Use of anti-claudin-1 antibodies to improve T cell availability

Abstract

The present disclosure relates to a method of promoting T cell-mediated anti-tumor activity in a subject having a fibrotic tumor, the method comprising administering an anti-claudin-1 antibody to the subject.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority benefit of U.S. Provisional Application No. 63 / 317,885, filed March 08, 2022, which is incorporated by reference in its entirety herein.

[0002] Sequence Listing Reference The contents of the electronically submitted sequence listing (Name: 4872_013PC01_Seqlisting_ST26, Size: 24,527 bytes, Creation Date: March 6, 2023) are incorporated by reference in their entirety into this specification.

[0003] According to various aspects of the present disclosure, the present disclosure relates to a method of promoting anti-tumor activity of T cells. [Background technology]

[0004] Cancer therapy with immune checkpoint inhibitors has greatly advanced physicians' ability to treat their subjects. However, many cancers are becoming resistant to immune checkpoint inhibitors. One phenotype associated with immune checkpoint inhibitor resistance is the lack of tumor T cell infiltration, also known as "cold tumors" or "T cell elimination" (Shuyue W. et al, Front. Immun., 12:690112 (2021); Christian et al., Front. Oncol., 11:712788 (Oct 2021)). Therefore, there is a need to develop therapies to treat cold tumors or T cell elimination so that therapies that overcome immune checkpoint inhibitor resistance can be developed. Summary of the Invention

[0005] The present disclosure provides a method of promoting T cell-mediated anti-tumor activity in a subject having a fibrotic tumor, the method comprising administering to the subject an anti-claudin-1 antibody.

[0006] In some embodiments, the method further comprises administering an immune checkpoint inhibitor.

[0007] In some embodiments, provided herein is a method of treating cancer in a subject having a solid tumor, the method comprising administering to the subject a therapeutically effective amount of an anti-claudin-1 antibody and an immune checkpoint inhibitor. In some embodiments, the anti-claudin-1 antibody promotes T cell-mediated anti-tumor activity in the tumor of the subject.

[0008] In some embodiments, provided herein is a method of enhancing the therapeutic efficacy of an immune checkpoint inhibitor in a subject having a fibrotic tumor, the method comprising: a) administering to the subject an anti-claudin-1 antibody, where the anti-claudin-1 antibody promotes T cell-mediated anti-tumor activity in the fibrotic tumor; and b) administering to the subject an immune checkpoint inhibitor.

[0009] In some embodiments, the anti-claudin-1 antibody is administered prior to administering the immune checkpoint inhibitor.

[0010] In some embodiments, the anti-claudin-1 antibody and the immune checkpoint inhibitor are administered simultaneously or sequentially.

[0011] In some embodiments, the anti-claudin-1 antibody and the immune checkpoint inhibitor are administered in the same composition.

[0012] In some embodiments, the anti-claudin-1 antibody and the immune checkpoint inhibitor are administered in different compositions.

[0013] In some embodiments, the anti-claudin-1 antibody and / or immune checkpoint inhibitor is administered intratumorally, intravenously, intraperitoneally, intramuscularly, intradermally, or subcutaneously.

[0014] In some embodiments, the immune checkpoint inhibitor is an antagonist of PD-1, PD-L1, CTLA-4, LAG-3, TIM-3, TIGIT, VISTA, B7-H3, BTLA, and / or Siglec-15.

[0015] In some embodiments, the immune checkpoint inhibitor is a small molecule inhibitor.

[0016] In some embodiments, the immune checkpoint inhibitor is an antibody.

[0017] In some embodiments, the immune checkpoint inhibitor is a PD-1 antagonist selected from the group consisting of nivolumab, pembrolizumab, semipilimab, and dostallimab.

[0018] In some embodiments, the immune checkpoint inhibitor is a PD-L1 antagonist selected from the group consisting of atezolizumab, durvalumab, and avelumab.

[0019] In some embodiments, the immune checkpoint inhibitor is a CTLA-4 antagonist selected from the group consisting of ipilimumab and tremelimumab.

[0020] In some embodiments, the immune checkpoint inhibitor is a TIGIT antagonist selected from the group consisting of tiragolumab, osipeliumab, domvanalimab, etigilimab, and vibostolimab.

[0021] In some embodiments, the cancer comprises a fibrotic tumor.

[0022] In some embodiments, the fibrotic tumor is characterized by higher expression of claudin-1 than the reference sample.

[0023] In some embodiments, the reference sample is a tissue sample of normal tissue, where the normal tissue is adjacent to the tumor.

[0024] In some embodiments, the tumor is selected from the group consisting of head and neck, lung, breast, melanoma, colon, pancreatic, esophageal, cholangiocarcinoma, and hepatocellular tumors.

[0025] In some embodiments, the anti-claudin-1 antibody is a monoclonal antibody comprising the six complementarity determining regions (CDRs) 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.

[0026] In some embodiments, the anti-claudin-1 antibody is humanized.

[0027] 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.

[0028] 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.

[0029] 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.

[0030] 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.

[0031] In some embodiments, 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.

[0032] In some embodiments, 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 "Gly Ala", and a CDR L3 comprising the amino acid sequence set forth in SEQ ID NO:10.

[0033] In some embodiments, the anti-claudin-1 antibody comprises a heavy chain sequence comprising the amino acid sequence set forth as SEQ ID NO:1.

[0034] In some embodiments, the anti-claudin-1 antibody comprises a light chain sequence comprising the amino acid sequence set forth as SEQ ID NO:2.

[0035] In some embodiments, the present disclosure provides a method for promoting T cell-mediated anti-tumor activity in a subject with a fibrotic tumor, comprising administering anti-claudin-1 chimeric antigen receptor (CAR) T cells to the subject. In some embodiments, the present disclosure comprises administering an immune checkpoint inhibitor.

[0036] In some embodiments, provided herein is a method of treating cancer in a subject having a solid tumor, the method comprising administering to the subject a therapeutically effective amount of anti-claudin-1 CAR T cells and an immune checkpoint inhibitor. In some embodiments, the anti-claudin-1 CAR T cells promote T cell-mediated anti-tumor activity in the tumor of the subject.

[0037] In some embodiments, provided herein is a method of enhancing the therapeutic efficacy of an immune checkpoint inhibitor in a subject having a fibrotic tumor, the method comprising administering anti-claudin-1 CAR T cells to the subject and administering an immune checkpoint inhibitor to the subject. In some embodiments, the anti-claudin-1 CAR T cells promote T cell-mediated anti-tumor activity in the fibrotic tumor. [Brief description of the drawings]

[0038] [Figure 1A]Figure 1 shows the expression of claudin-1 (CLDN1) in two different fibrotic tumor types. Immunohistochemistry (IHC) staining was performed to measure claudin-1 expression (left panel) and fibrotic markers (right panel). [Figure 1B] Figure 1 shows the expression of claudin-1 (CLDN1) in two different fibrotic tumor types. Immunohistochemistry (IHC) staining was performed to measure claudin-1 expression (left panel) and fibrotic markers (right panel). [Figure 2A] 1 shows T cell elimination in head and neck cancer. IHC staining for CLDN1 in multiple tumor samples and corresponding staining for CD3 illustrating fibrotic traps. [Figure 2B] 1 shows T cell elimination in head and neck cancer. The percentage of tumor samples that showed CLDN1 expression is shown. [Figure 2C] Figure 1. T cell elimination in head and neck cancer. Immunophenotypic breakdown of tumors with various CLDN1 expression levels. Immunophenotypes are hot (immune cells in the stroma and between cancer cells), elimination (immune cells mainly in the stroma), and cold (few immune cells visible). [Figure 3A] Figure 1 shows T cell elimination in esophageal cancer. Tumor immunophenotypes with various CLDN1 expression levels are shown: hot (immune cells in the stroma and between cancer cells), elimination (immune cells in the tumor but only in the stroma), and cold (few immune cells visible). [Figure 3B] IHC staining of CLDN1 was performed on esophageal tumor samples, demonstrating T cell exclusion in esophageal cancer. [Figure 3C] IHC staining for T cells (CD3 staining) was performed on esophageal tumor samples, demonstrating T cell exclusion in esophageal cancer. [Figure 3D] Showing the elimination of T cells in esophageal cancer. Esophageal tumor samples were subjected to IHC staining for fibrotic tissue (Sirius Red stain). [Figure 4A]We demonstrate immune escape driven by overexpression of mouse CLDN1 with human extracellular loops in liver mouse tumor cells Hepa 1-6 in vivo. [Figure 4B] We show that overexpression of mouse CLDN1 with human extracellular loops drives T cell elimination in liver mouse tumor cells Hepa 1-6 in vivo. [Diagram 5] Figure 1 shows the volume of Hepa 1-6 tumors after inoculation. Samples positive for mouse CLDN1 with human extracellular loops (CLDN1 hECL) showed larger tumor volume and exhibited immune evasion compared to samples without CLDN1 expression. [Figure 6] FIG. 1 shows an exemplary diagram for breaking checkpoint inhibitor resistance in cancer using anti-CLDN1 antibodies with direct anti-fibrotic effects. [Figure 7A] 1 shows overall survival probability over time in tumors (e.g., melanoma) with high or low levels of CLDN1 following administration of the checkpoint inhibitor aPD1. [Figure 7B] Time to treatment discontinuation in CLDN1-positive or CLDN1-negative patients is shown. [Figure 8A] A mechanistic model of CLDN-1 targeted therapy in the tumor microenvironment is presented. [Figure 8B] 1 shows a mechanistic model demonstrating the effect of anti-CLDN1 antibody administration on tumors involving T cell elimination. [Figure 9] We show CLDN1 expression in head and neck squamous cell carcinoma (HNSCC), colorectal cancer (CRC), esophageal cancer, squamous non-small cell lung cancer (squamous NSCLC), intrahepatic cholangiocarcinoma (iCCA), hepatocellular carcinoma (HCC), and urothelial carcinoma. [Figure 10A] 1 shows tumor volumes in Hepa1-6 CLDN1+ challenged mice after administration of anti-CLDN1 antibody alone, PD1 antagonist (aPD1) alone, and the combination of anti-CLDN1 antibody and PD1 antagonist. Mice were rechallenged on day 40. [Figure 10B]Immunocytochemistry measuring T cell dendritic cell infiltration after control treatment is shown. [Figure 10C] Immunocytochemistry measuring T cell dendritic cell infiltration after treatment with anti-CLDN1 antibody and PD1 antagonist is shown. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0039] 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.

[0040] Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described below. 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.

[0041] In order to further define this disclosure, the following terms and definitions are provided.

[0042] 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," may be 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."

[0043] The term "about" is used herein to mean approximately, roughly, around, or within the region of. When the term "about" is used in conjunction with a numerical range, "about" modifies that range by extending the boundaries above and below the stated value. In general, the term "about" is used herein to modify a numerical value above and below by a variance of 10 percent (%) above or below (higher or lower) the stated value.

[0044] Throughout this disclosure, various aspects of the invention 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 inflexible limitation on the scope of the invention. Thus, the description of a range should be considered to have specifically disclosed each individual numerical value within that range, in addition to all possible subranges. For example, a description of a range such as "1 to 6" should be considered to have specifically disclosed each individual numerical value within that range, e.g., 1, 2, 3, 4, 5, and 6, in addition to subranges such as "1 to 3", "1 to 4", "1 to 5", "2 to 4", "2 to 6", "3 to 6", etc. This applies regardless of the breadth of the range. The numerical ranges described include both endpoints of the numbers defining the range and include each integer within the defined range.

[0045] Units, prefixes, and symbols are shown in the form accepted by the International System of Units (SI). Numerical ranges are inclusive of the numeric endpoints defining the range. When a range of values ​​is described, it is understood that each intervening integer value and each fraction between the upper and lower limits of the range is specifically disclosed, along with each subrange between such values. The upper and lower limits of any range may be independently included or excluded within the range, and any range that includes either limit, does not include either limit, or includes both limits is encompassed within the disclosure. Thus, ranges described herein are understood to be shorthand for all values ​​within the range, including the stated 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, 10.

[0046] When a value is explicitly stated, it is understood that values ​​that are approximately the same amount or the same amount as the stated value are also within the scope of this disclosure. Where a combination is disclosed, each subcombination of the elements of the combination is also specifically disclosed and is within the scope of this disclosure. Conversely, where different elements or groups of elements are disclosed separately, these combinations are also disclosed. Where any element of this disclosure is disclosed as having multiple alternatives, examples of the disclosure in which each alternative is excluded alone or in any combination with other alternatives are also disclosed herein, and multiple elements of the disclosure may have such exclusions, and all combinations of elements with such exclusions are disclosed herein.

[0047] As used herein, "and / or" shall be construed as specifically disclosing two specified features or components with or without the other. Thus, the term "and / or" when used in phrases such as "A and / or B" is intended to include "A and B," "A or B," "A" (single), and "B" (single). Similarly, the term "and / or" when 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 (single); B (single); and C (single).

[0048] As used herein, the terms "treat" or "treatment" refer to the administration of a composition to a subject for therapeutic purposes. 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. Claudins are a family of approximately 18 proteins that play important structural and functional roles in tight junctions. They are transmembrane proteins that interact with other transmembrane proteins (e.g., junctional adhesion molecules (JAMs) and occludin), as well as the scaffolding proteins ZO-1, ZO-2, and ZO-3. Claudin-1 expression is widespread in epithelial cells, but in mesenchymal tissues, expression appears to be restricted to neural sheath cells. Claudin-1 expression has been reported to occur in 29% to 92% of tumors.

[0049] 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 not only encompasses 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 includes 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 the antibody binding to a given antigen. Typically, an antibody binds to at least 1×10 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 non-specific antigen (eg, BSA, casein).

