Treatment of drug-resistant hepatocellular carcinoma

Abstract

The present disclosure relates to a method of treating hepatocellular carcinoma in a human subject, comprising administering an anti-claudin-1 antibody to the human subject, wherein the hepatocellular carcinoma is resistant to sorafenib and / or a PD-1 antagonist.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of and priority to U.S. Provisional Patent Application No. 63 / 377,861, filed September 30, 2022, the contents of which are incorporated herein by reference in their entirety.

[0002] Electronic Sequence Listing Reference The contents of the electronic sequence listing (ALNT-011_001WO_SeqList_ST26.xml; size 14,194 bytes; and creation date: September 28, 2023) are incorporated herein by reference in their entirety.

[0003] The present disclosure relates to methods for treating sorafenib-resistant hepatocellular carcinoma and / or nivolumab-resistant hepatocellular carcinoma. [Background technology]

[0004] Hepatocellular carcinoma (HCC) poses a significant public health burden, becoming the fourth leading cause of cancer-related deaths worldwide and growing rapidly (Llovet JM, et al., Nat Rev Dis Primers 2021;7:6). HCC typically develops against a background of advanced liver fibrosis caused by viral or metabolic insults. Regardless of etiology, overactivation of oncogenic signaling pathways, such as the Ras / Raf / MAPK, PI3K / AKT / mTOR, Notch, and Wnt / β-catenin pathways, is a common event involved in the initiation and progression of HCC. Furthermore, the tumor microenvironment (TME) plays an important role in the outcome of HCC (Llovet et al.).

[0005] Current treatment options for advanced HCC remain unsatisfactory due to limited response rates (Llovet et al., and Finn RS, et al., N Engl J Med 2020;382:1894-905). Resistance to current systemic therapies is associated with tumor cell plasticity, such as epithelial-mesenchymal (EMT) transition and stemness, as well as an immune-exhausted or immune-excluded TME (Llovet et al., Calderaro J, et al., J Hepatol 2019;71:616-30, and Qin S, et al., Signal Transduct Target Ther 2020;5:228). Meanwhile, persistence of pro-tumorigenic signals within the fibrotic niche contributes to a high risk of tumor recurrence after curative treatment approaches (Llovet et al.). Novel HCC therapeutics that address the drawbacks of drug resistance and tumor recurrence are needed. Provided herein are methods and compositions that address this need. Summary of the Invention

[0006] Provided herein are methods for identifying a human subject with hepatocellular carcinoma (HCC) suitable for therapy with an anti-claudin-1 antibody or antigen-binding fragment thereof, the method comprising the steps of: a) obtaining a biological sample from the human subject with HCC; b) detecting expression of claudin-1; c) comparing the detected expression level of claudin-1 with a control expression level; and d) identifying the human subject as a responder if the detected expression level of claudin-1 is higher than the control expression level. In some embodiments, the method further comprises: e) administering an anti-claudin-1 antibody or antigen-binding fragment thereof in an amount sufficient to alleviate symptoms of HCC if the human subject is identified as a responder.

[0007] Provided herein is a method for treating a human subject with HCC, comprising: a) obtaining a biological sample from the human subject with HCC; b) detecting the expression level of claudin-1; c) comparing the detected expression level of claudin-1 with a control expression level; d) identifying the human subject as a responder if the detected expression level of claudin-1 is higher than the control expression level; and e) administering an anti-claudin-1 antibody or antigen-binding fragment thereof in an amount sufficient to alleviate symptoms of HCC if the human subject is identified as a responder.

[0008] In some embodiments, the control expression level is determined from a normal tissue sample, which is adjacent to a biological sample from a human subject with HCC.

[0009] Provided herein are methods for treating HCC in a human subject, comprising administering a therapeutically effective amount of an anti-claudin-1 antibody or antigen-binding fragment thereof. In some embodiments, the HCC is resistant to sorafenib, a PD-1 antagonist, and / or a PD-L1 antagonist. In some embodiments, the HCC is resistant to sorafenib. In some embodiments, the HCC is resistant to a PD-1 antagonist. In some embodiments, the HCC is resistant to a PD-L1 antagonist.

[0010] In some embodiments, the anti-claudin-1 antibody or antigen-binding fragment thereof comprises a CDRH1 comprising the amino acid sequence of SEQ ID NO: 5, a CDRH2 comprising the amino acid sequence of SEQ ID NO: 6, a CDRH3 comprising the amino acid sequence of SEQ ID NO: 7, a CDRL1 comprising the amino acid sequence of SEQ ID NO: 8, a CDRL2 comprising the amino acid sequence of GA, and a CDRL3 comprising the amino acid sequence of SEQ ID NO: 10.

[0011] In some embodiments, the anti-claudin-1 antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:3 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:4.

[0012] In some embodiments, the anti-claudin-1 antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 13 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 14.

[0013] In some embodiments, the anti-claudin-1 antibody is a humanized antibody.

[0014] In some embodiments, the HCC is resistant to sorafenib or a PD-1 antagonist.

[0015] In some embodiments, the human subject has been previously treated with sorafenib. In some embodiments, the human subject is administered a therapeutically effective amount of sorafenib simultaneously or sequentially.

[0016] In some embodiments, the human subject has previously been treated with a PD-1 antagonist. In some embodiments, the human subject is simultaneously or sequentially administered a therapeutically effective amount of a PD-1 antagonist. In some embodiments, the PD-1 antagonist is nivolumab, pembrolizumab, cemiplimab, dostarlimab, or a combination thereof. In some embodiments, the PD-1 antagonist is nivolumab.

[0017] In some embodiments, the human subject has previously been treated with a PD-L1 antagonist. In some embodiments, the human subject is simultaneously or sequentially administered a therapeutically effective amount of a PD-L1 antagonist. In some embodiments, the PD-1 antagonist is atezolizumab (TECENTRIQ; RG7446), or durvalumab (IMFINZI; MEDI4736), or avelumab (Bavencio), or a combination thereof.

[0018] The features of the present disclosure are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the disclosure are utilized, and the accompanying drawings of which: [Brief explanation of the drawings]

[0019] [Figure 1A] This shows that anti-claudin-1 monoclonal antibody ("H3L3" antibody) inhibits viability in a patient-derived ex vivo model of HCC. Representative photomicrographs show tumor spheroids generated from HCC liver tissue treated with anti-claudin-1 monoclonal antibody ("H3L3") or control mAb on day 6 after treatment. Scale bar: 200 μm. [Figure 1B] Anti-claudin-1 monoclonal antibody ("H3L3" antibody) inhibits viability in a patient-derived ex vivo model of HCC. A graph showing relative cell viability after 6 days of treatment with anti-claudin-1 mAb "H3L3" (Table 1) or sorafenib compared to control mAb-treated spheroids is shown (n=3 replicates per condition, p=0.003 and p=0.04, Student's t-test). Bars represent mean ± SEM. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001). [Figure 1C]This figure shows that anti-claudin-1 monoclonal antibody ("H3L3" antibody) inhibits viability in a patient-derived ex vivo model of HCC. A heat map showing cell viability of tumor spheroids (n=15 donors) treated with anti-claudin-1 mAb "H3L3," control Ab, sorafenib, or nivolumab is shown. The heat map shows % cell viability compared to control mAb-treated cells at day 6 using ATP quantification (n=15 different donors with at least duplicates per condition). Donors (#ST1, #S06, #S15, #S16, #S17, #S18, #R2, #R21, #R24, #R11, #381, #S19, #S22, #S349, #S394). DETAILED DESCRIPTION OF THE INVENTION

[0020] In many hepatocellular carcinoma (HCC) patients, the efficacy of prior therapy is hindered by the development of drug resistance. HCC exhibits high intratumor and interindividual heterogeneity, which affects disease progression, classification, prognosis, and, inevitably, cellular susceptibility to drug resistance. Furthermore, persistence of pro-tumorigenic signals in the tumor microenvironment may contribute to the high risk of tumor recurrence after prior therapy, such as sorafenib, PD-1 antagonists, and PD-L1 antagonists.

