Use of anti-tight connexin-1 antibodies to increase 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 to the subject an anti-tight junction protein-1 antibody.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of priority to U.S. Provisional Application No. 63 / 317,885, filed on March 8, 2022, which is hereby incorporated by reference in its entirety.

[0003] Sequence Listing References

[0004] The contents of the electronically submitted Sequence Listing (Name: 4872_013PC01_Seqlisting_ST26; Size: 24,527 bytes; and Creation Date: March 6, 2023) are incorporated herein by reference in their entirety. Technical Field

[0005] According to various aspects of the present disclosure, the present disclosure relates to methods of promoting T cell anti-tumor activity. Background Art

[0006] Cancer therapy using immune checkpoint inhibitors greatly improves the ability of physicians to treat subjects. However, many cancers have shown immune checkpoint inhibitor resistance. A phenotype associated with immune checkpoint inhibitor resistance is the lack of tumor T cell infiltration, also known as "cold tumors" or "T cell rejection" (see Shuyue W. et al., Front. Immun., 12: 690112 (2021); Christian et al., Front. Oncol., 11: 712788 (October 2021)). Therefore, there is a need to develop a therapy that allows therapy to overcome immune checkpoint inhibitor resistance to treat cold tumors or T cell rejection. Summary of the invention

[0007] 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 an anti-Claudin-1 antibody to the subject.

[0008] In some aspects, the method further comprises administering an immune checkpoint inhibitor.

[0009] In some aspects, the present invention provides a method of treating cancer in a subject with 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 aspects, the anti-claudin-1 antibody promotes T cell-mediated anti-tumor activity in the subject's tumor.

[0010] In some aspects, provided herein is a method for increasing the therapeutic efficacy of an immune checkpoint inhibitor in a subject having a fibrotic tumor, the method comprising a) administering an anti-claudin-1 antibody to the subject, wherein the anti-claudin-1 antibody promotes T cell-mediated anti-tumor activity in the fibrotic tumor; and b) administering an immune checkpoint inhibitor to the subject.

[0011] In some aspects, the anti-Claudin-1 antibody is administered prior to administration of the immune checkpoint inhibitor.

[0012] In some aspects, the anti-Claudin-1 antibody and the immune checkpoint inhibitor are administered simultaneously or sequentially.

[0013] In some aspects, the anti-Claudin-1 antibody and the immune checkpoint inhibitor are administered in the same composition.

[0014] In some aspects, the anti-Claudin-1 antibody and the immune checkpoint inhibitor are administered in different compositions.

[0015] In some aspects, the anti-Claudin-1 antibody and / or immune checkpoint inhibitor is administered intratumorally, intravenously, intraperitoneally, intramuscularly, intrathecally, or subcutaneously.

[0016] In some aspects, 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.

[0017] In some aspects, the immune checkpoint inhibitor is a small molecule inhibitor.

[0018] In some aspects, the immune checkpoint inhibitor is an antibody.

[0019] In some aspects, the immune checkpoint inhibitor is a PD-1 antagonist selected from the group consisting of nivolumab, pembrolizumab, cemiplimab, and dotarizumab.

[0020] In some aspects, the immune checkpoint inhibitor is a PD-L1 antagonist selected from the group consisting of atezolizumab, durvalumab, and avelumab.

[0021] In some aspects, the immune checkpoint inhibitor is a CTLA-4 antagonist selected from the group consisting of ipilimumab and tremelimumab.

[0022] In some aspects, the immune checkpoint inhibitor is a TIGIT antagonist selected from the group consisting of tisleliumab, osperlimumab, donavulimab, etilizumab, and vibrimab.

[0023] In some aspects, the cancer comprises a fibrotic tumor.

[0024] In some aspects, the fibrotic tumor is characterized by high expression of Claudin-1 relative to a reference sample.

[0025] In some aspects, the reference sample is a tissue sample from normal tissue, wherein the normal tissue is adjacent to the tumor.

[0026] In some aspects, the tumor is selected from the group consisting of a head and neck tumor, a lung tumor, a breast tumor, a melanoma tumor, a colorectal tumor, a pancreatic tumor, an esophageal tumor, a bile duct cancer, and a hepatocellular tumor.

[0027] In some aspects, 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 with the DSMZ on July 29, 2008 under accession number DSMACC2938.

[0028] In some aspects, the anti-Claudin-1 antibody is humanized.

[0029] In some aspects, the anti-Claudin-1 antibody comprises a VH comprising the amino acid sequence shown in SEQ ID NO:3 or SEQ ID NO:13.

[0030] In some aspects, the anti-Claudin-1 antibody comprises a VL comprising the amino acid sequence set forth in SEQ ID NO:4 or SEQ ID NO:14.

[0031] In some aspects, the anti-Claudin-1 antibody comprises a VH comprising the amino acid sequence shown in SEQ ID NO:3; and a VL comprising the amino acid sequence shown in SEQ ID NO:4.

[0032] In some aspects, the anti-Claudin-1 antibody comprises a VH comprising the amino acid sequence shown in SEQ ID NO:13; and a VL comprising the amino acid sequence shown in SEQ ID NO:14.

[0033] In some aspects, the anti-Claudin-1 antibody comprises a complementarity determining region (CDR) H1 comprising the amino acid sequence shown in SEQ ID NO:5, a CDR H2 comprising the amino acid sequence shown in SEQ ID NO:6, and a CDR H3 comprising the amino acid sequence shown in SEQ ID NO:7.

[0034] In some aspects, the anti-Claudin-1 antibody comprises a complementarity determining region (CDR) L1 comprising the amino acid sequence shown in SEQ ID NO:8, a CDR L2 comprising the amino acid sequence "Gly Ala", and a CDR L3 comprising the amino acid sequence shown in SEQ ID NO:10.

[0035] In some aspects, the anti-Claudin-1 antibody comprises a heavy chain sequence comprising the amino acid sequence shown in SEQ ID NO:1.

[0036] In some aspects, the anti-Claudin-1 antibody comprises a light chain sequence comprising the amino acid sequence shown in SEQ ID NO:2.

[0037] In some aspects, the present invention provides a method for promoting T cell-mediated anti-tumor activity in a subject with a fibrotic tumor, the method comprising administering anti-claudin-1 chimeric antigen receptor (CAR) T cells to the subject. In some aspects, including administering an immune checkpoint inhibitor.

[0038] In some aspects, the present invention provides a method for treating cancer in a subject with 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 aspects, the anti-claudin-1 CAR T cells promote T cell-mediated anti-tumor activity in the subject's tumor.

[0039] In some aspects, the present invention provides a method for increasing the therapeutic efficacy of an immune checkpoint inhibitor in a subject with a fibrotic tumor, the method comprising administering an anti-claudin-1 CAR T cell to the subject and administering an immune checkpoint inhibitor to the subject. In some aspects, the anti-claudin-1 CAR T cell promotes T cell-mediated anti-tumor activity in fibrotic tumors. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1A to Figure 1B (FIG.1A-1B) shows claudin-1 (CLDN1) expression in two different fibrotic tumor types. Immunohistochemistry (IHC) staining was performed to measure claudin-1 expression (left panel) and fibrosis markers (right panel).

[0041] FIG. 2A to FIG. 2C (FIG.2A-2C) show T cell exclusion in head and neck cancer. Figure 2A IHC staining of CLDN1 in a number of tumor samples is shown, with corresponding CD3 staining exemplifying the fibrosis trap. Figure 2B The percentage of tumor samples showing CLDN1 expression is shown. Figure 2CThe classification of the immunophenotypes of tumors with different CLDN1 expression levels is shown. The immunophenotypes are hot (immune cells in the stroma and among cancer cells), repellent (immune cells mainly in the stroma), and cold (hardly any immune cells can be seen).

[0042] FIG. 3A to FIG. 3D (FIG.3A-3D) show T cell exclusion in esophageal cancer. Figure 3A The immunophenotypes of tumors with different CLDN1 expression levels are shown. The immunophenotypes are hot (immune cells in the stroma and between cancer cells), repellent (immune cells within the tumor but only in the stroma), and cold (hardly any immune cells can be seen). Figure 3B )、T cells (CD3 staining) ( Figure 3C ) and fibrotic tissue (Sirius red staining) ( Figure 3D ) for IHC staining.

[0043] Figure 4 A to Figure 4 B (FIG.4A-4B) shows immune escape driven by overexpression of the human extracellular loop containing mouse CLDN1 in the hepatic mouse tumor cells Hepa 1-6 in vivo ( Figure 4 A) and T cell rejection ( Figure 4 B).

[0044] Figure 5 (FIG.5) shows the volume of Hepa 1-6 tumors after inoculation. Compared with samples without CLDN1 expression, positive samples containing human extracellular loop of mouse CLDN1 (CLDN1 hECL) showed larger tumor volume and exhibited immune evasion.

[0045] Figure 6 (FIG.6) shows an exemplary diagram of using anti-CLDN1 antibodies with direct anti-fibrotic effects to disrupt checkpoint inhibitor resistance in cancer.

[0046] Fig. 7A (FIG.7A) shows the overall survival probability over time in tumors with high or low levels of CLDN1 (such as melanoma) after administration of the checkpoint inhibitor aPD1.

[0047] Figure 7B (FIG.7B) shows the time to treatment discontinuation in CLDN1-positive or CLDN1-negative patients.

[0048] Fig. 8A (FIG.8A) shows a mechanistic model of CLDN-1 targeted therapy in the tumor microenvironment.

[0049] Figure 8B(FIG.8B) shows a mechanistic model showing the effect of administering anti-CLDN1 antibodies to tumors with T cell rejection.

[0050] Fig. 9 (FIG.9) shows CLDN1 expression in head and neck squamous cell carcinoma (HNSCC), colorectal cancer (CRC), esophageal cancer, squamous non-small cell lung cancer (Sq.NSCLC), intrahepatic cholangiocarcinoma (iCCA), hepatocellular carcinoma (HCC), and urothelial carcinoma.

[0051] Fig. 10A (FIG.10A) shows the tumor volume in Hepa1-6 CLDN1+ challenged mice after administration of anti-CLDN1 antibody alone, PD1 antagonist alone (aPD1), and a combination of anti-CLDN1 antibody and PD1 antagonist. Mice were challenged again on day 40.

[0052] FIG. 10B to FIG. 10C (FIG.10B-10C) shows the measurement control ( Fig. 10B ) and after treatment with anti-CLDN1 antibody and PD1 antagonist ( Fig. 10C ) of T cell dendritic cell infiltration. DETAILED DESCRIPTION

[0053] I. Definitions

[0054] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those of ordinary skill in the art to which the present disclosure belongs. In the event of a conflict, the present application (including definitions) shall prevail. In addition, unless the context otherwise requires, singular terms shall include plural terms and plural terms shall include singular terms. All publications, patents and other references mentioned herein are incorporated by reference in their entirety for all purposes, as if each individual publication or patent application was clearly and individually indicated to be incorporated by reference in its entirety.

[0055] Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described below. These materials, methods and examples are illustrative only and are not intended to be limiting. Other features and advantages of the present disclosure will be apparent from the detailed description and claims.

[0056] To further define the present disclosure, the following terms and definitions are provided.

[0057] Unless the context clearly indicates otherwise, the singular forms "a", "an", and "the" include plural referents. The term "a" (or "an") and the terms "one or more" and "at least one" are used interchangeably herein. In some aspects, the term "a" or "an" refers to "single". In other aspects, the term "a" or "an" includes "two or more" or "plurality".

[0058] The term "about" is used herein to mean approximately, roughly, around, or in the range of. When the term "about" is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values ​​set forth. Generally, the term "about" is used herein to modify numerical values ​​above and below the stated value by 10% up or down (higher or lower).

[0059] Throughout this disclosure, various aspects of the present invention are presented in range format.It should be understood that the description of range format is only for convenience and brevity, and should not be interpreted as an inflexible limitation to the scope of the invention.Therefore, the description of the scope should be considered to specifically disclose all possible sub-ranges and the single numerical values ​​in the scope.For example, the description of a scope such as 1 to 6 should be considered to specifically disclose sub-ranges, such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., and the single digits in the scope, such as 1, 2, 3, 4, 5 and 6.This is applicable regardless of the width of the scope.The numerical range listed includes the number defining the scope and includes each integer in the defined range.

[0060] Units, prefixes and symbols are expressed in a form acceptable to the Système International de Unites (SI). Numerical ranges include the number of limited ranges. In the case of enumerated numerical ranges, it is understood that each intermediate integer value and each fraction thereof between the upper and lower limits of the enumeration of the range, and each subrange between these values ​​are also specifically disclosed. The upper and lower limits of any range may be independently included in the range or excluded from the range, and each range including any one, zero or two limits is also encompassed in the present disclosure. Therefore, the ranges listed herein are understood to be a shorthand expression of all values ​​in the range including the enumerated endpoints. For example, a range of 1 to 10 is understood to include any number, combination of numbers or subranges in the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10.

[0061] In the case of explicitly enumerating values, it is understood that the values ​​of the amount or quantity approximately the same as the enumerated values ​​are also within the scope of the present disclosure. In the case of a disclosed combination, each sub-combination of the elements of the combination is also specifically disclosed and within the scope of the present disclosure. On the contrary, in the case of separately disclosing different elements or groups of elements, their combinations are also disclosed. In the case where any element of the present disclosure is disclosed as having multiple alternatives, the examples of the disclosure are also disclosed herein, wherein each alternative is excluded individually or excluded in any combination with other alternatives; more than one element of the present disclosure may have such exclusions, and all combinations of elements with such exclusions are disclosed herein.

[0062] The term "and / or" as used herein is considered to be a specific disclosure of each of the two specified features or components with or without the other. Thus, the term "and / or" as used herein in phrases such as "A and / or B" is meant to include: "both A and B"; "A or B"; "A" (alone); and "B" (alone). Similarly, the term "and / or" as used in phrases such as "A, B, and / or C" is intended to cover each of the following aspects: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0063] As used herein, the term "treating" or "treatment" refers to the administration of a composition to a subject for therapeutic purposes.

[0064] The term "human tight junction protein -1 (or CLDN1)" refers to a protein having the sequence shown in NCBI accession number NP_066924.1, or any naturally occurring variant commonly found in HCV-permissive populations. Tight junction proteins 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 such as: junctional adhesion molecules (JAMs) and occludens, as well as scaffold proteins ZO-1, ZO-2, and ZO-3. Tight junction protein -1 is widely expressed in epithelial cells, but in mesenchymal tissues, expression appears to be limited to perineurial cells. Tight junction protein -1 expression is reported to occur in 29% to 92% of tumors.