[0050] The term "monoclonal antibody" or antigen-binding fragment thereof refers to a population of homogeneous antibodies or antigen-binding fragments that highly specifically recognize and bind to a single antigenic determinant or epitope. This is in contrast to polyclonal antibodies, which typically contain different antibodies against different antigenic determinants. The term "monoclonal antibody" or antigen-binding fragment thereof encompasses both intact and full-length monoclonal antibodies, as well as antibody fragments (e.g., Fab, Fab', F(ab')2, Fv), single-chain (scFv) variants, fusion proteins containing antibody portions, and other modified immunoglobulin molecules that contain an antigen recognition site. Furthermore, "monoclonal antibody" or antigen-binding fragment thereof refers to antibodies and antigen-binding fragments thereof produced by any method, including but not limited to hybridoma, phage selection, recombinant expression, and transgenic animals.

[0051] As used herein, the term "humanized antibody" refers to a chimeric antibody that comprises amino acid residues from a non-human hypervariable region and amino acid residues from a human framework region (FR). In particular, a humanized antibody contains 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 contain 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.

[0052] Whenever an embodiment is described herein using the term "comprising," it is understood that otherwise similar embodiments described using the terms "consisting of" and / or "consisting essentially of" are also provided.

[0053] As used herein, the term "administration" refers to the physical introduction of a composition containing a therapeutic agent (e.g., an anti-claudin-1 antibody and / or a combination of immune checkpoint inhibitors) into a subject using any of a variety of methods and delivery systems known to those skilled in the art. Routes of administration include intravenous, intramuscular, subcutaneous, intraperitoneal, intraspinal, or other parenteral routes of administration (e.g., by injection or infusion). As used herein, the term "parenteral administration" refers to modes of administration other than enteral and topical administration, usually by injection, including, but 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, and in vivo electroporation. Other parenteral routes include topical, epidermal, or mucosal routes of administration, such as intranasal, vaginal, rectal, sublingual, or topical. Administration can also be, for example, once, multiple times, and / or over one or more extended periods of time.

[0054] The term "effective amount" refers to an amount of an agent that produces a desired biological, therapeutic, and / or prophylactic result. The result may be reduction, amelioration, alleviation, mitigation, and / or remission of one or more of the signs, symptoms, or causes of a disease, or any other desired change in a biological system. An effective amount in the context of a solid tumor includes an amount sufficient to shrink the tumor and / or slow the rate of tumor growth (e.g., inhibit 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 can (i) reduce the number of cancer cells; (ii) reduce tumor size; (iii) inhibit, delay, to some extent, or stop cancer cells from invading surrounding organs; (iv) inhibit (i.e., to some extent, slow or 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 anti-claudin-1 antibody and immune checkpoint inhibitor combined that has been clinically found to affect a significant reduction in cancer (e.g., aggressive solid tumors) or delay in the progression of cancer.

[0055] "Cancer" refers to a broad group of diverse diseases characterized by the uncontrolled growth of abnormal cells in the body. Unregulated cell division and growth leads to the formation of malignant tumors that can invade adjacent tissues and may even metastasize to distant parts of the body via the lymphatic system or bloodstream. "Cancer" or "cancerous tissue" can also include tumors.

[0056] As used herein, the term "tumor" refers to any mass of tissue resulting from the growth or proliferation of excess cells, either benign (non-cancerous) or malignant (cancerous), including pre-cancerous lesions.

[0057] As used herein, the term "cold tumor" refers to tumors that exhibit low levels of immune infiltration, tumors that respond poorly to immunotherapy, and / or tumor heterogeneity. Such cold tumors may have characteristics including, but not limited to, a substantial reduction or absence of intratumoral CD8+ T effector cells in number and / or activity, and / or a substantial increase in the number and / or activity of intratumoral immune suppressor cells. Cold tumors are also referred to as tumors with "T cell exclusion". Immunotherapy with immune checkpoint inhibitors (ICIs) has greatly improved the clinical efficacy of malignant tumor therapy, but ICI-mediated antitumor responses depend on the infiltration of T cells capable of recognizing and killing tumor cells. Therefore, ICIs may not be effective against "cold tumors" characterized by a lack of T cell infiltration.

[0058] 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), where 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.

[0059] As used herein, a "patient" includes any patient suffering from cancer (e.g., fibrotic cancer). The terms "subject" and "patient" are used interchangeably herein.

[0060] II. Anti-Claudin-1 Antibody The present invention relates to the use of anti-claudin-1 antibodies to promote T cell-mediated anti-tumor activity in subjects with fibrotic tumors, to treat cancer in subjects with solid tumors, and to enhance the therapeutic efficacy of immune checkpoint inhibitors in subjects with fibrotic tumors.

[0061] Antibodies against human claudin-1 have been previously described for the treatment of hepatitis C virus infection, hepatocellular carcinoma, and certain fibrotic diseases (e.g., pulmonary fibrosis) (see WO2010 / 034812, WO2016 / 146809, and WO2021 / 094469). Anti-claudin-1 antibodies that can be used in the practice of the invention include any antibody raised against claudin-1. Examples of these are disclosed in WO2010 / 034812 and WO2017 / 162678.

[0062] Other examples of suitable anti-claudin-1 antibodies include those disclosed in European Patent No. EP 1 167 389, U.S. Pat. No. 6,627,439, International Patent Applications published under Nos. WO2014 / 132307, WO2015 / 014659 and WO2015 / 014357, and Yamashita et al., J. Pharmacol. Exp. Ther., 2015, 353(1):112-118.

[0063] Anti-claudin-1 antibodies suitable for use in the present invention can be polyclonal or monoclonal antibodies.

[0064] Anti-claudin-1 antibodies suitable for use herein may be "humanized" and sequence differences between rodent antibodies and human sequences may be minimized by replacing residues that differ from the human sequence by site-directed mutagenesis of individual residues, or by grafting of entire regions, or by chemical synthesis. Humanized antibodies may 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 permissible as long as they do not significantly modify 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.

[0065] In some embodiments, the humanized anti-claudin-1 antibody for use in accordance with the present invention is an antibody previously described in WO2017 / 162678. Exemplary sequences of the antibodies or antigen-binding fragments provided herein are set forth in Table 1. [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4]

[0066] In some embodiments, 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.

[0067] In some embodiments, 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 "Gly Ala", and a CDR L3 comprising the amino acid sequence set forth in SEQ ID NO:10.

[0068] In some embodiments, the complementarity determining regions (CDRs) disclosed herein are defined according to IMGT®, although it is understood that other methods of defining CDRs in the art may also be used.

[0069] 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.

[0070] In some embodiments, the anti-claudin-1 antibody comprises a heavy chain variable region ("VH") comprising the amino acid sequence set forth as SEQ ID NO:3 or 13.

[0071] In some embodiments, the anti-claudin-1 antibody comprises a light chain variable region ("VL") comprising the amino acid sequence set forth as SEQ ID NO:4 or 14.

[0072] 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.

[0073] 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.

[0074] In some embodiments, the anti-claudin-1 antibody comprises a heavy chain sequence comprising the amino acid sequence set forth as SEQ ID NO:1.

[0075] In some embodiments, the anti-claudin-1 antibody comprises a light chain sequence comprising the amino acid sequence set forth as SEQ ID NO:2.

[0076] In some embodiments, the anti-claudin-1 antibody comprises a heavy chain sequence comprising the amino acid sequence set forth as SEQ ID NO:1, SEQ ID NO:11, or SEQ ID NO:21.

[0077] In some embodiments, the anti-claudin-1 antibody comprises a light chain sequence comprising the amino acid sequence set forth as SEQ ID NO:2 or SEQ ID NO:12.

[0078] The humanized anti-claudin-1 antibody may be a complete monoclonal antibody having an isotope selected from the group consisting of IgG1, IgG2, IgG3, and IgG4. Alternatively, the humanized anti-claudin-1 antibody may be a fragment of a monoclonal antibody selected from the group consisting of Fv, Fab, F(ab')2, Fab', dsFv, scFv, sc(Fv)2, and diabody.

[0079] The anti-claudin-1 antibody (or biologically active variant or fragment thereof) suitable for use according to the present invention may be functionally linked (e.g., by chemical conjugation, genetic fusion, non-covalent or other linkage) to one or more other molecular entities. 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 linked 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.

[0080] The antibody molecule and the molecular entity may be directly bound to each other by a covalent bond, or alternatively, the antibody molecule and the molecular entity may be covalently bound to each other via a linker group, which can be achieved by using any of a wide variety of stable bifunctional agents known in the art, including homofunctional and heterofunctional linkers.

[0081] In some embodiments, the anti-claudin-1 antibody (or biologically active fragment thereof) for use in accordance with the present invention is conjugated to a detection agent. Any of a wide variety of detectable agents can be used, including but not limited to various ligands, radionuclides (e.g., 3 H, 125 I, 131I, etc.), fluorescent dyes (e.g., fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthalaldehyde, and fluorescerin), chemiluminescent agents (e.g., luciferin, luciferase, and aequorin), microparticles (e.g., quantum dots, nanocrystals, fluorophores, etc.), enzymes (e.g., enzymes used in ELISA, i.e., horseradish peroxidase, beta-galactosidase, luciferase, alkaline phosphatase), colorimetric labels, magnetic labels, and biotin, dioxygenin, or other haptens, as well as proteins for which antisera or monoclonal antibodies are available.

[0082] Other molecular entities that can be conjugated to the anti-claudin-1 antibodies (or biologically active fragments thereof) of the present invention include, but are not limited to, linear or branched hydrophilic polymer groups, fatty acid groups, or fatty ester groups.

[0083] Thus, in the practice of the present invention, anti-claudin-1 antibodies can be used in the form of full-length antibodies, biologically active variants or fragments thereof, chimeric antibodies, humanized antibodies, and antibody-derived molecules that contain 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, single light chains of individual antibodies, individual antibody heavy chains, chimeric fusions of antibody chains with other molecules, and antibody conjugates (e.g., antibodies conjugated to therapeutic or detection agents). Preferably, the anti-claudin-1 antibody-related molecules according to the present invention retain the ability of the antibody to bind to its antigen, particularly the extracellular domain of claudin-1.

[0084] III. Chimeric Antigen Receptors Chimeric antigen receptor (CAR) T-cell therapy (or CAR T-cell therapy) is a cancer treatment based on the use of T cells genetically engineered to express synthetic receptors that bind to tumor antigens. Engineered CAR T cells are expanded in vitro and then infused into patients to attack and destroy chemotherapy-resistant cancers.

[0085] The term "chimeric antigen receptor" (CAR) refers to a molecule that combines a binding domain for a component present on a target cell (e.g., an antibody-based specificity for a desired antigen (e.g., a tumor antigen such as CLDN-1)) with a T cell receptor activating intracellular domain to generate a chimeric protein that exhibits specific anti-target cell immune activity.

[0086] As used herein, the "signal transduction domain" or "signal transduction domain" of a CAR is responsible for intracellular signal transduction following binding of the extracellular ligand binding domain to a target, resulting in immune cell activation and an immune response. In other words, the signal transduction domain is responsible for activating at least one of the normal effector functions of the immune cell in which the CAR is expressed. For example, the effector function of a T cell can be cytolytic activity or helper activity (including secretion of cytokines). Thus, the term "signal transduction domain" refers to a portion of a protein that transduces the signal of the effector function and instructs the cell to perform a specialized function. Examples of signal transduction domains used in CARs can be the cytoplasmic sequences of the T cell receptor (TCR) and co-receptors that act in concert to initiate signal transduction after antigen receptor binding, as well as any derivative or variant of these sequences, and any synthetic sequence with the same functional capabilities. In some cases, the signal transduction domain includes two different classes of cytoplasmic signaling sequences: those that initiate antigen-dependent primary activation, and those that act antigen-independently to provide secondary or costimulatory signals. The primary cytoplasmic signaling sequence may comprise a signaling motif known as an ITAM immunoreceptor tyrosine-based activation motif. ITAMs are well-defined signaling motifs found in the cytoplasmic tails of various receptors that function as binding sites for syk / zap70 class tyrosine kinases. Exemplary ITAMs include those derived from TCR zeta, FcR gamma, FcR beta, FcR epsilon, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d. In some embodiments, the signal transduction domain of the CAR may comprise a CD3 zeta signaling domain (SEQ ID NO: 15).

[0087] CARs are synthetic receptors that consist of one targeting moiety linked to one or more signaling domains in a single fusion molecule. In general, the binding site of a CAR consists of the antigen-binding domain of a single-chain antibody (scFv), which contains the variable fragments of the light and heavy chains of a monoclonal antibody linked by a flexible linker. This molecule is linked to an intracellular signaling molecule, which contains one or more intracellular signaling domains that mediate T cell activation. The signaling domain of first generation CARs is derived from the cytoplasmic region of the CD3 zeta chain or the Fc receptor gamma chain (or the intracellular signaling domain of another immunoreceptor tyrosine-based activation motif [ITAM]-containing protein). First generation CARs have been shown to successfully redirect the cytotoxicity of T cells. However, they have failed to produce long-term expansion and antitumor activity in vivo. Second, third, and fourth generation CARs have been formed by adding signaling domains from costimulatory molecules, as well as transmembrane and hinge domains. Second generation chimeric receptors also incorporate a costimulatory endodomain (e.g., 4-1BB / CD3ζ). Third generation CARs contain multiple costimulatory signaling modules. Fourth generation CARs are generated by adding IL-12 to the base of second generation constructs and are known as T cell redirected for universal cytokine-mediated killing (TRUCK). TRUCK enhances T cell activation and activates and attracts innate immune cells to eliminate antigen-negative cancer cells in the target lesion. Human therapeutic trials using CAR T cell therapy have shown some success. For example, CAR-redirected T cells specific for the B cell differentiation antigen CD19 have shown dramatic efficacy in the treatment of B cell malignancies, while TCR-redirected T cells have shown benefit in patients suffering from solid tumors.Stauss et al. describe strategies for modifying therapeutic CARs and TCRs to enhance antigen-specific effector function and limit toxicity of engineered T cells for use in treating cancer (Current Opinion in Pharmacology 2015, 24:113-118), for example.