[0021] Provided herein are methods for treating HCC in a subject, comprising administering a therapeutically effective amount of an anti-claudin-1 antibody. In some embodiments, the HCC is resistant to sorafenib therapy. In some embodiments, the HCC is resistant to PD-1 antagonist therapy (e.g., nivolumab). In some embodiments, the HCC is resistant to PD-L1 antagonist therapy. This is based in part on the observation that administration of exemplary anti-claudin-1 antibodies was effective in reducing the cell viability of HCC cells derived from sorafenib-resistant HCC and nivolumab-resistant HCC. Without being bound by theory, such resistant HCC cells may be targetable and sensitive to anti-claudin-1 antibody therapy because they exhibit high expression of claudin-1. In some cases, high expression of claudin-1 can be pharmacologically induced. This therefore provides advantageous properties corresponding to the high therapeutic potential of using anti-claudin-1 antibodies to treat subsets of HCC that are resistant to prior therapies such as sorafenib and nivolumab.

[0022] I. Definition 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 otherwise required by context, singular terms shall include the plural and plural terms shall include the singular. All publications, patents, and other references mentioned herein are incorporated by reference in their entirety for all purposes, as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. The techniques and procedures described herein are generally carried out according to conventional methods well known in the art and as described in the various general and more specific references cited and described throughout this specification. See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual (Third ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY 2000). See also Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates (1992). The nomenclature utilized in connection with the present description, and the testing procedures and techniques described herein, are those well known and commonly used in the art.

[0023] Exemplary methods and materials are described herein, although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure. The materials, methods, and examples are illustrative only and are not intended to be limiting. Other features and advantages of the present disclosure will be apparent from the detailed description and claims.

[0024] In order to further define this disclosure, the following terms and definitions are provided.

[0025] The singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. The terms "a" (or "an"), as well as "one or more" and "at least one," may be used interchangeably herein. In certain embodiments, the term "a" or "an" means "single." In some embodiments, the term "a" or "an" includes "two or more" or "plural."

[0026] The term "about" is used herein to mean approximately, roughly, roughly, or within a range thereof. 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 values. In general, the term "about" is used herein to modify numerical values ​​above and below the stated value by a variance of 10 percent above and below (plus or minus).

[0027] Various aspects of the present disclosure are 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 indefinite limitation on the scope of the present disclosure. Thus, the description of a range should be considered to specifically disclose not only individual numerical values ​​within that range, but also all possible subranges. For example, the description of a range such as 1 to 6 should be considered to specifically disclose subranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range. The numerical ranges described include the numerical boundaries defining the range and include each integer within the defined range.

[0028] Units, prefixes, and symbols are shown in the format recognized by the International System of Units (SI). Numerical ranges are intended to be inclusive of the numbers defining the range. When a range of values ​​is recited, it is understood that each intervening integer value, and each portion thereof, between the recited upper and lower limits of that range is also specifically disclosed, along with each subrange between such values. The upper and lower limits of any range may independently be included or excluded within the range, and ranges including either one of these limits, excluding any limit, or including both limits are included within the present disclosure. Thus, ranges provided herein are understood to be shorthand notations for all values ​​within that range, inclusive of the recited endpoints. For example, a range of 1 to 10 is understood to include any number, combination of numbers, or subrange from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10.

[0029] When a value is explicitly recited, it is understood that values ​​that are approximately the same amount or quantity as the recited value are also within the scope of the present disclosure. Where a combination is disclosed, each subcombination of the elements of that combination is also specifically disclosed and is within the scope of the present disclosure. Conversely, where different elements or groups of elements are individually disclosed, combinations thereof are also disclosed. Where any element of the present disclosure is disclosed as having multiple alternatives, examples of that disclosure in which each alternative is excluded, alone or in any combination with other alternatives, are also disclosed herein, and multiple elements of the present disclosure can have such exclusions, and all combinations of elements with such exclusions are disclosed herein.

[0030] As used herein, the term "and / or" should be interpreted as a specific disclosure of each of the two specified features or components, regardless of the presence or absence of the other. Thus, the term "and / or" used herein in phrases such as "A and / or B" is intended to include "A and B," "A or B," "A" (alone), and "B" (alone). Similarly, the term "and / or" used in phrases such as "A, B, and / or C" is intended to encompass 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).

[0031] As used herein, the terms "treat," "treating," and "treatment" are interchangeable and encompass partially or completely preventing, ameliorating, alleviating, and / or managing symptoms, secondary disorders, or conditions associated with hepatocellular carcinoma (HCC). In some aspects, the HCC is metastatic HCC, unresectable HCC, refractory HCC, or recurrent HCC. In some embodiments, the HCC is any of early stage HCC, non-metastatic HCC, primary HCC, advanced HCC, locally advanced HCC, metastatic HCC, HCC in remission, recurrent HCC, adjuvant HCC, or neoadjuvant HCC. In some aspects, the hepatocellular carcinoma is recurrent, refractory, or resistant to prior therapy. As used herein, the term "treating" refers to the application or administration of one or more active agents to a subject with a symptom, secondary disorder, or condition associated with HCC, with the intent of partially or completely alleviating, ameliorating, alleviating, delaying the onset of, inhibiting the progression of, reducing the severity of, and / or reducing the incidence of one or more symptoms, secondary disorders, or characteristics associated with HCC. Symptoms, secondary disorders, and / or conditions associated with HCC include, but are not limited to, abdominal pain, fatigue, loss of appetite, cachexia, ascites, and biliary obstruction. Treatment may also be administered to subjects who exhibit only early signs of a symptom, secondary disorder, and / or condition associated with HCC, with the intent of reducing the risk of developing such a symptom, secondary disorder, and / or condition.

[0032] The term human "claudin-1" (or "CLDN1") refers to the protein having the sequence set forth in NCBI accession number NP_066924.1, or any naturally occurring variant commonly found in HCV-permissive human populations.

[0033] As used herein, the term "antibody" refers to an immunoglobulin that contains an antigen-binding site that immunospecifically binds to an antigen. Thus, the term antibody encompasses not only whole antibody molecules, but also antibody fragments, and variants (including derivatives) of antibodies and antibody fragments, so long as the derivatives and fragments maintain specific binding ability. The term encompasses monoclonal and polyclonal antibodies. The term also encompasses any protein having a binding domain that is homologous or largely homologous to an immunoglobulin binding domain. These proteins may be derived from natural sources or may be partially or wholly synthetically produced. The term "specific binding," when used in reference to an antibody, refers to an antibody that binds to a predetermined antigen. Typically, an antibody has a specific binding capacity of at least 1 x 10 7 M 1 and binds to a given antigen with an affinity that is at least two-fold higher than its binding affinity to a nonspecific antigen (e.g., BSA, casein).

[0034] The terms "antigen-binding fragment," "antigen-binding region," or "antigen-binding site," or "binding portion" refer to the portion of an immunoglobulin molecule involved in antigen binding. The antigen-binding site is formed by amino acid residues from the N-terminal variable ("V") regions of the heavy ("H") and light ("L") chains. Within the V regions of the heavy and light chains, three highly diverse segments called "hypervariable regions" are interposed between highly conserved adjacent segments known as "framework regions" or "FRs." Thus, the term "FR" refers to the amino acid sequences naturally found between and adjacent to the hypervariable regions in immunoglobulins. In an antibody molecule, the three hypervariable regions of the light chain and the three hypervariable regions of the heavy chain are arranged relative to one another in three-dimensional space to form an antigen-binding surface. The antigen-binding surface is complementary to the three-dimensional surface of a bound antigen, and the three hypervariable regions of each of the heavy and light chains are referred to as "complementarity-determining regions" or "CDRs." Various methods are known in the art for numbering the amino acid sequence of an antibody and identifying the complementarity-determining regions. For example, the Kabat numbering system (see Kabat, E.A., et al., Sequences of Protein of Immunological Interest, Fifth Edition, US Department of Health and Human Services, US Government Printing Office (1991)) or the IMGT numbering system (see IMGT®, the international ImMunoGeneTics information system®, available online at http: / / www.imgt.org / ). The IMGT numbering system is routinely used in the art and is accepted as a reliable and accurate system for identifying amino acid positions in coding sequences, aligning alleles, and easily comparing sequences in immunoglobulins (IGs) and T-cell receptors (TRs) from any vertebrate species.The accuracy and consistency of IMGT data are based on the IMGT-ONTOLOGY, the first and first-of-its-kind ontology for immunogenetics and immunoinformatics (see Lefranc. MP et al., Biomolecules, 2014 December; 4(4), 1102-1139). IMGT tools and databases run against the IMGT reference directory, which was constructed from a large repository of sequences. In the IMGT system, IG V-DOMAINS and IG C-DOMAINS are delimited, taking into account exon delimitations where appropriate. Thus, with the availability of more sequences to the IMGT database, the IMGT exon numbering system can be, and is, used by those skilled in the art for reliable identification of amino acid positions within coding sequences and alignment of alleles. Furthermore, the correspondence between the IMGT proprietary numbering and other numbering systems (i.e., Kabat) is available in the IMGT Scientific Chart (see Lefranc. MP et al., Biomolecules, 2014 December; 4(4), 1102-1139).

