include the numbers defining the range. When a range of values is recited, it should also be understood that each integer value intervening between the recited upper and lower limits of that range, and each sub-range between such values, are specifically disclosed. It is also to be understood that the upper and lower limits of any range may independently be included or excluded within the range, and that each range including either, neither, or both of these limiting values is included in this application. Thus, the ranges provided herein are to be understood as a shorthand representation of all values within the range, including the recited end points. For example, the range of 1 to 10 is to be understood to include any number, combination of numbers, or sub-range from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10.

[0063] 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 sub-combination of the elements of that combination is also specifically disclosed and 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 the plurality of elements of the present disclosure can have such exclusions, and all combinations of elements having such exclusions are disclosed herein.

[0064] As used herein, the term "and / or" should be interpreted as a specific disclosure of each of two designated features or components, regardless of the presence or absence of 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).

[0065] The term "treating" or "treatment" as used herein refers to administering a composition to a subject for therapeutic purposes.

[0066] 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.

[0067] As used herein, the term "antibody" refers to any immunoglobulin containing an antigen-binding site that immunospecifically binds to an antigen. Thus, the term "antibody" encompasses not only the entire antibody molecule, but also antibody fragments, as well as variants (including derivatives) of antibodies and antibody fragments, as long as the derivatives and fragments maintain their specific binding ability. This term encompasses monoclonal antibodies and polyclonal antibodies. This term also encompasses 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 may be produced partially or wholly synthetically. As used with respect to an antibody, the term "specifically binds" refers to an antibody that binds to a given antigen. Typically, an antibody binds with an affinity of at least 1×10 7 M 1 and binds to the given antigen with an affinity that is at least 2-fold greater than its affinity for binding to non-specific antigens (e.g., BSA, casein).

[0068] 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 frameworks (FRs). 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 CDRs of human antibodies. 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.

[0069] It is understood that whenever a particular aspect is described herein using the language "comprising", a similar aspect is provided unless it is described using the terms "consisting of" and / or "consisting essentially of".

[0070] 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 administration by injection or infusion, intramuscular administration, subcutaneous administration, intraperitoneal administration, intraspinal administration, or other parenteral routes of administration. The phrase "parenteral administration" as used herein means administration by a route other than enteral and topical administration, usually by injection, and includes, but is not limited to, intravenous injection and infusion, intramuscular injection and infusion, arterial 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, transtracheal 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, as well as in vivo electroporation. Other parenteral routes of administration include local, epithelial, or mucosal routes of administration, for example, intranasal, intravaginal, rectal, sublingual, or topical routes of administration. Also, administration can be carried out, for example, once, multiple times, and / or over one or more extended periods of time.

[0071] The term "effective amount" refers to the 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 signs, symptoms, 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 tumor shrinkage and / or reduce the rate of tumor growth (e.g., suppress tumor growth) or prevent or delay other undesirable cell proliferation. In some embodiments, an effective amount is an amount sufficient to delay the onset of a tumor. In some embodiments, an effective amount is an amount sufficient to prevent or delay tumor recurrence. An 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 tumor size, (iii) prevent, delay, slow to some extent, or stop cancer cell infiltration into peripheral organs, (iv) prevent tumor metastasis (i.e., delay and / or stop to some extent), (v) prevent 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, an "effective amount" is the amount of an anti-claudin-1 antibody that has been clinically proven to result in a significant reduction in cancer or a slowing of cancer progression, such as in advanced solid tumors.

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

[0073] II. Anti-Claudin-1 Antibody The present invention relates to the use of anti-claudin-1 antibodies for the treatment of cholangiopathies. In some embodiments, the cholangiopathy is PSC. In some embodiments, the cholangiopathy is PBC. In some embodiments, the cholangiopathy is a hereditary cholangiopathy. In some embodiments, the cholangiopathy is an idiopathic cholangiopathy. In some embodiments, the cholangiopathy is a malignant cholangiopathy. In some embodiments, the cholangiopathy is secondary sclerosing cholangitis.

[0074] In some embodiments, hereditary cholangiopathy is selected from the group consisting of Alagille syndrome, Caroli syndrome, cystic fibrosis, progressive familial intrahepatic cholestasis, and polycystic liver disease.

[0075] In some embodiments, idiopathic cholangiopathy is selected from the group consisting of autoimmune cholangitis, biliary atresia, idiopathic pediatric or adult cholangiopathy, IgG4-related cholangitis, primary biliary cirrhosis, and primary sclerosing cholangitis.

[0076] In some embodiments, malignant cholangiopathy is cholangiocarcinoma.

[0077] In some embodiments, secondary sclerosing cholangitis is selected from the group consisting of ABCB4 deficiency, abdominal trauma (surgical or blunt), AIDS cholangiopathy, amyloidosis, chemicals / drugs (i.e., 5-fluorouracil), common bile duct stones, eosinophilic or mast cell cholangitis, graft-versus-host disease with liver, iatrogenic biliary stricture, portal hypertensive biliary disease, recurrent pyogenic cholangitis, sarcoidosis, sickle cell disease, and vascular / ischemic (i.e., hepatic artery stenosis after liver transplantation).

[0078] CLDN1 is a transmembrane protein with two major roles: (1) together with other proteins, it contributes to the barrier function by tight junctions, and (2) it is expressed outside the tight junctions of the basolateral membrane of epithelial cells. 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.

[0079] Antibodies against human Claudin-1 have been previously described for treating 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.

[0080] Other examples of suitable anti-Claudin-1 antibodies include those disclosed in European Patent No. EP1167389, US Patent No. 6,627,439, International Patent Application Publication Nos. WO2014 / 132307, WO2015 / 014659, and WO2015 / 014357, and Yamashita et al., J. Pharmacol. Exp. Ther., 2015, 353(1):112-118.

[0081] The anti-Claudin-1 antibodies suitable for use in the present invention may be polyclonal antibodies or monoclonal antibodies.

[0082] An anti-claudin-1 antibody suitable for use according to the present invention may also be "humanized". The sequence differences between the rodent antibody and the human sequence can be minimized by site-directed mutagenesis of individual residues, transplantation of entire regions, or chemical synthesis to replace residues different 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 human immunoglobulin molecules, 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.

[0083] 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

[0084] 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.

[0085] 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.

[0086] 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.

[0087] 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 accession number DSM ACC2938.

[0088] In some embodiments, the variable heavy chain ("VH") and variable light chain ("VL") 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 accession number DSM ACC2938.

[0089] 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.

[0090] 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.

[0091] 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.

[0092] 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.

[0093] 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.

[0094] 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.

[0095] 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.

[0096] 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.

[0097] 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.

[0098] 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.

[0099] 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.

[0100] 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.

[0101] 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.

[0102] 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.

[0103] 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.

[0104] 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.

[0105] 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.

[0106] In some embodiments, the anti-claudin-1 antibody comprises a heavy chain comprising the amino acid sequence shown in SEQ ID NO: 1.

[0107] 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.

[0108] 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.

[0109] In some embodiments, the anti-claudin-1 antibody comprises a light chain comprising the amino acid sequence shown in SEQ ID NO: 2.

[0110] 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.

[0111] 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.

[0112] In some embodiments, the anti-claudin-1 antibody comprises a heavy chain comprising the amino acid sequence shown in SEQ ID NO: 1, and a light chain comprising the amino acid sequence shown in SEQ ID NO: 2.

[0113] 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, 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.

[0114] 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, 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.

[0115] In some embodiments, the anti-claudin-1 antibody comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 11.

[0116] 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.

[0117] 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.

[0118] In some embodiments, the anti-claudin-1 antibody comprises a light chain comprising the amino acid sequence set forth in SEQ ID NO: 12.

[0119] 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.

[0120] 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.

[0121] 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.

[0122] 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.

[0123] 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.

[0124] 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.