[0065] As used herein, the term "antibody" refers to any immunoglobulin containing an antigen binding site that immunospecifically binds to an antigen. Thus, the term antibody encompasses not only whole antibody molecules, but also antibody fragments and variants (including derivatives) of antibodies and antibody fragments, as long as the derivatives and fragments retain specific binding ability. The term encompasses monoclonal antibodies and polyclonal antibodies. The term also encompasses any protein having a binding domain that is homologous or largely homologous to an immunoglobulin binding domain. These proteins may be from natural sources, or may be produced in part or in whole synthetically. When used with respect to antibodies, the term "specific binding" refers to the binding of an antibody to a predetermined antigen. Typically, an antibody binds to an antigen with at least 1 x 10 7 M 1 The antibody binds to the predetermined antigen with an affinity of at least twice that of the non-specific antigen (e.g., BSA, casein).

[0066] The term "monoclonal antibody" or its antigen binding fragment refers to a homogeneous antibody or antigen binding fragment population that participates in the highly specific recognition and binding of a single antigenic determinant or epitope. This is in contrast to polyclonal antibodies that typically include different antibodies for different antigenic determinants. The term "monoclonal antibody" or its antigen binding fragment covers complete and full-length monoclonal antibodies as well as antibody fragments (such as Fab, Fab', F(ab')2, Fv), single-chain (scFv) mutants, fusion proteins comprising antibody portions, and any other modified immunoglobulin molecules comprising antigen recognition sites. In addition, "monoclonal antibody" or its antigen binding fragment refers to such antibodies and antigen binding fragments thereof prepared in a variety of ways, including but not limited to hybridomas, phage selection, recombinant expression, and transgenic animals.

[0067] As used herein, the term "humanized antibody" refers to a chimeric antibody comprising amino acid residues from non-human hypervariable regions and amino acid residues from human framework regions (FRs). In particular, a humanized antibody comprises all or substantially all of at least one, usually two variable domains, wherein all or substantially all of the complementary determining regions (CDRs) are those of human antibodies. A humanized antibody optionally may comprise at least a portion of an antibody constant region derived from a human antibody. A "humanized form" of an antibody (e.g., a non-human antibody) refers to an antibody that has undergone humanization.

[0068] It should be understood that whenever an aspect is described herein with the term “comprising,” other similar aspects described with “consisting of” and / or “consisting essentially of” are also provided.

[0069] As used herein, the term "administer" refers to the physical introduction of a composition (e.g., a combination of anti-tight junction protein-1 antibodies and / or immune checkpoint inhibitors) comprising a therapeutic agent to a subject using any of the various methods and delivery systems known to those skilled in the art. Routes of administration include intravenous, intramuscular, subcutaneous, intraperitoneal, spinal or other parenteral routes of administration, such as by injection or infusion. As used herein, the phrase "parenteral administration" refers to a mode of administration other than enteral and topical administration, typically by injection, and includes but is not limited to intravenous, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcutaneous, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion and in vivo electroporation. Other non-parenteral routes include topical, epidermal or mucosal routes of administration, such as intranasal, vaginal, rectal, sublingual or topical administration. Administration can also be performed, for example, once, multiple times and / or over one or more extended time periods.

[0070] The term "effective amount" refers to the amount of a medicament that provides a desired biological, therapeutic and / or preventive result. The result can be a reduction, improvement, alleviation, reduction, delay and / or alleviation of one or more signs, symptoms or causes of disease, or any other desired change in a biological system. With regard to solid tumors, an effective amount includes an amount sufficient to cause tumor reduction and / or reduce tumor growth rate (such as inhibiting tumor growth) or prevent or delay other unwanted cell proliferation. In some aspects, an effective amount is an amount sufficient to delay tumor development. In some aspects, an effective amount is an amount sufficient to prevent or delay tumor recurrence. The effective amount can be administered once or multiple times. An effective amount of a drug or composition can be: (i) reducing the number of cancer cells; (ii) reducing tumor size; (iii) inhibiting, blocking, slowing down and possibly stopping cancer cell infiltration into peripheral organs to some extent; (iv) inhibiting (i.e., slowing down and possibly stopping) tumor metastasis to some extent; (v) inhibiting tumor growth; (vi) preventing or delaying the occurrence and / or recurrence of tumors; and / or (vii) alleviating to some extent one or more symptoms associated with cancer. In one example, an "effective amount" is a combination of an amount of an anti-claudin-1 antibody and an amount of an immune checkpoint inhibitor that has been clinically shown to affect a significant reduction in cancer or a slowing of the progression of cancer (such as advanced solid tumors).

[0071] "Cancer" refers to a broad variety of diseases characterized by the uncontrolled growth of abnormal cells in the body. Unregulated cell division and growth lead to the formation of malignant tumors that invade adjacent tissues and may also metastasize to distant parts of the body via the lymphatic system or bloodstream. "Cancer" or "cancer tissue" may include tumors.

[0072] As used herein, the term "tumor" refers to any mass of tissue, benign (non-cancerous) or malignant (cancerous), resulting from excessive cell growth or proliferation, including precancerous lesions.

[0073] As used herein, the term "cold tumor" refers to a tumor that exhibits low levels of immune infiltration, responds poorly to immunotherapy, and / or exhibits tumor heterogeneity. These cold tumors may have features including, but not limited to, the following: the number and / or activity of CD8+T effector cells in the tumor is significantly reduced or absent, and / or the number and / or activity of immunosuppressive cells in the tumor is significantly increased. Cold tumors are also referred to as tumors with "T cell rejection". Immunotherapy with immune checkpoint inhibitors (ICIs) has greatly improved the clinical efficacy of malignant tumor therapy, but the anti-tumor response mediated by ICIs depends on the infiltration of T cells that can recognize and kill tumor cells. Therefore, ICIs may be ineffective in "cold tumors" characterized by a lack of T cell infiltration.

[0074] The term "high expression of claudin-1" refers to the proportion of cells in a test tissue sample that are scored as expressing claudin-1. In some aspects, claudin-1 expression is determined by immunohistochemistry (IHC), wherein high expression of claudin-1 in a sample means that at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or 100% of the total number of cells in the test sample express claudin-1.

[0075] As used herein, "patient" includes any patient suffering from cancer (eg, fibrotic cancer). The terms "subject" and "patient" are used interchangeably herein.

[0076] II. Anti-claudin-1 Antibodies

[0077] The present invention relates to the use of anti-claudin-1 antibodies for promoting T cell-mediated anti-tumor activity in subjects with fibrotic tumors, treating cancer in subjects with solid tumors, and increasing the therapeutic efficacy of immune checkpoint inhibitors in subjects with fibrotic tumors.

[0078] Antibodies against human claudin-1 have been previously described for the treatment of hepatitis C virus infection, hepatocellular carcinoma, and certain fibrotic diseases, such as pulmonary fibrosis (see WO 2010 / 034812, WO 2016 / 146809, and WO 2021 / 094469). Anti-claudin-1 antibodies useful in the practice of the present invention include any antibodies raised against claudin-1. Examples are disclosed in WO 2010 / 034812 and WO 2017 / 162678.

[0079] Other examples of suitable anti-claudin-1 antibodies include those disclosed in European Patent No. EP 1 167 389, U.S. Patent No. 6,627,439, International Patent Application Published as WO 2014 / 132307, International Patent Application Published as WO 2015 / 014659 and WO 2015 / 014357, and Yamashita et al., J. Pharmacol. Exp. Ther., 2015, 353(1): 112-118.

[0080] The anti-Claudin-1 antibody suitable for use in the present invention may be a polyclonal antibody or a monoclonal antibody.

[0081] Anti-claudin-1 antibodies suitable for use according to the present invention may also be "humanized": sequence differences between rodent antibodies and human sequences can be minimized by replacing residues that differ from those in the human sequence with site-directed mutagenesis of single residues or by transplantation of entire regions or by chemical synthesis. Humanized antibodies may also be produced using recombinant methods. In a humanized form of an antibody, some, most or all of the amino acids outside the CDR regions are replaced with amino acids from a human immunoglobulin molecule, while some, most or all of the amino acids within one or more CDR regions remain unchanged. Small additions, deletions, insertions, substitutions or modifications of amino acids are permitted as long as they do not significantly modify the biological activity of the resulting antibody. Suitable human "alternative" immunoglobulin molecules include IgG1, IgG2, IgG2a, IgG2b, IgG3, IgG4, IgA, IgM, IgD or IgE molecules and fragments thereof.

[0082] In some aspects, the humanized anti-claudin-1 antibody used according to the present invention is an antibody previously described in WO 2017 / 162678. Exemplary sequences of antibodies or antigen-binding fragments provided herein are described in Table 1.

[0083] Table 1 - Antibodies or antigen-binding fragments

[0084]

[0085]

[0086]

[0087] In some aspects, the anti-Claudin-1 antibody comprises a complementarity determining region (CDR) H1 comprising the amino acid sequence shown in SEQ ID NO:5, a CDR H2 comprising the amino acid sequence shown in SEQ ID NO:6, and a CDR H3 comprising the amino acid sequence shown in SEQ ID NO:7.

[0088] In some aspects, the anti-Claudin-1 antibody comprises a complementarity determining region (CDR) L1 comprising the amino acid sequence shown in SEQ ID NO:8, a CDR L2 comprising the amino acid sequence "Gly Ala", and a CDR L3 comprising the amino acid sequence shown in SEQ ID NO:10.

[0089] In some aspects, the complementarity determining regions (CDRs) disclosed herein are according to Definition. However, it will be appreciated that other methods of defining CDRs known in the art may also be used.

[0090] In some aspects, the six complementarity determining regions (CDRs) of the anti-Claudin-1 antibody are identical to the anti-Claudin-1 monoclonal antibody secreted by the hybridoma cell line deposited with the DSMZ on July 29, 2008 under accession number DSMACC2938.

[0091] In some aspects, the anti-Claudin-1 antibody comprises a heavy chain variable region ("VH") comprising the amino acid sequence shown in SEQ ID NO:3 or 13.

[0092] In some aspects, the anti-Claudin-1 antibody comprises a light chain variable region ("VL") comprising the amino acid sequence shown in SEQ ID NO:4 or 14.

[0093] In some aspects, the heavy chain variable region ("VH") and light chain variable region ("VL") of the anti-Claudin-1 antibody are identical to the anti-Claudin-1 monoclonal antibody secreted by the hybridoma cell line deposited with the DSMZ on July 29, 2008 under accession number DSMACC2938.

[0094] In some aspects, the heavy and light chains of the anti-Claudin-1 antibody are identical to the anti-Claudin-1 monoclonal antibody secreted by the hybridoma cell line deposited with the DSMZ on July 29, 2008 under accession number DSMACC2938.

[0095] In some aspects, the anti-Claudin-1 antibody comprises a heavy chain sequence comprising the amino acid sequence shown in SEQ ID NO:1.

[0096] In some aspects, the anti-Claudin-1 antibody comprises a light chain sequence comprising the amino acid sequence shown in SEQ ID NO:2.

[0097] In some aspects, the anti-Claudin-1 antibody comprises a heavy chain sequence comprising the amino acid sequence shown in SEQ ID NO:1, SEQ ID NO:11, or SEQ ID NO:21.

[0098] In some aspects, the anti-Claudin-1 antibody comprises a light chain sequence comprising the amino acid sequence shown in SEQ ID NO:2 or SEQ ID NO:12.

[0099] The humanized anti-claudin-1 antibody may be a fully monoclonal antibody having an isotope selected from the group consisting of IgG1, IgG2, IgG3 and IgG4. Alternatively, the humanized anti-claudin-1 antibody may be a fragment of a monoclonal antibody selected from the group consisting of Fv, Fab, F(ab')2, Fab', dsFv, scFv, sc(Fv)2 and diabody.

[0100] Anti-claudin-1 antibodies (or biologically active variants or fragments thereof) suitable for use according to the present invention may be functionally linked (e.g., by chemical coupling, genetic fusion, non-covalent binding or other means) to one or more other molecular entities. Methods for preparing such modified antibodies (or conjugated antibodies) are known in the art (see, e.g., "Affinity Techniques. Enzyme Purification: Part B", Methods in Enzymol., 1974, Vol. 34, Jakoby and Wilneck (eds.), Academic Press: New York, NY; and Wilchek and Bayer, Anal. Biochem., 1988, 171: 1-32). Preferably, the molecular entity is linked to a position on the antibody molecule that does not interfere with the binding properties of the resulting conjugate, such as a position that does not participate in the specific binding of the antibody to its target.

[0101] The antibody molecule and the molecular entity can be covalently linked to each other, directly linked. Alternatively, the antibody molecule and the molecular entity can be covalently linked to each other via a linker group. This can be achieved by using any of a variety of stable bifunctional agents well known in the art, including homofunctional and heterofunctional linkers.

[0102] In some aspects, the anti-Claudin-1 antibodies (or biologically active fragments thereof) used in accordance with the present invention are conjugated to a detectable agent. Any of a variety of detectable agents may be used, including but not limited to various ligands, radionuclides (e.g., 3 H. 125 I. 131I, etc.), fluorescent dyes (e.g., fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthalaldehyde and fluorescamine), chemiluminescent agents (e.g., luciferin, luciferase and aequorin), microparticles (e.g., quantum dots, nanocrystals, phosphors, etc.), enzymes (e.g., those used in ELISA, i.e., horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase), colorimetric labels, magnetic labels and biotin, digoxigenin or other haptens and proteins for which antisera or monoclonal antibodies can be obtained.

[0103] 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.

[0104] Therefore, in the practice of the present invention, the anti-claudin-1 antibody can be used in the form of a full-length antibody, a biologically active variant or fragment thereof, a chimeric antibody, a humanized antibody, and an antibody-derived molecule comprising at least one complementarity determining region (CDR) from the heavy chain or light chain variable region of the anti-claudin-1 antibody, including molecules such as: Fab fragments, F(ab')2 fragments, Fd fragments, Fabc fragments, Sc antibodies (single chain antibodies), diabodies, single antibody light chain single chains, single antibody heavy chains, chimeric fusions between antibody chains and other molecules, and antibody conjugates, such as antibodies conjugated to therapeutic agents or detectable agents. Preferably, the anti-claudin-1 antibody-related molecules according to the present invention retain the ability of the antibody to bind to its antigen, particularly the extracellular domain of claudin-1.

[0105] III. Chimeric Antigen Receptors

[0106] Chimeric antigen receptor (CAR) T cell therapy, or CAR T cell therapy, is a cancer treatment based on the use of T cells that are genetically engineered to express synthetic receptors that bind to tumor antigens. The engineered CAR T cells are expanded in vitro and infused into the patient to attack and destroy chemotherapy-resistant cancers.