[0035] As used herein, the term "humanized antibody" refers to a chimeric antibody comprising amino acid residues derived from non-human hypervariable regions and amino acid residues derived from human framework regions (FRs). In particular, a humanized antibody comprises all or substantially all of at least one, and typically two, variable domains, with all or substantially all of the complementarity-determining regions (CDRs) being those of a human antibody. A humanized antibody may optionally comprise at least a portion of an antibody constant region derived from a human antibody. A "humanized form" of an antibody, e.g., a non-human antibody, refers to an antibody that has undergone humanization.

[0036] The term "humanization" refers to the fact that sequence differences between rodent antibodies and human sequences can be minimized by replacing residues that differ from those in the human sequence by site-directed mutagenesis of individual residues, or by grafting entire regions, or by chemical synthesis. Humanized antibodies can also be produced using recombinant methods. In humanized forms of antibodies, 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 "substituted" immunoglobulin molecules include IgG1, IgG2, IgG2a, IgG2b, IgG3, IgG4, IgA, IgM, IgD, or IgE molecules and fragments thereof.

[0037] Whenever an embodiment is described herein using the term "comprising," it is understood that similar embodiments are also provided except that they are described using the terms "consisting of" and / or "consisting essentially of."

[0038] As used herein, the term "administering" refers to the physical introduction of a composition containing a therapeutic agent (e.g., an anti-claudin-1 antibody) into a subject using any of a variety of methods and delivery systems known to those skilled in the art. Examples of routes of administration include intravenous, intramuscular, subcutaneous, intraperitoneal, spinal, or other parenteral routes, such as by injection or infusion. The phrase "parenteral administration," as used herein, refers to methods of administration, typically by injection, excluding enteral and topical administration, and includes, but is not limited to, intravenous, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injection and infusion, as well as in vivo electroporation. Other parenteral administration routes include topical, epithelial, or mucosal routes, such as intranasal, intravaginal, rectal, sublingual, or topical routes. Also, administration can be, for example, once, multiple times and / or over one or more extended periods of time.

[0039] 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, amelioration, alleviation, reduction, delay, and / or alleviation of one or more of the signs, symptoms, or causes of a disease, or any other desired alteration of 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 tumor onset. 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 may (i) reduce the number of cancer cells, (ii) reduce tumor size, (iii) inhibit, delay, slow to some extent, and stop cancer cell invasion into peripheral organs, (iv) inhibit (i.e., slow to some extent and stop) tumor metastasis, (v) inhibit tumor growth, (vi) prevent or delay tumor onset and / or recurrence, and / or (vii) alleviate to some extent one or more symptoms associated with cancer. In one example, an "effective amount" is an amount of an anti-claudin-1 antibody that has been clinically proven to result in a significant reduction in cancer, such as HCC, or a slowdown in the progression of cancer.

[0040] "Cancer" refers to a broad group of diseases characterized by the uncontrolled growth of abnormal cells in the body. Unregulated cell division and growth leads to the formation of malignant tumors, which can invade adjacent tissues and even metastasize to distant parts of the body via the lymphatic system or bloodstream. "Cancer" or "cancerous tissue" can also include tumors.

[0041] As used herein, the term "tumor" refers to any mass of tissue (including precancerous lesions) resulting from the growth or proliferation of excess cells, either benign (non-cancerous) or malignant (cancerous).

[0042] "Sorafenib" or 4-[4-[[4-chloro-3-(trifluoromethyl)phenyl]carbamoylamino]phenoxy]-N-methyl-pyridine-2-carboxamide (e.g., PubChem ID 216239) is a drug approved for the treatment of hepatocellular carcinoma (HCC). Its mechanism of action is mediated by its inhibitory activity against overexpressed kinases, particularly receptors with kinase activity and Raf kinase, in a series of molecular pathways involved in the transformation of normal cells into tumor cells.

[0043] Nivolumab (also known as OPDIVO®; formerly known as 5C4, BMS-936558, MDX-1106, or ONO-4538) is a fully human IgG4(S228P) PD-1 immune checkpoint inhibitor antibody that selectively prevents interaction with PD-1 ligands (PD-L1 and PD-L2) and prevents downregulation of anti-tumor T cell function (U.S. Patent No. 8,008,449; Wang et al., 2014 Cancer Immunol Res. 2(9):846-56).

[0044] "Antagonist" and "inhibitor" are used interchangeably herein and include any substance that blocks or inhibits the physiological action of another substance.

[0045] The term "high expression of claudin-1" refers to the percentage of cells in a test tissue sample that are scored as expressing claudin-1. In some embodiments, claudin-1 expression is assayed by immunohistochemistry (IHC), wherein high expression of claudin-1 in a sample means that at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or 100% of the total number of cells in the test sample express claudin-1.

[0046] As used herein, a "patient" includes any patient suffering from cancer (e.g., fibrosing cancer). The terms "subject" and "patient" are used interchangeably herein.

[0047] II. Anti-claudin-1 antibody The present disclosure provides use of an anti-claudin-1 antibody or an antigen-binding fragment thereof for treating drug-resistant HCC. In some embodiments, the HCC is sorafenib-resistant. In some embodiments, the HCC is resistant to a PD-1 antagonist. In some embodiments, the PD-1 antagonist is nivolumab.

[0048] Antibodies against human claudin-1 have previously been described to treat hepatitis C virus infection, hepatocellular carcinoma, and certain fibrotic diseases, such as pulmonary fibrosis (see WO2010 / 034812, WO2016 / 146809, and WO2021 / 094469).

[0049] Anti-claudin-1 antibodies that can be used in the practice of the present disclosure include any antibody raised against claudin-1. Exemplary anti-claudin-1 antibodies include, but are not limited to, those described in WO2010 / 034812 and WO2017 / 162678, the contents of each of which are incorporated herein by reference. Exemplary anti-claudin-1 antibodies include, but are not limited to, those described in European Patent No. EP1,167,389, U.S. Patent No. 6,627,439, PCT Publication Nos. WO2014 / 132307, WO2015 / 014659, and WO2015 / 014357, the contents of each of which are incorporated herein by reference. Exemplary anti-claudin-1 antibodies are also described in Yamashita et al., J. Pharmacol. Exp. Ther., 2015, 353(1):112-118.

[0050] In some embodiments, the anti-claudin-1 antibody is a polyclonal or monoclonal antibody. In some embodiments, the anti-claudin-1 antibody is a humanized antibody.

[0051] Exemplary anti-claudin-1 antibodies or antigen-binding fragments of the present disclosure are listed in Table 1. In some embodiments, the anti-claudin-1 antibody is "H1L1." In some embodiments, the anti-claudin-1 antibody is "H3L3." The CDRs listed in Table 1 are defined according to the IMGT nomenclature (see IMGT®, the international ImMunoGeneTics information system®, available online at http: / / www.imgt.org / ).

[0052] [Table 1-1] [Table 1-2]

[0053] In some embodiments, the "H1L1" anti-claudin-1 antibody comprises complementarity determining region (CDR) CDRH1 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, CDR H3 comprising the amino acid sequence set forth in SEQ ID NO: 7, CDR L1 comprising the amino acid sequence set forth in SEQ ID NO: 8, CDR L2 comprising the amino acid sequence GA, and CDR L3 comprising the amino acid sequence set forth in SEQ ID NO: 10.

[0054] In some embodiments, the "H1L1" anti-claudin-1 antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:3 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:4.

[0055] In some embodiments, the "H1L1" anti-claudin-1 antibody or antigen-binding fragment thereof comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:1 and a light chain comprising the amino acid sequence of SEQ ID NO:2.