[0125] The anti-claudin-1 antibody (or its biologically active variant or fragment) 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 methods). Methods for preparing such modified antibodies (or conjugated antibodies) are known in the art (see, for example, “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 at 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.

[0126] In some embodiments, the anti-claudin-1 antibodies described herein target the extracellular loop 1 of claudin-1 that is exposed outside of the tight junction of the basolateral membrane of epithelial cells (as described in Milly L. et al. Nature Biotech 2015).

[0127] The antibody molecules and molecular entities may be directly covalently bonded to each other. Alternatively, the antibody molecules and molecular entities 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.

[0128] In some embodiments, the anti-claudin-1 antibody (or a biologically active fragment thereof) for use in accordance with 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, 131 I), 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 for which antisera or monoclonal antibodies are available.

[0129] Other molecular entities that can be conjugated to the anti-claudin-1 antibody (or a biologically active fragment thereof) of the present invention include, but are not limited to, linear or branched hydrophilic polymer groups, fatty acid groups, or fatty acid ester groups.

[0130] Therefore, in the practice of the present invention, the anti-claudin-1 antibody can be used in the form of an antibody-derived molecule comprising a full-length antibody, a biologically active variant or fragment thereof, a chimeric antibody, a humanized antibody, and at least one complementarity-determining region (CDR) from either the heavy or light chain variable region of the anti-claudin-1 antibody, including 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 a therapeutic or detectable agent. 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.

[0131] III. Cholangiopathy Cholangiopathies are a heterogeneous group of diseases that affect the bile ducts, either intrahepatic or extrahepatic. Individually rare, they account for up to 80% of pediatric and up to 20% of adult liver transplant indications overall. Cholangiopathies include primary biliary cholangitis or primary biliary cirrhosis (PBC), and primary sclerosing cholangitis (PSC). Cholangiopathies can be genetic, idiopathic, malignant, or secondary sclerosing cholangitis. Genetic cholangiopathies include Alagille syndrome, Caroli syndrome, cystic fibrosis, and polycystic liver disease. Idiopathic cholangiopathies include autoimmune cholangitis, biliary atresia, idiopathic pediatric or adult cholangiopathy, IgG4-related cholangitis, primary biliary cirrhosis, and primary sclerosing cholangitis. Malignant cholangiopathies include cholangiocarcinoma. Secondary sclerosing cholangitis includes ABCB4 deficiency, abdominal trauma (surgical or blunt), AIDS cholangiopathy, amyloidosis, chemicals / drugs (i.e., 5-fluorouracil), common bile duct stones, eosinophilic or mast cell cholangitis, graft-versus-host disease with liver, iatrogenic biliary stricture, portal hypertensive biliary disease, recurrent pyogenic cholangitis, sarcoidosis, sickle cell disease, liver fluke infection, post-viral sclerosing cholangitis (i.e., after COVID-19 infection), extrinsic biliary obstruction (e.g., benign and malignant lymph node swelling, extrahepatic biliary tumors, pancreatic tumors, papillary tumors), and vascular / ischemic (i.e., hepatic artery stenosis after liver transplantation).

[0132] PSC has an estimated prevalence of 16.2 cases per 100,000 people in Europe. PSC is a rare chronic cholestatic liver disease characterized by inflammatory destruction of the intrahepatic and / or extrahepatic bile ducts, causing cholestasis, fibrosis, and ultimately cirrhosis, often requiring liver transplantation (LT). Most often, it occurs in association with inflammatory bowel disease (IBD), which often precedes the onset of PSC. PSC is usually diagnosed after detecting cholestasis during the health evaluation or screening of IBD patients (see Rabiee A, Silveira MG; Primary sclerosing cholangitis; Transl Gastroenterol Hepatol; vol.6:29 (April 5 2021)).

[0133] In patients presenting symptoms, abdominal pain is the most frequent symptom (20%), followed by itching (10%), jaundice (6%), and fatigue (6%) (3), but symptoms can vary widely among patients. Hepatomegaly and splenomegaly can be present in 44% and 39% of patients, respectively. Acute itching and / or cholangitis presenting with jaundice, fever, and abdominal pain can be the result of either benign or malignant biliary obstruction. Indeed, as the signs and symptoms of biliary stasis worsen, concern about cholangiocarcinoma (CCA), the most feared and not infrequent complication of PSC, increases. When patients progress to end-stage liver disease, symptoms associated with variceal bleeding, ascites, or hepatic encephalopathy may occur. In patients with associated IBD, abdominal pain, diarrhea, and gastrointestinal bleeding may be the only symptoms along with abnormal liver biochemistry. Elevated serum alkaline phosphatase (ALP) and γ-glutamyltransferase values in a biliary stasis pattern are biochemical features of PSC, but up to 30% - 40% of patients have normal ALP at diagnosis or during the course of the disease. Increases in serum aspartate and alanine aminotransferase levels are usually not as prominent and typically are less than five times the upper limit of normal (ULN). Serum total bilirubin levels are normal at diagnosis in most cases. (Silveria, Transl Gastroenterol Hepatol; vol.6:29 (April 5 2021)).

[0134] One of the histological features of PSC is the finding of concentric periductal fibrosis, also known as "onion-skin fibrosis", which is detected in less than 15% of liver biopsies from PSC patients. (Silveria, Transl Gastroenterol Hepatol; vol.6:29 (April 5 2021)).

[0135] After the progression of the disease, fibrotic strictures of both intrahepatic and extrahepatic bile ducts impair the function of the bile ducts, resulting in obstructive jaundice with intractable pruritus, an increased risk of life-threatening bacterial infections (i.e., ascending cholangitis), and the development of cirrhosis with end-stage liver disease. Since up to 20% of PSC patients develop cholangiocarcinoma, PSC itself is considered a pre-malignant disease.

[0136] A characteristic feature of cholangiopathies such as PSC is ductular reaction. During chronic intrahepatic cholangiocyte injury, the integrity of the bile duct epithelium is restored via the proliferation of hepatic progenitor cells and / or the differentiation conversion of hepatocytes into cholangiocytes. Reactive ductules and activated myofibroblasts maintain each other in a bidirectional manner. Previous transcriptome analyses of ductular reactions in liver tissues of HCV-infected and PSC patients have shown that CLDN1 is one of the top 20 genes upregulated in PSC versus HCV-induced liver disease, suggesting a potential disease association between CLDN1 and bile duct injury.

[0137] Primary biliary cholangitis (PBC), also known as primary biliary cirrhosis, is a chronic cholestatic liver disease. PBC is a multifactorial and enigmatic disease. An autoimmune attack targeting cholangiocytes through disruption of tolerance triggers the onset of the disease.

[0138] A characteristic feature of PBC is antimitochondrial antibodies, which are detected in 90 - 95% of PBC patients and are the most disease-specific autoantibodies in human immunopathology. Antimitochondrial antibodies recognize a family of enzymes located in the inner mitochondrial membrane. This is called the 2-oxoacid dehydrogenase complex (2-OADC) and mainly includes the pyruvate dehydrogenase complex E2 subunit (PDC-E2), the branched-chain 2-OADC E2 subunit (BCOADC-E2), the 2-oxoglutarate dehydrogenase complex E2 subunit (OGDC-E2), and the dihydrolipoamide dehydrogenase-binding protein (E3BP). Antimitochondrial antibodies and autoreactive CD4+ and CD8+ T cell epitopes are restricted within a common peptide sequence in the inner lipoyl domain of human PDC-E2.

[0139] Biliary epithelial cells and hepatocytes in PBC patients express large amounts of human leukocyte antigen (HLA) class I and II molecules. Biliary epithelial cells express adhesion molecules, cytokines, and chemokines, and recruit monocytes to the biliary tract of the liver. One example is fractalkine (CX3CL1), a chemokine with both chemotactic and cell adhesion functions. The predominance of type 1 helper T cytokines and lipopolysaccharide in the microenvironment of damaged bile ducts induces upregulation of fractalkine expression in biliary epithelial cells, subsequently causing chemotaxis of monocytes expressing its receptor (CX3CR1), including CD4+ and CD8+ T cells. Serum fractalkine levels in PBC are high in patients with prominent cholangitis activity (CA) at the initial stage and decrease in response to treatment.