[0088] In some embodiments of the present invention, a chimeric antigen receptor (CAR) specific for claudin-1 expressed on the surface of cancer cells is included. In some embodiments of the present invention, the CAR described herein comprises an extracellular target-specific binding domain, a transmembrane domain, an intracellular signaling domain (e.g., a signaling domain derived from CD3 zeta or FcR gamma), and / or one or more costimulatory signaling domains derived from a costimulatory molecule (e.g., but not limited to, 4-1BB). In some embodiments, the CAR comprises a hinge or spacer region (e.g., a CD8 alpha hinge) between the extracellular binding domain and the transmembrane domain. In some embodiments, the chimeric antigen receptor (CAR) comprises an extracellular target-specific binding domain, which is an anti-claudin single chain antibody (scFv), which can be a mouse, human, or humanized scFv. The single chain antibody can be cloned from the V region genes of a hybridoma specific for the desired target. Techniques that can be used for cloning the variable heavy (VH) and variable light (VL) chains are described, for example, in Orlandi et al., PNAS, 1989;86:3833-3837. Thus, in some embodiments, the binding domain comprises an antibody-derived binding domain, but may also be a non-antibody-derived binding domain. An antibody-derived binding domain may be a fragment of an antibody, or a product of engineering one or more fragments of an antibody, which fragment is responsible for binding to an antigen.

[0089] In some embodiments, the CAR of the present invention can include linkers between various domains, which are added for proper spacing and conformation of the molecule. For example, in some embodiments, there may be a linker between the binding domains VH or VL, which may be 1-10 amino acids long. In some embodiments, the linker between either domain of the chimeric antigen receptor may be 1-20 or 20 amino acids long. In this regard, the linker may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids long. In some embodiments, the linker may be 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids long. Ranges including the numerical values ​​described herein are also included herein, for example, the linker is 10-30 amino acids long.

[0090] In some embodiments, a linker suitable for use in the CAR described herein is a flexible linker. Suitable linkers can be readily selected and can be of any suitable different length, for example, 1 amino acid (e.g., Gly) to 20 amino acids, 2 to 15 amino acids, 3 to 12 amino acids, 4 to 10 amino acids, 5 to 9 amino acids, 6 to 8 amino acids, or 7 to 8 amino acids, and can be 1, 2, 3, 4, 5, 6, or 7 amino acids.

[0091] Exemplary flexible linkers include glycine polymers (G)n, glycine-serine polymers (where n is an integer of at least 1), glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art. Because glycine and glycine-serine polymers are relatively unstructured, they may be able to function as neutral tethers between domains of fusion proteins such as the CARs described herein. Glycine has access to significantly more si-si space than alanine and is much less restricted than residues with long side chains (see Scheraga, Rev. Computational Chem. 11173-142 (1992)). One of skill in the art will recognize that the design of a CAR may include a fully or partially flexible linker such that, in addition to the flexible linker, the design of a CAR may include one or more moieties that confer a non-flexible structure to provide a desired CAR structure. Specific linkers include (G4S)n linkers (where n=1-3). In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO: 16 or SEQ ID NO: 17.

[0092] The binding domain of a CAR may be followed by a "spacer" or "hinge". This refers to a region that moves the antigen binding domain away from the effector cell surface to allow for proper cell / cell contact, antigen binding, and activation (Patel et al., Gene Therapy, 1999;6:412-419). The hinge region of a CAR is generally between the transmembrane (TM) and the binding domain. In some embodiments, the hinge region is an immunoglobulin hinge region, which may be a wild-type immunoglobulin hinge region or a modified wild-type immunoglobulin hinge region. Other exemplary hinge regions used in the CARs described herein include hinge regions derived from the extracellular regions of type 1 membrane proteins such as CD8 alpha, CD4, CD28, and CD7, which may be the wild-type hinge region from these molecules or modified. In some embodiments, the hinge region comprises a CD8 alpha hinge (SEQ ID NO: 18).

[0093] The "transmembrane" region or domain is the portion of the CAR that anchors the extracellular binding moiety to the cell membrane of an immune effector cell and facilitates binding of the binding domain to a target antigen. The transmembrane domain can be a CD3 zeta transmembrane domain, although other transmembrane domains may be used, including those obtained from CD8 alpha, CD4, CD28, CD45, CD9, CD16, CD22, CD33, CD64, CD80, CD86, CD134, CD137, and CD154. In some embodiments, the transmembrane domain is the transmembrane domain of CD137. In some embodiments, the transmembrane domain comprises the amino acid sequence of SEQ ID NO: 19. In some embodiments, the transmembrane domain is synthetic, in which case it comprises predominantly hydrophobic residues such as leucine and valine.

[0094] The term "intracellular signaling domain" or "signaling domain" refers to a portion of a chimeric antigen receptor protein that transmits the message of effective CAR binding to a target antigen inside an immune effector cell to induce effector cell functions (e.g., activation, cytokine production, proliferation, and cytotoxic activity (including release of cytotoxic factors to the CAR-bound target cell), or other cellular responses elicited by antigen binding to the extracellular CAR domain). The term "effector function" refers to a specialized function of a cell. The effector function of a T cell can be, for example, an activity that includes cytolytic or help activity, or secretion of cytokines. Thus, the terms "intracellular signaling domain" or "signaling domain" are used interchangeably herein and refer to a portion of a protein that transduces an effector function signal and instructs a cell to carry out a specialized function. Usually, the entire intracellular signaling domain can be used, but in many cases it is not necessary to use the entire domain. To the extent that a truncated portion of an intracellular signaling domain is used, such a truncated portion can be used in place of the entire domain, so long as it transmits the effector function signal. The term intracellular signaling domain is intended to include any truncated portion of the intracellular signaling domain sufficient to transmit an effector function signal. The intracellular signaling domain is also known as the "signal transduction domain" and is typically derived from a portion of the human CD3 or FcR gamma chain.

[0095] It is known that signals generated through the T cell receptor alone are insufficient for full activation of T cells, and that secondary or costimulatory signals are also required. Thus, it can be said that T cell activation is mediated by two distinct classes of cytoplasmic signaling sequences, namely, those that initiate primary activation in an antigen-dependent manner through the T cell receptor (primary cytoplasmic signaling sequences), and those that act in an antigen-independent manner to provide secondary or costimulatory signals (secondary cytoplasmic signaling sequences). Cytoplasmic signaling sequences that act in a costimulatory manner can contain signaling motifs known as immunoreceptor tyrosine-based activation motifs, or ITAMs.

[0096] Examples of ITAMs comprising primary cytoplasmic signaling sequences of particular use in the present invention include those derived from TCR zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d. In some embodiments, the intracellular signaling domain of the anti-BCMA CAR described herein is derived from CD3 zeta. In some embodiments, the signaling domain comprises the amino acid sequence of SEQ ID NO: 15.

[0097] As used herein, the term "costimulatory signaling domain" or "costimulatory domain" refers to a portion of a CAR that includes the intracellular domain of a costimulatory molecule. A costimulatory molecule is a cell surface molecule other than an antigen receptor or an Fc receptor that provides a second signal required for efficient activation and function of T lymphocytes upon antigen binding. Examples of such costimulatory molecules include CD27, CD28, 4-1BB (CD137), OX40 (CD134), CD30, CD40, PD-1, ICOS (CD278), LFA-1, CD2, CD7, LIGHT, NKD2C, B7-H2, and ligands that specifically bind to CD83. Thus, although the present disclosure provides exemplary costimulatory domains derived from CD3 zeta and 4-1BB, other costimulatory domains are contemplated for use with the CARs described herein. The inclusion of one or more costimulatory signaling domains may enhance the efficacy and expansion of T cells expressing a CAR receptor. The intracellular signaling domain and the costimulatory signaling domain can be linked in tandem in any order to the carboxyl terminus of the transmembrane domain. In some embodiments, the costimulatory domain comprises the amino acid sequence of SEQ ID NO:20.

[0098] In some embodiments, an anti-claudin-1 CAR of the present disclosure comprises any of the elements of Table 2. [Table 2]

[0099] Although scFv-based CARs engineered to contain signaling domains from CD3 or FcR gamma have been shown to deliver potent signals for T cell activation and effector function, in the absence of concomitant costimulatory signals they are not sufficient to induce signals that promote T cell survival and expansion. Other CARs that contain the binding domain, hinge, transmembrane domain, and signaling domain derived from CD3 zeta or FcR gamma, along with one or more costimulatory signaling domains (e.g., intracellular costimulatory domains derived from CD28, CD137, CD134, and CD278), may more effectively induce anti-tumor activity, as well as increased cytokine secretion, lytic activity, survival, and proliferation in CAR-expressing T cells in vitro, in animal models, and in cancer patients (Milone et al., Molecular Therapy, 2009; 17:1453-1464; Zhong et al., Molecular Therapy, 2010; 18:413-420; Carpenito et al., PNAS, 2009; 106:3360-3365).

[0100] In some embodiments, the anti-claudin-1 CAR of the present invention comprises (a) an anti-claudin-1 binding domain (e.g., an scFv having a binding region (e.g., CDR or variable domain) from any one or more sequences identified in Table 1), (b) a hinge region from human CD8 alpha, (c) a human CD8 alpha transmembrane domain, (d) a human T cell receptor CD3 zeta chain (CD3) intracellular signaling domain, and optionally one or more costimulatory signaling domains (e.g., 4-1BB). In some embodiments, the distinct protein domains are arranged in the following order from amino-terminus to carboxy-terminus: anti-claudin-1 binding domain, hinge region, and transmembrane domain. The intracellular signaling domain and optional costimulatory signaling domain are linked in tandem in any order to the transmembrane carboxy-terminus to form a single-chain chimeric polypeptide. In some embodiments, the nucleic acid construct encoding the anti-claudin-1 CAR is a chimeric nucleic acid molecule comprising a nucleic acid molecule comprising different coding sequences (e.g., (5'→3') coding sequences for an anti-claudin-1 scFv, a human CD8 alpha-hinge, a human CD8 alpha transmembrane domain, and a CD3 zeta intracellular signaling domain). In some embodiments, the nucleic acid construct encoding the anti-claudin-1 CAR is a chimeric nucleic acid molecule comprising a nucleic acid molecule comprising different coding sequences (e.g., (5'→3') coding sequences for an anti-claudin-1 scFv, a human CD8 alpha-hinge, a human CD8 alpha transmembrane domain, a 4-1BB costimulatory domain, and a CD3 zeta costimulatory domain).

[0101] In some embodiments, the polynucleotide encoding the CAR described herein is inserted into a vector. To express the anti-claudin-1 CAR, the vector can be introduced into a host cell to allow expression of the polypeptide in the host cell. The expression vector can include various elements for controlling expression, including but not limited to promoter sequences, transcription initiation sequences, enhancer sequences, selection markers, and signal sequences. These elements can be appropriately selected by those skilled in the art, as described above. For example, the promoter sequence can be selected to promote transcription of the polynucleotide in the vector. Suitable promoter sequences include but are not limited to T7 promoter, T3 promoter, SP6 promoter, beta-actin promoter, EF1a promoter, CMV promoter, and SV40 promoter. The enhancer sequence can be selected to enhance transcription of the polynucleotide. The selection marker can be selected to allow selection of host cells inserted with the vector from host cells that are not, for example, the selection marker can be a gene that confers antibiotic resistance. The signal sequence can be selected to allow the expressed polypeptide to be transported out of the host cell.

[0102] The CAR of the present invention is introduced into a host cell using transfection and / or transduction techniques known in the art. As used herein, the terms "transfection" and "transduction" refer to the process by which an exogenous nucleic acid sequence is introduced into a host cell. The nucleic acid may be integrated into the host cell DNA or may be maintained extrachromosomally. The nucleic acid may be maintained transiently or may be stably introduced. Transfection can be achieved by various means known in the art, including but not limited to calcium phosphate-DNA co-precipitation, DEAE-dextran mediated transfection, polybrene mediated transfection, electroporation, microinjection, liposome fusion, lipofection, protoplast fusion, retroviral infection, and gene guns. Transduction refers to the delivery of a gene(s) using a viral or retroviral vector by means of viral infection rather than by transfection.

[0103] As used herein, the terms "genetically engineered" or "genetically modified" refer to the addition of additional genetic material in the form of DNA or RNA to the total genetic material in a cell. The terms "genetically modified cell," "modified cell," and "redirected cell" are used interchangeably.

[0104] In some embodiments, the CAR of the present invention is introduced into and expressed in immune effector cells to redirect their specificity to a target antigen of interest (e.g., claudin-1).

[0105] The present invention provides a method for producing immune effector cells expressing a CAR as described herein. In some embodiments, the method includes transfecting or transducing immune effector cells isolated from a subject (e.g., a subject having a claudin-1 expressing tumor cell) so that the immune effector cells express one or more CARs as described herein. In some embodiments, the immune effector cells are isolated from an individual and genetically modified in vitro without further manipulation. Such cells can then be directly re-administered to the patient. In some embodiments, the immune effector cells are first activated and stimulated to proliferate in vitro, and then genetically modified to express a CAR. In this regard, the immune effector cells can be cultured before or after being genetically modified (i.e., transduced or transfected to express a CAR as described herein).

[0106] Prior to the in vitro manipulation or genetic modification of immune effector cells as described herein, a source of cells can be obtained from a subject. In some embodiments, the immune effector cells used with the CARs described herein include T cells. T cells can be obtained from multiple sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and tumors. In some embodiments, T cells can be obtained from a unit of blood collected from a subject using any technique known to those skilled in the art (e.g., FICOLL separation). In some embodiments, cells from an individual's circulating blood are obtained by apheresis. The apheresis product typically includes lymphocytes (including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets). In some embodiments, cells collected by apheresis can be washed to remove the plasma fraction and place the cells in an appropriate buffer or medium for further processing. In some embodiments, the cells are washed with PBS. In some embodiments, the washed solution lacks calcium and may lack magnesium or many, if not all, divalent cations. As will be appreciated by those skilled in the art, the washing step can be accomplished by methods known to those skilled in the art (e.g., by using a semi-automated flow-through centrifuge). After washing, the cells can be resuspended in a variety of biocompatible buffers or other saline solutions with or without buffers. In some embodiments, undesirable components of the apheresis sample can be removed directly in the culture medium in which the cells are resuspended.