[0056] In some embodiments, the "H3L3" anti-claudin-1 antibody comprises complementarity determining regions (CDRs) CDRH1 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, CDR H3 comprising the amino acid sequence set forth in SEQ ID NO: 7, CDR L1 comprising the amino acid sequence set forth in SEQ ID NO: 8, CDR L2 comprising the amino acid sequence GA, and CDR L3 comprising the amino acid sequence set forth in SEQ ID NO: 10.

[0057] In some embodiments, the "H3L3" anti-claudin-1 antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 13 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 14.

[0058] In some embodiments, the "H3L3" anti-claudin-1 antibody or antigen-binding fragment thereof comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:11 and a light chain comprising the amino acid sequence of SEQ ID NO:12.

[0059] 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 at DSMZ on July 29, 2008 under accession number DSM ACC2938.

[0060] In some embodiments, the heavy chain variable region ("VH") and light chain variable region ("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 at DSMZ on July 29, 2008 under accession number DSM ACC2938.

[0061] 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 at DSMZ on July 29, 2008 under accession number DSM ACC2938.

[0062] In some embodiments, the anti-claudin-1 antibody can be a complete monoclonal antibody having an isotype selected from the group consisting of IgG1, IgG2, IgG3, and IgG4. In some embodiments, the 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.

[0063] In some embodiments, an anti-claudin-1 antibody (or biologically active variant or fragment thereof) suitable for use according to the present disclosure may be operably linked (e.g., by chemical conjugation, genetic fusion, non-covalent bonding, or otherwise) to one or more other molecular entities. Methods for preparing such modified antibodies (or conjugated antibodies) are known in the art (see, e.g., "Affinity Techniques. Enzyme Purification: Part B," Methods in Enzymol., 1974, Vol. 34, Jakoby and Wilneck (Eds.), Academic Press: New York, NY, and Wilchek and Bayer, Anal. Biochem., 1988, 171:1-32). Preferably, the molecular entity is attached to a position on the antibody molecule that does not interfere with the binding properties of the resulting conjugate, e.g., a position that is not involved in the specific binding of the antibody to its target.

[0064] In some embodiments, the antibody molecule (e.g., an anti-claudin-1 antibody) and the molecular entity may be directly covalently bound to each other. In some embodiments, the antibody molecule and the molecular entity may be covalently bound to each other via a linker group. This can be achieved by using any of a wide variety of stable bifunctional agents known in the art, including homofunctional and heterofunctional linkers.

[0065] In some embodiments, the anti-claudin-1 antibody (or biologically active fragment thereof) for use in accordance with the present disclosure is conjugated to a detectable agent. Any of a wide variety of detectable agents may be used, including various ligands, radionuclides (e.g., 3 H, 125 I, 131 I, etc.), fluorescent dyes (e.g., fluorescein isothiocyanate, rhodamine, phycoerytherin, phycocyanin, allophycocyanin, o-phthalaldehyde, and fluorescamine), chemiluminescent agents (e.g., luciferin, luciferase, and aequorin), microparticles (e.g., quantum dots, nanocrystals, phosphors, etc.), enzymes (e.g., those used in ELISA, i.e., horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase, etc.), colorimetric labels, magnetic labels, and biotin, digoxigenin, or other haptens and proteins for which antisera or monoclonal antibodies are available.

[0066] Other molecular entities that can be conjugated to the anti-claudin-1 antibodies (or biologically active fragments thereof) of the present disclosure include, but are not limited to, linear or branched hydrophilic polymer groups, fatty acid groups, or fatty acid ester groups.

[0067] In some embodiments, the anti-claudin-1 antibody is a full-length antibody, a biologically active variant thereof or a fragment thereof, a chimeric antibody, a humanized antibody, or an antibody-derived molecule comprising at least one complementarity-determining region (CDR) from either the heavy or light chain variable region of an anti-claudin-1 antibody, including molecules such as Fab fragments, F(ab')2 fragments, Fd fragments, Fabc fragments, Sc antibodies (single-chain antibodies), diabodies, individual antibody light chains, individual antibody heavy chains, chimeric fusions between antibody chains and other molecules, and antibody conjugates such as antibodies conjugated to therapeutic or detectable agents. Anti-claudin-1 antibody-related molecules according to the present disclosure retain the ability to bind to their antigen (e.g., claudin-1), particularly the extracellular domain of claudin-1.

[0068] III. How to use The present disclosure provides a method for administering an effective amount of an anti-claudin-1 antibody, or an antigen-binding fragment thereof, or a pharmaceutical composition thereof to a subject in need thereof (i.e., a subject with hepatocellular carcinoma).The present disclosure provides a method for inhibiting hepatocellular carcinoma (HCC) in a subject, the method comprising administering an anti-claudin-1 antibody or an antigen-binding fragment thereof to the subject.

[0069] The methods of the disclosure can be accomplished using an effective amount of an anti-claudin-1 antibody, or antigen-binding fragment thereof, or a pharmaceutical composition comprising such an antibody or fragment.

[0070] In some embodiments, the methods provided herein are used to treat subjects with sorafenib-resistant HCC and / or nivolumab-resistant HCC. Sorafenib remains a globally accepted systemic first-line treatment for advanced HCC (Llovet JM, et al. N. Engl. J. Med. 2008;359:378-390). Its approval in 2007 is one of the landmark achievements in HCC treatment, even though it only slightly improved overall median survival. Sorafenib is a molecular targeted agent that acts on vascular endothelial growth factors (VEGFR1, 2, 3), platelet-derived growth factor receptor-β (PDGFRβ), and Raf family kinases (mainly C-Raf, not B-Raf) (Liu L., et al., Cancer Res. 2006;66:11851-11858). However, its efficacy has been observed to be hindered by drug resistance in many patients. HCC is highly heterogeneous both within tumors and among individuals, which affects disease progression, classification, prognosis, and consequently cellular susceptibility to drug resistance.

[0071] Nivolumab is a fully human IgG4 anti-programmed death-1 (PD-1) monoclonal antibody that inhibits immune checkpoint signaling. Nivolumab treatment improves survival compared with chemotherapy in several tumor types, including melanoma, non-small cell lung cancer, and renal cell carcinoma. Nivolumab has also been approved in various countries for patients with advanced HCC who have been previously treated with sorafenib.

[0072] The present disclosure provides a method for treating HCC in a subject, the method comprising administering an anti-claudin-1 antibody or antigen-binding fragment thereof to the subject. The present disclosure also provides a method for treating sorafenib-resistant HCC in a subject, the method comprising administering an anti-claudin-1 antibody or antigen-binding fragment thereof to the subject. The present disclosure also provides a method for treating anti-PD-1-resistant HCC in a subject, the method comprising administering an anti-claudin-1 antibody or antigen-binding fragment thereof. The present disclosure also provides a method for treating anti-PD-L1-resistant HCC in a subject, the method comprising administering an anti-claudin-1 antibody or antigen-binding fragment thereof.

[0073] In some embodiments, the anti-claudin-1 antibody or antigen-binding fragment thereof is administered to a subject simultaneously with or sequentially with sorafenib. In some embodiments, the anti-claudin-1 antibody or antigen-binding fragment thereof is administered after administration of sorafenib, a PD-1 antagonist, or a PD-L1 antagonist.

[0074] In some embodiments, the subject has been previously treated with a PD-1 antagonist.

[0075] In some embodiments, the PD-1 antagonist is a small molecule PD-1 inhibitor. In some embodiments, the PD-1 antagonist is an anti-PD-1 antibody or antigen-binding fragment thereof. In some embodiments, the PD-1 antagonist is selected from the group consisting of nivolumab, pembrolizumab, cemiplimab, and dostarlimab.

[0076] In some embodiments, the subject has been previously treated with a PD-L1 antagonist.

[0077] In some embodiments, the PD-L1 antagonist is a small molecule PD-L1 inhibitor. In some embodiments, the PD-L1 antagonist is an anti-PD-L1 antibody or antigen-binding fragment thereof. In some embodiments, the PD-L1 antagonist is selected from the group consisting of atezolizumab (TECENTRIQ; RG7446; MPDL3280A; RO5541267), durvalumab (MEDI4736), BMS-936559, avelumab (bavencio), LY3300054, CX-072 (Proclaim-CX-072), FAZ053, KN035, and MDX-1105.