[0140] In some embodiments, the cholangiopathy is biliary atresia.

[0141] In some embodiments, the cholangiopathy is Alagille syndrome.

[0142] Other factors associated with the progression of PBC include genetic predisposition, environmental triggers, and the composition of the microbiota.

[0143] IV. Methods of Use The methods of the present invention can be achieved using the anti-claudin-1 antibodies described herein, or biologically active fragments thereof, or pharmaceutical compositions comprising such antibodies or fragments. These methods generally involve administering an effective amount of an anti-claudin-1 antibody, or a biologically active fragment thereof, or a pharmaceutical composition thereof, to a subject in need thereof (i.e., a subject having a fibrotic tumor). Administration can be carried out using any of the administration methods known to those skilled in the art.

[0144] The present disclosure provides a method of treating cholangiopathy in a human subject in need thereof, the method comprising administering a therapeutically effective amount of an anti-claudin-1 antibody to the human subject.

[0145] In some embodiments, the cholangiopathy is primary sclerosing cholangitis (PSC).

[0146] In some embodiments, the cholangiopathy is primary biliary cirrhosis (PBC) (also referred to as primary biliary cholangitis).

[0147] In some embodiments, the cholangiopathy is a hereditary cholangiopathy. In some embodiments, the cholangiopathy is an idiopathic cholangiopathy. In some embodiments, the cholangiopathy is a malignant cholangiopathy. In some embodiments, the cholangiopathy is secondary sclerosing cholangitis.

[0148] In some embodiments, the hereditary cholangiopathy is selected from the group consisting of Alagille syndrome, Caroli syndrome, cystic fibrosis, and polycystic liver disease.

[0149] In some embodiments, the idiopathic cholangiopathy is selected from the group consisting of autoimmune cholangitis, biliary atresia, idiopathic pediatric or adult cholangiopathy, IgG4-related cholangitis, primary biliary cirrhosis, and primary sclerosing cholangitis.

[0150] In some embodiments, the malignant cholangiopathy is cholangiocarcinoma.

[0151] In some embodiments, the secondary sclerosing cholangitis is selected from the group consisting of ABCB4 deficiency, abdominal trauma (surgical or blunt), AIDS cholangiopathy, amyloidosis, chemicals / drugs (i.e., 5-fluorouracil), common bile duct stones, eosinophilic or mast cell cholangitis, graft-versus-host disease with liver, iatrogenic biliary stricture, portal hypertensive biliary disease, recurrent pyogenic cholangitis, sarcoidosis, sickle cell disease, and vascular / ischemic (i.e., hepatic artery stenosis after liver transplantation).

[0152] In some embodiments, provided herein is a method of effecting restoration of the integrity of the bile duct epithelium 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. In some embodiments, the administration results in the proliferation of hepatic progenitor cells and / or the differentiation conversion of hepatocytes into ductal cells.

[0153] In some embodiments, provided herein is a method of reducing PSC-related ulcerative colitis, the method comprising administering an anti-claudin-1 antibody to a human subject.

[0154] In some embodiments, provided herein is a method of reducing biliary fibrosis, the method comprising administering an anti-claudin-1 antibody to a human subject.

[0155] In some embodiments, claudin-1 (CLDN1) is overexpressed in the human subject compared to its expression level in a normal subject.

[0156] 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.

[0157] In some embodiments, the anti-claudin-1 antibody is humanized.

[0158] 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.

[0159] In some embodiments, the anti-claudin-1 antibody comprises a VH having an amino acid sequence with 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.

[0160] 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.

[0161] 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.

[0162] 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.

[0163] 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.

[0164] 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.

[0165] 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.

[0166] In some embodiments, the anti-claudin-1 antibody comprises a VL 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: 4.

[0167] In some embodiments, the anti-claudin-1 antibody comprises a VL 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: 14.

[0168] 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.

[0169] 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.

[0170] 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.

[0171] 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.

[0172] 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.

[0173] 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.

[0174] In some embodiments, the anti-claudin-1 antibody comprises complementarity-determining region (CDR) H1 comprising the amino acid sequence set forth in SEQ ID NO:5, CDR H2 comprising the amino acid sequence set forth in SEQ ID NO:6, and CDR H3 comprising the amino acid sequence set forth in SEQ ID NO:7.

[0175] In some embodiments, the anti-claudin-1 antibody comprises complementarity-determining region (CDR) L1 comprising the amino acid sequence set forth in SEQ ID NO:8, CDR L2 comprising the amino acid sequence shown as GAS, and CDR L3 comprising the amino acid sequence set forth in SEQ ID NO:10.

[0176] In some embodiments, the anti-claudin-1 antibody is administered intratumorally, intravenously, intraperitoneally, intramuscularly, intrathecally, or subcutaneously.

[0177] In some embodiments, provided herein are methods for reducing cholestasis and improving hepatitis and liver function.

[0178] In some embodiments, provided herein are methods for improving survival in cholangiopathy.

[0179] In some embodiments, provided herein are methods for preventing cholangiocarcinoma.

[0180] V. Administration An 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. A variety of 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, e.g., infusion or bolus injection. Administration may be systemic or local.

[0181] In some embodiments, the anti-claudin-1 antibody is administered intratumorally, intravenously, intraperitoneally, intramuscularly, intrathecally or subcutaneously.

[0182] 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 at a dosage such that the delivered amount is 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.

[0183] Treatment according to the present invention can consist of a single dose or multiple doses. Thus, administration of the anti-claudin-1 antibody, or a biologically active fragment thereof (or its pharmaceutical composition) can be constant over a particular period, or at regular and specific intervals, for example, hourly, daily, weekly (or other multi-day intervals), monthly, annually (e.g., in a time-release form). Alternatively, delivery can be carried out multiple times during a given period, for example, two or more times per week, two or more times per month, etc. Delivery can be continuous delivery over a period, for example, intravenous delivery.

[0184] Generally, the amount of the anti-claudin-1 antibody, or a biologically active fragment thereof (or its pharmaceutical composition) administered is preferably in the range of about 1 ng / kg to about 100 mg / kg of subject body weight, for example, about 100 ng / kg to about 50 mg / kg of subject body weight, or about 1 μg / kg to about 10 mg / kg of subject body weight, or about 100 μg / kg to about 1 mg / kg of subject body weight.

[0185] VI. Pharmaceutical Composition 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 the anti-Claudin-1 antibody described herein, or a biologically active fragment thereof, and at least one pharmaceutically acceptable carrier or excipient.

[0186] In some embodiments, provided herein is a pharmaceutical composition for the treatment of cholangiopathy, the pharmaceutical composition comprising a therapeutically effective amount of an anti-Claudin-1 antibody.

[0187] In some embodiments, the cholangiopathy is primary sclerosing cholangitis (PSC).

[0188] In some embodiments, the cholangiopathy is primary biliary cirrhosis (PBC).

[0189] In some embodiments, the cholangiopathy is hereditary cholangiopathy. In some embodiments, the cholangiopathy is idiopathic cholangiopathy. In some embodiments, the cholangiopathy is malignant cholangiopathy. In some embodiments, the cholangiopathy is secondary sclerosing cholangitis.

[0190] In some embodiments, the hereditary cholangiopathy is selected from the group consisting of Alagille syndrome, Caroli syndrome, cystic fibrosis, and polycystic liver disease.

[0191] In some embodiments, the idiopathic cholangiopathy is selected from the group consisting of autoimmune cholangitis, biliary atresia, idiopathic pediatric or adult cholangiopathy, IgG4-related cholangitis, primary biliary cirrhosis, and primary sclerosing cholangitis.