[0107] 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 produce a chimeric protein that exhibits specific anti-target cell immune activity.

[0108] As used herein, the "signal transduction domain" or "signaling domain" of CAR is responsible for intracellular signaling after the extracellular ligand binding domain binds to the target, resulting in the activation and immune response of immune cells. In other words, the signal transduction domain is responsible for activating at least one normal effector function of immune cells expressing CAR. For example, the effector function of T cells can be cytolytic activity or auxiliary activity, including the secretion of cytokines. Therefore, the term "signal transduction domain" refers to a protein portion that transduces effector function signals and instructs cells to perform specialized functions. Examples of signal transduction domains for CARs can be cytoplasmic sequences of T cell receptors and co-receptors, which act synergistically to initiate signal transduction after antigen receptor engagement, as well as any derivatives or variants of these sequences and any synthetic sequences with the same functional capabilities. In some cases, the signaling domain contains two different types of cytoplasmic signaling sequences, i.e., those sequences that initiate antigen-dependent primary activation and those sequences that act in an antigen-independent manner to provide secondary or costimulatory signals. The primary cytoplasmic signaling sequence may contain a signaling motif, which is referred to as an immunoreceptor tyrosine-based activation motif of ITAM. ITAM is a clear signaling motif found in the cytoplasmic tail of a variety of receptors used as binding sites for syk / zap70 class tyrosine kinases. Exemplary ITAMs include those derived from: TCR ζ, FcR γ, FcR β, FcR ε, CD3 γ, CD3 δ, CD3 ε, CD5, CD22, CD79a, CD79b and CD66d. In some aspects, the signal transduction domain of CAR can include a CD3 ζ signaling domain (SEQ ID NO: 15).

[0109] CAR is a synthetic receptor composed of a targeting moiety, which is associated with one or more signaling domains in a single fusion molecule. Typically, the binding portion of CAR is composed of the antigen binding domain of a single-chain antibody (scFv), which comprises a light chain and a heavy chain variable fragment of a monoclonal antibody connected by a flexible joint. This molecule is connected to an intracellular signaling molecule comprising one or more intracellular signaling domains that mediate T cell activation. The signaling domain of the first generation of CAR is derived from the cytoplasmic region of CD3ζ or Fc receptor γ chain (or another intracellular signaling domain of a protein containing an activation motif [ITAM] based on immunoreceptor tyrosine). The first generation of CAR has been shown to successfully redirect T cell toxicity. However, they cannot provide extended amplification and anti-tumor activity in vivo. Signaling domains from costimulatory molecules, as well as transmembrane and hinge domains have been added to form second, third and fourth generation CARs. The second generation chimeric receptor is also incorporated into a costimulatory intracellular domain (e.g., 4-1BB / CD3ζ). A third generation CAR containing multiple costimulatory signaling modules. The fourth generation CAR is produced by adding IL-12 to the second generation construct, and is referred to as T cells (TRUCK) that are redirected for universal cytokine-mediated killing. TRUCK enhances T cell activation and activates and attracts innate immune cells to eliminate antigen-negative cancer cells in targeted lesions. Therapeutic trials using CAR T cell therapy in humans have shown some success. For example, T cells redirected by CARs specific for B cell differentiation antigen CD19 have shown significant efficacy in the treatment of B cell malignancies, and TCR redirected T cells have shown benefits in patients with solid cancers. Stauss et al. describe strategies for modifying therapeutic CARs and TCRs for treating cancer, for example, enhancing antigen-specific effector functions and limiting the toxicity of engineered T cells (Current Opinion in Pharmacology 2015, 24: 113-118).

[0110] In some aspects of the present invention, a chimeric antigen receptor (CAR) is included that is specific for tight junction protein-1 expressed on the surface of cancer cells. In some aspects of the present invention, CAR as described herein comprises an extracellular target-specific binding domain, a transmembrane domain, an intracellular signaling domain (such as a signaling domain derived from CD3ζ or FcRγ) and / or one or more costimulatory signaling domains derived from costimulatory molecules (such as but not limited to 4-1BB). In some aspects, CAR includes a hinge region or a spacer region between the extracellular binding domain and the transmembrane domain, such as a CD8α hinge. In some aspects, a chimeric antigen receptor (CAR) comprises an extracellular target-specific binding domain, which is an anti-tight junction protein single-chain antibody (scFv), and can be a mouse, human or humanized scFv. Single-chain antibodies can be cloned from V region genes of hybridomas that are specific to the desired target. Techniques that can be used to clone variable region heavy chains (VH) and variable region light chains (VL) have been described, for example, in Orlandi et al., PNAS, 1989; 86: 3833-3837. Thus, in some aspects, the binding domain comprises an antibody-derived binding domain, but may be a non-antibody-derived binding domain. An antibody-derived binding domain may be a fragment of an antibody or a genetically engineered product of one or more fragments of an antibody that participate in binding to an antigen.

[0111] In some aspects, the CAR of the present invention may include a joint between various domains, added for the appropriate spacing and conformation of the molecule. For example, in some aspects, there may be a joint with a length of 1-10 amino acids between the binding domains VH or VL. In some aspects, the length of the joint between any domains of the chimeric antigen receptor may be 1-20 amino acids or 20 amino acids. In this regard, the length of the joint 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. In some aspects, the length of the joint may be 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 amino acids. The range including the numbers described herein is also included herein, such as a joint with a length of 10-30 amino acids.

[0112] In some aspects, the joint suitable for CAR described herein is a flexible joint. Suitable joints can be easily selected, and can be any suitable different lengths, such as 1 amino acid (eg, Gly) to 20 amino acids, 2 amino acids to 15 amino acids, 3 amino acids to 12 amino acids, including 4 amino acids to 10 amino acids, 5 amino acids to 9 amino acids, 6 amino acids to 8 amino acids or 7 amino acids to 8 amino acids, and can be 1,2,3,4,5,6 or 7 amino acids.

[0113] Exemplary flexible linkers include glycine polymers (G) n, glycine-serine polymers (where n is an integer of at least one), glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art. Glycine and glycine-serine polymers are relatively unstructured and can therefore be used as neutral tethers between domains of fusion proteins (such as CAR described herein). Glycine even obtains significantly more phi-psi space than alanine, and is much less restricted than residues with longer side chains (see Scheraga, Rev. Computational Chem. 11173-142 (1992)). Ordinary technicians will recognize that the design of CAR may include all or part of a flexible linker, so that the linker may include a flexible linker and one or more parts of a less flexible structure to provide a desired CAR structure. Specific linkers include (G4S) n linkers, where n = 1-3. In some aspects, the linker comprises SEQ ID NO: 16 or SEQ ID NO: 17 amino acid sequence.

[0114] The binding domain of CAR can be followed by a "spacer" or "hinge", which refers to the region where the antigen binding domain is moved away from the effector cell surface to achieve appropriate cell / cell contact, antigen binding and activation (Patel et al., Gene Therapy, 1999; 6: 412-419). The hinge region in CAR is generally between the transmembrane (TM) and the binding domain. In some aspects, the hinge region is an immunoglobulin hinge region, and may be a wild-type immunoglobulin hinge region or a changed wild-type immunoglobulin hinge region. Other exemplary hinge regions for CAR described herein include hinge regions derived from the extracellular region of type 1 membrane proteins (such as CD8α, CD4, CD28 and CD7), which may be wild-type hinge regions from these molecules or may be changed. In some aspects, the hinge region includes a CD8α hinge (SEQ ID NO: 18).

[0115] "Transmembrane" region or domain is a part of CAR, which anchors the extracellular binding part to the plasma membrane of immune effector cells and promotes the binding of the binding domain to the target antigen. The transmembrane domain can be a CD3ζ transmembrane domain, but other transmembrane domains that can be used include those obtained from: CD8α, CD4, CD28, CD45, CD9, CD16, CD22, CD33, CD64, CD80, CD86, CD134, CD137 and CD154. In some aspects, the transmembrane domain is the transmembrane domain of CD137. In some aspects, the transmembrane domain includes SEQ ID NO:19 amino acid sequence. In some aspects, the transmembrane domain is synthetic, in which case it will mainly include hydrophobic residues, such as leucine and valine.

[0116] "Intracellular signaling domain" or "signaling domain" refers to a portion of a chimeric antigen receptor protein that is involved in transducing information that effectively binds CAR to a target antigen to the interior of an immune effector cell to elicit effector cell functions, such as activation, cytokine production, proliferation, and cytotoxic activity, including the release of cytotoxic factors to CAR-bound target cells, or other cellular responses triggered by antigen binding to an extracellular CAR domain. The term "effector function" refers to a specialized function of a cell. The effector function of a T cell may be, for example, cytolytic activity or auxiliary or activity, including the secretion of cytokines. Therefore, the terms "intracellular signaling domain" or "signaling domain" used interchangeably herein refer to portions of proteins that transduce effector function signals and instruct cells to perform specialized functions. Although the entire intracellular signaling domain can generally be used, the entire domain may not be used in many cases. In terms of using a truncated portion of an intracellular signaling domain, such a truncated portion may be used instead of the entire domain, as long as it transduces an effector function signal. The term intracellular signaling domain means any truncated portion of an intracellular signaling domain that is sufficient to transduce an effector function signal. The intracellular signaling domain is also referred to as a "signaling domain" and is typically derived from a portion of the human CD3 or FcRγ chain.

[0117] It is known that the signal generated by the T cell receptor alone is not enough to fully activate T cells, and secondary or costimulatory signals are required. Therefore, T cell activation can be said to be mediated by two different types of cytoplasmic signaling sequences: those that initiate antigen-dependent primary activation 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, which are referred to as immunoreceptor tyrosine-based activation motifs or ITAMs.

[0118] Examples of ITAMs containing primary cytoplasmic signaling sequences that are particularly useful in the present invention include those derived from: TCRζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, and CD66d. In certain aspects, the signaling domain of the anti-BCMACAR described herein is derived from CD3ζ. In some aspects, the signaling domain comprises the amino acid sequence of SEQ ID NO: 15.

[0119] As used herein, the term "costimulatory signaling domain" or "costimulatory domain" refers to a CAR portion of an intracellular domain comprising a costimulatory molecule. Costimulatory molecules are cell surface molecules other than antigen receptors or Fc receptors that provide a second signal required for the effective activation and function of T lymphocytes when bound to an antigen. 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. Therefore, although the present disclosure provides exemplary costimulatory domains derived from CD3ζ and 4-1BB, other costimulatory domains are contemplated for use with CAR described herein. Including one or more costimulatory signaling domains can enhance the efficacy and amplification of T cells expressing CAR receptors. Intracellular signaling and costimulatory signaling domains can be connected in series to the carboxyl terminus of the transmembrane domain in any order. In some aspects, the costimulatory domain comprises the amino acid sequence of SEQ ID NO:20.

[0120] In some aspects, the anti-Claudin-1 CAR of the present disclosure comprises any element of Table 2.

[0121] Table 2 - CAR elements

[0122]

[0123] Although engineered to contain the signaling domain from CD3 or FCRγ, scFv-based CARs have been shown to deliver potent signals for T cell activation and effector functions, they are not sufficient to trigger signals that promote T cell survival and amplification in the absence of accompanying costimulatory signals. Other CARs containing binding domains, hinges, transmembrane and signaling domains derived from CD3ζ or FcRγ and one or more costimulatory signaling domains (e.g., derived from CD28, CD137, CD134 and CD278 intracellular costimulatory domains) can more effectively guide the anti-tumor activity in T cells expressing CAR in vitro and in animal models and cancer patients, as well as increased cytokine secretion, lytic activity, survival and proliferation (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).

[0124] In some aspects, the anti-claudin-1 CAR of the present invention comprises: (a) an anti-claudin-1 binding domain (e.g., a 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 derived from human CD8α, (c) a human CD8α transmembrane domain, and (d) a human T cell receptor CD3ζ chain (CD3) intracellular signaling domain, and optionally one or more costimulatory signaling domains, such as 4-1BB. In some aspects, the different protein domains are arranged in the following order from the amino terminus to the carboxyl terminus: an anti-claudin-1 binding domain, a hinge region, and a transmembrane domain. The intracellular signaling domain and the optional costimulatory signaling domain are connected in series to the transmembrane carboxyl terminus in any order to form a single-chain chimeric polypeptide. In some aspects, the nucleic acid construct encoding anti-tight junction protein -1 CAR is a chimeric nucleic acid molecule comprising a nucleic acid molecule, and the nucleic acid molecule comprises different coding sequences, such as (5' to 3') the coding sequences of each of the following: anti-tight junction protein -1 scFv, human CD8 α-hinge, human CD8 α transmembrane domain and CD3 ζ intracellular signaling domain. In some aspects, the nucleic acid construct encoding anti-tight junction protein -1 CAR is a chimeric nucleic acid molecule comprising a nucleic acid molecule, and the nucleic acid molecule comprises different coding sequences, such as (5' to 3') the coding sequences of each of the following: anti-tight junction protein -1 scFv, human CD8 α-hinge, human CD8 α transmembrane domain, 4-1BB costimulatory domain and CD3 ζ costimulatory domain.

[0125] In some aspects, the polynucleotide encoding the CAR described herein is inserted into a vector. In order to express the anti-tight junction protein-1 CAR, the vector can be introduced into the host cell to allow the polypeptide to be expressed in the host cell. The expression vector may contain a variety of elements for controlling expression, including but not limited to promoter sequences, transcription start sequences, enhancer sequences, selection markers, and signal sequences. As described above, those of ordinary skill in the art can appropriately select these elements. For example, a promoter sequence can be selected to promote the transcription of polynucleotides in the vector. Suitable promoter sequences include but are not limited to T7 promoters, T3 promoters, SP6 promoters, β-actin promoters, EF1a promoters, CMV promoters, and SV40 promoters. Enhancer sequences can be selected to enhance the transcription of polynucleotides. A selection marker can be selected to allow the host cell into which the vector is inserted to be selected from a host cell into which the vector is not inserted, for example, a selection marker can be a gene that confers antibiotic resistance. A signal sequence can be selected to allow the expressed polypeptide to be transported outside the host cell.

[0126] 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 of introducing an exogenous nucleic acid sequence into a host cell. Nucleic acids can be integrated into host cell DNA or can be maintained outside the chromosome. Nucleic acids can be transiently maintained or can be stably introduced. Transfection can be achieved by a variety of means known in the art, including but not limited to calcium phosphate-DNA coprecipitation, DEAE-dextran-mediated transfection, polybrene-mediated transfection, electroporation, microinjection, liposome fusion, lipofection, protoplast fusion, retroviral infection, and gene gun. Transduction refers to the use of viral or retroviral vectors to deliver genes by viral infection rather than by transfection.