[0078] In some embodiments, HCC is characterized by high expression of claudin-1. In some embodiments, the methods disclosed herein further comprise detecting the expression level of claudin-1 in an HCC tumor sample from the subject. In some embodiments, the methods disclosed herein further comprise comparing the expression level of claudin-1 with the expression level of claudin-1 in a reference sample, and if the expression level of claudin-1 in the HCC tumor sample is elevated relative to the expression level of claudin-1 in the reference sample, administering to the subject an anti-claudin-1 antibody, or an anti-claudin-1 antibody and sorafenib.

[0079] In some embodiments, the expression level of claudin-1 is quantified by immunohistochemistry (IHC). In some embodiments, the IHC test is graded with a score of 0 to +3. In some embodiments, high expression of claudin-1 is graded by IHC test as +1, +2, or +3.

[0080] To improve the treatment of HCC, in some embodiments, the present disclosure provides for identifying patients exhibiting high expression of claudin-1 and providing them with an anti-claudin-1 antibody or an antigen-binding fragment thereof, or an anti-claudin-1 antibody or an antigen-binding fragment thereof and sorafenib.

[0081] In some embodiments, the present disclosure relates to identifying patients with HCC that exhibit high expression of claudin-1 and treating the HCC by administering an anti-claudin-1 antibody or an antigen-binding fragment thereof, or a combination of an anti-claudin-1 antibody or an antigen-binding fragment thereof and sorafenib.

[0082] In some embodiments, the present disclosure includes a method for selecting an HCC patient for immunotherapy, the method comprising: (a) determining the expression level of claudin-1 in a tumor sample; and (b) selecting the tumor for immunotherapy if the tumor sample shows high expression of claudin-1. In some embodiments, the immunotherapy is administration of an anti-claudin-1 antibody or antigen-binding fragment thereof, or an anti-claudin-1 antibody or antigen-binding fragment thereof and sorafenib.

[0083] In some embodiments, the present disclosure includes a method for identifying an HCC tumor in a human patient as eligible for immunotherapy, the method comprising: (a) determining the expression level of claudin-1 in a tumor sample; and (b) identifying the tumor as eligible for immunotherapy if the tumor sample exhibits high expression of claudin-1. In some embodiments, the immunotherapy is administration of an anti-claudin-1 antibody or antigen-binding fragment thereof, or an anti-claudin-1 antibody or antigen-binding fragment thereof and sorafenib.

[0084] In some embodiments, the present disclosure includes a method for identifying an HCC tumor in a human patient as eligible for immunotherapy, the method comprising: (a) determining whether the tumor is resistant to sorafenib therapy; and (b) identifying the tumor as eligible for immunotherapy if the tumor sample is resistant to sorafenib treatment. In some embodiments, the immunotherapy is administration of an anti-claudin-1 antibody or antigen-binding fragment thereof, or an anti-claudin-1 antibody or antigen-binding fragment thereof and sorafenib.

[0085] In some embodiments, the present disclosure includes a method for identifying an HCC tumor in a human patient as eligible for immunotherapy, the method comprising: (a) determining whether the tumor is resistant to PD-1 antagonist therapy; and (b) identifying the tumor as eligible for immunotherapy if the tumor sample is resistant to nivolumab treatment. In some embodiments, the immunotherapy is administration of an anti-claudin-1 antibody or antigen-binding fragment thereof, or an anti-claudin-1 antibody or antigen-binding fragment thereof and sorafenib.

[0086] A cancer may be diagnosed as sorafenib-resistant and / or PD-1 antagonist-resistant after treatment with sorafenib and / or a PD-1 antagonist has begun. Alternatively, a cancer may be diagnosed as sorafenib-resistant and / or PD-1 antagonist-resistant before treatment with such compounds has begun. Sorafenib-resistant and / or PD-1 antagonist-resistant tumors may occur, for example, after six months or more of sorafenib and / or PD-1 antagonist treatment. Alternatively, sorafenib-resistant and / or PD-1 antagonist-resistant tumors may be diagnosed less than six months after initiation of sorafenib and / or PD-1 antagonist treatment.

[0087] Diagnosis of sorafenib resistance and / or PD-1 antagonist resistance can be achieved by monitoring tumor progression during sorafenib and / or PD-1 antagonist treatment. Tumor progression can be identified by comparing tumor status between multiple time points after the start of treatment, or by comparing tumor status at time points after the start of treatment with time points before the start of sorafenib and / or PD-1 antagonist treatment. Tumor progression can be monitored visually during treatment with sorafenib and / or PD-1 antagonists, for example, by radiography, e.g., X-ray, CT scan, or other monitoring methods known to those skilled in the art, including palpation of the cancer or methods for monitoring tumor biomarker levels. Cancer progression during treatment with sorafenib and / or PD-1 antagonists indicates sorafenib resistance and / or PD-1 antagonist resistance. Detection of new tumors or metastases indicates tumor progression. Cessation of tumor shrinkage indicates tumor progression. Cancer growth is indicated, for example, by an increase in tumor size, detection of metastasis or new cancers, and / or elevated levels of tumor biomarkers.

[0088] In some embodiments, the healthy sample / control sample / reference sample is a sample derived from normal tissue. In some embodiments, the normal tissue is tissue adjacent to a cancer in a subject. For example, when assaying expression by staining, the protein level is above a level that would be considered "negative" by one skilled in the art for a particular assay. Exemplary assays include, but are not limited to, RNA-based assays, FACS, and IHC, each of which has appropriate levels of positive and negative results. Any method for detecting the level of a protein in a sample is contemplated. Those skilled in the art can select a suitable method depending on the type of sample being analyzed and the identity and number of proteins to be detected. Non-limiting exemplary methods include immunohistochemistry, ELISA, Western blotting, multi-analyte detection (e.g., using Luminex technology), mass spectrometry, etc. Similarly, any method for detecting the level of mRNA in a sample is contemplated. Those skilled in the art can select a suitable method depending on the type of sample being analyzed and the identity and number of mRNAs to be detected. Non-limiting exemplary methods include RT-PCR, quantitative RT-PCR, and microarray-based methods.

[0089] In some embodiments, the expression of claudin-1 is determined by receiving the results of an assay capable of determining the expression of claudin-1.

[0090] To evaluate the expression of claudin-1, in some embodiments, a test tissue sample is obtained from a patient in need of therapy. In some embodiments, the test tissue sample is any clinically relevant tissue sample, such as, but not limited to, a tumor biopsy, a core biopsy tissue sample, a fine needle aspirate, or a sample of a bodily fluid such as blood, plasma, serum, lymph, ascites, cyst fluid, or urine. In some embodiments, the test tissue sample is derived from a primary tumor. In some embodiments, the test tissue sample is derived from a metastasis. In some embodiments, the test tissue sample is collected from a subject at multiple time points, for example, before, during, and / or after treatment. In some embodiments, the test tissue samples are collected from different sites in the subject (e.g., a sample from the primary tumor and a sample from a distant metastasis).

[0091] In some embodiments, the test tissue sample is a paraffin-embedded, fixed tissue sample. In some embodiments, the test tissue sample is a formalin-fixed, paraffin-embedded (FFPE) tissue sample. In some embodiments, the test tissue sample is a fresh tissue (e.g., tumor) sample. In some embodiments, the test tissue sample is a frozen or cryopreserved tissue sample. In some embodiments, the test tissue sample is a fresh-frozen (FF) tissue (e.g., tumor) sample. In some embodiments, the test tissue sample is an archived tissue sample. In some embodiments, the test tissue sample is an archived tissue sample with a known diagnosis, treatment, and / or outcome history. In some embodiments, the sample is a block of tissue. In some embodiments, the test tissue sample is dispersed cells. In some embodiments, the sample size is between about 1 cell and about 1 x 10 cells. 6 In some embodiments, the sample size is from about 1 cell to about 1 x 10 cells or more. 5In some embodiments, the sample size is from about 1 cell to about 10,000 cells. In some embodiments, the sample size is from about 1 cell to about 1,000 cells. In some embodiments, the sample size is from about 1 cell to about 100 cells. In some embodiments, the sample size is from about 1 cell to about 10 cells. In some embodiments, the sample size is a single cell.

[0092] In some embodiments, assessing claudin-1 expression can be accomplished without obtaining a test tissue sample. In some embodiments, selecting a suitable patient includes (i) optionally preparing a test tissue sample obtained from a patient with cancer of the tissue, where the test tissue sample contains tumor cells and / or tumor-infiltrating inflammatory cells, and (ii) assessing the proportion of cells in the test tissue sample that express claudin-1 on their cell surface based on assessing that the proportion of cells in the test tissue sample that express claudin-1 on their cell surface is greater than a predetermined threshold level.

