Biomarkers for cancer treatment using anti-claudin-1 antibodies

Abstract

In various aspects of this disclosure, the disclosure relates to a method for treating cancer, comprising (a) administering an anti-claudin-1 antibody to a subject, (b) determining the level of PRO-C3 in a test sample from the subject, and (c) comparing the level of PRO-C3 in the test sample to the level of PRO-C3 in a control sample. In some aspects, if the level of PRO-C3 in the test sample is not decreased compared to the level of PRO-C3 in the control sample, the dose of anti-claudin-1 antibody administered to the subject is increased. In some aspects, if the level of PRO-C3 in the test sample is decreased compared to the control sample, the dose of anti-claudin-1 antibody administered to the subject is not increased.

Description

Technical Field

[0001] Cross - Reference to Related Applications This patent application claims the benefit of U.S. Provisional Patent Application No. 63 / 491,860, filed Mar. 23, 2023, which is hereby incorporated by reference in its entirety.

[0002] Incorporation by Reference of Sequence Listing The sequence listing XML related to this application is provided electronically in XML file format and is hereby incorporated by reference. The name of the XML file containing the sequence listing XML is "ALNT - 012 / 001WO_SeqList.xml". The XML file is 20,878 bytes, was created on Mar. 7, 2024, and was electronically submitted via the USPTO Patent Center.

[0003] According to various aspects of the present disclosure, the present disclosure relates to a treatment method including administration of an anti - Claudin - 1 antibody and quantification of the N - terminal propeptide of type III collagen ("PRO - C3") or the granzyme B - cleaved type IV collagen product ("C4G").

Background Art

[0004] Anti - Claudin - 1 antibodies have been used to treat a series of diseases such as hepatocellular carcinoma (e.g., U.S. Patent No. 10,815,298), non - alcoholic fatty liver disease (e.g., U.S. Patent No. 10,927,170), and fibrotic diseases such as kidney fibrosis, lung fibrosis, and skin fibrosis (e.g., WO2021 / 094469A1).

[0005] Claudin 1 (CLDN1) is a protein limited to the inside of normal epithelial tight junctions in various tissues. Upon malignant transformation, CLDN1 is overexpressed and the epitope becomes exposed outside the tight junction (non - junctional CLDN1).

[0006] There is a need to identify biomarkers that improve the treatment with anti - Claudin - 1 antibodies (e.g., cancer treatment). [Overview of the project]

[0007] Certain embodiments of this disclosure provide a method for treating cancer, comprising: (a) administering an anti-claudin-1 antibody to a subject; (b) determining the level of PRO-C3 in a test sample from the subject; and (c) comparing the level of PRO-C3 in the test sample to the level of PRO-C3 in a control sample. In some embodiments, if the level of PRO-C3 in the test sample is not decreased compared to the level of PRO-C3 in the control sample, the dose of anti-claudin-1 antibody administered to the subject is increased. In some embodiments, if the level of PRO-C3 in the test sample is decreased compared to the control sample, the dose of anti-claudin-1 antibody administered to the subject is not increased.

[0008] Certain embodiments of this disclosure provide a method for treating cancer in a subject, wherein the subject is determined to have a level of PRO-C3 in a test sample from the subject that has not decreased compared to the level of PRO-C3 in a control sample, and the method comprises administering an increased dose of an anti-claudin-1 therapeutic agent to the subject. In some embodiments, the subject has previously been administered an anti-claudin-1 therapeutic agent.

[0009] Certain aspects of the present disclosure provide a method for monitoring cancer progression in a subject, comprising: (a) quantifying the level of PRO-C3 in a test sample from the subject, indicating that the subject has previously been administered an anti-claudin-1 therapeutic agent; and (b) comparing the level of PRO-C3 in the test sample to the level of PRO-C3 in a control sample. In some aspects, a level of PRO-C3 in the test sample that is not decreased compared to the level of PRO-C3 in the control sample indicates that the subject is sensitive to an increased dose of the anti-claudin-1 therapeutic agent.

[0010] Certain aspects of the present disclosure provide a method for monitoring cancer progression in a subject, comprising: (a) determining the level of PRO-C3 in a test sample from the subject, determining whether the subject has previously been administered an anti-claudin-1 antibody; and (b) comparing the level of PRO-C3 in the test sample to the level of PRO-C3 in a control sample. In some aspects, a level of PRO-C3 in the test sample that is not decreased compared to the level of PRO-C3 in the control sample indicates that the subject is sensitive to an increased dose of anti-claudin-1 antibody.