[0192] In some embodiments, the malignant cholangiopathy is cholangiocarcinoma.

[0193] In some embodiments, secondary sclerosing cholangitis is selected from the group consisting of ABCB4 deficiency, abdominal trauma (surgical or blunt), AIDS cholangiopathy, amyloidosis, chemicals / drugs (i.e., 5-fluorouracil), common bile duct stones, eosinophilic or mast cell cholangitis, graft-versus-host disease with liver, iatrogenic biliary stricture, portal hypertensive biliary disease, recurrent pyogenic cholangitis, sarcoidosis, sickle cell disease, and vascular / ischemic (i.e., hepatic artery stenosis after liver transplantation).

[0194] The pharmaceutical composition can be administered in any amount effective to achieve the desired prophylactic and / or therapeutic effect using any route of administration. The optimal pharmaceutical formulation may 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.

[0195] 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.

[0196] 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, which enables the administration of an anti-claudin-1 antibody, or a biologically active fragment thereof.

[0197] 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.

[0198] A notice or attachment in a form determined by a government agency that regulates the manufacture, use, or sale of a pharmaceutical or biological product may optionally be 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.

[0199] 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, and tracking of movement between workstations.

[0200] In some aspects, provided herein is a kit for treating a subject suffering from cholangiopathy, the kit comprising a therapeutically effective amount of an anti-claudin-1 antibody and an attachment comprising instructions for use of the kit.

[0201] In some aspects, the cholangiopathy is primary sclerosing cholangitis (PSC).

[0202] In some aspects, the cholangiopathy is primary biliary cirrhosis (PBC).

[0203] In some aspects, the cholangiopathy is hereditary cholangiopathy. In some aspects, the cholangiopathy is idiopathic cholangiopathy. In some aspects, the cholangiopathy is malignant cholangiopathy. In some aspects, the cholangiopathy is secondary sclerosing cholangitis.

[0204] In some aspects, the hereditary cholangiopathy is selected from the group consisting of Alagille syndrome, Caroli syndrome, cystic fibrosis, and polycystic liver disease.

[0205] In some aspects, the idiopathic cholangiopathy is selected from the group consisting of autoimmune cholangitis, biliary atresia, idiopathic pediatric or adult cholangiopathy, IgG4-related cholangitis, primary biliary cirrhosis, and primary sclerosing cholangitis.

[0206] In some embodiments, the malignant cholangiopathy is cholangiocarcinoma.

[0207] In some embodiments, secondary sclerosing cholangitis is selected from the group consisting of ABCB4 deficiency, abdominal trauma (surgical or blunt), AIDS cholangiopathy, amyloidosis, chemicals / drugs (i.e., 5-fluorouracil), common bile duct stones, eosinophilic or mast cell cholangitis, graft-versus-host disease with liver, iatrogenic biliary stricture, portal hypertensive biliary disease, recurrent pyogenic cholangitis, sarcoidosis, sickle cell disease, and vascular / ischemic (i.e., hepatic artery stenosis after liver transplantation).

Example

[0208] The following examples are illustrative and do not limit the scope of the claimed embodiments.

[0209] Example 1. Improvement of cholestasis and fibrosis by treatment with a CLDN1-specific monoclonal antibody in a DDC mouse model of cholangiopathy A proof-of-concept study using a CLDN1-specific mAb was conducted in a 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) mouse model, i.e., a well-recognized animal model of PSC in which C57BL / 6 mice were supplemented with 0.1% DDC in their diet. DDC impairs heme metabolism in mouse hepatocytes, causing the secretion of insoluble crystals of protoporphyrin IX into the bile canaliculi and leading to bile flow obstruction. Within 3 to 6 weeks, the DDC diet causes cholestasis, inflammation, fibrosis, and activation of hepatic progenitor cells. Fibrosis was evaluated by collagen and fibronectin staining of liver tissue.

[0210] All experiments were conducted in the animal facility of Inserm U1110, in accordance with local laws, approval from the ethics committee, and approval by the French Ministry of Higher Education and Research. Forty 7-week-old C57BL / 6J mice (Charles River Laboratories, France) were fed a 0.1% DDC-supplemented diet (SAFE, Augy, France) for 4 weeks. After the first week, when periductal fibrosis was established, the mice were randomized into groups that received intraperitoneal injection of CLDN1-specific H3L3 mAb (25 mg / kg, n = 20) or vehicle control (n = 20) once a week for 3 weeks (a total of 3 Ab injections). Mice were sacrificed at the end of the 4-week diet, and plasma and liver were collected for subsequent analysis (Figure 1). The content of fibrotic tissue was evaluated by Sirius red staining of formalin-fixed paraffin-embedded sections of the whole liver lobe. Quantification of the positive area was performed using the threshold method with ImageJ v1.53c software (National Institutes of Health, Bethesda, USA).

[0211] DDC impairs heme metabolism in mouse hepatocytes, and insoluble crystals of protoporphyrin IX are secreted into bile canaliculi, resulting in bile flow obstruction. The expression of CLDN1 in cholangiocytes, reactive ductules, and surrounding hepatocytes identified these cells as therapeutic targets for CLDN1 mAb (Figure 2). Based on the expression and functional data of CLDN1 in the DDC model, treatment with anti-CLDN1 antibody may improve the phenotypes of damaged hepatocytes, cholangiocytes, or reactive ductular cells, leading to reduction of fibrosis, improvement of cholestasis, and prevention of carcinogenesis. Furthermore, anti-CLDN1 antibody treatment may lead to the restoration of a physiological healing response rather than "inappropriate regeneration".

[0212] First, the publicly available RNA-Seq database was used to analyze CLDN1 expression in the DDC mouse model. As shown in FIGS. 3A-3D, CLDN1 is overexpressed in both progenitor cells (PROM1+) and non-progenitor cells in the regenerative mouse model, but not in the physiological regeneration model with progenitor cell proliferation and activation, nor in the physiological regeneration model with fibrosis deposition.

[0213] In the in vivo proof-of-concept study, diseased animals were treated with CLDN1-specific mAb. Seven-week-old C57BL / 6J mice were fed a 0.1% DDC-supplemented diet for 4 weeks. After the first week, when periductal fibrosis had already been established, the mice were randomized into groups that received intraperitoneal injections of CLDN1 mAb (25 mg / kg, n = 20) or vehicle control (n = 20) once a week for 3 weeks. The mice were sacrificed after 4 weeks of 0.1% DDC diet and 3 weeks of mAb treatment. The livers were harvested for subsequent analysis. CLDN1 mAb treatment resulted in a decrease in cholestasis, a characteristic of PSC, as indicated by a decrease in plasma alkaline phosphatase (2398 U / L vs. 2119 U / L, FIG. 5C). Automated analysis of Sirius red staining (FIG. 4) and collagen proportion area (CPA) (FIG. 5A) revealed a significant decrease in fibrosis in DDC mice treated with CLDN1 mAb compared to mice treated with vehicle control (p < 0.0001, t-test). The total CPA was 7.79% in mice treated with CLDN1 mAb, compared to 10.66% in the vehicle control (t-test p < 0.0001, FIG. 5A), clearly demonstrating the robustness of the efficacy. Furthermore, determination of the Ishak score, one of the most relevant fibrosis scoring systems applied to patients, confirmed a significant improvement in fibrosis in mice treated with CLDN1 mAb (FIG. 5B). Finally, no significant increase in major side effects or mortality was observed in animals treated with CLDN1 mAb compared to vehicle control animals.

[0214] These results suggest a functional role of CLDN1 in the etiology of PSC and provide robust preclinical proof-of-concept for CLDN1-specific mAbs as a novel therapeutic approach for PSC.