[0093] However, it should be understood that in any of the methods involving measuring claudin-1 expression in a test tissue sample, the step involving preparing a test tissue sample obtained from a patient is an optional step. In some embodiments, the method includes this step, while in other embodiments, it does not. It should also be understood that in some embodiments, the "measuring" or "assessing" step to identify or determine the number or percentage of cells expressing claudin-1 in the test tissue sample is performed by a modified method of assaying claudin-1 expression, for example, by performing a reverse transcriptase-polymerase chain reaction (RT-PCR) assay or an IHC assay. In some embodiments, no modified step is involved, and claudin-1 expression is assessed by, for example, reviewing a test result report from a laboratory. In some embodiments, claudin-1 expression is assessed by reviewing the results of an immunohistochemistry assay from a laboratory. In some embodiments, the method steps leading up to and including the assessment of claudin-1 expression provide intermediate results that can be provided to a physician or other healthcare provider for use in selecting suitable candidates for combination therapy with a claudin-1 inhibitor and an immune checkpoint inhibitor. In some embodiments, the method steps leading up to and including assessing claudin-1 expression provide intermediate results that can be provided to a physician or other healthcare provider for use in selecting suitable candidates for immune checkpoint inhibitor therapy. In some embodiments, the steps of providing the intermediate results are performed by a healthcare professional or someone acting under the direction of a healthcare professional. In some embodiments, these steps are performed by an independent laboratory or by an independent person, such as a laboratory technician.

[0094] In some embodiments of any of the present methods, the proportion of cells expressing claudin-1 is assessed by performing an assay to detect the presence of claudin-1 RNA. In some embodiments, the presence of claudin-1 RNA is detected by RT-PCR, in situ hybridization, or RNase protection. In some embodiments, the presence of claudin-1 RNA is detected by an RT-PCR-based assay. In some embodiments, scoring the RT-PCR-based assay comprises assessing the claudin-1 RNA expression level in the test tissue sample relative to a predetermined level.

[0095] In some embodiments, the proportion of cells expressing claudin-1 is assessed by performing an assay to detect the presence of claudin-1 polypeptide. In some embodiments, the presence of claudin-1 polypeptide is detected by IHC, enzyme-linked immunosorbent assay (ELISA), in vivo imaging, or flow cytometry. In some embodiments, claudin-1 expression is assayed by IHC. In some embodiments of all of these methods, cell surface expression of claudin-1 is assayed using, for example, IHC or in vivo imaging.

[0096] In some embodiments, the immunohistochemistry assay is scored at low magnification. In some embodiments, the low magnification is about 20x. In some embodiments, the immunohistochemistry assay is scored at high magnification. In some embodiments, the high magnification is about 40x.

[0097] In some embodiments, the immunohistochemistry assay is scored by image analysis software. In some embodiments, the immunohistochemistry assay is scored by a pathologist's visual immunoscore. In some embodiments, the immunohistochemistry assay is scored manually.

[0098] IV. PD-1 Antagonists and PD-L1 Antagonists Immune checkpoint proteins, such as PD-1 and PD-L1, interact with specific ligands that send signals to T cells that inhibit their function. Cancer cells exploit this by driving high levels of expression of checkpoint proteins on their surface to suppress anti-cancer immune responses.

[0099] In some embodiments, the PD-1 antagonist is an antibody. In some embodiments, the PD-1 antagonist is an antibody or fragment thereof that specifically binds to PD-1.

[0100] In some embodiments, the subject with HCC is resistant to treatment with a PD-1 antagonist. In some embodiments, the HCC is resistant to anti-PD-1 antibody therapy. In some embodiments, the anti-PD-1 antibody is nivolumab. In some embodiments, an anti-claudin-1 antibody or antigen-binding fragment thereof is administered to a subject with HCC that is resistant to treatment with a PD-1 antagonist.

[0101] In some embodiments, the HCC is treated with a pharmaceutical composition comprising an anti-claudin-1 antibody or antigen-binding fragment thereof.

[0102] The complete nucleotide and amino acid sequences of human PD-1 can be found in GenBank accession numbers NG_012110.1 and NP_005009.2. In some embodiments, the anti-PD-1 antibody is nivolumab. Nivolumab (also known as "OPDIVO®"; BMS-936558; formerly known as 5C4, BMS-936558, MDX-1106, or ONO-4538) is a fully human IgG4 (S228P) PD-1 immune checkpoint inhibitor antibody that selectively prevents interaction with PD-1 ligands (PD-L1 and PD-L2) and prevents down-regulation of anti-tumor T cell function (U.S. Patent No. 8,008,449; Wang et al., 2014 Cancer Immunol Res. 2(9):846-56). In some embodiments, the anti-PD-1 antibody or fragment thereof cross-competes with nivolumab. In some embodiments, the anti-PD-1 antibody or fragment thereof binds to the same epitope as nivolumab. In certain embodiments, the anti-PD-1 antibody has the same CDRs as nivolumab.

[0103] In some embodiments, the anti-PD-1 antibody is pembrolizumab. Pembrolizumab is a humanized monoclonal IgG4 (S228P) antibody directed against the human cell surface receptor PD-1 (programmed death-1 or programmed cell death-1). Pembrolizumab is described, for example, in U.S. Patent Nos. 8,354,509 and 8,900,587, the contents of which are incorporated herein by reference in their entireties.

[0104] Other exemplary anti-PD-1 antibodies include, but are not limited to, monoclonal antibodies 5C4 (herein referred to as nivolumab or BMS-936558), 17D8, 2D3, 4H1, 4A11, 7D3, and 5F4, which are described in WO2006 / 121168 (the contents of which are incorporated by reference in their entireties). Other exemplary anti-PD-1 antibodies include, but are not limited to, lambrolizumab (MK-3475), which is described in WO2008 / 156712, and AMP-514, which is described in WO2012 / 145493 (the contents of which are incorporated by reference in their entireties). Exemplary anti-PD-1 antibodies and other PD-1 inhibitors include, but are not limited to, those described in WO2009 / 014708, WO03 / 099196, WO2009 / 114335, and WO2011 / 161699, the contents of which are incorporated by reference in their entireties. In some embodiments, the anti-PD-1 antibody is REGN2810. In some embodiments, the anti-PD-1 antibody is PDR001. In some embodiments, the anti-PD-1 antibody is pidilizumab (CT-011).

[0105] Other exemplary anti-PD-1 monoclonal antibodies are described in, e.g., U.S. Patent Nos. 6,808,710, 7,488,802, 8,168,757, and 8,354,509, U.S. Publication No. 2016 / 0272708, and PCT Publication Nos. WO2012 / 145493, WO2008 / 156712, WO2015 / 11290, and ...6,808,710, 6,488,802, 6,168,757, and 8,354,509, U.S. Publication No. 2016 / 0272708, and PCT Publication Nos. WO2012 / 145493, WO2008 / 156712, WO2015 / 11290, and U.S. Patent Nos. 6,808,710, 6,808,710, 6,808,710, 6,808,710, and 6,808,710. 0, WO2012 / 145493, WO2015 / 112800, WO2014 / 206107, WO2015 / 35606, WO2015 / 085847, WO2014 / 179664, WO2017 / 020291, WO2017 / 020858, WO2016 / 197367, W O2017 / 024515, WO2017 / 025051, WO2017 / 123557, WO2016 / 106159, WO2014 / 19430 No. 2, WO2017 / 040790, WO2017 / 133540, WO2017 / 132827, WO2017 / 024465, WO2017

[0013] Examples of such techniques include, but are not limited to, those described in WO2017 / 025016, WO2017 / 106061, WO2017 / 19846, WO2017 / 024465, WO2017 / 025016, WO2017 / 132825, and WO2017 / 133540, each of which is incorporated herein by reference.