[0127] As used herein, the term "genetically engineered" or "genetically modified" refers to the addition of additional genetic material in the form of DNA or RNA to the total genetic material of a cell. The terms "genetically modified cell," "modified cell," and "redirected cell" are used interchangeably.

[0128] In some aspects, the CARs of the invention are introduced into and expressed in immune effector cells in order to redirect their specificity to a target antigen of interest, such as Claudin-1.

[0129] The present invention provides a method for preparing an immune effector cell expressing CAR as described herein. In some aspects, the method includes transfection or transduction of immune effector cells separated from a subject (such as a subject with a tumor cell expressing tight junction protein-1) so that the immune effector cells express one or more CARs as described herein. In some aspects, immune effector cells are separated from individuals and genetically modified without further in vitro manipulation. These cells can then be directly re-applied to individuals. In some aspects, before being genetically modified to express CAR, immune effector cells are first activated and stimulated to proliferate in vitro. In this regard, immune effector cells can be cultured before or after genetic modification (i.e., transduction or transfection to express CAR as described herein).

[0130] Prior to in vitro manipulation or genetic modification of immune effector cells as described herein, cell sources can be obtained from subjects. In some aspects, immune effector cells for use with CAR as described herein include T cells. T cells can be obtained from many sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from infection sites, ascites, pleural effusion, spleen tissue and tumors. In some aspects, T cells can be obtained from blood units collected from subjects using any number of techniques known to those skilled in the art (such as FICOLL separation). In some aspects, cells from individual circulating blood are obtained by apheresis. Apheresis products generally contain lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated leukocytes, red blood cells and platelets. In some aspects, cells collected by apheresis can be washed to remove the plasma portion and the cells are placed in a suitable buffer or medium for subsequent processing. In some aspects, cells are washed with PBS. In some aspects, the washed solution lacks calcium, and may lack magnesium or may lack many (if not all) divalent cations. As will be appreciated by one of ordinary skill in the art, the washing steps can be accomplished by methods known to those skilled in the art, such as by using a semi-automatic flow-through centrifuge. After washing, the cells can be resuspended in various biocompatible buffers or other saline solutions with or without buffers. In some aspects, unwanted components in the apheresis sample can be removed in the directly resuspended cell culture medium.

[0131] In some aspects, the erythrocytes are lysed and the monocytes are depleted, for example by PERC OLL TM Gradient centrifugation is used to separate T cells from peripheral blood mononuclear cells (PBMC). Specific subpopulations of T cells, such as CD28+, CD4+, CD8+, CD45RA+ and CD45RO+T cells, can be further separated by positive selection or negative selection techniques. For example, enrichment of T cell colonies by negative selection can be achieved with a combination of antibodies for surface markers unique to negatively selected cells. A method for this article is to perform cell sorting and / or selection by using negative magnetic immunoadhesion or flow cytometry of a mixture of monoclonal antibodies for cell surface markers present on cells for negative selection. For example, in order to enrich CD4+ cells by negative selection, monoclonal antibody mixtures generally include antibodies for CD14, CD20, CD1b, CD16, HLA-DR and CD8. Flow cytometry and cell sorting can also be used to separate the cell colonies of interest for use in the present invention.

[0132] Using the method as described herein, PBMC can be directly used for genetic modification with CAR. In some aspects, after separating PBMC, T lymphocytes are further separated, and in some aspects, before or after genetic modification and / or amplification, cytotoxic T lymphocytes and helper T lymphocytes can be classified into initial T cell subsets, memory T cell subsets, and effector T cell subsets. CD8+ cells can be obtained by using standard methods. In some aspects, by identifying the cell surface antigens associated with each of these types of CD8+ cells, CD8+ cells are further classified into initial cells, central memory cells, and effector cells. In some aspects, memory T cells are present in CD62L+ and CD62L- subsets of CD8+ peripheral blood lymphocytes. After staining with anti-CD8 and anti-CD62L antibodies, PBMCs are classified into CD62L-CD8+ and CD62L+CD8+ parts. In some aspects, the expression of phenotypic markers of central memory TCMs includes CD45RO, CD62L, CCR7, CD28, CD3 and CD127, and is negative for granzyme B. In some aspects, central memory T cells are CD45RO+, CD62L+, CD8+T cells. In some aspects, effector T cells are negative for CD62L, CCR7, CD28 and CD127, and are positive for granzyme B and perforin. In some aspects, naive CD8+T lymphocytes are characterized by the expression of phenotypic markers of naive T cells, including CD62L, CCR7, CD28, CD3, CD 127 and CD45RA.

[0133] In some aspects, CD4+T cells are further classified into subgroups. For example, by identifying a cell population with a cell surface antigen, CD4+T helper cells can be classified into naive cells, central memory cells, and effector cells. CD4+ lymphocytes can be obtained by standard methods. In some aspects, naive CD4+T lymphocytes are CD45RO-, CD45RA+, CD62L+CD4+T cells. In some aspects, central memory CD4+ cells are CD62L positive and CD45RO positive. In some aspects, effector CD4+ cells are CD62L and CD45RO negative.

[0134] Immune effector cells (such as T cells) can be genetically modified after being separated using known methods, or immune effector cells can be activated and amplified in vitro (or in the case of progenitor cells, differentiated) before being genetically modified. In some aspects, immune effector cells (such as T cells) are genetically modified with chimeric antigen receptors as described herein (for example, transduced with a viral vector comprising a nucleic acid encoding CAR), and then activated and amplified in vitro. Methods for activating and amplifying T cells are known in the art and are described in, for example, U.S. Patent Nos. 6,905,874, 6,867,041, 6,797,514; WO2012079000. Typically, such methods include contacting PBMC or separated T cells with stimulants and co-stimulators (such as anti-CD3 and anti-CD28 antibodies, usually attached to beads or other surfaces) in a culture medium with appropriate cytokines (such as IL-2). Anti-CD3 and anti-CD28 antibodies attached to the same beads are used as "alternative" antigen presenting cells (APC). In some aspects, T cell proliferation can be activated and stimulated using feeder cells and appropriate antibodies and cytokines using methods as described in US Pat. Nos. 6,040,177; 5,827,642; and WO2012129514.

[0135] The present invention provides a population of modified immune effector cells for treating a patient suffering from a malignancy caused by a tumor expressing Claudin-1, wherein the modified immune effector cells comprise an anti-Claudin-1 CAR as disclosed herein.

[0136] IV. How to use

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

[0138] Fibrotic tumors typically have a dense collagen network that causes small inter-fiber spacing in the stroma to delay the movement of particles greater than 10 nanometers (Netti PA et al. (2000) Cancer Res 60:2497-2503; Pluen A et al. (2001) Proc Natl Acad Sci 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 aspects, there is provided herein a method for promoting T cell-mediated anti-tumor activity in a subject with fibrotic tumors, the method comprising administering an anti-tight junction protein-1 antibody to the subject. In some aspects, the method for promoting T cell-mediated anti-tumor activity in a subject with fibrotic tumors also includes administering an immune checkpoint inhibitor.

[0139] 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 subject's tumor.

[0140] In some aspects, provided herein is a method for increasing the therapeutic efficacy of an immune checkpoint inhibitor in a subject having a fibrotic tumor, the method comprising a) administering an anti-claudin-1 antibody to the subject, wherein the anti-claudin-1 antibody promotes T cell-mediated anti-tumor activity in the fibrotic tumor; and b) administering an immune checkpoint inhibitor to the subject.

[0141] In some aspects, the anti-Claudin-1 antibody is administered prior to administration of the immune checkpoint inhibitor.

[0142] In some aspects, the anti-Claudin-1 antibody and the immune checkpoint inhibitor are administered simultaneously or sequentially.

[0143] In some aspects, the anti-Claudin-1 antibody and the immune checkpoint inhibitor are administered in the same composition.

[0144] In some aspects, the anti-Claudin-1 antibody and the immune checkpoint inhibitor are administered in different compositions.

[0145] In some aspects, the anti-Claudin-1 antibody and / or immune checkpoint inhibitor is administered intratumorally, intravenously, intraperitoneally, intramuscularly, intrathecally, or subcutaneously.

[0146] In some aspects, 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.

[0147] In some aspects, the immune checkpoint inhibitor is a small molecule inhibitor.

[0148] In some aspects, the immune checkpoint inhibitor is an antibody or an antigen-binding fragment thereof.

[0149] In some aspects, the immune checkpoint inhibitor is a PD-1 antagonist. In some aspects, the PD-1 antagonist is selected from the group consisting of nivolumab, pembrolizumab, cemiplimab, and dotalimumab.

[0150] In some aspects, the immune checkpoint inhibitor is a PD-L1 antagonist. In some aspects, the PD-L1 antagonist is selected from the group consisting of: atezolizumab, durvalumab, and avelumab.

[0151] In some aspects, the immune checkpoint inhibitor is a CTLA-4 antagonist. In some aspects, the CTLA-4 antagonist is selected from the group consisting of: ipilimumab and tremelimumab.

[0152] In some aspects, the immune checkpoint inhibitor is a LAG-3 antagonist (e.g., BI754111).

[0153] In some aspects, the immune checkpoint inhibitor is a TIM-3 antagonist (e.g., TSR-022 and LY3321367).

[0154] In some aspects, the immune checkpoint inhibitor is a VISTA (inhibitor of T-cell activated V-domain-containing immunoglobulin (Ig)) antagonist (e.g., CA-170 (anti-PD-L1 / L2 and anti-VISTA small molecule) and JNJ-61610588).

[0155] In some aspects, the immune checkpoint inhibitor is a B7-H3 antagonist.

[0156] In some aspects, the immune checkpoint inhibitor is a BTLA antagonist.

[0157] In some aspects, the immune checkpoint inhibitor is a Siglec-15 antagonist.

[0158] In some aspects, the immune checkpoint inhibitor is a TIGIT antagonist (e.g., BMS-986207, OMP-313M32, COM902 (CGEN-15137), and AB154). In some aspects, the TIGIT antagonist is selected from the group consisting of tisleliumab, osperlimab, dongvanalimumab, etilizumab, and vibrinomab.

[0159] In some aspects, the cancer comprises a fibrotic tumor.

[0160] In some aspects, fibrotic tumors are characterized by high expression of tight junction protein -1. In some aspects, the methods disclosed herein also include detecting the tight junction protein -1 expression level in a fibrotic tumor sample from a subject. In some aspects, the methods disclosed herein also include comparing the tight junction protein -1 expression level with the tight junction protein -1 expression level in a reference sample, wherein if the tight junction protein -1 expression level in the fibrotic tumor sample is increased relative to the tight junction protein -1 expression level in the reference sample, then the subject is administered an anti-tight junction protein -1 antibody and / or an immune checkpoint inhibitor as described herein.

[0161] In some aspects, the expression level of tight junction protein-1 in fibrotic tumor samples and / or reference samples is quantitatively tested by immunohistochemistry (IHC). In some aspects, the IHC test is calculated by the H-score method (see, for example, Parris, Toshima Z et al., BMC cancer Vol. 14: 324 (2014)). In some aspects, the H-score requires that each cell obtain a score of 0 to +3 (0 = negative staining; +1 = weak staining or low expression of the target antigen; +2 = medium staining or medium expression of the target antigen; and +3 = strong staining or high expression of the target antigen). In some aspects, the H-score is within the range of 0 to 300, wherein the H-score is calculated by adding the following: i) the percentage of cells scored as +1 in the sample, ii) twice the percentage of cells scored as +2 in the sample, and iii) three times the percentage of cells scored as +3 in the sample (i.e., H-score = (1*% 1+ cells) + (2*% 2+ cells) + (3*% 3+ cells)). In some aspects, high expression of Claudin-1 as graded by the H-score is between about 150 and about 300. In some aspects, moderate expression of Claudin-1 as graded by the H-score is between about 50 and about 149. In some aspects, low expression of Claudin-1 as graded by the H-score is between about 1 and about 49.

[0162] In some aspects, the expression of Claudin-1 is considered positive when the H-score is between about 1 and about 300. In some aspects, the expression of Claudin-1 is considered positive when the H-score is between about 50 and about 300. In some aspects, the expression of Claudin-1 is considered positive when the H-score is between about 150 and about 300.

[0163] To improve the treatment of fibrotic tumors, in some aspects, the present invention provides for identifying patients as having high expression of Claudin-1 and providing immunotherapy of anti-Claudin-1 antibodies and immune checkpoint inhibitors.

[0164] In another aspect, the present invention relates 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 aspects, the present invention includes methods 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.

[0165] In some aspects, the invention includes a method for selecting a fibrotic tumor in a human patient for immunotherapy, the method comprising: (a) determining the level of expression of Claudin-1 in a tumor sample; and (b) selecting the tumor for immunotherapy if the tumor sample has high expression of Claudin-1.

[0166] In some aspects, the invention includes a method for identifying a fibrotic tumor in a human patient as being amenable to immunotherapy, the method comprising: (a) determining the level of Claudin-1 expression in a tumor sample; and (b) identifying the tumor as being amenable to immunotherapy if the tumor sample has high Claudin-1 expression.

[0167] In some aspects, the invention includes a method for identifying a fibrotic tumor in a human patient that may be responsive to immunotherapy, the method comprising: (a) determining the level of expression of Claudin-1 in a tumor sample; and (b) identifying the tumor as likely to respond to treatment if the tumor has high expression of Claudin-1.

[0168] In some aspects, the invention includes a method for classifying a fibrotic tumor in a human patient as being likely to respond to immunotherapy, the method comprising: (a) determining the level of tight junction protein-1 expression in a tumor sample; and (b) if the tumor has high expression of tight junction protein-1, classifying the tumor as being likely to respond to immunotherapy. In some aspects, the immunotherapy comprises contacting the tumor with a therapeutically effective amount of an anti-tight junction protein-1 antibody and an immune checkpoint inhibitor.

[0169] In some aspects, the invention includes a method for identifying a patient having a fibrotic tumor who may respond to immunotherapy, the method comprising: (a) determining the level of expression of Claudin-1 in a tumor sample; and (b) identifying the patient who may respond to treatment if the tumor has high expression of Claudin-1.