[0215] To investigate whether the effect of CLDN1 mAb on cholangiofibrosis is dose-dependent, the experiment was repeated by conducting a single escalating dose study using anti-CLDN1 mAb ALE-F02 (Roehlen et al., 2022) in the DDC model (Fickert et al., 2007). Since CLDN1 mAb ALE-F02 selectively binds to human CLDN1, a humanized transgenic mouse model (h / mCLDN1 Tg mouse) in which mice were engineered to express a humanized extracellular loop 1 was used to enable strong binding of ALE-F02 CLDN1 mAb to CLDN1 in mouse tissues. Compared with the previous study shown in Figure 1, the DDC feeding scheme was modified to comply with the ethical requirements regarding weight loss.

[0216] Eighty 20- to 22-week-old h / mCLDN1 Tg mice were fed a 0.1% DDC-supplemented diet 5 days a week and a standard diet 2 days a week for the first 3 weeks. In the fourth week, the mice were fed a DDC-supplemented diet for 7 days. If periportal fibrosis was established in the first week (Figure 13), the mice were randomly assigned to four groups (1:1:1:1) and received either 5, 10, or 25 mg / kg of ALE.F02 CLDN1 mAb or a vehicle control (PBS) once a week by intraperitoneal injection. The total study period was 4 weeks. At the time of sacrifice, the livers were harvested for subsequent analysis.

[0217] Automated analysis of the collagen proportion area (CPA) of Sirius red-stained liver (Figure 13C) (Figure 13B) revealed that fibrosis was significantly reduced in a dose-dependent manner in mice treated with CLDN1 ALE.F02 mAb compared to mice treated with vehicle control. The relative reduction in CPA was -10.3% (p = 0.016, Mann-Whitney U test) relative to the control at ALE.F02 5 mg / kg, -19.2% (p < 0.0001, Student's t test) relative to the control at ALE.F02 10 mg / kg, and -21.9% (p < 0.0001, Student's t test) relative to the control at ALE.F02 25 mg / kg.

[0218] These results indicate that treatment with ALE.F02 anti-CLDN1 mAb dose-dependently reduces biliary fibrosis in a state-of-the-art mouse model of cholangiopathy and biliary fibrosis. Considering that there are similar biliary fibrosis mechanisms in the DDC model and patients with cholangiopathies such as PBC and PSC, treatment with CLDN1-specific Ab is likely to improve biliary fibrosis in patients with cholangiopathy.

[0219] Example 2. CLDN1 expression in liver tissue of patients Human liver tissues fixed with formaldehyde and embedded in paraffin were selected from the files of the pathology service of the University Hospital Geneva. These included cirrhotic specimens obtained from patients who had undergone hepatectomy during liver transplantation. Standard analysis using an optical microscope was performed on all specimens. The study was conducted in accordance with the principles of the Declaration of Helsinki. Immunohistochemistry was performed as follows: After antigen activation by heat, 3-μm sections of formaldehyde-fixed and paraffin-embedded specimens were incubated with rabbit polyclonal anti-human Claudin-1 antibody (Elabscience, Texas, USA) at a dilution of 1:250 for 1 hour at room temperature, followed by incubation with anti-rabbit antibody for 30 minutes (room temperature), and then with a liquid diaminobenzidine substrate-chromogen system (DakoCytomation, Glostrup, Denmark). In co-localization experiments, double immunostaining was performed on serial sections (3-μm thick) using Claudin-1 and each of these five different antibodies: rabbit polyclonal anti-human CK19 (ABCAM, Cambridge, UK) at a dilution of 1:400, rabbit polyclonal anti-human EPCAM (Invitrogen, Massachusetts, USA) at a dilution of 1:250. 3-μm serial sections of paraffin-embedded kidney were subjected to appropriate antigen activation, incubated with Claudin-1 at a dilution of 1:250 for 1 hour at room temperature, followed by incubation with anti-rabbit antibody for 30 minutes (room temperature), and then with a liquid diaminobenzidine substrate-chromogen system (DakoCytomation, Glostrup, Denmark). Subsequently, the sections were incubated with the appropriate antibody for 1 hour at room temperature, followed by incubation with the appropriate secondary antibody for 30 minutes, and then with a phosphatase alkaline-fast red enzyme system (DakoCytomation, Glostrup, Denmark). Counterstaining was performed using Mayer's hematoxylin.

[0220] Data on CLDN1 gene expression in mouse models were obtained from bulk RNA-seq datasets deposited in Gene Expression Omnibus (GSE20427, GSE77503, GSE28892, GSE29121). This bulk RNA-seq data from mouse models of liver regeneration revealed that CLDN1 is overexpressed by both PROM1+ liver progenitor cells and non-progenitor cells in the DDC mouse model (Figs. 3A-3D). On the other hand, CLDN1 is not overexpressed in a model of physiological liver regeneration, i.e., partial hepatectomy (Fig. 3A).

[0221] Data on CLDN1 expression in humans were obtained from publicly available datasets of single-cell resolution transcriptomics studies derived from either healthy liver (GSE124395) or fibrotic liver (GSE136103) based on the original datasets deposited in GEO (Gene Expression Omnibus, NIH, Bethesda, USA). Single-cell resolution transcriptome analysis in the human hepatocyte atlas and cirrhotic liver revealed high-level expression of CLDN1 in hepatocytes, EPCAM+ cells and cholangiocytes, as well as stellate cells and myofibroblasts (Figure 6A). Furthermore, CLDN1 expression co-clustered with known markers of cholangiocytes, i.e., bipotent epithelial progenitor cells of the liver (Figure 6B). Cells with the highest expression of CLDN1 also expressed markers of cholangiocytes (bipotent liver progenitor cells), suggesting an association between high CLDN1 expression and a highly plastic cell phenotype (Figure 6C). These observations were confirmed at the protein level, and double-immunofluorescence staining of CLDN1 / CK19 (Figure 7A) and CLDN1 / EPCAM (Figure 7B) by immunohistochemistry revealed CLDN1 expression by ductular reactive cells, cholangiocytes and hepatocytes within cirrhotic nodules. Using immunohistochemistry of patient tissues with advanced hepatic fibrosis, co-expression of CLDN1 and CK19 in the surrounding ductular reaction, and co-expression of CLDN1 and EPCAM in the membranes of ductular reactive cells and hepatocytes were observed (Figures 7A-7B). Since these cell types have been shown to play important roles in the pathogenesis of cholangiopathies, including PSC and PBC, CLDN1 is a therapeutic target for cholangiopathies.

[0222] To study the expression of CLDN1 as a target for therapeutic intervention across various cholangiopathies, CLDN1 expression was examined in cholangiopathy patients from transcriptome datasets obtained from public repositories. These included Gene Expression Omnibus (National Center for Biotechnology Information, NIH, Bethesda, MD, GSE46960, GSE206364, GSE118373) and Array Express (European Bioinformatics Institute, EMBL, Heidelberg, Germany, E-GEOD-61260).

[0223] CLDN1 was robustly and significantly upregulated in the livers of patients with primary biliary cholangitis (p = 0.0001, Mann-Whitney U test, E-GEOD-61260, Figure 14A) (5), biliary atresia (p < 0.0001, Mann-Whitney U test, GSE46960, Figure 14B) (Bessho, K., et al., Hepatology 60(1):211-223(2014)), Alagille syndrome (p = 0.001, Mann-Whitney U test, GSE206364, Figure 14B) (Kriegermeier, A., et al., PLoS One 17(12):e0279016(2022)) and primary sclerosing cholangitis (p = 0.018, Mann-Whitney U test, E-GEOD-61260, Figure 14D) (Bessho et al. 2014). Furthermore, CLDN1 was one of the top 20 genes upregulated in the laser microdissected ductular reaction of primary sclerosing cholangitis patients compared to the ductular reaction of HCV-related liver disease (p = 0.004, Mann-Whitney U test, GSE118373, Figure 14E) (Govaere, O., et al., J Pathol. 248(1):66-76(2019)).