[0106] In some embodiments, the anti-PD-1 antibody is selected from the group consisting of nivolumab (also known as OPDIVO®, 5C4, BMS-936558, MDX-1106, and ONO-4538), pembrolizumab (Merck; also known as KEYTRUDA®, lambrolizumab, and MK-3475; see WO2008 / 156712), PDR001 (Novartis; see WO2015 / 112900), MEDI-0680 (AstraZeneca; also known as AMP-514; see WO2012 / 145493), cemiplimab (Regeneron; also known as REGN-2810; see WO2015 / 112800), JS001 (TAIZHOU JUNSHI PHARMA; Si-Yang Liu et al. al., J.Hematol.Oncol.10:136(2017)), BGB-A317 (Beigene; see WO2015 / 35606 and US2015 / 0079109), INCSHR1210 (Jiangsu Hengrui Medicine; also known as SHR-1210; see WO2015 / 085847; Si-Yang Liu et al. al., J.Hematol.Oncol.10:136(2017)), TSR-042 (Tesaro Biopharmaceutical; also known as ANB011; see WO2014 / 179664), GLS-010 (Wuxi / Harbin Gloria Pharmaceuticals; also known as WBP3055; Si-Yang Liu et al. al., J. Hematol. Oncol. 10:136 (2017)), AM-0001 (Armo), STI-1110 (Sorrento Therapeutics; see WO2014 / 194302), AGEN2034 (Agenus; see WO2017 / 040790), MGA012 (Macrogenics; see WO2017 / 19846), and IBI308 (Innovent; see WO2017 / 024465, WO2017 / 025016, WO2017 / 132825, and WO2017 / 133540) (the contents of each of which are incorporated by reference in their entirety).

[0107] In some embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof cross-competes with pembrolizumab. In some embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof binds to the same epitope as pembrolizumab. In some embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof has the same CDRs as pembrolizumab. In some embodiments, the anti-PD-1 antibody is pembrolizumab. Pembrolizumab (also known as "KEYTRUDA®," lambrolizumab, and MK-3475) is a humanized monoclonal IgG4 antibody directed against the human cell surface receptor PD-1 (programmed death-1 or programmed cell death-1). Pembrolizumab is described, for example, in U.S. Patent Nos. 8,354,509 and 8,900,587. Pembrolizumab is FDA-approved for the treatment of recurrent or refractory melanoma.

[0108] In some embodiments, the PD-1 antagonist is selected from the group consisting of nivolumab, pembrolizumab, cemiplimab, and dostarlimab.

[0109] In some embodiments, the PD-L1 antagonist is an antibody. In some embodiments, the PD-L1 antagonist is an antibody or fragment thereof that specifically binds to PD-L1.

[0110] In some embodiments, the subject with HCC is resistant to treatment with a PD-L1 antagonist. In some embodiments, the HCC is resistant to anti-PD-L1 antibody therapy. In some embodiments, the anti-PD-L1 antibody is nivolumab. In some embodiments, an anti-claudin-1 antibody or antigen-binding fragment thereof is administered to a subject with HCC that is resistant to treatment with a PD-L1 antagonist.

[0111] Exemplary PD-L1 antagonists include BMS-936559 (also known as 12A4, MDX-1105; see, e.g., U.S. Pat. No. 7,943,743 and WO 2013 / 173223), atezolizumab (Roche; also known as TECENTRIQ®; MPDL3280A, RG7446; see U.S. Pat. No. 8,217,149; see also Herbst et al. (2013) J Clin Oncol 31(suppl):3000), durvalumab (AstraZeneca; also known as IMFINZI™, MEDI-4736; see WO2011 / 066389), avelumab (Pfizer; also known as BAVENCIO®, MSB-0010718C; see WO2013 / 079174), STI-1014 (Sorrento; see WO2013 / 181634), CX-072 (Cytomx; see WO2016 / 149201), KN035 (3D Med / Alphamab; Zhang et al., Cell Discov. 7:3 (March 2017), LY3300054 (Eli Lilly Co.; see, e.g., WO2017 / 034916), and CK-301 (Checkpoint Therapeutics; Gorelik et al., AACR: Abstract 4606 (April 2016)).

[0112] As will be appreciated by those skilled in the art, alternative and / or equivalent names may be used for certain antibodies described above, and such alternative and / or equivalent names are interchangeable in the context of this disclosure.

[0113] V. Administration The anti-claudin-1 antibody can be administered to a subject in need thereof by any suitable route. In some embodiments, the anti-claudin-1 antibody is administered in combination with sorafenib as described above. Various delivery systems, including tablets, capsules, injections, encapsulation in liposomes, microparticles, microcapsules, etc., are known and can be used to administer the antibody or sorafenib. Administration methods include, but are not limited to, cutaneous, intradermal, intramuscular, intraperitoneal, intralesional, intravenous, subcutaneous, intranasal, pulmonary, epidural, and oral routes. The anti-claudin-1 antibody may be administered by any convenient route or other suitable route, such as infusion or bolus injection, via absorption through epithelial or mucosal layers (e.g., oral mucosa, bronchial mucosa, rectal, and intestinal mucosa, etc.). Administration can be systemic or local.

[0114] As will be appreciated by those skilled in the art, in aspects in which an antibody is administered in combination with an additional therapeutic agent (e.g., sorafenib), the antibody and therapeutic agent may be administered by the same route (e.g., intravenous) or by different routes (e.g., intravenous, oral, or subcutaneous). Such combination therapy, as described above, encompasses combined administration (two or more therapeutic agents in the same or separate formulations) and separate administration, in which case administration of an antibody of the invention can occur before, simultaneously with, and / or after administration of the additional therapeutic agent(s). In one embodiment, administration of the bispecific antibody and administration of the additional therapeutic agent occur within about one month, or within about one, two, or three weeks, or within about one, two, three, four, five, or six days of each other.

[0115] VI. Kits The present disclosure provides a pharmaceutical pack or kit comprising one or more containers (e.g., vials, ampoules, test tubes, flasks, or bottles) holding one or more of the ingredients of the pharmaceutical compositions of the invention, allowing for administration of an anti-claudin-1 antibody and / or sorafenib.

[0116] The various components of the pharmaceutical pack or kit may 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 may include a medium for reconstituting the lyophilized components. The individual containers of the kit are preferably maintained in close confinement for commercial sale.

[0117] In some embodiments, the pharmaceutical pack or kit contains one or more additional therapeutic agent(s) as described above. Optionally, such container(s) may be accompanied by a notice or package insert in the form prescribed by a government agency that regulates the manufacture, use, or sale of pharmaceuticals or biological products. This notice reflects that the agency has approved the manufacture, use, or sale for human administration. The package insert notice may include instructions for using the pharmaceutical composition according to the treatment methods disclosed herein.

[0118] An identifier, e.g., a bar code, radio frequency, ID tag, etc., may be present in or on the kit and can be used to uniquely identify the kit, e.g., for purposes of quality control, inventory control, tracking movement between workstations, etc. [Example]

[0119] Any examples provided herein are offered by way of example, not by way of limitation. The following examples are illustrative and do not limit the scope of the claimed embodiments.

[0120] Example 1. Perturbation studies in spheroids derived from HCC patients

[0121] Tumor tissue fragments (5x5mm to 8x8mm in size) were excised from tumor masses of HCC patients (n = 15 different donors, Table 2) and then treated with enzymatic and mechanical dissociation to generate tumor cell clusters, similar to recent reports (Crouchet E, et al., Nat Commun 2021;12:5525; Juehling F, et al., Gut 2021;70:157-69). Cell aggregates were organized into spheroids in 96-well black / clear-bottom low-flange ultra-low attachment microplates (Corning) and cultured in MammoCult® basal medium (STEMCELL Technologies) supplemented with human growth supplement (3.4%), hydrocortisone (0.056%), heparin (0.011%), and 50% patient-specific serum.

[0122] Tumorspheres were treated with 10 μg / mL of isotype control mAb, 10 μg / mL of anti-claudin-1 mAb "H3L3" (Table 1), sorafenib (10 μM, Selleckchem), or nivolumab (10 μg / mL, Selleckchem) for 6 days in at least duplicates per condition. On day 6, tumorsphere viability was assessed using CellTiterGlo 3D (Promega) according to the manufacturer's instructions.