[0107] In some embodiments, T cells are isolated from peripheral blood mononuclear cells (PBMCs) by lysing red blood cells and depleting monocytes, for example by centrifugation through a PERCOLL™ gradient. Specific T cell subpopulations (e.g., CD28+, CD4+, CD8+, CD45RA+, and CD45RO+ T cells) can be further isolated by positive or negative selection techniques. For example, enrichment of T cell populations by negative selection can be achieved by combining antibodies directed to surface markers unique to the negatively selected cells. One method used herein is cell sorting and / or selection by negative magnetic immunoadhesion or flow cytometry, which uses a cocktail of monoclonal antibodies directed to cell surface markers present on the negatively selected cells. For example, to enrich for CD4+ cells by negative selection, the monoclonal antibody cocktail typically includes antibodies against CD14, CD20, CD1b, CD16, HLA-DR, and CD8. Flow cytometry and cell sorting can also be used to isolate cell populations of interest for use in the present invention.

[0108] PBMCs can be used directly for genetic modification with CARs using the methods described herein. In some embodiments, after isolation of PBMCs, T lymphocytes are further isolated, and in some embodiments, both cytotoxic and helper T lymphocytes can be sorted into naive, memory, and effector T cell subpopulations, either before or after genetic modification and / or expansion. CD8+ cells can be obtained using standard methods. In some embodiments, CD8+ cells are further sorted into naive, central memory, and effector cells, by identifying cell surface antigens associated with each of these types of CD8+ cells. In some embodiments, memory T cells are present in both the CD62L+ and CD62L subsets of CD8+ peripheral blood lymphocytes. PBMCs are sorted into CD62L-CD8+ and CD62L+CD8+ fractions after staining with anti-CD8 and anti-CD62L antibodies. In some embodiments, expression of phenotypic markers of central memory TCMs includes CD45RO, CD62L, CCR7, CD28, CD3, and CD127, which are negative for granzyme B. In some embodiments, central memory T cells are CD45RO+, CD62L+, CD8+ T cells. In some embodiments, effector T cells are negative for CD62L, CCR7, CD28, and CD127, and positive for granzyme B and perforin. In some embodiments, naive CD8+ T lymphocytes are characterized by expression of phenotypic markers of naive T cells, including CD62L, CCR7, CD28, CD3, CD127, and CD45RA.

[0109] In some embodiments, CD4+ T cells are further sorted into subpopulations. For example, CD4+ T helper cells can be sorted into naive, central memory, and effector cells by identifying cell populations with cell surface antigens. CD4+ lymphocytes can be obtained by standard methods. In some embodiments, naive CD4+ T lymphocytes are CD45RO-, CD45RA+, CD62L+ CD4+ T cells. In some embodiments, central memory CD4+ cells are CD62L positive and CD45RO positive. In some embodiments, effector CD4+ cells are CD62L and CD45RO negative.

[0110] Immune effector cells (e.g., T cells) can be genetically modified after isolation using known methods, or immune effector cells can be activated and expanded (or differentiated in the case of precursor cells) in vitro prior to genetic modification. In some embodiments, immune effector cells (e.g., T cells) are genetically modified with a chimeric antigen receptor described herein (e.g., transduced with a viral vector containing a nucleic acid encoding a CAR) and then activated and expanded in vitro. Methods for activating and expanding T cells are known in the art and described, for example, in U.S. Pat. Nos. 6,905,874, 6,867,041, 6,797,514, and WO2012079000. Generally, such methods include contacting PBMCs or isolated T cells with stimulatory and costimulatory agents (e.g., anti-CD3 and anti-CD28 antibodies), typically attached to beads or other surfaces, in a culture medium containing appropriate cytokines (e.g., IL-2). Anti-CD3 and anti-CD28 antibodies attached to the same bead function as "surrogate" antigen-presenting cells (APCs). In some embodiments, T cells can be activated and stimulated to proliferate with feeder cells, appropriate antibodies, and cytokines using methods such as those described in U.S. Patent Nos. 6,040,177; 5,827,642; and WO2012129514.

[0111] The present invention provides a population of modified immune effector cells for the treatment of patients having malignant tumors caused by claudin-1-expressing tumors, wherein the modified immune effector cells comprise an anti-claudin-1 CAR as disclosed herein.

[0112] IV.How to use The methods of the present invention can be achieved using an anti-claudin-1 antibody or a biologically active fragment thereof, or a pharmaceutical composition comprising such an antibody or fragment (see below). In general, these methods include 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 performed using any of the administration methods known to those skilled in the art (see below).

[0113] Typically, fibrotic tumors have a dense collagen network that causes small interfibrillar separations in the interstitium, slowing the movement of particles larger than 10 nanometers (Netti PA, et al. (2000) Cancer Res 60:2497-2503; Pluen A, et al. (2001) Proc Natl AcadSci USA 98:4628-4633; Ramanujan S, et al. (2002) Biophys J 83:1650-1660; and Brown E, et al. (2003) Nat Med 9:796-800). In some embodiments, provided herein is a method of promoting T cell-mediated anti-tumor activity in a subject having a fibrotic tumor, the method comprising administering an anti-claudin-1 antibody to the subject. In some embodiments, the method of promoting T cell-mediated anti-tumor activity in a subject having a fibrotic tumor further comprises administering an immune checkpoint inhibitor.

[0114] In some aspects, provided herein is a method of treating cancer in a subject having a solid tumor, the method comprising administering to the subject a therapeutically effective amount of an anti-claudin-1 antibody and an immune checkpoint inhibitor, wherein the anti-claudin-1 antibody promotes T cell-mediated anti-tumor activity in the tumor of the subject.

[0115] In some embodiments, provided herein is a method of enhancing the therapeutic efficacy of an immune checkpoint inhibitor in a subject having a fibrotic tumor, the method comprising: a) administering to the subject an anti-claudin-1 antibody, where the anti-claudin-1 antibody promotes T cell-mediated anti-tumor activity in the fibrotic tumor; and b) administering to the subject an immune checkpoint inhibitor.

[0116] In some embodiments, the anti-claudin-1 antibody is administered prior to administering the immune checkpoint inhibitor.

[0117] In some embodiments, the anti-claudin-1 antibody and the immune checkpoint inhibitor are administered simultaneously or sequentially.

[0118] In some embodiments, the anti-claudin-1 antibody and the immune checkpoint inhibitor are administered in the same composition.

[0119] In some embodiments, the anti-claudin-1 antibody and the immune checkpoint inhibitor are administered in different compositions.

[0120] In some embodiments, the anti-claudin-1 antibody and / or immune checkpoint inhibitor is administered intratumorally, intravenously, intraperitoneally, intramuscularly, intradermally, or subcutaneously.

[0121] In some embodiments, the immune checkpoint inhibitor is an antagonist of PD-1, PD-L1, CTLA-4, LAG-3, TIM-3, TIGIT, VISTA, B7-H3, BTLA, and / or Siglec-15.

[0122] In some embodiments, the immune checkpoint inhibitor is a small molecule inhibitor.

[0123] In some embodiments, the immune checkpoint inhibitor is an antibody or an antigen-binding fragment thereof.

[0124] In some embodiments, the immune checkpoint inhibitor is a PD-1 antagonist. In some embodiments, the PD-1 antagonist is selected from the group consisting of nivolumab, pembrolizumab, cemipillimab, and dostarlimab.

[0125] In some embodiments, the immune checkpoint inhibitor is a PD-L1 antagonist. In some embodiments, the PD-L1 antagonist is selected from the group consisting of atezolizumab, durvalumab, and avelumab.

[0126] In some embodiments, the immune checkpoint inhibitor is a CTLA-4 antagonist. In some embodiments, the CTLA-4 antagonist is selected from the group consisting of ipilimumab and tremelimumab.

[0127] In some embodiments, the immune checkpoint inhibitor is a LAG-3 antagonist (e.g., BI754111).

[0128] In some embodiments, the immune checkpoint inhibitor is a TIM-3 antagonist (e.g., TSR-022 and LY3321367).

[0129] In some embodiments, the immune checkpoint inhibitor is a VISTA (V-domain immunoglobulin (Ig)-containing suppressor of T-cell activation) antagonist (e.g., CA-170 (anti-PD-L1 / L2 and anti-VISTA small molecule) and JNJ-61610588).

[0130] In some embodiments, the immune checkpoint inhibitor is a B7-H3 antagonist.

[0131] In some embodiments, the immune checkpoint inhibitor is a BTLA antagonist.

[0132] In some embodiments, the immune checkpoint inhibitor is a Siglec-15 antagonist.

[0133] In some embodiments, the immune checkpoint inhibitor is a TIGIT antagonist (e.g., BMS-986207, OMP-313M32, COM902 (CGEN-15137), and AB154). In some embodiments, the TIGIT antagonist is selected from the group consisting of tiragolumab, osipellilimab, domvanalimab, etigilimab, and vibostolimab.

[0134] In some embodiments, the cancer comprises a fibrotic tumor.

[0135] In some embodiments, the fibrotic tumor 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 a fibrotic tumor sample from the subject. In some embodiments, the methods disclosed herein further comprise comparing the expression level of claudin-1 to the expression level of claudin-1 in a reference sample, and if the expression level of claudin-1 in the fibrotic tumor sample is increased relative to the expression level of claudin-1 in the reference sample, the subject is administered an anti-claudin-1 antibody and / or an immune checkpoint inhibitor described herein.

[0136] In some embodiments, the expression level of claudin-1 in the fibrotic tumor sample and / or the reference sample is quantified by immunohistochemistry (IHC) testing. In some embodiments, the IHC testing is calculated by the H-score method (see, for example, Parris, Toshima Z et al., BMC cancer vol.14:324(2014)). In some embodiments, the H-score requires that each cell receive a score of 0 to +3 (0=negative staining, +1=weak staining or low expression of the target antigen, +2=moderate staining or medium expression of the target antigen, and +3=strong staining or high expression of the target antigen). In some embodiments, the H-score ranges from 0 to 300, and the H-score is calculated by adding i) the percentage of cells in the sample with a score of +1, ii) twice the percentage of cells in the sample with a score of +2, and iii) three times the percentage of cells in the sample with a score of +3 (i.e., H-score=(1*% of 1+ cells)+(2*% of 2+ cells)+(3*% of 3+ cells). In some embodiments, high expression of claudin-1 in grading by the H-score method is about 150 to about 300. In some embodiments, medium expression of claudin-1 in grading by the H-score method is about 50 to about 149. In some embodiments, low expression of claudin-1 in grading by the H-score method is about 1 to about 49.

[0137] In some embodiments, claudin-1 expression is considered positive when the H-score is about 1 to about 300. In some embodiments, claudin-1 expression is considered positive when the H-score is about 50 to about 300. In some embodiments, claudin-1 expression is considered positive when the H-score is about 150 to about 300.

[0138] To improve the treatment of fibrotic tumors, in some embodiments, the invention provides for identifying patients as having high expression of claudin-1 and providing immunotherapy with anti-claudin-1 antibodies and immune checkpoint inhibitors.

[0139] In another embodiment, the invention is directed to identifying a patient as having a fibrotic tumor with high expression of claudin-1 and treating the fibrotic tumor by administering an anti-claudin-1 antibody or a combination of an anti-claudin-1 antibody and an immune checkpoint inhibitor. In some embodiments, the invention includes a method of identifying a patient as having a fibrotic tumor with high expression of claudin-1 and administering an anti-claudin-1 antibody to the patient.

[0140] In some embodiments, the invention includes a method of selecting a fibrotic tumor in a human patient for immunotherapy, the method comprising: (a) quantifying the expression level of claudin-1 in a tumor sample; and (b) selecting the tumor for immunotherapy if the tumor sample has high expression of claudin-1.

[0141] In some embodiments, the invention includes a method of identifying a fibrotic tumor in a human patient as eligible for immunotherapy, the method including: (a) quantifying the expression level of claudin-1 in a tumor sample; and (b) identifying the tumor as eligible for immunotherapy if the tumor sample has high expression of claudin-1.

[0142] In some embodiments, the invention includes a method for identifying a fibrotic tumor in a human patient that is likely to respond to immunotherapy, the method comprising: (a) quantifying the expression level of claudin-1 in a tumor sample; and (b) identifying the tumor as likely to respond to the treatment if the tumor has high expression of claudin-1.

[0143] In some embodiments, the invention includes a method of classifying a fibrotic tumor in a human patient as likely to respond to immunotherapy, the method comprising: (a) quantifying the expression level of claudin-1 in a tumor sample; and (b) classifying the tumor as likely to respond to immunotherapy if the tumor has high expression of claudin-1. In some embodiments, the immunotherapy comprises contacting the tumor with a therapeutically effective amount of an anti-claudin-1 antibody and an immune checkpoint inhibitor.

[0144] In some embodiments, the invention includes a method for identifying a patient having a fibrotic tumor that is likely to respond to immunotherapy, the method comprising: (a) quantifying the expression level of claudin-1 in a tumor sample; and (b) identifying a patient that is likely to respond to the treatment if the tumor has high expression of claudin-1.

[0145] In some embodiments, the invention includes a method of selecting a patient having a fibrotic tumor for immunotherapy, the method comprising: (a) quantifying the expression level of claudin-1 in a tumor sample; and (b) selecting the patient for immunotherapy if the tumor has high expression of claudin-1. In some embodiments, the immunotherapy comprises contacting the tumor with a therapeutically effective amount of an anti-claudin-1 antibody and an immune checkpoint inhibitor.

[0146] In some embodiments, the identifying comprises determining claudin-1 expression in the fibrotic tumor.

[0147] In some embodiments, claudin-1 expression is determined by receiving results of an assay capable of determining claudin-1 expression.

[0148] To evaluate the expression of claudin-1, in some embodiments, test tissue samples are obtained from patients who need therapy. In some embodiments, the test tissue samples include, but are not limited to, any clinically relevant samples, such as tumor biopsy, core biopsy tissue samples, fine needle aspirates, or bodily fluid samples, such as blood, plasma, serum, lymph, ascites, cyst fluid, or urine. In some embodiments, the test tissue samples are from primary tumors. In some embodiments, the test tissue samples are from metastases. In some embodiments, the test tissue samples are taken from subjects at multiple time points, such as before, during, and / or after treatment. In some embodiments, the test tissue samples are taken from different locations of the subject (e.g., a sample from the primary tumor and a sample from a distant metastasis).