[0170] In some aspects, the present invention includes a method for selecting a patient with a fibrotic tumor for immunotherapy, the method comprising: (a) determining the level of tight junction protein-1 expression in a tumor sample; and (b) if the tumor has high expression of tight junction protein-1, selecting the patient for immunotherapy. In some aspects, immunotherapy includes contacting the tumor with a therapeutically effective amount of an anti-tight junction protein-1 antibody and an immune checkpoint inhibitor.

[0171] In some aspects, the identifying comprises determining Claudin-1 expression in the fibrotic tumor.

[0172] In some aspects, Claudin-1 expression is determined by receiving the results of an assay capable of determining Claudin-1 expression.

[0173] In order to assess tight junction protein-1 expression, in some aspects, a test tissue sample is obtained from a patient in need of therapy. In some aspects, the test tissue sample includes but is not limited to any clinically relevant tissue sample, such as a tumor biopsy, a core biopsy tissue sample, a fine needle aspirate, or a body fluid sample, such as blood, plasma, serum, lymph, ascites, cystic fluid, or urine. In some aspects, the test tissue sample is from a primary tumor. In some aspects, the test tissue sample is from a metastatic lesion. In some aspects, at multiple time points, such as before treatment, during treatment, and / or after treatment, a test tissue sample is obtained from a subject. In some aspects, test tissue samples are obtained from different locations of a subject, such as a sample from a primary tumor and a sample from a distant metastatic lesion.

[0174] In some aspects, the test tissue sample is a paraffin-embedded fixed tissue sample. In some aspects, the test tissue sample is a formalin-fixed paraffin-embedded (FFPE) tissue sample. In some aspects, the test tissue sample is a fresh tissue (e.g., tumor) sample. In some aspects, the test tissue sample is a frozen or cryopreserved tissue sample. In some aspects, the test tissue sample is a fresh frozen (FF) tissue (e.g., tumor) sample. In some aspects, the test tissue sample is an archived tissue sample. In some aspects, the test tissue sample is an archived tissue sample with a known diagnosis, treatment, and / or outcome history. In some aspects, the sample is a tissue block. In some aspects, the test tissue sample is a dispersed cell. In some aspects, the sample size is about 1 cell to about 1x 10 6In some aspects, the sample size is from about 1 cell to about 1 x 10 5 In some aspects, the sample size is about 1 cell to about 10,000 cells. In some aspects, the sample size is about 1 cell to about 1,000 cells. In some aspects, the sample size is about 1 cell to about 100 cells. In some aspects, the sample size is about 1 cell to about 10 cells. In some aspects, the sample size is a single cell.

[0175] In another aspect, the assessment of the expression of Claudin-1 can be achieved without obtaining a test tissue sample. In some aspects, selecting a suitable patient comprises (i) optionally providing a test tissue sample obtained from a patient with tissue cancer, the test tissue sample comprising tumor cells and / or tumor-infiltrating inflammatory cells; and (ii) assessing the proportion of cells expressing Claudin-1 on the cell surface in the test tissue sample based on an assessment that the proportion of cells expressing Claudin-1 on the cell surface in the test tissue sample is above a predetermined threshold level.

[0176] However, in any method including measuring the expression of tight junction protein -1 in a test tissue sample, it should be understood that the step of providing a test tissue sample obtained from a patient is an optional step. That is, in some aspects, the method includes this step, and in other aspects, this step is not included in the method. It should also be understood that in some aspects, the "measurement" or "assessment" step of identifying or determining the number or ratio of cells expressing tight junction protein -1 in a test tissue sample is performed by a conversion method for determining tight junction protein -1 expression, such as by performing reverse transcriptase-polymerase chain reaction (RT-PCR) assay or IHC assay. In some other aspects, the conversion step is not involved, and tight junction protein -1 expression is assessed by, for example, checking the test result report from the laboratory. In some aspects, tight junction protein -1 expression is assessed by checking the results of immunohistochemical assays from the laboratory. In some aspects, the step of the method for assessing tight junction protein -1 expression provides an intermediate result, which can be provided to a physician or other health care provider for selecting a candidate for a combination therapy suitable for tight junction protein -1 inhibitors and immune checkpoint inhibitors. In some aspects, the step of providing an intermediate result until and including the method for assessing tight junction protein-1 expression can provide a doctor or other health care provider for selecting a candidate suitable for immune checkpoint inhibitor therapy. In some aspects, the step of providing an intermediate result is performed by a practitioner or a person acting under the guidance of a practitioner. In other aspects, these steps are performed by an independent laboratory or by an independent person (such as a laboratory technician).

[0177] In certain aspects of any of the methods of the invention, the proportion of cells expressing claudin-1 is assessed by an assay for detecting the presence of claudin-1 RNA. In another aspect, the presence of claudin-1 RNA is detected by RT-PCR, in situ hybridization, or RNase protection. In some aspects, the presence of claudin-1 RNA is detected by an RT-PCR-based assay. In some aspects, scoring the RT-PCR-based assay includes assessing the level of claudin-1 RNA expression in the test tissue sample relative to a predetermined level.

[0178] In other aspects, the proportion of cells expressing Claudin-1 is assessed by an assay for detecting the presence of Claudin-1 polypeptides. In another aspect, the presence of Claudin-1 polypeptides is detected by IHC, enzyme-linked immunosorbent assay (ELISA), in vivo imaging, or flow cytometry. In some aspects, Claudin-1 expression is determined by IHC. In other aspects of all of these methods, cell surface expression of Claudin-1 is determined using, for example, IHC or in vivo imaging.

[0179] In some aspects, the immunohistochemical assay is scored at low magnification. In some aspects, the low magnification is about 20X. In some aspects, the immunohistochemical assay is scored at high magnification. In some aspects, the high magnification is about 40X.

[0180] In some aspects, the immunohistochemical assays are scored by image analysis software. In some aspects, the immunohistochemical assays are scored by pathologist visual immune scoring. In some aspects, the immunohistochemical assays are scored manually.

[0181] In some aspects, the tumor is selected from the group consisting of a head and neck tumor, a lung tumor, a breast tumor, a melanoma tumor, a colorectal tumor, a pancreatic tumor, an esophageal tumor, a bile duct cancer, and a hepatocellular tumor.

[0182] In some aspects, the immune effector cells expressing CAR prepared as described herein can be used for methods and compositions of adoptive immunotherapy according to known techniques, or variations thereof that will be apparent to those skilled in the art based on the present 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.

[0183] In some aspects, the cells are formulated by first harvesting the cells from their culture medium, then washing and concentrating the cells in a culture medium and container system suitable for administration in a therapeutically effective amount (a "pharmaceutically acceptable" carrier). Suitable infusion media can be any isotonic medium formulation, typically normal saline, Normosol R (Abbott) or Plasma-Lyte A (Baxter), but 5% dextrose in water or Ringer's lactate may also be used. The infusion medium can be supplemented with human serum albumin.

[0184] In some aspects, the pharmaceutical composition comprises an anti-claudin-1 CAR-T cell and an immune checkpoint inhibitor. In some aspects, 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.

[0185] In some aspects, the immune checkpoint inhibitor is an inhibitor of PD-1, as described herein. In some aspects, the immune checkpoint inhibitor is an inhibitor of PD-L1, as described herein. In some aspects, the immune checkpoint inhibitor is an inhibitor of PD-L2, as described herein. In some aspects, the immune checkpoint inhibitor is an inhibitor of CTLA-4, as described herein. In some aspects, the immune checkpoint inhibitor is an inhibitor of LAG-3, as described herein. In some aspects, the immune checkpoint inhibitor is an inhibitor of TIM-3, as described herein. In some aspects, the immune checkpoint inhibitor is an inhibitor of TIGIT, as described herein. In some aspects, the immune checkpoint inhibitor is an inhibitor of VISTA, as described herein. In some aspects, the immune checkpoint inhibitor is an inhibitor of B7-H3, as described herein. In some aspects, the immune checkpoint inhibitor is an inhibitor of B7-H4, as described herein. In some aspects, the immune checkpoint inhibitor is an inhibitor of HVEM, as described herein. In some aspects, the immune checkpoint inhibitor is an inhibitor of GAL-9, as described herein. In some aspects, the immune checkpoint inhibitor is an inhibitor of KIR, as described herein. In some aspects, the immune checkpoint inhibitor is an inhibitor of BTLA, as described herein. In some aspects, the immune checkpoint inhibitor is an inhibitor of IDO, as described herein. In some aspects, the immune checkpoint inhibitor is an inhibitor of Siglec-15, as described herein.

[0186] In some aspects, the immune effector cell colony expressing CAR of the present invention can be applied alone, or as a pharmaceutical composition combined with a diluent and / or with other components (such as immune checkpoint inhibitors as described herein). In short, the pharmaceutical composition of the present invention may include a combination of immune effector cell colonies (such as T cells as described herein) expressing CAR and one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients. Such compositions may include: buffer, such as neutral buffered saline, phosphate buffered saline, etc.; carbohydrates, such as glucose, mannose, sucrose or dextran, mannitol; protein; polypeptide or amino acid, such as glycine; antioxidant; chelating agent, such as EDTA or glutathione; adjuvant (e.g., aluminum hydroxide); and preservative. The composition of the present invention is preferably formulated for intravenous administration.

[0187] In some aspects, 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 cellular immune responses mediated by cytotoxic T cells capable of killing infected cells, regulatory T cell responses, and helper T cell responses. Humoral immune responses mediated primarily by helper T cells capable of activating B cells to produce antibodies may also be induced. Various techniques can be used to analyze the types of immune responses induced by the compositions of the present invention, which are fully described in the art; for example, Current Protocols in Immunology, Editors: John E. Coligan, Ada M. Kruisbeek, David H. Margulies, Ethan M. Shevach, Warren Strober (2001) John Wiley & Sons, NY, NY.

[0188] In some aspects, the present invention provides a method for promoting T cell-mediated anti-tumor activity in a subject with a fibrotic tumor, the method comprising administering anti-claudin-1 chimeric antigen receptor (CAR) T cells to the subject. In some aspects, including administering an immune checkpoint inhibitor.

[0189] In some aspects, the present invention provides a method for treating cancer in a subject with 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 aspects, the anti-claudin-1 CAR T cells promote T cell-mediated anti-tumor activity in the subject's tumor.

[0190] In some aspects, the present invention provides a method for increasing the therapeutic efficacy of an immune checkpoint inhibitor in a subject with a fibrotic tumor, the method comprising administering an anti-claudin-1 CAR T cell to the subject and administering an immune checkpoint inhibitor to the subject. In some aspects, the anti-claudin-1 CAR T cell promotes T cell-mediated anti-tumor activity in fibrotic tumors.

[0191] V. Immune checkpoint inhibitors

[0192] Immune checkpoint proteins interact with specific ligands that send signals to T cells that inhibit T cell function. Cancer cells take advantage of this by driving high levels of checkpoint proteins on their surface, thereby suppressing anti-cancer immune responses.

[0193] Immune checkpoint inhibitors include any compound that can inhibit the function of immune checkpoint proteins. Inhibition includes reduced function and complete blocking. In some aspects, immune checkpoint proteins are human checkpoint proteins. Therefore, in some aspects, immune checkpoint inhibitors are preferably inhibitors of human immune checkpoints.

[0194] In some aspects, 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. The pathways involving LAG-3, BTLA, B7-H3, B7-H4, TIM-3, and KIR constitute immune checkpoint pathways similar to CTLA-4 and PD-1 dependent pathways (see, e.g., Pardoll, 2012, Nature Rev Cancer 12: 252-264; Mellman et al., 2011, Nature 480: 480-489). In some aspects, 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 aspects, 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.

[0195] In some aspects, the immune checkpoint inhibitor is an inhibitor of PD-1. In some aspects, the immune checkpoint inhibitor is an inhibitor of PD-L1. In some aspects, the immune checkpoint inhibitor is an inhibitor of CTLA-4. In some aspects, the immune checkpoint inhibitor is an inhibitor of LAG-3. In some aspects, the immune checkpoint inhibitor is an inhibitor of TIM-3. In some aspects, the immune checkpoint inhibitor is an inhibitor of TIGIT. In some aspects, the immune checkpoint inhibitor is an inhibitor of VISTA. In some aspects, the immune checkpoint inhibitor is an inhibitor of B7-H3. In some aspects, the immune checkpoint inhibitor is an inhibitor of BTLA. In some aspects, the immune checkpoint inhibitor is an inhibitor of Siglec-15.

[0196] In some aspects, the immune checkpoint inhibitor is an antibody.

[0197] In some aspects, 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 aspects, the immune checkpoint inhibitor is a monoclonal antibody, a fully human antibody, a chimeric antibody, a humanized antibody, or a fragment thereof that can at least partially antagonize the following: 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.

[0198] In some aspects, the immune checkpoint inhibitor is an antibody or fragment thereof that specifically binds to PD-1. In some aspects, the immune checkpoint inhibitor is an antibody or fragment thereof that specifically binds to PD-L1. In some aspects, the immune checkpoint inhibitor is an antibody or fragment thereof that specifically binds to CTLA-4. In some aspects, the immune checkpoint inhibitor is an antibody or fragment thereof that specifically binds to LAG-3. In some aspects, the immune checkpoint inhibitor is an antibody or fragment thereof that specifically binds to TIM-3. In some aspects, the immune checkpoint inhibitor is an antibody or fragment thereof that specifically binds to TIGIT. In some aspects, the immune checkpoint inhibitor is an antibody or fragment thereof that specifically binds to VISTA. In some aspects, the immune checkpoint inhibitor is an antibody or fragment thereof that specifically binds to B7-H3. In some aspects, the immune checkpoint inhibitor is an antibody or fragment thereof that specifically binds to BTLA. In some aspects, the immune checkpoint inhibitor is an antibody or fragment thereof that specifically binds to Siglec-15.

[0199] In some aspects, the pharmaceutical composition comprises an anti-claudin-1 antibody or a biologically active fragment thereof and a CTLA-4 inhibitor, preferably a monoclonal antibody that specifically binds to (and inhibits) CTLA-4. The complete human CTLA-4 nucleic acid sequence can be found under GenBank accession number NG_011502.1. Monoclonal antibodies that specifically bind to CTLA-4 include, but are not limited to, ipilimumab ( BMS) and tremelimumab (AstraZeneca / MedImmune); and 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 U.S. Patent Nos. 5,811,097, 5,855,887, 6,05 No. 1,227, No. 6,984,720, No. 6,682,736, No. 6,207,156, No. 5,977,318, No. 6,682,736, No. 7,109,003, No. 7,132,281 and No. 8,491,895, the contents of each of which are incorporated herein by reference; or an antibody comprising the heavy chain and light chain variable regions of any of these antibodies. Human monoclonal antibodies that specifically bind to CTLA-4 with high affinity are disclosed in U.S. Pat. No. 6,984,720. Other anti-CTLA-4 monoclonal antibodies have been described in, for example, U.S. Pat. No. 7,034,121 and International Publication Nos. WO 2012 / 122444, WO 2007 / 113648, WO 2016 / 196237, and WO 2000 / 037504. In some aspects, the immune checkpoint inhibitor is a CTLA-4 antagonist. In some aspects, the CTLA-4 antagonist is selected from the group consisting of: ipilimumab and tremelimumab.