[0011] Certain embodiments of this disclosure provide a method for treating a subject having cancer, comprising the steps of obtaining a test sample from the subject and determining whether the subject has a reduced level of PRO-C3 by comparing the level of PROC3 in the test sample with that of a control sample, wherein the subject has previously been administered a certain dose of anti-claudin-1 antibody. In some embodiments, if the subject has a reduced level of PROC3 in the test sample compared to a control sample, the subject is not administered an increased dose of anti-claudin-1 antibody. In some embodiments, if the subject does not have a reduced level of PRO-C3 in the test sample compared to a control sample, the subject is administered an increased dose of anti-claudin-1 antibody.

[0012] Certain aspects of the present disclosure provide a method for designing a personalized therapy for a subject with cancer, comprising: (a) quantifying the level of PRO-C3 in a test sample from the subject; (b) comparing the level of PRO-C3 in the test sample obtained in step (a) with the level of PRO-C3 in a control sample; (c) determining that the subject has an undecreased level of PRO-C3 compared with the level of PRO-C3 in the control sample; and (d) administering an increased dose of an anti-claudin-1 antibody.

[0013] In some embodiments, the subject has previously been administered a certain dose of anti-claudin-1 antibody. In some embodiments, if the level of PRO-C3 in the test sample obtained in step (a) is not decreased compared to the level of PRO-C3 in the control sample, the subject is sensitive to administration of an increased dose of anti-claudin-1 antibody.

[0014] Certain embodiments of this disclosure provide a method for classifying subjects with cancer into a cohort and treating said subjects, comprising: (a) quantifying the level of PROC3 in a test sample from the subject; (b) comparing the level of PRO-C3 in the test sample obtained in step (a) with the level of PRO-C3 in a control sample; (c) determining that the subject has an undecreased level of PRO-C3 compared to the level of PRO-C3 in a control sample; (d) classifying the subject into a cohort based on the level of PRO-C3 in the test sample compared with a control sample; and (e) administering an increased dose of anti-claudin-1 antibody. In some embodiments, the subject has previously been administered a certain dose of anti-claudin-1 antibody. In some embodiments, if the level of PRO-C3 in the test sample obtained in step (a) is undecreased compared to the level of PRO-C3 in a control sample, the subject is sensitive to administration of an increased dose of anti-claudin-1 antibody.

[0015] Certain embodiments of this disclosure provide a method for treating cancer, comprising (a) administering an anti-claudin-1 antibody to a subject; (b) determining the level of C4G in a test sample from the subject; and (c) comparing the level of C4G in the test sample to the level of C4G in a control sample. In some embodiments, if the level of C4G in the test sample is not increased compared to the level of C4G in the control sample, the dose of the anti-claudin-1 antibody administered to the subject is increased. In some embodiments, if the level of C4G in the test sample is increased compared to the control sample, the dose of the anti-claudin-1 antibody administered to the subject is not increased.

[0016] Certain embodiments of this disclosure provide a method for treating cancer in a subject, wherein the subject is determined to have a level of C4G in a test sample from the subject that has not increased compared to the level of C4G in a control sample, and the method comprises administering an increased dose of an anti-claudin-1 therapeutic agent to the subject. In some embodiments, the subject has previously been administered an anti-claudin-1 therapeutic agent.

[0017] Certain aspects of the present disclosure provide a method for monitoring cancer progression in a subject, comprising (a) quantifying the level of C4G in a test sample from the subject, indicating that the subject has previously been administered an anti-claudin-1 therapeutic agent, and (b) comparing the level of C4G in the test sample to the level of C4G in a control sample, wherein the level of C4G in the test sample that has not increased compared to the level of C4G in the control sample indicates that the subject is sensitive to an increased dose of the anti-claudin-1 therapeutic agent.

[0018] Certain aspects of the present disclosure provide a method for monitoring cancer progression in a subject, comprising: (a) determining the level of C4G in a test sample from the subject, determining whether the subject has previously been administered an anti-claudin-1 antibody; and (b) comparing the level of C4G in the test sample to the level of C4G in a control sample. In some aspects, a level of C4G in the test sample that is not elevated compared to the level of C4G in the control sample indicates that the subject is sensitive to an increased dose of anti-claudin-1 antibody.