[0149] 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 6 In some embodiments, the sample size is from about 1 cell to about 1 x 10 5In some embodiments, the sample size is about 1 cell to about 10,000 cells. In some embodiments, the sample size is about 1 cell to about 1,000 cells. In some embodiments, the sample size is about 1 cell to about 100 cells. In some embodiments, the sample size is about 1 cell to about 10 cells. In some embodiments, the sample size is a single cell.

[0150] In another embodiment, the assessment of claudin-1 expression can be accomplished without obtaining a test tissue sample. In some embodiments, selecting a suitable patient includes (i) preparing a test tissue sample, optionally obtained from a patient having cancer of the tissue, the test tissue sample including tumor cells and / or tumor-infiltrating inflammatory cells, and (ii) assessing the percentage of cells in the test tissue sample that express claudin-1 on their cell surface based on assessing that the percentage of cells in the test tissue sample that express claudin-1 on their cell surface is greater than a predetermined threshold level.

[0151] However, it should be understood that in any method involving the measurement of claudin-1 expression in a test tissue sample, the step involving the preparation of a test tissue sample obtained from a patient is an optional step. That is, in certain embodiments, the method includes this step, and in other embodiments, this step is not included in the method. It should also be understood that in certain embodiments, the "measuring" or "assessing" step to identify or quantify 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 certain other embodiments, no modified step is involved, and claudin-1 expression is evaluated, for example, by reviewing a test result report from a laboratory. In some embodiments, claudin-1 expression is evaluated by reviewing the results of an immunohistochemistry assay from a laboratory. In certain embodiments, the method steps leading up to and including the evaluation of claudin-1 expression provide intermediate results that can be provided to a physician or other health care provider for use in selecting candidates suitable for combination therapy with a claudin-1 inhibitor and an immune checkpoint inhibitor. In certain embodiments, the steps of the method 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 immune checkpoint inhibitor therapy. In certain embodiments, the steps of providing intermediate results are performed by a healthcare professional or by a person acting under the direction of a healthcare professional. In other embodiments, these steps are performed by an independent laboratory or by an independent person (e.g., a laboratory technician).

[0152] In certain embodiments of the methods of the present invention, the percentage of cells expressing claudin-1 is assessed by performing an assay to detect the presence of claudin-1 RNA. In further 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.

[0153] In other embodiments, the percentage of cells expressing claudin-1 is assessed by performing an assay to detect the presence of claudin-1 polypeptide. In further 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, the expression of claudin-1 is assayed by IHC. In all other embodiments of these methods, the cell surface expression of claudin-1 is assayed, for example, using IHC or in vivo imaging.

[0154] 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.

[0155] 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.

[0156] In some embodiments, the tumor is selected from the group consisting of head and neck, lung, breast, melanoma, colon, pancreatic, esophageal, cholangiocarcinoma, and hepatocellular tumors.

[0157] In some embodiments, CAR-expressing immune effector cells prepared as described herein can be utilized in methods and compositions for adoptive immunotherapy according to known techniques, or variations thereof that will become apparent to those of skill in the art based on this disclosure. See, e.g., U.S. Patent Application Publication No. 2003 / 0170238 to Gruenberg et al. See also U.S. Patent No. 4,690,915 to Rosenberg.

[0158] In some embodiments, the cells are formulated by first harvesting them from their culture medium, then washing and concentrating a therapeutically effective amount of the cells in a medium and container system (a "pharmaceutical acceptable" carrier) suitable for administration. A suitable infusion medium can be any isotonic medium formulation, typically saline, Normosol R (Abbott) or Plasma-Lyte A (Baxter), although 5% dextrose in water or lactated Ringer's solution can also be utilized. The infusion medium may be supplemented with human serum albumin.

[0159] In some embodiments, the pharmaceutical composition comprises anti-claudin-1 CAR-T cells and an immune checkpoint inhibitor, in some embodiments, the immune checkpoint inhibitor is selected from the group consisting of CTLA-4, PD-1, PD-L1, PD-L2, B7-H3, B7-H4, HVEM, TIM-3, GAL-9, LAG-3, VISTA, KIR, BTLA, TIGIT, IDO, and / or Siglec-15 inhibitors as described herein.

[0160] In some embodiments, the immune checkpoint inhibitor is an inhibitor of PD-1 as described herein. In some embodiments, the immune checkpoint inhibitor is an inhibitor of PD-L1 as described herein. In some embodiments, the immune checkpoint inhibitor is an inhibitor of PD-L2 as described herein. In some embodiments, the immune checkpoint inhibitor is an inhibitor of CTLA-4 as described herein. In some embodiments, the immune checkpoint inhibitor is an inhibitor of LAG-3 as described herein. In some embodiments, the immune checkpoint inhibitor is an inhibitor of TIM-3 as described herein. In some embodiments, the immune checkpoint inhibitor is an inhibitor of TIGIT as described herein. In some embodiments, the immune checkpoint inhibitor is an inhibitor of VISTA as described herein. In some embodiments, the immune checkpoint inhibitor is an inhibitor of B7-H3 as described herein. In some embodiments, the immune checkpoint inhibitor is an inhibitor of B7-H4 as described herein. In some embodiments, the immune checkpoint inhibitor is an inhibitor of HVEM as described herein. In some embodiments, the immune checkpoint inhibitor is an inhibitor of GAL-9 as described herein. In some embodiments, the immune checkpoint inhibitor is an inhibitor of KIR as described herein. In some embodiments, the immune checkpoint inhibitor is an inhibitor of BTLA as described herein. In some embodiments, the immune checkpoint inhibitor is an inhibitor of IDO as described herein. In some embodiments, the immune checkpoint inhibitor is an inhibitor of Siglec-15 as described herein.

[0161] In some embodiments, the CAR-expressing immune effector cell population of the present invention can be administered as a pharmaceutical composition, either alone or in combination with a diluent and / or other components (e.g., immune checkpoint inhibitors as described herein). Briefly, the pharmaceutical composition of the present invention can include the CAR-expressing immune effector cell population described herein (e.g., T cells) in combination with one or more pharma- ceutical or physiologically acceptable carriers, diluents, or excipients. Such compositions can include buffers (e.g., neutral buffered saline, phosphate buffered saline, etc.), carbohydrates (e.g., glucose, mannose, sucrose, or dextran, mannitol), proteins, polypeptides, or amino acids (e.g., glycine), antioxidants, chelating agents (e.g., EDTA or glutathione), adjuvants (e.g., aluminum hydroxide), and preservatives. The compositions of the present invention are preferably formulated for intravenous administration.

[0162] In some embodiments, the anti-tumor immune response induced in a subject by administering the CAR-expressing T cells described herein using the methods described herein or other methods known in the art may include a cellular immune response mediated by cytotoxic T cells, regulatory T cells, and helper T cell responses that can kill infected cells. A humoral immune response can also be induced that is primarily mediated by helper T cells that can activate B cells to result in antibody production. Various techniques can be used to analyze the type of immune response induced by the compositions of the present invention, and these are well described in the art. See, for example, Current Protocols in Immunology, Edited by: John E. Coligan, Ada M. Kruisbeek, David H. Margulies, Ethan M. Shevach, Warren Strober (2001) John Wiley & Sons, NY, NY.

[0163] In some embodiments, the present disclosure provides a method for promoting T cell-mediated anti-tumor activity in a subject with a fibrotic tumor, comprising administering anti-claudin-1 chimeric antigen receptor (CAR) T cells to the subject. In some embodiments, the present disclosure comprises administering an immune checkpoint inhibitor.

[0164] In some embodiments, provided herein is a method of treating cancer in a subject having a solid tumor, the method comprising administering to the subject a therapeutically effective amount of anti-claudin-1 CAR T cells and an immune checkpoint inhibitor. In some embodiments, the anti-claudin-1 CAR T cells promote T cell-mediated anti-tumor activity in the tumor of the subject.

[0165] In some embodiments, provided herein is a method of enhancing the therapeutic efficacy of an immune checkpoint inhibitor in a subject having a fibrotic tumor, the method comprising administering anti-claudin-1 CAR T cells to the subject and administering an immune checkpoint inhibitor to the subject. In some embodiments, the anti-claudin-1 CAR T cells promote T cell-mediated anti-tumor activity in the fibrotic tumor.

[0166] V. Immune Checkpoint Inhibitors Immune checkpoint proteins interact with specific ligands that send signals to T cells that inhibit their function, and cancer cells exploit this by driving high expression of checkpoint proteins on their surface to suppress anti-cancer immune responses.

[0167] Immune checkpoint inhibitors include any compound capable of inhibiting the function of an immune checkpoint protein. Inhibition includes reduction and complete blocking of function. In some embodiments, the immune checkpoint protein is a human checkpoint protein. Thus, in some embodiments, the immune checkpoint inhibitor is preferably an inhibitor of a human immune checkpoint.

[0168] In some embodiments, checkpoint proteins include, but are not limited to, CTLA-4, PD-1 (and its ligands PD-L1 and PD-L2), B7-H3, B7-H4, HVEM, TIM-3, GAL-9, LAG-3, VISTA, KIR, BTLA, TIGIT, IDO, and / or Siglec-15. Pathways involving LAG-3, BTLA, B7-H3, B7-H4, TIM-3, and KIR constitute immune checkpoint pathways similar to those dependent on CTLA-4 and PD-1 (see, e.g., Pardoll, 2012, Nature Rev Cancer 12:252-264; Mellman et al, 2011, Nature 480:480-489). In some embodiments, the immune checkpoint inhibitor is an inhibitor of CTLA-4, PD-1, PD-L1, PD-L2, B7-H3, B7-H4, HVEM, TIM-3, GAL-9, LAG-3, VISTA, KIR, BTLA, TIGIT, IDO, and / or Siglec-15. In some embodiments, the immune checkpoint inhibitor is an inhibitor of PD-1, PD-L1, CTLA-4, LAG-3, TIM-3, TIGIT, VISTA, B7-H3, BTLA, and / or Siglec-15.

[0169] In some embodiments, the immune checkpoint inhibitor is an inhibitor of PD-1. In some embodiments, the immune checkpoint inhibitor is an inhibitor of PD-L1. In some embodiments, the immune checkpoint inhibitor is an inhibitor of CTLA-4. In some embodiments, the immune checkpoint inhibitor is an inhibitor of LAG-3. In some embodiments, the immune checkpoint inhibitor is an inhibitor of TIM-3. In some embodiments, the immune checkpoint inhibitor is an inhibitor of TIGIT. In some embodiments, the immune checkpoint inhibitor is an inhibitor of VISTA. In some embodiments, the immune checkpoint inhibitor is an inhibitor of B7-H3. In some embodiments, the immune checkpoint inhibitor is an inhibitor of BTLA. In some embodiments, the immune checkpoint inhibitor is an inhibitor of Siglec-15.

[0170] In some embodiments, the immune checkpoint inhibitor is an antibody.

[0171] In some embodiments, the immune checkpoint inhibitor comprises an antibody or fragment thereof that specifically binds to an immune checkpoint protein selected from the group consisting of CTLA-4, PD-1, PD-L1, PD-L2, B7-H3, B7-H4, HVEM, TIM-3, GAL-9, LAG-3, VISTA, KIR, BTLA, TIGIT, IDO, and Siglec-15. In some embodiments, the immune checkpoint inhibitor is a monoclonal antibody, fully human antibody, chimeric antibody, humanized antibody, or fragment thereof capable of at least partially antagonizing CTLA-4, PD-1, PD-L1, PD-L2, B7-H3, B7-H4, HVEM, TIM-3, GAL-9, LAG-3, VISTA, KIR, BTLA, TIGIT, IDO, and / or Siglec-15.

[0172] In some embodiments, the immune checkpoint inhibitor is an antibody or fragment thereof that specifically binds to PD-1. In some embodiments, the immune checkpoint inhibitor is an antibody or fragment thereof that specifically binds to PD-L1. In some embodiments, the immune checkpoint inhibitor is an antibody or fragment thereof that specifically binds to CTLA-4. In some embodiments, the immune checkpoint inhibitor is an antibody or fragment thereof that specifically binds to LAG-3. In some embodiments, the immune checkpoint inhibitor is an antibody or fragment thereof that specifically binds to TIM-3. In some embodiments, the immune checkpoint inhibitor is an antibody or fragment thereof that specifically binds to TIGIT. In some embodiments, the immune checkpoint inhibitor is an antibody or fragment thereof that specifically binds to VISTA. In some embodiments, the immune checkpoint inhibitor is an antibody or fragment thereof that specifically binds to B7-H3. In some embodiments, the immune checkpoint inhibitor is an antibody or fragment thereof that specifically binds to BTLA. In some embodiments, the immune checkpoint inhibitor is an antibody or fragment thereof that specifically binds to Siglec-15.

[0173] In some embodiments, the pharmaceutical composition comprises an anti-claudin-1 antibody or biologically active fragment thereof and a CTLA-4 inhibitor, preferably a monoclonal antibody that specifically binds (inhibits) CTLA-4. The complete human CTLA-4 nucleic acid sequence can be found in GenBank Accession No. NG_011502.1. Monoclonal antibodies that specifically bind to CTLA-4 include, but are not limited to, ipilimumab (Yervoy®; BMS) and tremelimumab (AstraZeneca / MedImmune), as well as antibodies disclosed in U.S. Patent Application Publication Nos. 2005 / 0201994, 2002 / 0039581, and 2002 / 0086014, the contents of each of which are incorporated herein by reference. and antibodies disclosed in U.S. Patent Nos. 5,811,097, 5,855,887, 6,051,227, 6,984,720, 6,682,736, 6,207,156, 5,977,318, 6,682,736, 7,109,003, 7,132,281, and 8,491,895, or antibodies comprising the heavy and light chain variable regions of any of these antibodies. A human monoclonal antibody that specifically binds to CTLA-4 with high affinity is disclosed in U.S. Patent No. 6,984,720. Other anti-CTLA-4 monoclonal antibodies are described, for example, in U.S. Patent No. 7,034,121, International Publication Nos. WO2012 / 122444, WO2007 / 113648, WO2016 / 196237, and WO2000 / 037504. In some embodiments, the immune checkpoint inhibitor is a CTLA-4 antagonist. In some embodiments, the CTLA-4 antagonist is selected from the group consisting of ipilimumab and tremelimumab.