[0224] These data indicate that CLDN1 is strongly upregulated across multiple cholangiopathies, and confirm its role as a therapeutic target for CLDN1-specific mAbs across a number of diverse and distinct cholangiopathies, including, for example, PBC, PSC, biliary atresia, and Alagille syndrome.

[0225] Example 3. CLDN1 in the DDC model of human liver chimeric mice FRG-NOD Fah- / - mice are used as a further proof of concept for non-conjugated CLDN-1 specific monoclonal antibodies in a humanized model (Figure 8). Primary human hepatocytes are injected on day 0. Twelve weeks after injection, the mice are checked for successful engraftment and humanization. If humanization is successful, the mice receive a pulsed DDC diet and weekly injections of anti-CLDN-1 monoclonal antibody (25 mg / kg) or control. The mice are sacrificed 12 - 20 weeks later. After sacrifice, the mice are studied using standard techniques in the art, such as immunohistochemistry, for fibrosis, ductular reaction, cholestasis, biliary dysplasia, and plasticity of human progenitor cells.

[0226] Example 4. CLDN1 as a target for proof of concept studies in the bile duct ligation model: Treatment with anti-CLDN1 mAb H3L3 improves survival, cholestasis, liver function, and fibrosis. To examine whether CLDN1 is generally a therapeutic target for cholangiopathy, a bile duct ligation (BDL) model was applied (Figure 9). In the BDL model, the common bile duct of mice is surgically ligated. Bile duct obstruction causes cholangiopathy and subsequent liver diseases including fibrosis and inflammation. When bile flow is completely obstructed, acute cholestasis occurs, followed by activation of hepatic progenitor cells and development of biliary fibrosis (Georgiev, P., et al., Br J Surg 95(5):646-656 (2008), Takahashi, Y., et al., Chapter 13 - Animal Models of Liver Disease. In: Conn PM, editor. Animal Models for the Study of Human Disease (Second Edition) Academic Press; p. 313-339 (2017)). Despite the high mortality rate, this model is frequently used as a universal model of cholangiopathy including PBC, PSC, biliary atresia, and other cholangiopathies because cholangiopathy is rapidly induced.

[0227] Using transgenic mice expressing the human extracellular loop 1 of CLDN1, the efficacy of anti-CLDN1 mAb in the BDL model was investigated. Considering the mortality of mice after surgical ligation of the common bile duct in this model, the protocol was shortened to 7 days after surgical ligation of the common bile duct. Forty mice were subjected to bile duct ligation and then randomly assigned 1:1 to two treatment groups, and either anti-CLDN1 mAb H3L3 (Colpitts et al. 2018) or vehicle control (PBS) was administered intraperitoneally. Each treatment was administered immediately after surgery and on the 4th day after surgery (Figure 15A). Surviving mice (n = 10 in the control group and n = 15 in the H3L3 group) were euthanized on the 7th day after surgery, and the liver and plasma were collected for subsequent analysis. Analysis of the Kaplan-Meier survival curve showed a survival advantage in the treatment group (HR = 0.39 for death in the treatment group, 95% CI 0.14-1.15, p = 0.08) (Figure 15B). Liver function tests revealed that markers of liver injury, liver function, and cholestasis were significantly and markedly improved in animals treated with CLDN1 H3L3 mAb compared to animals treated with control. These included a significant and marked decrease in the levels of alanine aminotransferase ALT (306 vs 1591 U / l, relative change -80.8%, p = 0.016, Mann-Whitney U test) and aspartate aminotransferase AST (372 vs 3069 U / l, relative change -87.9%, p = 0.019, Mann-Whitney U test) as markers of hepatocyte injury and inflammation (Figure 15C), a significant and marked decrease in the levels of total bilirubin (174.6 vs 318.4 μmol / l, relative change -45.2%, p = 0.012, Student's t test) and alkaline phosphatase (598.4 vs 913.9 U / l, relative change -34.5%, p = 0.017, Student's t test) indicating a significant and marked improvement in cholestasis (Figure 15D). Furthermore, albumin levels were increased in animals treated with CLDN1 mAb compared to animals treated with control, indicating a significant and marked improvement in liver function by CLDN1 mAb treatment (24 vs 14 g / l, relative change +71.4%, p = 0.0006, Mann-Whitney U test) (Figure 15E).Automated analysis of the collagen proportion area (CPA) of Sirius red-stained liver (Figure 15F) revealed that liver fibrosis in mice treated with CLDN1 mAb was significantly and markedly reduced compared to mice treated with the control (4.08% vs. 6.45%, relative change -36.8%, p < 0.0001, Student's t-test).

[0228] In summary, these results demonstrate that anti-CLDN1 mAb treatment improves survival and significantly and markedly improves liver function, cholestasis, and biliary fibrosis in a state-of-the-art animal model of obstructive cholestasis and cholangiopathy. Considering that there are similar pathological mechanisms in the bile duct ligation model and cholangiopathy patients, treatment with CLDN1-specific Ab is likely to improve the bile ducts and liver function, as well as the overall outcome, of cholangiopathy patients.

[0229] Example 5. CLDN1 mAb treatment in a long-term model of progression to PSC and cholangiocarcinoma MDR2- / -(Abcb4- / -) mice are used to model PSC and cholangiocarcinoma (CCA) (Figure 10). Mdr2 (Abcb4) is the mouse ortholog of the human MDR3 (ABCB4) gene, which encodes a canalicular phospholipid transporter. Gene disruption of the Mdr2 gene in mice causes liver fibrosis, sclerosing cholangitis, and cholelithiasis due to the complete absence of phosphatidylcholine from bile. Mdr2- / -(Abcb4- / - mice) spontaneously develop biliary fibrosis and reproduce the important histological features and carcinogenesis of PSC over 10 - 12 months. Six weeks after the start of the study, mice are initiated with anti-CLDN-1 monoclonal antibody (25 mg / kg) or a control once-weekly injection. Mice are sacrificed at week 48. After sacrifice, the mice are studied using standard techniques in the art, such as immunohistochemistry, for fibrosis, ductular reaction, and plasticity of human progenitor cells.

[0230] Example 6. Effect of CLDN1 mAb on HepaRG cells as a model of cell fate: Treatment with the anti-CLDN1 mAb H3L3 promotes the maturation of progenitor cells into hepatocyte-like or cholangiocyte-like cells. The HepaRG cell line, a hepatoblast cell line with the ability to differentiate into both hepatocytes and cholangiocyte-like cells, is treated with an anti-CLDN1 monoclonal antibody (Figure 11).

[0231] In the first experiment, HepaRG cells are grown until they are completely confluent and then treated with either an anti-CLDN1 monoclonal antibody or a control with 2% DMSO every three days for 14 days to obtain a mixed population of hepatocyte-like and cholangiocyte-like cells. Fluorescence-activated cell sorting (FACS) is used to determine the ratio of hepatocyte-like cells to cholangiocyte-like cells (Figure 12A).

[0232] In the second experiment, completely confluent HepaRG cells are reseeded at a low density (2×10 4 / cm 2 ). After reseeding, the cells are treated with an anti-CLDN1 monoclonal antibody (H3L3) or a control for 5 - 14 days. After treatment, the cells are harvested and analyzed for the efficiency of dedifferentiation to return to the progenitor state by evaluating the expression of CK19 via FACS or immunofluorescence (Figure 12B).

[0233] In the third experiment, HepaRG cells are seeded on day 0 and differentiated into cholangiocyte-like cells by day 10. On day 10, CLDN1 expression is confirmed by FACS. Then, these cholangiocyte-like cells are treated with TNFα or an anti-CLDN1 monoclonal antibody for 72 hours. After 72 hours, the cells are harvested and RNA sequencing is performed on these two populations (Figure 12C).

[0234] For all three experiments, it is expected that after treatment with the anti-CLDN1 monoclonal antibody, a phenotype with low fibrotic promotion and low carcinogenicity will be observed.