[0200] In some aspects, 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 (and inhibits) PD-1. The complete nucleotide sequence and amino acid sequence of human PD-1 can be found under GenBank accession numbers NG_012110.1 and NP_005009.2. In some aspects, the anti-PD-1 antibody is nivolumab. Nivolumab (also known as BMS-936558; formerly known as 5C4, BMS-936558, MDX-1106 or ONO-4538) is a fully human IgG4 (S228P) PD-1 immune checkpoint inhibitor antibody that selectively prevents interaction with PD-1 ligands (PD-L1 and PD-L2), thereby blocking 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 aspect, the anti-PD-1 antibody or fragment thereof cross-competes with nivolumab. In other aspects, the anti-PD-1 antibody or fragment thereof binds to the same epitope as nivolumab. In certain aspects, the anti-PD-1 antibody has the same CDRs as nivolumab.

[0201] In another aspect, 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. Pat. Nos. 8,354,509 and 8,900,587.

[0202] Anti-human PD-1 antibodies (or VH and / or VL domains derived therefrom) suitable for use in the present invention can be produced using methods well known in the art. Alternatively, art-recognized anti-PD-1 antibodies can be used. For example, monoclonal antibodies 5C4 (referred to herein as nivolumab or BMS-936558), 17D8, 2D3, 4H1, 4A11, 7D3, and 5F4 described in WO 2006 / 121168 can be used, the teachings of which are incorporated herein by reference. Other known PD-1 antibodies include lambrolizumab (MK-3475) described in WO 2008 / 156712 and AMP-514 described in WO 2012 / 145493, the teachings of which are incorporated herein by reference. Other known anti-PD-1 antibodies and other PD-1 inhibitors include those described in WO 2009 / 014708, WO 03 / 099196, WO 2009 / 114335, and WO 2011 / 161699, the teachings of which are incorporated herein by reference. In some aspects, the anti-PD-1 antibody is REGN2810. In some aspects, 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 may also be used.

[0203] Other anti-PD-1 monoclonal antibodies have been described, for example, in 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. WO 2012 / 145493, WO 2008 / 156712, WO 2015 / 112900, WO 2012 / 145493, WO 2015 / 112800, WO 2014 / 206107, WO 2015 / 35606, WO 2015 / 085847, WO 2014 / 179664, WO 2017 / 020291, WO 2018 / 19491; and PCT Publication Nos. WO 2014 / 156712, WO 2015 / 112900, WO 2012 / 145493, WO 2015 / 112800, No. 2017 / 020858, No. WO 2016 / 197367, No. WO2017 / 024515, No. WO 2017 / 025051, No. WO 2017 / 123557, No. WO 2016 / 106159, No. WO2014 / 194302, No. WO No. 2017 / 040790, No. WO 2017 / 133540, No. WO 2017 / 132827, No. WO2017 / 024465, No. WO 2017 / 025016, No. WO 2017 / 106061, No. WO No. 2017 / 19846, No. WO2017 / 024465, No. WO 2017 / 025016, WO 2017 / 132825, and WO 2017 / 133540, each of which is incorporated herein by reference.

[0204] In some aspects, the anti-PD-1 antibody is selected from the group consisting of: nivolumab (also known as 5C4, BMS-936558, MDX-1106, and ONO-4538), pembrolizumab (Merck; also known as ), JS001 (TAIZHOU JUNSHIPHARMA; see Si-Yang Liu et al., J. Hematol. Oncol. 10:136 (2017)), BGB-A317 (Beigene; see WO 2015 / 35606 and US 2015 / 0079109), INCSHR1210 (Jiangsu Hengrui Medicine; also known as SHR-1210; see WO 2015 / 145493), and MK-3475 (lambda3 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), Cemiplimab (Regeneron; also known as REGN-2810; see WO 2015 / 112800), 2015 / 085847; Si-Yang Liu et al., J. Hematol. Oncol. 10:136 (2017)), TSR-042 (Tesar Biopharmaceutical; also known as ANB011; see WO 2014 / 179664), GLS-010 (Wuxi / Harbin Gloria Pharmaceuticals; also known as WBP3055; see Si-Yang Liu et al., J. Hematol. Oncol. 10:136 (2017)), AM-0001 (Armo), STI-1110 (Sorrento Therapeutics; see WO 2014 / 194302), AGEN2034 (Agenus; see WO 2017 / 040790), MGA012 (Macrogenics, see WO 2017 / 19846) and IBI308 (Innovent; see WO 2017 / 024465, WO 2017 / 025016, WO 2017 / 132825 and WO 2017 / 133540), which are incorporated herein by reference.

[0205] In another aspect, the anti-PD-1 antibody or antigen-binding fragment thereof cross-competes with pembrolizumab. In some aspects, the anti-PD-1 antibody or antigen-binding fragment thereof binds to the same epitope as pembrolizumab. In certain aspects, the anti-PD-1 antibody or antigen-binding fragment thereof has the same CDRs as pembrolizumab. In another aspect, the anti-PD-1 antibody is pembrolizumab. Pembrolizumab (also known as Pembrolizumab (Pembro and MK-3475) is a humanized monoclonal IgG4 antibody against the human surface receptor PD-1 (programmed death-1 or programmed cell death-1). Pembrolizumab is described in, for example, U.S. Pat. Nos. 8,354,509 and 8,900,587. Pembrolizumab has been approved by the FDA for the treatment of relapsed or refractory melanoma.

[0206] In some aspects, the PD-1 antagonist is selected from the group consisting of nivolumab, pembrolizumab, cemiplimab, and dotarizumab.

[0207] In some aspects, 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 (and 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 can be used, the contents of which are incorporated herein by reference. 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 the following: U.S. Pat. Nos. 7,635,757 and 8,217,149; U.S. Publication No. 2009 / 0317368; and PCT Publication Nos. WO 2011 / 066389 and WO 2012 / 145493, the teachings of which are also incorporated by reference herein. 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 with any of these art-recognized antibodies or inhibitors for binding to PD-L1 can also be used.

[0208] In certain aspects, the anti-PD-L1 antibody is BMS-936559 (formerly known as 12A4 or MDX-1105) (see, e.g., U.S. Pat. No. 7,943,743; WO 2013 / 173223). In other aspects, 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. Pat. No. 8,217,149), MEDI4736 (Khleif, 2013, Proceedings from the European Cancer Congress 2013; September 27-October 1, 2013; Amsterdam, The Netherlands. Abstract 802) or MSB0010718C (also known as avelumab; see US2014 / 0341917). In certain aspects, an antibody that cross-competes with the above-mentioned PD-L1 antibodies for binding to human PD-L1 or binds to the same epitope region of human PD-L1 as the above-mentioned PD-L1 antibodies is a mAb. For administration to human subjects, these cross-competing antibodies may be chimeric antibodies, or may be humanized antibodies or human antibodies. Such chimeric, humanized or human mAbs may be prepared and isolated by methods well known in the art. In certain aspects, 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 MPDL3280A, RG7446; see US 8,217,149; see also Herbst et al. (2013) J Clin Oncol 31(Suppl):3000), durvalumab (AstraZeneca; also known as IMFINZI TM , MEDI-4736; see WO 2011 / 066389), Avelumab (Pfizer; also known as MSB-0010718C; see WO 2013 / 079174), STI-1014 (Sorrento; see WO 2013 / 181634), CX-072 (Cytomx; see WO 2016 / 149201), KN035 (3D Med / Alphamab; see Zhang et al., Cell Discov. 7:3 (March 2017), LY3300054 (Eli Lilly Co.; see, e.g., WO 2017 / 034916), and CK-301 (Checkpoint Therapeutics; see Gorelik et al., AACR: Abstract 4606 (April 2016)).

[0209] In some aspects, the pharmaceutical composition comprises an anti-Claudin-1 antibody or a biologically active fragment thereof and a PD-L2 inhibitor, such as MIH18 (described in Pfistershammer et al., Eur J Immunol. 36: 1104-1113 (2006).

[0210] In some aspects, the pharmaceutical composition comprises an anti-Claudin-1 antibody or a biologically active fragment thereof and a LAG-3 inhibitor. In some aspects, the LAG-3 inhibitor is an anti-LAG-3 antibody.

[0211] Anti-human LAG-3 antibodies (or VH / VL domains derived therefrom) suitable for use in the present invention can be produced using methods well known in the art. Alternatively, art-recognized anti-LAG-3 antibodies can be used. For example, the anti-human LAG-3 antibodies described in US2011 / 0150892A1 can be used, the teachings of which are incorporated herein by reference, and are referred to as monoclonal antibody 25F7 (also referred to as "25F7" and "LAG3.1"). Other art-recognized anti-LAG-3 antibodies that can be used include IMP731 (H5L7BW) described in US2011 / 007023, MK-4280 (28G-10) described in WO 2016028672, Journal for ImmunoTherapy of Cancer, (2016) Vol. 4, Supplement. REGN3767 described in Supplement 1 Abstract No. P195, BAP050 described in WO2017 / 019894, IMP-701 (LAG-525), IMP321 (eftilagimod alpha), Sym022, TSR-033, MGD 013, BI754111, FS118, AVA-017 and GSK2831781. These and other anti-LAG-3 antibodies useful in the claimed invention can be found, for example, in WO2016 / 028672, WO2017 / 106129, WO2017 / 062888, WO2009 / 044273, WO2018 / 069500, WO2016 / 126858, WO2014 / 179664, WO2016 / 200782, WO2015 / 200119, WO2017 / 019846, WO2017 / 198741, WO2017 / 2205 55, WO2017 / 220569, WO2018 / 071500, WO2017 / 015560, WO2017 / 025498, WO2017 / 087589, WO2017 / 087901, WO2018 / 083087, WO2017 / 149143, WO2017 / 219995, US2017 / 0260271, WO2017 / 086367, WO / 2017 / 086419, WO2018 / 034227 and WO2014 / 140180. The contents of each of these references are incorporated herein by reference.

[0212] In some aspects, the pharmaceutical composition comprises an anti-Claudin-1 antibody or a biologically active fragment thereof and a BLTA inhibitor, such as antibody 4C7 disclosed in US Pat. No. 8,563,694, which is incorporated herein by reference.

[0213] In some aspects, the pharmaceutical composition comprises an anti-claudin-1 antibody or a biologically active fragment thereof and a B7-H4 checkpoint inhibitor, such as an antibody disclosed in U.S. Patent Application Publication No. 2014 / 0294861 (incorporated herein by reference) or a soluble recombinant form of B7-H4 as disclosed in U.S. Patent Application Publication No. 2012 / 0177645 (incorporated herein by reference).

[0214] In some aspects, the pharmaceutical composition comprises an anti-Claudin-1 antibody or a biologically active fragment thereof and a B7-H3 checkpoint inhibitor, such as antibody MGA271 disclosed as BRCA84D or a derivative as disclosed in U.S. Patent Application Publication No. 2012 / 0294796 (incorporated herein by reference).

[0215] In some aspects, the pharmaceutical composition comprises an anti-claudin-1 antibody or a biologically active fragment thereof and a TIM-3 checkpoint inhibitor, such as an antibody disclosed in U.S. Pat. No. 8,841,418 (incorporated herein by reference) or an anti-human TIM-3 blocking antibody F38-2E2 disclosed in Jones et al., J. Exp. Med., 205(12):2763-79 (2008).

[0216] In some aspects, the pharmaceutical composition comprises an anti-Claudin-1 antibody or a biologically active fragment thereof and a KIR checkpoint inhibitor, such as the antibody lirilumab (described in Romagne et al., Blood, 114(13):2667-2677 (2009)).

[0217] In some aspects, the pharmaceutical composition comprises an anti-tight junction protein-1 antibody or its biologically active fragment and a TIGIT inhibitor.TIGIT checkpoint inhibitors preferably inhibit the interaction of TIGIT with poliovirus receptor (CD155), and include but are not limited to antibodies targeting human TIGIT, such as U.S. Patent No. 9,499,596 (incorporated herein by reference) and U.S. Patent Application Publication No. 2016 / 0355589, No. 2016 / 0176963 (incorporated herein by reference), and poliovirus receptor variants, such as U.S. Patent No. 9,327,014 (incorporated herein by reference) disclosed in those. In some aspects, immune checkpoint inhibitors are TIGIT antagonists. In some aspects, TIGIT antagonists are selected from the group consisting of: tirilimumab, osperlimumab, dongwanali monoclonal antibody, etilimonumab and webolimumab.

[0218] In some aspects, the pharmaceutical composition comprises an anti-claudin-1 antibody or a biologically active fragment thereof and an IDO inhibitor (indoleamine-pyrrole 2,3-dioxygenase. IDO is considered 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 resistance to anti-CTLA-4 therapy. In some aspects, IDO inhibitors for use according to the methods described herein include, but are not limited to, tryptophan mimetics such as D-1MT (D isoform of 1-methyl-DL-tryptophan (MT)), L-1MT (L isoform of MT), MTH-trp (methylthiohydantoin-dl-tryptophan; a transcriptional inhibitor of IDO) and β-carboline; indole mimetics such as naphthoquinone-based agents S-allyl-brassinin, S-benzyl-brassinin, 5-bromo-brassinin, and phenylimidazole-based agents 4-phenylimidazole, exiguamine A (exiguamine A), icadolstat, rosmarinic acid, norharman and NSC401366. In some aspects, IDO inhibitors include INCB 024360 (icadolstat; N'-(3-bromo-4-fluorophenyl)-N-hydroxy-4-[2-(sulfamoylamino)ethylamino]-1,2,5-oxadiazole-3-carboxamidine), indolemod ((2R)-2-amino-3-(1-methylindol-3-yl)propionic acid), IDO peptide vaccine (Copenhagen University) and NLG919 (NewLink Genetics; 1-cyclohexyl-2-(5H-imidazo[5,1-a]isoindol-5-yl)ethanol). In some aspects, IDO inhibitors preferably inhibit metabolic pathways and include, but are not limited to, norharman (see Chiarugi A et al., "Combined inhibition of indoleamine 2,3-dioxygenase and nitric oxide synthase modulates neurotoxin release by interfero n-gamma-activated macrophages", Journal of Leukocyte Biology. 68(2):260-6.(2000)), rosmarinic acid (see Lee HJ et al., "Rosmarinic acid inhibits indoleamine2,3-dioxygenase expression in murine dendritic cells", BiochemicalPharmacology.73(9):1412-21(2007)), COX-2 inhibitors (see Cesario A et al., “The interplay between indoleamine 2,3-dioxygenase 1(IDO1)and cyclooxygenase(COX)-2in 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-tryptophancorrelates with antitumor responses”. Cancer Research.67(2):792-801(2007) and Chauhan N et al., (April 2009), “Reassessment of the reaction mechanismin the hemedioxygenases”.Journal of the American Chemical Society Society. 131(12):4186-7(2009)), including, for example, a specific racemic 1-methyl-D-tryptophan (called indomod), icaridostat (INCB 24360), navoximod (GDC-0919) (see Jochems C et al., "The IDO1 selective inhibitorepacadostat enhances dendritic cell imm unogenicity and lytic ability of tumor antigen-specific T cells", Onc otarget. 7(25):37762-37772. (2016)) and or BMS-986205. In some aspects, the IDO inhibitor is selected from norharman, rosmarinic acid, a COX-2 inhibitor, 1-methyltryptophan, indomod, icaridostat (INCB24360), navoximod (GDC-0919) and / or BMS-986205. .