[0174] In some embodiments, the pharmaceutical composition comprises an anti-claudin-1 antibody or a biologically active fragment thereof and a PD-1 inhibitor, preferably a monoclonal antibody that specifically binds to (inhibits) PD-1. The complete nucleotide and amino acid sequences of human PD-1 can be found in GenBank Accession Nos. 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 5C4, BMS-936558, MDX-1106, or ONO-4538) is a fully human IgG4 (S228P) PD-1 immune checkpoint inhibitor antibody that selectively blocks interaction with PD-1 ligands (PD-L1 and PD-L2) to inhibit downregulation of anti-tumor T cell function (U.S. Pat. No. 8,008,449; Wang et al., 2014 Cancer Immunol Res. 2(9):846-56). In another embodiment, the anti-PD-1 antibody or fragment thereof cross-competes with nivolumab. In other 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.

[0175] In another embodiment, 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.

[0176] Anti-human PD-1 antibodies (or VH and / or VL domains derived therefrom) suitable for use in the present invention can be generated using methods well known in the art. Alternatively, art-recognized anti-PD-1 antibodies can be used. For example, monoclonal antibodies 5C4 (herein referred to as nivolumab or BMS-936558), 17D8, 2D3, 4H1, 4A11, 7D3, and 5F4, described in WO2006 / 121168 (the teachings of which are incorporated herein by reference), can be used. Other known PD-1 antibodies include lambrolizumab (MK-3475), described in WO2008 / 156712, and AMP-514, described in WO2012 / 145493 (the teachings of which are incorporated herein by reference). Additional known anti-PD-1 antibodies and other PD-1 inhibitors include those described in WO2009 / 014708, WO 03 / 099196, WO2009 / 114335, and WO2011 / 161699, the teachings of which are incorporated herein by reference. In some embodiments, the anti-PD-1 antibody is REGN2810. In some embodiments, the anti-PD-1 antibody is PDR001. Another known anti-PD-1 antibody is pidilizumab (CT-011). Antibodies or antigen-binding fragments thereof that compete with any of these antibodies or inhibitors for binding to PD-1 can also be used.

[0177] For other anti-PD-1 monoclonal antibodies, see, e.g., U.S. Pat. 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 / 1 12900, WO2012 / 145493, WO2015 / 112800, WO2014 / 206107, WO2015 / 35606, W O2015 / 085847, WO2014 / 179664, WO2017 / 020291, WO2017 / 020858, WO2016 / 197 367, WO2017 / 024515, WO2017 / 025051, WO2017 / 123557, WO2016 / 106159, WO 2014 / 194302, WO2017 / 040790, WO2017 / 133540, WO2017 / 132827, WO2017 / 0244 65, WO2017 / 025016, WO2017 / 106061, WO2017 / 19846, WO2017 / 024465, WO2017 / 025016, WO2017 / 132825, and WO2017 / 133540, each of which is incorporated herein by reference.

[0178] 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 WO 2008 / 156712), PDR001 (Novartis; see WO 2015 / 112900), MEDI-0680 (AstraZeneca; also known as AMP-514; see WO 2012 / 145493), semipilimab (Regeneron; also known as REGN-2810; see WO 2015 / 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) (these references are incorporated herein by reference).

[0179] In another embodiment, 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 certain embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof has the same CDRs as pembrolizumab. In another embodiment, 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.

[0180] In some embodiments, the PD-1 antagonist is selected from the group consisting of nivolumab, pembrolizumab, semipilimab, and dostarlimab.

[0181] In some embodiments, the pharmaceutical composition comprises an anti-claudin-1 antibody or a biologically active fragment thereof and a PD-L1 inhibitor, preferably a monoclonal antibody that specifically binds to (inhibits) PD-L1. Any recognized anti-PD-L1 antibody can be used. For example, the human anti-PD-L1 antibody disclosed in U.S. Patent No. 7,943,743 (the contents of which are incorporated herein by reference) can be used. Such anti-PD-L1 antibodies include 3G10, 12A4 (also known as BMS-936559), 10A5, 5F8, 10H10, 1B12, 7H1, 11E6, 12B7, and 13G4. Other art-recognized anti-PD-L1 antibodies that can be used include, for example, those described in U.S. Patent Nos. 7,635,757 and 8,217,149, and PCT Publication Nos. WO2011 / 066389 and WO2012 / 145493, the teachings of which are also incorporated herein by reference. Other examples of anti-PD-L1 antibodies include atezolizumab (TECENTRIQ; RG7446), or durvalumab (IMFINZI; MEDI4736), or avelumab (Bavencio). Antibodies or antigen-binding fragments thereof that compete for binding to PD-L1 with any of these art-recognized antibodies or inhibitors can also be used.

[0182] In certain embodiments, the anti-PD-L1 antibody is BMS-936559 (formerly known as 12A4 or MDX-1105) (see, e.g., U.S. Patent No. 7,943,743; WO2013 / 173223). In other embodiments, the anti-PD-L1 antibody is MPDL3280A (also known as RG7446 and atezolizumab) (see, e.g., Herbst et al. 2013 J Clin Oncol 31(suppl):3000; U.S. Patent No. 8,217,149), MEDI4736 (Khleif, 2013, In: Proceedings from the European Cancer Congress 2013; September 27-October 1, 2013; Amsterdam, The Netherlands. Abstract 802), or MSB0010718C (also known as avelumab; see US 2014 / 0341917). In certain embodiments, the antibody that cross-competes for binding to human PD-L1 or binds to the same epitope region of human PD-L1 as the PD-L1 antibodies of the above references is a mAb. For administration to humans, these cross-competing antibodies may be chimeric, humanized or human antibodies. Such chimeric, humanized or human mAbs can be prepared and isolated by methods well known in the art.In certain embodiments, the anti-PD-L1 antibody is selected from the group consisting of 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 US Pat. No. 8,217,149; see also Herbst et al. (2013) J Clin Oncol see also 31(suppl):3000), durvalumab (AstraZeneca; also known as IMFINZI™, MEDI-4736; see WO 2011 / 066389), avelumab (Pfizer; also known as BAVENCIO®, MSB-0010718C; see WO 2013 / 079174), STI-1014 (Sorrento; see WO 2013 / 181634), CX-072 (Cytomx; see WO 2016 / 149201), KN035 (3D Med / Alphamab; Zhang et al., Cell Discov. 7:3 (March 2017), LY3300054 (Eli Lilly Co.; see, e.g., WO 2017 / 034916), and CK-301 (Checkpoint Therapeutics; Gorelik et al., AACR: Abstract 4606 (April 2016).

[0183] In some embodiments, the pharmaceutical composition comprises an anti-claudin-1 antibody, or a biologically active fragment thereof, and a PD-L2 inhibitor, e.g., MIH18 (described in Pfistershammer et al., Eur J Immunol. 36:1104-1113 (2006)).

[0184] In some embodiments, the pharmaceutical composition comprises an anti-claudin-1 antibody or a biologically active fragment thereof and a LAG-3 inhibitor. In some embodiments, the LAG-3 inhibitor is an anti-LAG-3 antibody.

[0185] Anti-human LAG-3 antibodies (or VH / VL domains derived therefrom) suitable for use in the present invention can be generated using methods well known in the art. Alternatively, art-recognized anti-LAG-3 antibodies can be used. For example, the anti-human LAG-3 antibody described in US2011 / 0150892A1 (the teachings of which are incorporated herein by reference) and designated monoclonal antibody 25F7 (also known as "25F7" and "LAG3.1") can be used. Other art-recognized anti-LAG-3 antibodies that may be used include IMP731 (H5L7BW) described in US2011 / 007023, MK-4280 (28G-10) described in WO2016028672, REGN3767 described in Journal for ImmunoTherapy of Cancer, (2016) Vol. 4, Supplement 1 Abstract Number: P195, BAP050 described in WO2017 / 019894, IMP-701 (LAG-525), IMP321 (eftiragimodo alfa), Sym022, TSR-033, MGD013, BI754111, FS118, AVA-017, and GSK2831781. These and other anti-LAG-3 antibodies useful in the claimed invention are described in, e.g., WO2016 / 028672, WO2017 / 106129, WO2017 / 062888, WO2009 / 044273, WO2018 / 069500, WO2016 / 126858, WO2014 / 179664, WO2016 / 200782, WO2015 / 200119, WO2017 / 019846, WO2017 / 198741, WO2017 / 220555, WO2017 / 220562, WO2017 / 220571, WO2017 / 220572, WO2017 / 220573, WO2017 / 220574, WO2017 / 220575, WO2017 / 220576, WO2017 / 220577, WO2017 / 220578, WO2017 / 220579 ... US2017 / 0260271, WO2017 / 086367, WO2017 / 086419, WO2018 / 034227, and WO2014 / 140180, the contents of each of which are incorporated herein by reference.

[0186] In some embodiments, the pharmaceutical composition comprises an anti-claudin-1 antibody or a biologically active fragment thereof and a BLTA inhibitor (e.g., antibody 4C7 disclosed in U.S. Pat. No. 8,563,694, incorporated herein by reference).

[0187] In some embodiments, the pharmaceutical composition comprises an anti-claudin-1 antibody or biologically active fragment thereof and a B7-H4 checkpoint inhibitor (e.g., an antibody disclosed in U.S. Patent Application Publication No. 2014 / 0294861, which is incorporated by reference herein, or a soluble recombinant form of B7-H4 disclosed in U.S. Patent Application Publication No. 2012 / 0177645, which is incorporated by reference herein).

[0188] In some embodiments, the pharmaceutical composition comprises an anti-claudin-1 antibody or biologically active fragment thereof and a B7-H3 checkpoint inhibitor (e.g., the antibody MGA271, disclosed as BRCA84D, or a derivative disclosed in U.S. Patent Application Publication No. 2012 / 0294796, which is incorporated herein by reference).

[0189] In some embodiments, the pharmaceutical composition comprises an anti-claudin-1 antibody or biologically active fragment thereof and a TIM-3 checkpoint inhibitor (e.g., an antibody disclosed in U.S. Pat. No. 8,841,418, incorporated herein by reference, or the anti-human TIM-3 blocking antibody F38-2E2 (disclosed by Jones et al., J. Exp. Med., 205(12):2763-79 (2008)).

[0190] In some embodiments, the pharmaceutical composition comprises an anti-claudin-1 antibody or biologically active fragment thereof and a KIR checkpoint inhibitor (e.g., the antibody lirilumab (described in Romagne et al., Blood, 114(13):2667-2677 (2009))).

[0191] In some embodiments, the pharmaceutical composition comprises an anti-claudin-1 antibody or its biologically active fragment and a TIGIT inhibitor.The TIGIT checkpoint inhibitor preferably inhibits the interaction between TIGIT and poliovirus receptor (CD155), and includes, but is not limited to, antibodies targeting human TIGIT (e.g., those disclosed in U.S. Patent No. 9,499,596 (incorporated herein by reference) and U.S. Patent Application Publication No. 2016 / 0355589, U.S. Patent Application Publication No. 2016 / 0176963 (incorporated herein by reference)), and poliovirus receptor variants (e.g., those disclosed in U.S. Patent No. 9,327,014 (incorporated herein by reference)).In some embodiments, the immune checkpoint inhibitor is a TIGIT antagonist. In some embodiments, the TIGIT antagonist is selected from the group consisting of tiragolumab, osipellimab, domvanalimab, etigilimab, and vibostolimab.

[0192] In some embodiments, the pharmaceutical composition comprises anti-claudin-1 antibody or its biologically active fragment and IDO inhibitor (indoleamine-pyrrole 2,3-dioxygenase).IDO is recognized as an immune checkpoint protein, and its expression in tumor cells contributes to immune tolerance by shutting down effector T cells.IDO is believed to contribute to the resistance of anti-CTLA-4 therapy. In some embodiments, IDO inhibitors for use in accordance with the methods described herein include, but are not limited to, tryptophan mimetics, such as D-1MT (the D isoform of 1-methyl-DL-tryptophan (MT)), L-1MT (the L isoform of MT), MTH-Trp (methylthiohydantoin-dl-tryptophan; a transcriptional repressor of IDO), and β-carbolines, indol mimetics, such as naphthoquinones, S-allyl-brassinin, S-benzyl-brassinin, 5-bromo-brassinin, and phenylimidazoles, 4-phenylimidazole, exiguamine A, epacadostat, rosmarinic acid, norharman, and NSC401366. In some embodiments, IDO inhibitors include INCB 024360 (epacadostat; N'-(3-bromo-4-fluorophenyl)-N-hydroxy-4-[2-(sulfamoylamino)ethylamino]-1,2,5-oxadiazole-3-carboximidamide), indoximod ((2R)-2-amino-3-(1-methylindol-3-yl)propanoic acid), IDO peptide vaccine (Copenhagen University), and NLG919 (NewLink Genetics; 1-cyclohexyl-2-(5H-imidazo[5,1-a]isoindol-5-yl)ethanol). In some embodiments, the IDO inhibitor preferably inhibits a metabolic pathway, including but not limited to norharman (Chiarugi A, et al., “Combined inhibition of indoleamine 2,3-dioxygenase and nitric oxide synthase modulates neurotoxin release by interferon-gamma-activated macrophages”, Journal of Leukocyte Biology.68(2):260-6.(2000)), rosmarinic acid (Lee HJ,et al., “Rosmarinic acid inhibits indoleamine 2,3-dioxygenase expression in murine dendritic cells”, Biochemical Pharmacology.73(9):1412-21(2007)), COX-2 inhibitors (Cesario A,et al., “The interplay between indoleamine 2,3-dioxygenase 1(IDO1)and cyclooxygenase(COX)-2 in chronic inflammation and cancer”, Current Medicinal Chemistry.18(15):2263-71(2011)), 1-methyltryptophan (Hou DY,et al.,“Inhibition of indoleamine 2,3-dioxygenase in dendritic cells by stereoisomers of 1-methyl-tryptophan correlates with antitumor responses” Cancer Research.67(2):792-801(2007) and Chauhan N,et al.,(April 2009),“Reassessment of the reaction mechanism in the heme dioxygenases”.Journal of the American Chemical Society.131(12):4186-7(2009)) (including, for example, certain racemers 1-methyl-D-tryptophan (aka indoximod), Epacadostat (INCB24360), navoximod (GDC-0919) (see Jochems C, et al., “The IDO1 selective inhibitor epacadostat enhances dendritic cell immunogenicity and lytic ability of tumor antigen-specific T cells”, Oncotarget.7(25):37762-37772.(2016))), and / or BMS-986205. In some embodiments, the IDO inhibitor is selected from the group consisting of norharman, rosmarinic acid, COX-2 inhibitors, 1-methyltryptophan, indoximod, epacadostat (INCB24360), navoximod (GDC-0919), and / or BMS-986205.