[0019] Certain embodiments of this disclosure provide a method for treating a subject having cancer, comprising the step of determining whether the subject has an elevated level of C4G by obtaining a test sample from the subject and comparing the level of C4G in the test sample with that of a control sample. In some embodiments, the subject has previously been administered a certain dose of anti-claudin-1 antibody. In some embodiments, if the subject has an elevated level of C4G in the test sample compared to a control sample, the subject is not administered an elevated dose of anti-claudin-1 antibody. In some embodiments, if the subject does not have an elevated level of C4G in the test sample compared to a control sample, the subject is administered an elevated dose of anti-claudin-1 antibody.

[0020] Certain aspects of the present disclosure provide a method for designing an individualized therapy for a subject with cancer, comprising: (a) quantifying the level of C4G in a test sample from the subject; (b) comparing the level of C4G in the test sample obtained in step (a) with the level of C4G in a control sample; (c) determining that the subject has an unincreased level of C4G compared to the level of C4G in the control sample; and (d) administering an increased dose of anti-claudin-1 antibody. In some aspects, the subject has previously been administered a certain dose of anti-claudin-1 antibody. In some aspects, if the level of C4G in the test sample obtained in step (a) is unincreased compared to the level of C4G in the control sample, the subject is sensitive to administration of an increased dose of anti-claudin-1 antibody.

[0021] Certain embodiments of this disclosure provide a method for classifying subjects with cancer into a cohort and treating said subjects, comprising: (a) quantifying the level of C4G in a test sample from the subject; (b) comparing the level of C4G in the test sample obtained in step (a) with the level of C4G in a control sample; (c) determining that the subject has an elevated level of C4G compared to the level of C4G in a control sample; (d) classifying the subject into a cohort based on the level of C4G in the test sample compared to a control sample; and (e) administering an increased dose of anti-claudin-1 antibody. In some embodiments, the subject has previously been administered a certain dose of anti-claudin-1 antibody. In some embodiments, if the level of C4G in the test sample obtained in step (a) is not elevated compared to the level of C4G in a control sample, the subject is sensitive to administration of an increased dose of anti-claudin-1 antibody.

[0022] In some embodiments, if the subject is sensitive to an increased dose of anti-claudin-1 antibody, the anti-claudin-1 antibody is administered to the subject.

[0023] In some embodiments, the anti-claudin-1 therapeutic agent is an anti-claudin-1 antibody.

[0024] In some embodiments, anti-claudin-1 antibodies are administered intratumorally, intravenously, intraperitoneally, intramuscularly, intrathecally, or subcutaneously.

[0025] In some embodiments, cancer is selected from the group consisting of head and neck cancer (e.g., head and neck squamous cell carcinoma), lung cancer, breast cancer, melanoma, colorectal cancer, pancreatic cancer, esophageal cancer, bile duct cancer, and hepatocellular carcinoma.

[0026] In some embodiments, the control sample is a sample from a subject before administration of an anti-claudin-1 antibody or anti-claudin-1 therapeutic agent.

[0027] In some embodiments, the level of PRO-C3 C4G in the control sample is from about 10 ng / mL to about 20 ng / mL.

[0028] In some embodiments, the level of C4G in the control sample is from about 30 ng / mL to about 40 ng / mL.

[0029] In some embodiments, the anti-claudin-1 antibody is a monoclonal antibody comprising six complementarity-determining regions (CDRs) of an anti-claudin-1 monoclonal antibody secreted by a hybridoma cell line deposited with DSMZ on July 29, 2008 under accession number DSM ACC2938.

[0030] In some embodiments, the anti-claudin-1 antibody is humanized.

[0031] In some embodiments, the anti-claudin-1 antibody comprises a VH comprising the amino acid sequence set forth in SEQ ID NO: 3 or SEQ ID NO: 13.

[0032] In some embodiments, the anti-claudin-1 antibody comprises a VL comprising the amino acid sequence set forth in SEQ ID NO: 4 or SEQ ID NO: 14.

[0033] In some embodiments, the anti-claudin-1 antibody comprises a VH comprising the amino acid sequence set forth in SEQ ID NO: 3 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 4.

[0034] In some embodiments, the anti-claudin-1 antibody comprises a VH comprising the amino acid sequence set forth in SEQ ID NO: 13 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 14.

[0035] In some embodiments, the anti-claudin-1 antibody comprises complementarity-determining region (CDR) H1 comprising the amino acid sequence set forth in SEQ ID NO: 5, CDR H2 comprising the amino acid sequence set forth in SEQ ID NO: 6, and CDR H3 comprising the amino acid sequence set forth in SEQ ID NO: 7.