[0123] [Table 2]

[0124] We also evaluated the effect of anti-claudin-1 antibody (anti-claudin-1 mAb "H3L3") on tumor growth in a fully patient-derived culture system that models tumor heterogeneity. Primary HCC tumorspheres cultured as multicellular microtissues maintain their original cell-cell contacts and recapitulate non-parenchymal cells of the TME, including T cells associated with tumor progression and treatment resistance (Song Y, et al., J Exp Clin Cancer Res 2018;37:109). Treatment with anti-claudin-1 mAb "H3L3" significantly disrupted the structure of HCC spheroids (Figure 1A). Furthermore, anti-claudin-1 mAb "H3L3" showed a significant effect on cell viability of sorafenib-resistant HCC spheroids (p=0.003 and p=0.04, Student's t-test, Figure 1B). Subsequent screening in HCC spheroids derived from 15 different HCC patients (patient characteristics are shown in Table 2) confirmed that the anti-claudin-1 mAb "H3L3" exerted a superior effect on tumor cell viability compared with sorafenib and nivolumab (47% vs. 33% and 15%, respectively, defined as a mean decrease in cell viability of less than 15%; Figure 1C).

[0125] Taken together, these data demonstrate the potent inhibitory effect of the anti-claudin-1 mAb "H3L3" on the growth of HCC, including sorafenib- and nivolumab-resistant tumors.

[0126] The practice of the present disclosure will employ, 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 those in the art and are fully explained in the literature.

[0127] Incorporation by Reference Throughout this application, various publications, patents, and / or patent applications are referenced, the disclosures of which are hereby incorporated by reference in their entireties into this application in order to more fully describe the state of the art to which this disclosure pertains.

[0128] Embodiment Additional embodiments of the present disclosure include the following.

[0129] Embodiment 1. A method of treating hepatocellular carcinoma (HCC) in a subject, comprising administering a therapeutically effective amount of an anti-claudin-1 antibody, wherein the HCC is resistant to sorafenib therapy.

[0130] Embodiment 2. A method of treating sorafenib-resistant hepatocellular carcinoma in a subject, comprising administering a therapeutically effective amount of an anti-claudin-1 antibody, wherein the HCC is resistant to PD-1 antagonist therapy.

[0131] Embodiment 3. The method of embodiment 1, wherein the subject has been previously treated with sorafenib.

[0132] Embodiment 4. The method of embodiment 2, wherein the subject has been previously treated with a PD-1 antagonist.

[0133] Embodiment 5. The method of embodiment 2 or 4, wherein the PD-1 antagonist is an antibody.

[0134] Embodiment 6. The method of embodiment 5, wherein the PD-1 antagonist is selected from the group consisting of nivolumab, pembrolizumab, cemiplimab, and dostarlimab.

[0135] Embodiment 7. The method of embodiment 6, wherein the PD-1 antagonist is nivolumab.

[0136] Embodiment 8. The method of any one of embodiments 1 to 7, wherein the HCC is characterized by high expression of claudin-1 relative to a reference sample or resistance to sorafenib and / or nivolumab.

[0137] Embodiment 9 The method of embodiment 8, wherein the reference sample is a tissue sample derived from a normal tissue, and the normal tissue is adjacent to the HCC tumor.

[0138] Embodiment 10. The method of any one of embodiments 1 to 9, wherein the anti-claudin-1 antibody is a monoclonal antibody comprising six complementarity-determining regions (CDRs) of an anti-claudin-1 monoclonal antibody secreted by a hybridoma cell line deposited at DSMZ on July 29, 2008 under accession number DSM ACC2938.

[0139] Embodiment 11. The method of any one of embodiments 1 to 10, wherein the anti-claudin-1 antibody is humanized.

[0140] Embodiment 12. The method of any one of embodiments 1 to 11, wherein 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.

[0141] Embodiment 13. The method of any one of embodiments 1 to 12, wherein 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.

[0142] Embodiment 14. The method described in any one of embodiments 1 to 13, 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.

[0143] Embodiment 15. The method described in any one of embodiments 1 to 14, 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.

[0144] Embodiment 16. The method of any one of embodiments 1 to 15, wherein the anti-claudin-1 antibody comprises a 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.

[0145] Embodiment 17. The method of any one of embodiments 1 to 16, wherein the anti-claudin-1 antibody comprises a 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 GA, and a CDR L3 comprising the amino acid sequence set forth in SEQ ID NO: 10.

[0146] Embodiment 18 The method of embodiment 1, further comprising a therapeutically effective amount of sorafenib for said subject.

Claims

1. 1. A method of treating hepatocellular carcinoma (HCC) in a human subject, comprising administering a therapeutically effective amount of an anti-claudin-1 antibody or antigen-binding fragment thereof, wherein the anti-claudin-1 antibody or antigen-binding fragment thereof: heavy chain complementarity determining region 1 (CDRH1) comprising the amino acid sequence of SEQ ID NO:5; a heavy chain complementarity determining region 2 (CDRH2) comprising the amino acid sequence of SEQ ID NO: 6; a heavy chain complementarity determining region 3 (CDRH3) comprising the amino acid sequence of SEQ ID NO: 7; a light chain complementarity determining region 1 (CDRL1) comprising the amino acid sequence of SEQ ID NO: 8; a light chain complementarity-determining region 2 (CDRL2) comprising the amino acid sequence of GA; and a light chain complementarity-determining region 3 (CDRL3) comprising the amino acid sequence of SEQ ID NO: 10; The method, wherein the HCC is resistant to sorafenib or a PD-1 antagonist.

2. The method of claim 1, wherein the anti-claudin-1 antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 3 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:

4.

3. The method of claim 1, wherein the anti-claudin-1 antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 13 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:

14.

4. 1. A method for identifying a human subject with HCC suitable for therapy with an anti-claudin-1 antibody or antigen-binding fragment thereof, comprising: a) obtaining a biological sample from a human subject with HCC; b) detecting the expression of claudin-1; c) comparing the detected expression level of the claudin-1 with a control expression level; and d) identifying the human subject as a responder if the detected expression level of claudin-1 is higher than the control expression level. The method comprising:

5. 5. The method of claim 4, e) if the human subject is identified as a responder, administering an anti-claudin-1 antibody or antigen-binding fragment thereof in an amount sufficient to reduce the symptoms of HCC. The method comprising:

6. 1. A method for treating a human subject having HCC, comprising: a) obtaining a biological sample from a human subject with HCC; b) detecting the expression level of claudin-1; c) comparing the detected expression level of the claudin-1 with a control expression level; d) identifying the human subject as a responder if the detected expression level of claudin-1 is higher than the control expression level; and e) if the human subject is identified as a responder, administering an anti-claudin-1 antibody or antigen-binding fragment thereof in an amount sufficient to reduce the symptoms of HCC. The method comprising:

7. The method of any one of claims 4 to 6, wherein the control expression level is determined from a normal tissue sample, the normal tissue sample being adjacent to the biological sample from the human subject with HCC.

8. The method of any one of claims 4 to 7, wherein the HCC is resistant to sorafenib or a PD-1 antagonist.

9. The method according to any one of claims 4 to 8, wherein the anti-claudin-1 antibody or antigen-binding fragment thereof is CDRH1 comprising the amino acid sequence of SEQ ID NO: 5, CDRH2 comprising the amino acid sequence of SEQ ID NO: 6; a CDRH3 comprising the amino acid sequence of SEQ ID NO: 7; CDRL1 comprising the amino acid sequence of SEQ ID NO: 8, a CDRL2 comprising the amino acid sequence of GA, and CDRL3 comprising the amino acid sequence of SEQ ID NO: 10 The method comprising:

10. The method according to any one of claims 4 to 9, wherein the anti-claudin-1 antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 3 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:

4.

11. The method according to any one of claims 4 to 9, wherein the anti-claudin-1 antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 13 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:

14.

12. The method according to any one of claims 1 to 11, wherein the anti-claudin-1 antibody is a humanized antibody.

13. The method of any one of claims 1 to 12, wherein the human subject has been previously treated with sorafenib.

14. 14. The method of any one of claims 1 to 13, wherein the human subject is simultaneously or sequentially administered a therapeutically effective amount of sorafenib.

15. The method of any one of claims 1 to 12, wherein the human subject has been previously treated with a PD-1 antagonist.

16. 16. The method of any one of claims 1 to 12 or 15, wherein the human subject is simultaneously or sequentially administered a therapeutically effective amount of a PD-1 antagonist.

17. 17. The method of any one of claims 1-12, 15, or 16, wherein the PD-1 antagonist is nivolumab, pembrolizumab, cemiplimab, dostarlimab, or a combination thereof.

18. 18. The method of claim 17, wherein the PD-1 antagonist is nivolumab.

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