[0193] In some embodiments, the pharmaceutical composition comprises an anti-claudin-1 antibody or its biologically active fragment and a TIGIT inhibitor. The TIGIT checkpoint inhibitor preferably inhibits the interaction between TIGIT and poliovirus receptor (CD155), and includes, but is not limited to, antibodies targeting human TIGIT (e.g., those disclosed in U.S. Patent No. 9,499,596 (incorporated herein by reference) and U.S. Patent Application Publication No. 2016 / 0355589, U.S. Patent Application Publication No. 2016 / 0176963 (incorporated herein by reference)), and poliovirus receptor variants (e.g., those disclosed in U.S. Patent No. 9,327,014 (incorporated herein by reference)).

[0194] In some embodiments, the immune checkpoint inhibitor is an antagonist of: IDO1 (indoleamine-2,3-dioxygenase 1) (e.g., indoximod (NLG8189, 1-methyl-D-TRP), epacadostat (INCB-024360), KHK2455, PF-06840003 (PCT Publication No. WO2016 / 181348A1), pyrrolizidine-2,5-dione derivatives (PCT Publication No. WO2015 / 1 No. 73764A1), naboximod (RG6078, GDC-0919, NLG919, and BMS-986205 (F001287)); KIR (killer cell immunoglobulin-like receptors) (e.g., lirilumab (I-7F9, BMS-980615, or IPH2101) and IPH4102 (anti-KIR3DL2 monoclonal antibody); TDO (tryptophan 2,3-dioxygenase) (e.g., 4-(indol-3-yl)-pyrazole derivatives (U.S. Patent No. 9,126,984B2 and U.S. Publication No. 2016 / 0263087A1); 3-indole substituted derivatives (PCT Publication Nos. WO2015140717A1, WO2017025868A1, WO2016147144A1), 3-(indol-3-yl)-pyridine derivatives (U.S. Publication No. 20150225367A1 and PCT Publication No. WO2015121812A1); dual IDO / TDO (e.g., small Dual IDO / TDO inhibitors (PCT Publication Nos. WO2015150097A1, WO2015082499A2, WO2016026772A1, WO2016071283A1, WO2016071293A2, and WO2017007700A1); CD40 (e.g., series BMS3h-56 (U.S. Patent No. 9,475,879), lucatumumab (HCD122 and CHIR-12.12), CHIR-5.9, and dacetuzumab (huS2C6, PRO 64553, RG 3636, SGN 14, SGN-40); adenosine A2a receptor (A2aR) (e.g., CPI-444, PBF-509, istradefylline (KW-6002), preladenant (SCH420814), tozadenant (SYN115), bipadenant (BIIB014), HTL-1071, ST1535, SCH412348, SCH442416, SCH58261, ZM241385, and AZD4635 (small molecule A2aR inhibitors); CEACAM1 (CD66a) (e.g., CM-24 (MK-6018)); CEA (carcinoembryonic antigen) (e.g., sergituzumab amnaleukin (RG7813, RO-6895882), RG7802 (RO6958688)); CD47 (e.g., HuF9-G4, CC-90002, TTI-621, ALX148, NI-1701, NI-1801, SRF231, and Effi-DEM); PVRIG (poliovirus receptor-related immunoglobulin domain containing, CD122R) (e.g., COM701); GARP (glycoprotein A repeat dominant) (e.g., ARGX-115); CD80 (e.g., galiximab (IDEC-114) and AV 1142742 (RhuDex);CD86;and CD96.

[0195] In some embodiments, the immune checkpoint inhibitor is an agonist of STING (stimulator of IFN genes) (e.g., 2' or 3'-monofluoro substituted, or 2'3'-difluoro substituted mixed linkage 2',5'-3',5' cyclic-dinucleotides (PCT Publication No. WO2017 / 075477A1); 2'-fluoro substituted, bis-3',5' cyclic-dinucleotides and 2',2"-diF-Rp,Rp, bis-3',5' cyclic-dinucleotides (PCT Publication No. WO2016 / 145102A1); and fluorinated cyclic-dinucleotides (PCT Publication No. WO2016 / 096174A1); or CD20 (e.g., RITUXAN® and ABP 798).

[0196] As one of skill in the art will recognize, alternative and / or equivalent names may be used for certain antibodies referred to above, and such alternative and / or equivalent names are interchangeable in the context of the present invention.

[0197] VI. Administration The anti-claudin-1 antibody, its biologically active fragment, or anti-claudin-1 CAR T cells (optionally after formulation with one or more suitable pharma- ceutically acceptable carriers or excipients) can be administered to a subject in need thereof by any suitable route at a desired dosage. In some embodiments, the anti-claudin-1 antibody, its biologically active fragment, or anti-claudin-1 CAR T cells are delivered in combination with an immune checkpoint inhibitor as described above. Various delivery systems are known and can be used to administer the antibody, including tablets, capsules, injection solutions, encapsulation in liposomes, microparticles, microcapsules, etc. Methods of administration include, but are not limited to, transdermal, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, pulmonary, epidural, and oral routes. The anti-claudin-1 antibody, biologically active fragment thereof, anti-claudin-1 CAR T cell, or pharmaceutical composition thereof, and immune checkpoint inhibitor can be administered by any convenient or other suitable route, for example, by infusion or bolus injection, by absorption through epithelial or mucosal linings (e.g., oral mucosa, bronchial mucosa, rectal and intestinal mucosa, etc.). Administration can be systemic or local. As will be appreciated by those skilled in the art, in embodiments in which the antibody is administered in combination with an additional therapeutic agent (e.g., an immune checkpoint inhibitor), the antibody and the therapeutic agent can be administered by the same route (e.g., intravenously) or by different routes (e.g., intravenously, orally, or subcutaneously).

[0198] In some embodiments, the anti-claudin-1 antibody or anti-claudin-1 T cells and the immune checkpoint inhibitor are administered intratumorally, intravenously, intraperitoneally, intramuscularly, intradermally, or subcutaneously.

[0199] In some embodiments, the anti-claudin-1 antibody or anti-claudin-1 CAR T cells are administered prior to administering the immune checkpoint inhibitor.

[0200] In some embodiments, the anti-claudin-1 antibody, or anti-claudin-1 CAR T cells, and the immune checkpoint inhibitor are administered simultaneously or sequentially.

[0201] In some embodiments, the anti-claudin-1 antibody or anti-claudin-1 CAR T cells and the immune checkpoint inhibitor are administered in the same composition.

[0202] In some embodiments, the anti-claudin-1 antibody or anti-claudin-1 CAR T cells and the immune checkpoint inhibitor are administered in different compositions.

[0203] The anti-claudin-1 antibody or biologically active fragment thereof (optionally after formulation with one or more suitable pharma- ceutically acceptable carriers or excipients), or anti-claudin-1 CAR T cells, is administered in a dosage such that the amount delivered is effective for the intended purpose. The route of administration, formulation, and dosage administered depend on the desired therapeutic effect, the severity of the condition to be treated (if already present), the presence or absence of any infection, the age, sex, weight, and general health of the patient, as well as the potency, bioavailability, and in vivo half-life of the antibody or composition used, the use (or not) of concomitant therapy, and other clinical factors. These factors can be readily determined by the attending physician during the course of treatment. Alternatively or additionally, the dosage to be administered can be determined from tests using animal models (e.g., non-human primates or rodents). Adjusting the dose to achieve maximum efficacy based on these or other methods is well known in the art and within the capabilities of a trained physician. As trials are conducted with anti-claudin-1 antibodies or anti-claudin-1 CAR T cells, more information will become available about appropriate dosage levels and duration of treatment.

[0204] Treatment according to the present invention may consist of a single dose or multiple doses. Thus, administration of anti-claudin-1 antibody or its biologically active fragment, or anti-claudin-1 CAR T cell (or pharmaceutical composition thereof) may be constant for a certain period of time, or may be regular and at a certain interval (e.g., hourly, daily, weekly (or at some other multiple day interval), monthly, yearly (e.g., in time-release form)). Alternatively, delivery may be multiple times during a given period of time (e.g., more than once a week, more than once a month, etc.). Delivery may be continuous delivery (e.g., intravenous administration) for a certain period of time.

[0205] Generally, the amount of anti-claudin-1 antibody or biologically active fragment thereof, or anti-claudin-1 CAR T cell, (or a pharmaceutical composition thereof) administered will preferably be in the range of about 1 ng / kg to about 100 mg / kg of the subject's body weight, e.g., about 100 ng / kg to about 50 mg / kg of the subject's body weight, or about 1 μg / kg to about 10 mg / kg of the subject's body weight, or about 100 μg / kg to about 1 mg / kg of the subject's body weight.

[0206] VII. Pharmaceutical Compositions As mentioned above, anti-claudin-1 antibodies (and related molecules) or anti-claudin-1 CAR T cells can be administered per se or as a pharmaceutical composition. Thus, the present invention provides pharmaceutical compositions comprising an effective amount of an anti-claudin-1 antibody or biologically active fragment thereof described herein and at least one pharma- ceutical acceptable carrier or excipient. In some embodiments, the composition further comprises one or more additional biologically active agents. In some embodiments, the one or more additional biologically active agents are immune checkpoint inhibitors.

[0207] In some embodiments, the pharmaceutical composition is used in a method for promoting T cell-mediated anti-tumor activity in a subject having a fibrotic tumor.

[0208] In some embodiments, the pharmaceutical composition is used in a method of treating cancer in a subject having a solid tumor.

[0209] In some embodiments, the pharmaceutical composition is used in a method for enhancing the therapeutic efficacy of an immune checkpoint inhibitor in a subject having a fibrotic tumor.

[0210] The pharmaceutical composition can be administered in any amount and by any route of administration effective for achieving the desired prophylactic and / or therapeutic effect. The optimal pharmaceutical formulation can vary depending on the route of administration and the desired dosage. Such formulations can affect the physical state, stability, in vivo release rate, and in vivo clearance rate of the administered active ingredient.

[0211] The pharmaceutical composition of the present invention can be formulated in a dosage unit form for ease of administration and uniformity of dosage.In some embodiments, the anti-claudin-1 antibody and the immune checkpoint inhibitor are formulated together.In some embodiments, the anti-claudin-1 antibody and the immune checkpoint inhibitor are formulated separately.However, it should be understood that the total daily dosage of the composition is determined by the attending physician within the scope of sound medical judgment.

[0212] VIII. 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 of the components of the inventive pharmaceutical composition to allow for administration of an anti-claudin-1 antibody or a biologically active fragment thereof, or an anti-claudin-1 CAR T cell.

[0213] The different components of the pharmaceutical pack or kit can be supplied in solid (e.g. lyophilized) or liquid form. Each component is generally suitably provided in its respective container in aliquots or in concentrated form. The pharmaceutical pack or kit may also include media for reconstituting the lyophilized components. The individual containers of the kit are preferably kept in close confinement for commercial sale.

[0214] 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 a format 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 administration to humans. The package insert notice may include instructions for using the pharmaceutical composition according to the treatment method disclosed herein.

[0215] An identifier (e.g., a bar code, radio frequency, ID tag, etc.) may be present in or on the kit. The identifier can be used to uniquely identify the kit, for example, for purposes of quality control, inventory control, tracking movement between workstations, etc. EXAMPLES

[0216] The following examples are illustrative, and are not intended to limit the scope of the claimed embodiments.

[0217] Example 1. Claudin-1 expression in the tumor microenvironment High expression of CLDN1 is associated with immune-compromised or inactive tumor microenvironment. Changes in tumor cell phenotype by mAb treatment lead to activation of T cells in the tumor microenvironment, possibly through alterations in tumor cell secretome and / or metalloproteases (Figure 8A). Figure 8B shows a mechanism by which administration of anti-CLDN1 antibodies can disrupt the tumor barrier and help transition the tumor state from T cell elimination to T cell infiltration permissive.

[0218] Example 2. Claudin-1 expression in fibrotic tumor types Over 1200 paraffin-embedded tumor biopsies from 12 different indications were stained by immunohistochemistry to analyze the expression of Claudin-1 (CLDN1), T-cell marker (C3), and fibrosis (Sirius Red staining) in head and neck squamous cell carcinoma (HNSCC), colorectal cancer (CRC), esophageal cancer, squamous non-small cell lung cancer (squamous NSCLC), intrahepatic cholangiocarcinoma (iCCA), hepatocellular carcinoma (HCC), and urothelial carcinoma (see FIG. 9). Samples were scored using the semiquantitative H-score method, which calculates the percentage and sum of the intensity of positively stained tumor cells within the infiltrating tissue component (negative staining=0; weak staining=1+; moderate staining=2+; strong staining=3+). A score of 0 corresponds to negative staining, a score of 1 to 49 corresponds to low expression, a score of 50 to 149 corresponds to moderate expression, and a score of 150 to 300 corresponds to high expression (see, for example, Parris, Toshima Z et al., BMC cancer vol. 14: 324 (2014)).