[0219] In some aspects, the pharmaceutical composition comprises an anti-claudin-1 antibody or a biologically active fragment thereof and a TIGIT inhibitor. TIGIT checkpoint inhibitors preferably inhibit the interaction of TIGIT with the poliovirus receptor (CD155), and include but are not limited to antibodies targeting human TIGIT, such as those disclosed in U.S. Patent No. 9,499,596 (incorporated herein by reference) and U.S. Patent Application Publication No. 2016 / 0355589, No. 2016 / 0176963 (incorporated herein by reference), and poliovirus receptor variants, such as those disclosed in U.S. Patent No. 9,327,014 (incorporated herein by reference).

[0220] In some aspects, the immune checkpoint inhibitor is an antagonist of IDO1 (indoleamine-2,3-dioxygenase 1) (e.g., indomod (NLG8189, 1-methyl-D-TRP), icadolstat (INCB-024360), KHK2455, PF-06840003 (PCT Publication No. WO 2016 / 181348A1), pyrrolidine-2,5-dione derivatives (PCT Publication No. WO 2016 / 181348A1), 2015 / 173764A1), navomod (RG6078, GDC-0919, NLG919) and BMS-986205 (F001287)); KIR (killer cell immunoglobulin-like receptor) (e.g., lirelumab (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. WO2015140717 A1, WO 2017025868A1, WO 2016147144 A1), 3-(indol-3-yl)-pyridine derivatives (U.S. Publication No. 20150225367A1 and PCT Publication No. WO 2015121812A1); dual IDO / TDO (e.g., small molecule dual IDO / TDO inhibitors disclosed in PCT Publication Nos. WO 2015150097 A1, WO 2015082499A2, WO 2016026772A1, WO2016071283A1, WO 2016071293 A2, and WO 2017007700 A1); CD40 (e.g., Lineage BMS3h-56 (U.S. Patent No. 9,475,879), Lukatumomab (HCD122 and CHIR-12.12), CHIR-5.9 and daclizumab (huS2C6, PRO 64553, RG 3636, SGN 14, SGN-40)); adenosine A2a receptor (A2aR) (e.g., CPI-444, PBF-509, istradefylline (KW-6002), rendant (SCH420814), tozanatin (SYN115), vepadnan (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., cetulimumab amunalegin (cer gutuzumabamunaleukin (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-associated immunoglobulin domain-containing protein, CD122R) (e.g., COM701); GARP (major glycoprotein A repeat) (e.g., ARGX-115); CD80 (e.g., galiximab (IDEC-114) and AV 1142742 (RhuDex); CD86; and CD96.

[0221] In some aspects, 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-linked 2',5'-3',5' cyclic dinucleotides (PCT Publication No. WO 2017 / 075477 A1); 2'-fluoro-substituted bis-3',5' cyclic dinucleotides and 2',2"-diF-Rp,Rp, bis-3',5' cyclic dinucleotides (PCT Publication No. WO 2016 / 145102 A1); and fluorinated cyclic dinucleotides (PCT Publication No. WO 2016 / 096174 A1); or CD20 (e.g., and ABP 798).

[0222] As known to those skilled in the art, alternative and / or equivalent names may be used for some of the above antibodies. In the context of the present invention, such alternative and / or equivalent names are interchangeable.

[0223] VI. Application

[0224] The required dose of anti-tight junction protein-1 antibody, its biologically active fragment or anti-tight junction protein-1 CAR T cell (optionally after being formulated with one or more suitable pharmaceutically acceptable carriers or excipients) can be administered to a subject in need by any suitable route. In some aspects, anti-tight junction protein-1 antibody, its biologically active fragment or anti-tight junction protein-1 CAR T cell is delivered in combination with immune checkpoint inhibitors as described above. Various delivery systems are known and can be used to administer antibodies, including tablets, capsules, injectable solutions, capsules in liposomes, microparticles, microcapsules, etc. The method of administration includes but is not limited to skin, intradermal, intramuscular, intraperitoneal, intralesional, intravenous, subcutaneous, intranasal, pulmonary, epidural and oral routes. Anti-tight junction protein-1 antibody, its biologically active fragment, anti-tight junction protein-1 CART cell or its pharmaceutical composition and immune checkpoint inhibitor can be administered by any convenient or other appropriate route, such as by infusion or push injection, absorbed by epithelial or mucosal lining (e.g., oral mucosa, bronchial mucosa, rectal and intestinal mucosa, etc.). Administration can be systemic or local. As will be appreciated by those of ordinary skill in the art, in the context of an antibody being administered in combination with another therapeutic agent (e.g., an immune checkpoint inhibitor), the antibody and therapeutic agent can be administered by the same route (e.g., intravenously) or by different routes (e.g., intravenously, orally or subcutaneously).

[0225] In some aspects, the anti-Claudin-1 antibody or anti-Claudin-1 T cells and immune checkpoint inhibitor are administered intratumorally, intravenously, intraperitoneally, intramuscularly, intrathecally, or subcutaneously.

[0226] In some aspects, the anti-Claudin-1 antibody or anti-Claudin-1 CAR T cells are administered prior to administration of the immune checkpoint inhibitor.

[0227] In some aspects, the anti-Claudin-1 antibody, or anti-Claudin-1 CAR T cells and the immune checkpoint inhibitor are administered simultaneously or sequentially.

[0228] In some aspects, the anti-Claudin-1 antibody, or anti-Claudin-1 CAR T cell, and the immune checkpoint inhibitor are administered in the same composition.

[0229] In some aspects, the anti-Claudin-1 antibody, or anti-Claudin-1 CAR T cells and the immune checkpoint inhibitor are administered in different compositions.

[0230] Anti-claudin-1 antibodies or biologically active fragments thereof (optionally after formulation with one or more appropriate pharmaceutically acceptable carriers or excipients), or anti-claudin-1 CAR T cells, will be administered at a dose that makes the amount delivered effective for the intended purpose. The route of administration, the dosage of formulation and administration will depend on the desired therapeutic effect, the severity of the condition to be treated (if already present), the presence of any infection, the age, sex, weight and general health of the patient, as well as the efficacy, bioavailability and half-life in vivo of the antibody or composition used, the use (or non-use) of concomitant therapy and other clinical factors. These factors can be easily determined by the attending physician during the course of treatment. Alternatively or in addition, the dose to be administered can be determined based on studies using animal models (e.g., non-human primates or rodents). Adjusting the dose based on these or other methods to achieve maximum efficacy is well known in the art and is within the capabilities of trained physicians. When anti-claudin-1 antibodies or anti-claudin-1 CAR T cells are used for research, further information about appropriate dose levels and duration of treatment will appear.

[0231] Treatment according to the present invention may consist of a single dose or multiple doses. Therefore, the administration of anti-claudin-1 antibodies or their biologically active fragments, or anti-claudin-1 CAR T cells (or their pharmaceutical compositions) may be continued for a period of time or periodically administered and administered at specific intervals, such as hourly, daily, weekly (or at some other multi-day intervals), monthly, annually (e.g., in a time-release form). Alternatively, delivery may occur multiple times within a given time period, such as twice a week or more; twice a month or more, etc. Delivery may be continuous delivery for a period of time, such as intravenous delivery.

[0232] Typically, the amount of anti-claudin-1 antibody or biologically active fragment thereof, or anti-claudin-1 CAR T cell (or 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, for example, between about 100 ng / kg to about 50 mg / kg of the subject's body weight; or between about 1 μg / kg to about 10 mg / kg of the subject's body weight, or between about 100 μg / kg and about 1 mg / kg of the subject's body weight.

[0233] VII. Pharmaceutical Compositions

[0234] As described above, anti-claudin-1 antibodies (and related molecules), or anti-claudin-1 CAR T cells, can be administered alone or as a pharmaceutical composition. Therefore, the present invention provides a pharmaceutical composition comprising an effective amount of an anti-claudin-1 antibody as described herein or a biologically active fragment thereof and at least one pharmaceutically acceptable carrier or excipient. In some aspects, the composition further comprises one or more additional bioactive agents. In some aspects, one or more additional bioactive agents are immune checkpoint inhibitors.

[0235] In some aspects, the pharmaceutical composition is for use in a method of promoting T cell-mediated anti-tumor activity in a subject having a fibrotic tumor.

[0236] In some aspects, the pharmaceutical composition is for use in a method of treating cancer in a subject having a solid tumor.

[0237] In some aspects, the pharmaceutical composition is used in a method of increasing the therapeutic efficacy of an immune checkpoint inhibitor in a subject having a fibrotic tumor.

[0238] The pharmaceutical composition can be administered in any amount and using any administration route that is effective to achieve the desired preventive and / or therapeutic effect. The optimal pharmaceutical formulation can vary depending on the route of administration and the desired dosage. Such formulations can affect the physical state, stability, in vivo release rate, and in vivo clearance rate of the administered active ingredient.

[0239] The pharmaceutical composition of the present invention can be formulated into a dosage unit form that is easy to apply and uniform in dosage. In some aspects, anti-claudin-1 antibodies and immune checkpoint inhibitors are formulated together. In some aspects, anti-claudin-1 antibodies and immune checkpoint inhibitors are formulated separately. However, it should be understood that the total daily dose of the composition will be determined by the attending physician within the scope of reasonable medical judgment.

[0240] VIII. Kit

[0241] 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 ingredients of the pharmaceutical compositions of the present invention, thereby allowing for administration of anti-claudin-1 antibodies or biologically active fragments thereof, or anti-claudin-1 CAR T cells.

[0242] The different components of a pharmaceutical package or test kit can be supplied in solid (e.g., lyophilized) or liquid form. Each component is generally suitable for being equally divided in its respective container or provided in a concentrated form. The pharmaceutical package or test kit may include a medium for reconstructing the lyophilized components. Each container of the test kit preferably remains tightly closed for commercial sale.

[0243] In some aspects, the pharmaceutical package or kit includes one or more additional therapeutic agents as described above. Optionally, associated with the container may be a notice or package insert in a form prescribed by a governmental agency that regulates the manufacture, use or sale of a drug or biological product, which notice reflects the agency's approval of manufacture, use or sale for human administration. The notice or package insert may contain instructions for use of the pharmaceutical composition according to the methods of treatment disclosed herein.

[0244] An identifier (e.g., barcode, radio frequency, ID tag, etc.) may be present in or on the reagent kit. For example, the identifier may be used to uniquely identify the reagent kit for purposes such as quality control, inventory control, tracking movement between workstations, etc.

[0245] Example

[0246] The following embodiments are illustrative and do not limit the scope of the claimed aspects.

[0247] Example 1. Expression of Claudin-1 in the Tumor Microenvironment

[0248] High CLDN1 expression is associated with an immunologically low or inactive tumor microenvironment. The alteration of tumor cell phenotype by mAb treatment may lead to the activation of T cells in the tumor microenvironment through changes in the tumor cell secretome and / or metalloproteinases. Fig. 8A ). Figure 8B The authors show that administration of anti-CLDN1 antibodies can disrupt the tumor barrier to help switch the tumor state from one that excludes T cells to one that allows T cell infiltration.

[0249] Example 2. Claudin-1 expression in fibrotic tumor types

[0250] More than 1200 paraffin-embedded tumor biopsies from 12 different indications were stained by immunohistochemistry for expression of tight junction protein-1 (CLDN1), T cell markers (CD3), and fibrosis (Sirius red staining) in head and neck squamous cell carcinoma (HNSCC), colorectal cancer (CRC), esophageal cancer, squamous non-small cell lung cancer (Sq. NSCLC), intrahepatic cholangiocarcinoma (iCCA), hepatocellular carcinoma (HCC), and urothelial carcinoma (see Fig. 9Samples were scored using a semiquantitative H-score, which calculates the sum of the percentage and intensity of positively stained tumor cells within the invasive tissue component (negative staining = 0; weak staining = 1+; moderate staining = 2+; strong staining = 3+), where 0 indicates negative staining, 1-49 indicates low expression, 50-149 indicates moderate expression, and 150-300 indicates high expression (see, e.g., Parris, Parris, Toshima Z et al., BMC cancer Vol. 14: 324 (2014)).

[0251] Slides were stained with a Roche Discovery Ultraautostainer autostainer. Slides were baked at 60 ° C for 1 hour and then dewaxed using a standard autostainer protocol. Heat-induced epitope retrieval (HIER) was performed at 95 ° C for 48 minutes using Roche CC1 (high pH). Peroxidase inhibitors were applied. The primary antibody (anti-CLDN1, Sigma-Aldrich HPA048319, 1:50) was incubated at 37 ° C for 24 minutes. The secondary antibody Roche anti-rabbit HQ was incubated at 37 ° C for 8 minutes, followed by Roche anti-HQ HRP incubated at 37 ° C for 8 minutes. DAB was applied using the Roche ChromoMap DAB kit, followed by Roche hematoxylin II.

[0252] In head and neck tumors, 60 samples were analyzed and 90% were determined to be CLDN1 positive tumors ( Figure 1A In esophageal cancer tumors, 40 samples were analyzed and 78% were determined to be CLDN1 positive tumors ( Figure 1B ). It was finally determined that CLDN1 was overexpressed in different fibrotic tumors other than liver cancer.