[0235] Treatment with the CLDN1 mAb inhibits inflammatory, fibrogenic, and oncogenic signaling observed in PSC patients in the cholangiocyte model. To identify the cellular circuits and signaling pathways targeted by CLDN1 mAb therapy in cholangiopathies, the transcriptional signatures of inflammation, fibrosis, and cancer-related signaling were analyzed in a cholangiocyte-based model, and the regulation induced by the antibody was compared with changes in gene expression observed in the livers of PSC patients. The cholangiocyte model consisted of HepaRG differentiated cholangiocyte-like cells, incubated with tumor necrosis factor α (TNF-α) as previously described (Dianat, N., et al., Hepatology 60(2):700-714(2014)). Gene expression in the treated cells was evaluated by RNASeq and compared with gene expression and signaling pathways in the livers of PSC versus control patients as described in (Horvath et al. 2014)(E-GEOD-61260).

[0236] HepaRG progenitor cells were cultured in William's E medium supplemented with 2% FBS, 1 μg / ml insulin, and 1 μM hydrocortisone hemisuccinate as previously described (Dianat et al. 2014). To induce cholangiocyte differentiation, on day 4 after seeding, the cells were treated with 10 ng / ml IL-6 for 2 days, then with 10 nM sodium taurocholate hydrate for 2 days, and then with 10 nM sodium taurocholate hydrate and 1.8 μM sodium butyrate for 2 days. After completion of cholangiocyte differentiation, the cells were treated with either 10 ng / ml TNF-α and 10 μg / ml H3L3 (Colpitts et al. 2018) anti-CLDN1 mAb or 10 ng / ml TNF-α and 10 μg / ml isotype control antibody for 96 hours. Four days later, the cells were harvested and RNA was extracted for RNAseq. Gene Set Enrichment Analysis (GSEA) was used for unbiased pathway analysis using the Molecular Signatures Database (MSigDB) (Subramanian, A., et al., Proc Natl Acad Sci USA 102(43):15545-15550 (2005)). Unbiased evaluation of gene sets included in MSigDB was also used for analysis of clinically observed gene expression changes in PSC liver tissue microarray data (E-GEOD-61260) (Horvath et al. 2014), and then compared with RNA-Seq gene expression in a cholangiocyte-like cell-based model treated with CLDN1-specific mAb or isotype control mAb. An FDR < 0.05 was considered statistically significant (Figure 17A).

[0237] As shown in Figure 17, in the cholangiocyte-like cell-based model, H3L3 CLDN1 mAb treatment reversed the gene expression of a wide range of signaling pathways that were upregulated in the diseased livers of PSC patients. These included the inflammatory pathways observed in the TNF-α-NFkB and STAT3 gene expression pathways, which showed the most significant inhibitory effect of CLDN1 Ab compared to the control Ab (Figure 17B). Furthermore, the gene expression of the profibrotic TGFβ signaling pathway, which was highly upregulated in the livers of PSC patients, was downregulated by CLDN1 mAb treatment in the cell-based model (Figure 17B). Interestingly, several oncogenic signaling pathways that were highly upregulated in the livers of PSC patients were also downregulated in cells treated with CLDN1 mAb compared to cells treated with the isotype mAb. These pathways included EGFR, HRAS, and AKT / MTOR signaling (Figure 17B).

[0238] Collectively, these data indicate that CLDN1 mAb treatment potently inhibits the expression of inflammatory, fibrogenic, and oncogenic signaling pathways in the cholangiocyte-based model, which were upregulated in the liver tissue of PSC patients. Furthermore, these data suggest that the inhibitory effect of CLDN1 mAb treatment on inflammatory, profibrotic, and pro-oncogenic pathways is likely to be clinically relevant in PSC and cholangitis patients. These data suggest that treatment with CLDN1 mAb may improve biliary diseases, inflammation, fibrosis, and cancer in cholangitis patients.

[0239] Example 8. TNFα induces upregulation of CLDN1 protein expression targeted by CLDN1 mAb, and treatment with CLDN1 mAb inhibits TNFα-NFkB signaling. To understand the regulation of CLDN1 expression as a target of CLDN1 mAb in human cholangiocytes and cholangiopathies, CLDN1 expression was studied in a cell-based model consisting of primary human cholangiocytes and treated with tumor necrosis factor alpha (TNFα) to model cholangitis and injury. Since TNFα was identified as a clinically relevant pro-inflammatory cytokine in the sera of PSC patients, it was selected as an inducer of the inflammatory response (Zweers, SJ., et al. Liver In. 36(9):1370-1377(2016)). Furthermore, the NFkB (nuclear factor kappa-light-chain enhancer of activated B cells) signaling (a downstream effector pathway induced by TNFα) was identified as one of the top upregulated signaling pathways in PSC livers as shown in Figure 17B.

[0240] Primary human cholangiocytes were purchased from Innoprot, Bizkaia, Spain (Human Biliary Epithelial Cells, HBEpiC, ref. P10654, batch number 21614) and cultured on a poly-1-lysine-coated surface according to the manufacturer's instructions. After stimulation with 10 ng / ml of TNFα for 24 hours, cells were harvested for the evaluation of CLDN1 expression by flow cytometry using anti-CLDN1 mAb H3L3 (Colpitts et al. 2018) and an anti-human AF647-conjugated secondary antibody.

[0241] As shown in Figure 18A, TNFα stimulation significantly increased CLDN1 protein expression in primary human cholangiocytes that strongly bound to CLDN1 mAb H3L3 on the cholangiocyte surface as shown by flow cytometry (p = 0.0006, Mann-Whitney U test). These data indicate that CLDN1 expression is upregulated by defined pro-inflammatory cytokines, supporting the possibility that CLDN1 is targeted by CLDN1-specific mAbs on human cholangiocytes in inflammatory cholangiopathies such as PSC and cholangitis.

[0242] To examine the effect of CLDN1 mAb H3L3 on pro-inflammatory signaling in cholangiocytes, NFκB signaling was studied in primary human cholangiocytes (HBEpiC). Cholangiocytes were treated with either CLDN1 H3L3 (Colpitts et al. (2018)) or isotype control mAb (both at 10 μg / ml) for 72 hours, and the antibodies were renewed after 48 hours. NFκB signaling was induced by treatment with TNFα (10 ng / ml) in the presence of antibody treatment. Cells were harvested for protein extraction and immunoblotting for IKBα (nuclear factor of κ light polypeptide gene enhancer in B-cells inhibitor), pIKBα (phosphorylated nuclear factor of κ light polypeptide gene enhancer in B-cells inhibitor), P65, and pP65 (phosphorylated P65) 60 or 120 minutes after incubation with TNFα. GAPDH staining was used as a loading control. Protein signals were then quantified by imaging. As shown in FIGS. 18B - C, CLDN1 mAb H3L3 treatment inhibited TNF-α-mediated phosphorylation of IKBα (p = 0.003, Mann-Whitney U test, n = 10, FIG. 18B) and P65 (p = 0.0002, Mann-Whitney U test, n = 10, FIG. 18C) in primary human cholangiocytes. These data indicate that treatment with CLDN1 inhibits TNFα-NFκB signaling in a cell-based model of human cholangiocytes compared to control mAb. Inhibition of TNFα-NFκB signaling by CLDN1 antibody may contribute to its therapeutic effect in patients with cholangiopathy.