[0219] Slides were stained with a Roche Discovery Ultra autostainer. Slides were baked at 60°C for 1 h and then deparaffinized using standard autostaining protocols. Heat-induced epitope retrieval (HIER) was performed with Roche CC1 (high pH) for 48 min at 95°C. Peroxidase inhibitors were applied. Primary antibody (anti-CLDN1, Sigma-Aldrich HPA048319, 1:50) was incubated for 24 min at 37°C. Secondary antibody Roche anti-rabbit HQ was incubated for 8 min at 37°C, followed by Roche anti-HQ HRP for 8 min at 37°C. DAB was applied using the Roche ChromoMap DAB kit, followed by staining with Roche Hematoxylin II.

[0220] Sixty head and neck tumor samples were analyzed, and 90% were determined to be CLDN1 positive (Figure 1A). Forty esophageal cancer samples were analyzed, and 78% were determined to be CLDN1 positive (Figure 1B). Finally, CLDN1 was determined to be overexpressed in various fibrotic tumors other than liver cancer.

[0221] Furthermore, data indicate that nonfunctional CLDN1 (NJ-CLDN1) is frequently overexpressed in solid tumors. Notably, CLDN1 expression in tumor cells positively correlates with T cell localization within the fibrotic tissue environment. T cell elimination is one of the mechanisms described to interfere with the efficacy of checkpoint inhibitors (CPIs).

[0222] Example 3. Claudin-1 expression and T cell exclusion in head and neck cancer Tumor tissue samples were obtained from patients with head and neck cancer. Immunohistochemistry was used to measure the expression of CLDN1 and CD3 (representing fibrosis and "fibrotic traps") (Figure 2A). 90% of the tumor samples analyzed were CLDN1 positive (Figure 2B). Figure 2C shows the immunophenotypic breakdown of tumors with various CLDN1 expression levels. The immunophenotypes are hot (immune cells in the stroma and between cancer cells), exclusion (immune cells in the tumor but only in the stroma), and cold (few immune cells visible). The data indicate that T-cell exclusion is the predominant immunophenotype in head and neck cancer, with 30-80% of CLDN1-positive tumors having a T-cell exclusion phenotype.

[0223] Example 4. Claudin-1 expression and T cell exclusion in esophageal cancer Tumor tissue samples were obtained from patients with esophageal cancer. Figure 3A shows the immunophenotypes in tumors with various CLDN1 expression levels. The immunophenotypes are hot (immune cells in the stroma and between cancer cells), excluded (immune cells in the tumor but only in the stroma), and cold (few immune cells visible). Immunohistochemistry was used to measure CLDN1 expression (Figure 3B), the presence of T cells (Figure 3C), and fibrotic tissue (Figure 3D). Taken together, the data indicate that CLDN1 expression correlates with T cell exclusion in cancer. Example 5. Overexpression of mouse claudin-1 with human extracellular loops drives immune escape and T cell elimination in liver mouse tumor cells Hepa1-6 in vivo.

[0224] The data shown in Figures 4A, 4B, and 5 indicate a direct role of claudin-1 overexpression in driving immune evasion and T cell elimination in vivo. In wild-type Hepa 1-6 tumor cells (no claudin-1; Figure 5, line with square markers), tumors are rejected by the immune system over time, whereas claudin-1 overexpression (claudin-1 hECL; Figure 5, line with triangle markers) drives immune evasion and tumor growth. Anti-CD3 (T cell marker) IHC analysis performed on tumor samples taken at the end of the experiment (day 20) shows how claudin-1 (Cldn1 hECL) overexpression drives T cell elimination from the tumor bed and accumulation in the interstitium (Figure 4B) compared to no claudin-1 expression (Figure 4A).

[0225] Example 6. Use of anti-claudin-1 antibodies to break checkpoint inhibitor resistance in cancer The data presented in Examples 1-4 show that fibrosis is a common factor in checkpoint inhibitor resistance and T cell elimination in cancer. Anti-claudin-1 antibodies (e.g., any of those described herein) are administered to subjects with fibrotic tumors. Anti-claudin-1 antibodies have a direct antifibrotic effect that promotes T cell-mediated antitumor activity. Importantly, tumors such as melanoma (Figure 7A) and head and neck squamous cell carcinoma (Figure 7B) with high levels of CLDN1 respond poorly to checkpoint inhibitor aPD1. Anti-claudin-1 antibodies are administered in combination with checkpoint inhibitor aPD1 in the Hepa1-6 synergistic tumor model of liver cancer (overexpression of murine CLDN1 promotes immune escape in vivo). Furthermore, a patient-derived xenograft (PDX) model of head and neck cancer (with confirmed high expression of CLDN1 and T cell elimination) is used to demonstrate the synergistic effect of anti-claudin-1 antibodies and checkpoint inhibitor aPD1 administration. Administration of immune checkpoint inhibitors simultaneously with or after administration of anti-claudin-1 antibodies can improve the therapeutic efficacy of immune checkpoint inhibitors in fibrotic tumors.

[0226] The data in Figures 10A-10C show that overexpression of CLDN1 in Hepa1-6 mouse liver tumor cells promoted T cell elimination and resistance to anti-PD1 treatment. Importantly, the anti-CLDN1 antibody of the present disclosure restored both T cell infiltration and anti-PD1 efficacy in Hepa1-6 CLDN1+ tumors. Figure 10A shows that the tumor volume in the cohort of anti-CLDN1 antibody and PD1 antagonist was significantly reduced compared to the cohorts of isotype control, anti-CLDN1 antibody alone, and PD1 antagonist alone. Figures 10B and 10C show that after administration of the combination of anti-CLDN1 antibody and PD1 antagonist, a much higher number of T cells successfully infiltrated into the CLDN1+ tumor.

[0227] Mechanistically, NJ-CLDN1 interacts with various components involved in extracellular matrix remodeling to establish a physical barrier that excludes immune cells from tumor nests. Anti-CLDN1 antibodies have a direct antifibrotic effect by disrupting the interface between CDLN1+ tumor cells and the stroma, restoring immune cell infiltration.

[0228] Example 7. Generation of anti-claudin-1 CAR T cells (hypothetical) T cell isolation and activation The patient is connected to the device, which transfers peripheral blood through a single-use disposable tubing set. Centrifugal force induced by an optical sensor separates the blood into bands of appropriate density for isolation and collection of the desired cell layer. The uncollected blood components are then returned to the patient. Alternatively, apheresis is used as a method of T cell collection. Manipulated T cells are isolated from the patient and reintroduced into the same individual (autologous therapy) or isolated from a donor and introduced into a different individual (xenogeneic therapy). The collected cells are cryopreserved or processed without prior freezing. The isolated cells are optionally processed for T cell (specific T cell subset) enrichment using antibody-coupled magnetic beads for positive or negative selection. For example, T cells can be enriched based on expression of CD62L, CD4, and CD8. The isolated T cells are activated, for example, by polyclonal stimulation with soluble anti-CD3 antibodies or immobilized CD3 and CD28 antibodies. CD3 and CD28 antibodies can be immobilized by coating tissue culture flasks. Paramagnetic beads (e.g., Dynabeads) can also be coated with these antibodies. In suspension, the coated beads provide adequate stimulation for much larger T cell cultures. Before formulating the final cell product, the beads are removed as they could be dangerous if injected into a patient. Removal is achieved by disrupting T cell / bead aggregates via agitation and then passing the suspension through a strong magnetic field, retaining the beads and allowing the cells to flow through. An alternative is to use a stimulation reagent such as Transact, which utilizes humanized anti-CD3 and anti-CD28 antibodies conjugated to a colloidal polymer nanomatrix. The nanomatrix is ​​washed with a centrifugation step before final product formulation. An alternative is to use a similar T cell stimulation method using a hydrogel "stimulation matrix" incorporating the antibodies. This matrix can also be removed by washing after stimulation and expansion. Other approaches, such as soluble activation proteins, lipid microbubbles, soluble microspheres, and conjugated antibodies, are also promising options for use in activating isolated T cells.

[0229] Genetic engineering of isolated T cells to achieve expression of anti-claudin-1 CAR After activation, T cells are engineered to express anti-claudin-1 CAR. Viral or alternatively non-viral systems are used to engineer isolated T cells. Plasmid-based transposon / transposase systems and viral vectors (including but not limited to gammaretroviral and lentiviral vectors), as well as genome editing (e.g., CRISPR / Cas9-based gene editing) and naked DNA electroporation are applied into isolated T cells for gene transfer to anti-claudin-1 coding regions and associated regulatory sequences.

[0230] Expansion of anti-claudin-1 CAR T cells Engineered CAR T cells expressing anti-claudin-1 CAR are then expanded in vitro by standard culture techniques or alternative methods including rocking bioreactors (e.g., Xuri™ Cell Expansion System and WAVE™ Bioreactor System), which utilize a perfusion regime to add nutrients and remove growth inhibitors, simplifying the manufacturing process. Engineered anti-claudin-1 CAR T cells are also stimulated with supplemented gamma chain cytokines, including but not limited to IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21. Optionally, anti-claudin-1 CAR T cells are also treated with specific pathway inhibitors, including but not limited to GSK3β, mTOR, AKT, and PI3K.

[0231] The engineered CAR T cells expressing the anti-claudin-1 CAR are then cryopreserved for quality control testing before being administered to patients.

[0232] 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.

[0233] All references cited above, and all references cited herein, are hereby incorporated by reference in their entirety.

[0234] Any examples provided herein are offered by way of illustration and not as limitations.

Claims

1. A pharmaceutical composition comprising an anti-claudin-1 antibody for use in combination with an immune checkpoint inhibitor in the treatment of solid tumors in a subject, The anti-claudin-1 antibody comprises a complementation-determining region (CDR) H1 containing the amino acid sequence described in SEQ ID NO: 5, CDR H2 containing the amino acid sequence described in SEQ ID NO: 6, CDR H3 containing the amino acid sequence described in SEQ ID NO: 7, CDR L1 containing the amino acid sequence described in SEQ ID NO: 8, CDR L2 containing the amino acid sequence "Gly Ala", and CDR L3 containing the amino acid sequence described in SEQ ID NO:

10. Pharmaceutical composition.

2. The pharmaceutical composition according to claim 1, wherein the anti-claudin-1 antibody is humanized.

3. The pharmaceutical composition according to claim 1, wherein the anti-claudin-1 antibody comprises VH containing the amino acid sequence described in SEQ ID NO: 3 or SEQ ID NO: 13 and / or VL containing the amino acid sequence described in SEQ ID NO: 4 or SEQ ID NO:

14.

4. The pharmaceutical composition according to claim 1, wherein the anti-claudin-1 antibody comprises VH containing the amino acid sequence described in SEQ ID NO: 3 and VL containing the amino acid sequence described in SEQ ID NO:

4.

5. The pharmaceutical composition according to claim 1, wherein the anti-claudin-1 antibody comprises VH containing the amino acid sequence described in SEQ ID NO: 13 and VL containing the amino acid sequence described in SEQ ID NO:

14.

6. The pharmaceutical composition according to claim 1, wherein the anti-claudin-1 antibody comprises a heavy chain sequence including the amino acid sequence described as SEQ ID NO:

1.

7. The pharmaceutical composition according to claim 1, wherein the anti-claudin-1 antibody comprises a light chain sequence including the amino acid sequence described as SEQ ID NO:

2.

8. The pharmaceutical composition according to any one of claims 1 to 7, wherein the pharmaceutical composition is administered before administering the immune checkpoint inhibitor.

9. A pharmaceutical composition comprising anti-claudin-1 chimeric antigen receptor (CAR) T cells for use in combination with an immune checkpoint inhibitor in the treatment of solid tumors in a subject, The CAR T cell includes a binding domain comprising a complementation-determining region (CDR) H1 containing the amino acid sequence described in SEQ ID NO: 5, CDR H2 containing the amino acid sequence described in SEQ ID NO: 6, CDR H3 containing the amino acid sequence described in SEQ ID NO: 7, CDR L1 containing the amino acid sequence described in SEQ ID NO: 8, CDR L2 containing the amino acid sequence "Gly Ala", and CDR L3 containing the amino acid sequence described in SEQ ID NO:

10. Pharmaceutical composition.

10. The pharmaceutical composition according to any one of claims 1 to 7 and 9, wherein the pharmaceutical composition and the immune checkpoint inhibitor are administered simultaneously or sequentially.

11. The pharmaceutical composition according to any one of claims 1 to 7 and 9, wherein the pharmaceutical composition and / or the immune checkpoint inhibitor are administered intratumorally, intravenously, intraperitoneally, intramuscularly, intrathecally, or subcutaneously.

12. The pharmaceutical composition according to any one of claims 1 to 7 and 9, wherein the immune checkpoint inhibitor is an antagonist of PD-1, PD-L1, CTLA-4, LAG-3, TIM-3, TIGIT, VISTA, B7-H3, BTLA, and / or Siglex-15.

13. The pharmaceutical composition according to any one of claims 1 to 7 and 9, wherein the immune checkpoint inhibitor is a small molecule inhibitor.

14. The pharmaceutical composition according to any one of claims 1 to 7 and 9, wherein the immune checkpoint inhibitor is an antibody.

15. The pharmaceutical composition according to claim 14, wherein the immune checkpoint inhibitor is (a) a PD-1 antagonist selected from the group consisting of nivolumab, pembrolizumab, semipirimab, and dostallimab; (b) a PD-L1 antagonist selected from the group consisting of atezolizumab, durvalumab, and avelumab; (c) a CTLA-4 antagonist selected from the group consisting of ipilimumab and tremelimumab; or (d) a TIGIT antagonist selected from the group consisting of tilagorumab, osperimab, dombanarimab, etigirimab, and vivostrimab.

16. The pharmaceutical composition according to any one of claims 1 to 7 and 9, wherein the cancer includes a fibrotic tumor.

17. The pharmaceutical composition according to claim 16, wherein the fibrotic tumor is characterized by high expression of claudin-1.

18. The pharmaceutical composition according to any one of claims 1 to 7 and 9, wherein the tumor is selected from the group consisting of head and neck, lung, breast, melanoma, colon, pancreas, esophagus, bile duct, and hepatocellular carcinoma.

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