[0253] Furthermore, the data showed that non-junctional CLDN1 (NJ-CLDN1) is often overexpressed in solid tumors. Notably, CLDN1 expression on tumor cells was positively correlated with T cell localization into fibrotic tissue environments. T cell exclusion is one of the mechanisms described to hinder the efficacy of checkpoint inhibitors (CPIs).

[0254] Example 3. Claudin-1 expression and T cell rejection in head and neck cancer

[0255] Tumor tissue samples were obtained from subjects with head and neck cancer. Immunohistochemistry was used to measure the expression of CLDN1 and CD3 (representing fibrosis and "fibrosis trap") ( Figure 2A 90% of the analyzed tumor samples were CLDN1 positive ( Figure 2B ). Figure 2CThe classification of the immunophenotype of tumors with different CLDN1 expression levels is shown. The immunophenotypes are hot (immune cells in the stroma and between cancer cells), rejection (immune cells within the tumor but only in the stroma), and cold (almost no immune cells can be seen). This data shows that T cell rejection is the main immunophenotype in head and neck cancer, with 30-80% of CLDN1-positive tumors having a T cell rejection phenotype.

[0256] Example 4. Claudin-1 expression and T cell rejection in esophageal cancer

[0257] A tumor tissue sample is obtained from a subject having esophageal cancer. Figure 3A The immunophenotypes of tumors with different CLDN1 expression levels are shown. The immunophenotypes are hot (immune cells in the stroma and between cancer cells), repellent (immune cells within the tumor but only in the stroma), and cold (hardly any immune cells can be seen). CLDN1 expression was measured using immunohistochemistry ( Figure 3B ), T cell presence ( Figure 3C ) and fibrotic tissue ( Figure 3D ). In conclusion, the data show that CLDN1 expression is associated with T cell rejection in cancer.

[0258] Example 5. Overexpression of a human extracellular loop containing mouse claudin-1 drives immune escape and T cell rejection in the hepatic mouse tumor cells Hepa1-6 in vivo.

[0259] Figure 4 A. Figure 4 B and Figure 5 The data presented in show a direct role for overexpression of claudin-1 in driving immune evasion and T cell rejection in vivo. In wild-type Hepa1-6 tumor cells (no claudin-1; Figure 5 , line with square markers), tumors are rejected by the immune system over time, and claudin-1 (claudin-1hECL; Figure 5 , lines with triangle markers) drive immune evasion and tumor growth. Anti-CD3 (T cell marker) IHC analysis of tumor samples obtained at the end of the experiment (day 20) showed that compared with those that did not express claudin-1 ( Figure 4 A) Compared to Claudin-1 (Cldn1 hECL), overexpression of claudin-1 (Claudin-1 hECL) drives T cell exclusion from the tumor bed and accumulation in the stroma ( Figure 4 B).

[0260] Example 6. Disruption of checkpoint inhibitor resistance in cancer using anti-claudin-1 antibodies

[0261] The data provided in Examples 1-4 show that fibrosis is a common feature of checkpoint inhibitor resistance and T cell rejection in cancer. Anti-claudin-1 antibodies (such as any of the antibodies described herein) are administered to subjects with fibrotic tumors. Anti-claudin-1 antibodies have direct anti-fibrotic effects that promote T cell-mediated anti-tumor activity. Importantly, tumors with high levels of CLDN1, such as melanoma ( Fig. 7A ) and head and neck squamous cell carcinoma ( Figure 7B )) responds poorly to the checkpoint inhibitor aPD1. Anti-claudin-1 antibodies will be administered in combination with the checkpoint inhibitor aPD1 in the Hepa1-6 synergistic tumor model of liver cancer, in which overexpression of mouse CLDN1 drives immune escape in vivo. In addition, a patient-derived xenograft (PDX) model for head and neck cancer, in which high expression of CLDN1 and T cell rejection have been confirmed, will be used to show the synergistic effect of administering anti-claudin-1 antibodies with the checkpoint inhibitor aPD1. Administration of immune checkpoint inhibitors simultaneously with or after administration of anti-claudin-1 antibodies increases the therapeutic efficacy of immune checkpoint inhibitors in fibrotic tumors.

[0262] FIG. 10A to FIG. 10C The data in show that overexpression of CLDN1 in Hepa1-6 mouse liver tumor cells promotes T cell rejection and resistance to anti-PD1 therapy. Importantly, the anti-CLDN1 antibodies of the present disclosure restore T cell infiltration and anti-PD1 efficacy in Hepa1-6 CLDN1+ tumors. Fig. 10A It was shown that the tumor volume was dramatically reduced in the anti-CLDN1 antibody and PD1 antagonist groups compared with the isotype control, anti-CLDN1 antibody alone, and PD1 antagonist alone groups. Fig. 10B and Fig. 10C showed that after administration of a combination of anti-CLDN1 antibody and PD1 antagonist, T cells were able to successfully infiltrate CLDN1+ tumors in much greater numbers.

[0263] Mechanistically, NJ-CLDN1 interacts with different components involved in extracellular matrix remodeling, thereby establishing a physical barrier that excludes immune cells from tumor nests. Anti-CLDN1 antibodies have a direct antifibrotic effect by interfering with the interface between CDLN1+ tumor cells and the matrix, thereby restoring immune cell infiltration.

[0264] Example 7. Generation of anti-claudin-1 CAR T cells (prospective)

[0265] Isolation and activation of T cells

[0266] The patient is connected to a device that moves peripheral blood through a disposable tubing set. The centrifugal force guided by an optical sensor separates the blood into appropriate density bands for separating and collecting the required cell layers. The uncollected blood components are then returned to the patient. Alternatively, apheresis will be used as a method for T cell collection. Engineered T cells will be separated from the patient and reintroduced into the same individual (autologous therapy), or separated from a donor and subsequently introduced into different individuals (xenotherapy). The collected cells will be cryopreserved or processed without prior freezing. Positive or negative selection is performed using antibody-conjugated magnetic beads, and the separated cells are optionally processed to enrich T cells (specific T cell subsets). For example, T cells can be enriched based on the expression of CD62L, CD4 and CD8. For example, by polyclonal stimulation, soluble anti-CD3 antibodies or fixed CD3 and CD28 antibodies are used to activate the separated T cells. CD3 and CD28 antibodies can be fixed by coating tissue culture bottles. Paramagnetic beads (such as Dynabeads) can also be coated with these antibodies; in suspension, the coated beads provide appropriate stimulation for much larger T cell cultures. Before preparing the final cell product, the beads will be removed because they may cause harm if they are infused into the patient. The T cell / bead aggregates are destroyed by agitation, and then the suspension is passed through a strong magnetic field to achieve removal, which retains the beads but allows the cells to flow through. Alternatively, a stimulator (such as Transact) is used, which utilizes humanized anti-CD3 antibodies and anti-CD28 antibodies conjugated to colloidal polymer nanomatrices. Before the final product is prepared, the nanomatrix is ​​washed away in a centrifugation step. Alternatively, a similar T cell stimulation method will be used, which uses a hydrogel "stimulation matrix" incorporated with antibodies, which can also be removed by washing after stimulation and amplification. Other methods (such as soluble activation proteins, lipid microbubbles, soluble microspheres, and connected antibodies) are also potential options for activating isolated T cells.

[0267] Genetic engineering of isolated T cells to express anti-claudin-1 CAR

[0268] After activation, T cells are genetically engineered to express anti-tight junction protein-1 CAR. Use a viral system or a non-viral system to genetically engineer the T cells separated. Based on the transposon / transposase system and viral vector of plasmid, including but not limited to γ-retrovirus and lentiviral vector and genome editing (for example, gene editing based on CRISPR / Cas9) and electroporation of naked DNA, it will be used to deliver the gene of anti-tight junction protein-1 coding region and related regulatory sequence into the T cells separated.

[0269] Expansion of anti-claudin-1 CAR T cells

[0270] The engineered CAR T cells expressing the anti-Claudin-1 CAR are then expanded in vitro by standard culture techniques or by alternative methods, including rocking motion bioreactors such as Xuri TM Cell Expansion Systems and WAVE TM Bioreactor system) (which utilizes a perfusion scheme to add nutrients and remove growth inhibitory substances to simplify the manufacturing process). Engineered anti-tight junction protein-1 CAR T cells will also be stimulated with supplemented γ chain cytokines, including but not limited to IL-2, IL-4, IL-7, IL-9, IL-15 and IL-21. Optionally, anti-tight junction protein-1 CAR T cells will also be treated with specific pathway inhibitors, including but not limited to GSK3β, mTOR, AKT and PI3K.

[0271] The engineered CAR T cells expressing the anti-Claudin-1 CAR were then cryopreserved for quality control testing before administration to patients.

[0272] ***

[0273] Unless otherwise indicated, the practice of the present disclosure will employ conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art. Such techniques are fully explained in the literature.

[0274] All references cited above and all references cited herein are incorporated by reference in their entirety.

[0275] Any examples provided herein are provided by way of illustration and not limitation.

Claims

1. A method for 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.

2. The method of claim 1, further comprising administering an immune checkpoint inhibitor.

3. A method for 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 subject's tumor.

4. A method for increasing the therapeutic efficacy of an immune checkpoint inhibitor in a subject with a fibrotic tumor, the method comprising: a. administering an anti-claudin-1 antibody to the subject, wherein the anti-claudin-1 antibody promotes T cell-mediated anti-tumor activity in the fibrotic tumor; and b. administering the immune checkpoint inhibitor to the subject.

5. The method of any one of claims 2 to 4, wherein the anti-Claudin-1 antibody is administered prior to administration of the immune checkpoint inhibitor.

6. The method of any one of claims 2 to 4, wherein the anti-Claudin-1 antibody and the immune checkpoint inhibitor are administered simultaneously or sequentially.

7. The method of any one of claims 2 to 4, wherein the anti-Claudin-1 antibody and the immune checkpoint inhibitor are administered in the same composition.

8. The method of any one of claims 2 to 4, wherein the anti-Claudin-1 antibody and the immune checkpoint inhibitor are administered in different compositions.

9. The method of any one of claims 2 to 4, wherein the anti-claudin-1 antibody and / or the immune checkpoint inhibitor is administered intratumorally, intravenously, intraperitoneally, intramuscularly, intrathecally or subcutaneously.

10. The method of any one of claims 2 to 4, 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 Siglec-15.

11. The method of any one of claims 2 to 4, wherein the immune checkpoint inhibitor is a small molecule inhibitor.

12. The method of any one of claims 2 to 4, wherein the immune checkpoint inhibitor is an antibody.

13. The method of claim 12, wherein the immune checkpoint inhibitor is a PD-1 antagonist selected from the group consisting of nivolumab, pembrolizumab, cemiplimab, and dotalimumab.

14. The method of claim 12, wherein the immune checkpoint inhibitor is a PD-L1 antagonist selected from the group consisting of atezolizumab, durvalumab, and avelumab.

15. The method of claim 12, wherein the immune checkpoint inhibitor is a CTLA-4 antagonist selected from the group consisting of ipilimumab and tremelimumab.

16. The method of claim 12, wherein the immune checkpoint inhibitor is a TIGIT antagonist selected from the group consisting of tisleliumab, osperlimumab, donavulizumab, etilizumab, and vembrolizumab.

17. The method of claim 3, wherein the cancer comprises a fibrotic tumor.

18. The method of any one of claims 1, 2, or 4 to 17, wherein the fibrotic tumor is characterized by high expression of Claudin-1 relative to a reference sample.

19. The method of claim 18, wherein the reference sample is a tissue sample from normal tissue, wherein the normal tissue is adjacent to the tumor.

20. The method of any one of claims 1 to 19, wherein the tumor is selected from the group consisting of head and neck tumors, lung tumors, breast tumors, melanoma tumors, colorectal tumors, pancreatic tumors, esophageal tumors, bile duct cancer, and hepatocellular tumors.

21. The method of any one of claims 1 to 20, wherein 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 with the DSMZ on July 29, 2008 under the accession number DSMACC2938.

22. The method of any one of claims 1 to 21, wherein the anti-Claudin-1 antibody is humanized.

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

13. 24 . The method of any one of claims 1 to 23 , wherein the anti-Claudin-1 antibody comprises a VL comprising the amino acid sequence shown in SEQ ID NO: 4 or SEQ ID NO:

14. 25 . The method of any one of claims 1 to 24 , wherein the anti-Claudin-1 antibody comprises a VH comprising the amino acid sequence shown in SEQ ID NO: 3; and a VL comprising the amino acid sequence shown in SEQ ID NO:

4. 26 . The method of any one of claims 1 to 25 , wherein the anti-Claudin-1 antibody comprises a VH comprising the amino acid sequence shown in SEQ ID NO: 13; and a VL comprising the amino acid sequence shown in SEQ ID NO:

14.

27. The method of any one of claims 1 to 26, wherein the anti-Claudin-1 antibody comprises a complementarity determining region (CDR) H1 comprising the amino acid sequence shown in SEQ ID NO:5, a CDR H2 comprising the amino acid sequence shown in SEQ ID NO:6, and a CDR H3 comprising the amino acid sequence shown in SEQ ID NO:

7.

28. The method of any one of claims 1 to 27, wherein the anti-Claudin-1 antibody comprises a complementarity determining region (CDR) L1 comprising the amino acid sequence shown in SEQ ID NO:8, a CDR L2 comprising the amino acid sequence "Gly Ala", and a CDR L3 comprising the amino acid sequence shown in SEQ ID NO:

10.

29. The method of any one of claims 1 to 28, wherein the anti-Claudin-1 antibody comprises a heavy chain sequence comprising the amino acid sequence shown in SEQ ID NO:

1.

30. The method of any one of claims 1 to 29, wherein the anti-Claudin-1 antibody comprises a light chain sequence comprising the amino acid sequence shown in SEQ ID NO:

2.

31. A method of promoting T cell-mediated anti-tumor activity in a subject having a fibrotic tumor, the method comprising administering anti-claudin-1 chimeric antigen receptor (CAR) T cells to the subject.

32. The method of claim 31 , further comprising administering an immune checkpoint inhibitor.

33. A method for 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, wherein the anti-claudin-1 CAR T cells promote T cell-mediated anti-tumor activity in the subject's tumor.

34. A method of increasing the therapeutic efficacy of an immune checkpoint inhibitor in a subject with a fibrotic tumor, the method comprising: a. administering anti-claudin-1 CAR T cells to the subject, wherein the anti-claudin-1 CAR T cells promote T cell-mediated anti-tumor activity in the fibrotic tumor; and b. administering the immune checkpoint inhibitor to the subject.

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