[0243] Example 9. Effect of CLDN1 mAb on HepaRG cells as a model of cell fate: Treatment with anti-CLDN1 mAb H3L3 promotes the maturation of progenitor cells into hepatocyte-like cells. To further understand the mechanism of action of CLDN1 therapy in improving liver function in biliary tract diseases, the effect of CLDN1 mAb was investigated using a HepaRG cell-based hepatic progenitor cell model (Laurent, V., et al., Methods Mol Biol. 987:295-314 (2013)). The HepaRG progenitor model consists of hepatoblast-like cells with the ability to differentiate into hepatocytes (Marion, MJ, et al., Methods Mol Biol. 640:261-272 (2010)) or cholangiocytes (Marion et al. 2010). Using a defined protocol, HepaRG can be differentiated into hepatocyte-like cells (Marion et al. 2010), which enables the study of the effect of compounds on the hepatocyte maturation process. Here, the HepaRG model was used to study the effect of CLDN1 H3L3 mAb treatment on the differentiation of HepaRG hepatic progenitor cells into hepatocyte-like cells compared to isotype control mAb treatment (Figure 16A).

[0244] HepaRG progenitor cells were cultured as previously described (Laurent et al. 2013). After growing for 14 days until they were completely confluent, the cells were treated with 2% DMSO to induce hepatocyte differentiation. Anti-CLDN1 mAb (10 μg / ml) (Colpitts et al. 2018) or an isotype control (10 μg / ml) was added to the differentiation medium. Fresh antibody was added during medium changes twice a week. After 2 weeks of differentiation, the cells were harvested to examine the protein and gene expression markers of mature hepatocytes. The abundance of mature hepatocyte-like cells was evaluated by flow cytometry using a fluorescent-conjugated preS1 HBV peptide that specifically binds to the Na+-taurocholate cotransporting polypeptide (NTCP) protein on target cells, which is a marker of mature hepatocytes (Ni, Y., et al. Methods Mol Biol. 1540:15-25 (2017)). The gene expression of typical hepatocyte and duct markers was evaluated by RT-qPCR.

[0245] Treatment of HepaRG cells with CLDN1 mAb significantly increased the abundance of NTCP+ cells (n = 3 independent experiments, p = 0.002, Student's t-test) (Figure 16B), indicating that anti-CLDN1 mAb treatment promotes the maturation of progenitor cells into hepatocytes (Ni Y., et al. 2017). Furthermore, as shown in Figure 16C, anti-CLDN1 mAb treatment upregulated the expression of hepatocyte markers albumin (ALB) (n = 4 independent experiments, p = 0.0004, Student's t-test), hepatocyte nuclear factor 4 alpha (HNF4A) (n = 4 independent experiments, p = 0.005, Student's t-test), cytochrome P450 3A4 (CYP3A4) (n = 4 independent experiments, p = 0.018, Student's t-test), transthyretin (TTR) (n = 4 independent experiments, p = 0.0001, Student's t-test), UDP glucuronosyltransferase family 1 member A1 (UGT1A1) (n = 4 independent experiments, p = 0.007, Mann-Whitney U test), bile acid-CoA:amino acid N-acyltransferase (BAAT) (p = 0.0009, Student's t-test), and transferrin (TF) (n = 4 independent experiments, p = 0.0016, Student's t-test). In contrast, the expression of secreted phosphoprotein 1 (SPP1), a marker of immature cells or duct markers, was significantly downregulated (p < 0.0001, Student's t-test).

[0246] These results indicate that anti-CLDN1 mAb treatment significantly upregulates the expression of genes associated with a wide range of hepatocyte-specific functions, including hepatocyte-specific protein biosynthesis, bile acid transport and metabolism, and xenobiotic metabolism. Downregulation of SPP1 gene expression further supports that CLDN1 mAb treatment regulates cell plasticity and the fate of hepatocytes towards maturity from immaturity. Collectively, these findings show that anti-CLDN1 mAb treatment promotes the maturation of immature hepatic progenitor cells into mature hepatocyte-like cells in a cell-based model, which may account for part of its effect on the improvement of liver function as shown in DDC and bile duct ligation animal models, respectively. Considering that these mechanisms have been shown to play a functional role in patients, these observations are clinically relevant and are likely to contribute to the improvement of liver diseases in cholestatic patients treated with CLDN1-specific Abs. ***

[0247] In the practice of 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.

[0248] All cited references are hereby incorporated by reference in their entirety.

[0249] All examples provided in this specification are provided by way of example and not by way of limitation.

Claims

1. A method for treating cholangiopathy in a human subject in need thereof, the method comprising administering to the human subject a therapeutically effective amount of an anti-claudin-1 antibody.

2. A method for restoring the integrity of biliary epithelium 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, wherein the administration results in the proliferation of hepatic progenitor cells and / or the differentiation and conversion of hepatocytes into ductal cells.

3. A method for reducing PSC-related ulcerative colitis, the method comprising administering an anti-claudin-1 antibody to a human subject.

4. A method for reducing biliary fibrosis, the method comprising administering an anti-claudin-1 antibody to a human subject.

5. The method according to claim 1, wherein the cholangiopathy is primary sclerosing cholangitis (PSC).

6. The method according to claim 1, wherein the cholangiopathy is primary biliary cirrhosis (PBC).

7. The method according to any one of claims 1 to 6, wherein claudin-1 (CLDN1) is overexpressed in the human subject compared to the expression level in a normal subject.

8. The method according to any one of claims 1 to 7, 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.

9. The method according to any one of claims 1 to 8, 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.

10. The method according to any one of claims 1 to 9, wherein the anti-claudin-1 antibody is humanized.

11. The method according to any one of claims 1 to 10, 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.

12. The method according to any one of claims 1 to 11, 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.

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: 3 and a VL comprising the amino acid sequence shown in SEQ ID NO:

4.

14. The method according to any one of claims 1 to 13, 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.

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 cholangiopathy in a human subject, wherein the method comprises administering an effective amount of the anti-claudin-1 antibody or the pharmaceutical composition thereof to the human subject, the anti-claudin-1 antibody or the pharmaceutical composition thereof.

17. Use according to claim 16, wherein the cholangiopathy is primary sclerosing cholangitis (PSC).

18. Use according to claim 16, wherein the cholangiopathy is primary biliary cirrhosis (PBC).

19. Use according to any one of claims 16 to 18, 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 deposit number DSM ACC2938.

20. Use according to any one of claims 16 to 19, 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.

21. Use according to any one of claims 16 to 20, wherein the anti-claudin-1 antibody is humanized.

22. Use according to any one of claims 16 to 21, 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.

23. Use according to any one of claims 16 to 22, 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.

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 and a VL comprising the amino acid sequence shown in SEQ ID NO:

4.

25. Use according to any one of claims 16 to 24, 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.

26. Use according to any one of claims 16 to 25, wherein 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.

27. Use according to any one of claims 16 to 26, wherein 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.

28. Use according to any one of claims 16 to 27, wherein the anti-claudin-1 antibody is administered intratumorally, intravenously, intraperitoneally, intramuscularly, intrathecally or subcutaneously.

29. The method according to claim 1, wherein the cholangiopathy is biliary fibrosis.

30. The method according to claim 29, wherein the biliary fibrosis is caused by cystic fibrosis.

31. The method according to claim 29, wherein the biliary fibrosis is caused by IgG4-related disease.

32. Use according to claim 16, wherein the cholangiopathy is biliary fibrosis.

33. The use according to claim 32, wherein the biliary fibrosis is caused by cystic fibrosis.

34. The use according to claim 32, wherein the biliary fibrosis is caused by IgG4-related disease.

35. The method according to claim 1, wherein the biliary disease is biliary atresia.

36. The method according to claim 1, wherein the biliary disease is Alagille syndrome.

37. The use according to claim 16, wherein the biliary disease is biliary atresia.

38. The use according to claim 16, wherein the biliary disease is Alagille syndrome.

39. A method for reducing bile stasis in a human subject in need thereof, the method comprising administering a therapeutically effective amount of an anti-claudin-1 antibody to the human subject.

40. A method for improving hepatitis and liver function in a human subject in need thereof, the method comprising administering a therapeutically effective amount of an anti-claudin-1 antibody to the human subject.

41. A method for improving survival from biliary disease in a human subject in need thereof, the method comprising administering a therapeutically effective amount of an anti-claudin-1 antibody to the human subject.

Images