[0036] In some embodiments, the anti-claudin-1 antibody comprises a complementation-determining region (CDR) L1 containing the amino acid sequence described in SEQ ID NO: 8, a CDR L2 containing the amino acid sequence GA, and a CDR L3 containing the amino acid sequence described in SEQ ID NO: 10.

[0037] In some embodiments, the anti-claudin-1 antibody comprises a heavy chain having the amino acid sequence described in SEQ ID NO: 1.

[0038] In some embodiments, the anti-claudin-1 antibody comprises a light chain having the amino acid sequence described in SEQ ID NO: 2. [Brief explanation of the drawing]

[0039] [Figure 1] This shows CLDN1 mRNA expression in solid tumors. [Figure 2] This shows the results of anti-CLDN1 antibody treatment of head and neck squamous cell carcinoma (HNSCC) in patient-derived xenograft models, compared to IgG1 control, as measured by tumor volume (mm3). [Figure 3A] This shows the PRO-C3 levels (ng / mL) after treatment with anti-CLDN1 antibody or IgG1 in patient-derived xenograft models of head and neck squamous cell carcinoma (HNSCC). [Figure 3B] This shows the C4G levels (ng / mL) after treatment with anti-CLDN1 antibody or IgG1 in patient-derived xenograft models of head and neck squamous cell carcinoma (HNSCC). [Figure 4A] This shows in vitro ADCC measurements between WT IgG1 Fc or anti-CLDN1 antibodies with Fc variants compared to an IgG1 control. [Figure 4B] This shows tumor growth inhibition (TGI) in the CAL27 HNSCC CDX model after treatment with IgG1 control, anti-CLDN1 antibody (WT IgG1 Fc), or anti-CLDN1 antibody (Fc variant). [Modes for carrying out the invention]

[0040] In some embodiments, this specification provides a method for treating cancer, comprising: (a) administering an anti-claudin-1 antibody to a subject; (b) determining the level of PRO-C3 in a test sample from the subject; and (c) comparing the level of PRO-C3 in the test sample to the level of PRO-C3 in a control sample. In some embodiments, if the level of PRO-C3 in the test sample is not decreased compared to the level of PRO-C3 in the control sample, the dose of anti-claudin-1 antibody administered to the subject is increased. In some embodiments, if the level of PRO-C3 in the test sample is decreased compared to the control sample, the dose of anti-claudin-1 antibody administered to the subject is not increased.

[0041] In some embodiments, the Specified Method for Treating Cancer in Subjects Determined to Have PRO-C3 Levels in a Test Sample from Subjects Not Decreased Compared to PRO-C3 Levels in a Control Sample. In some embodiments, Subjects Have Previously Received an Anti-Claudin-1 Therapeutic Agent, and the Method comprises Administering an Increased Dose of the Anti-Claudin-1 Therapeutic Agent to Subjects.

[0042] In some embodiments, the Specified provides a method for monitoring cancer progression in a subject, comprising: (a) quantifying the level of PRO-C3 in a test sample from the subject, indicating that the subject has previously been administered an anti-claudin-1 therapeutic agent; and (b) comparing the level of PRO-C3 in the test sample to the level of PRO-C3 in a control sample. In some embodiments, a level of PRO-C3 in the test sample that has not decreased compared to the level of PRO-C3 in the control sample indicates that the subject is sensitive to an increased dose of the anti-claudin-1 therapeutic agent.

[0043] In some embodiments, the Specified provides a method for monitoring cancer progression in a subject, comprising: (a) determining the level of PRO-C3 in a test sample from the subject, determining whether the subject has previously been administered an anti-claudin-1 antibody; and (b) comparing the level of PRO-C3 in the test sample to the level of PRO-C3 in a control sample. In some embodiments, a level of PRO-C3 in the test sample that is not decreased compared to the level of PRO-C3 in the control sample indicates that the subject is sensitive to an increased dose of anti-claudin-1 antibody.

[0044] In some embodiments, the Specified Method is provided for treating a subject having cancer, comprising the step of obtaining a test sample from the subject and determining whether the subject has a reduced level of PRO-C3 by comparing the level of PRO-C3 in the test sample with that of a control sample. In some embodiments, the subject has previously been administered a certain dose of anti-claudin-1 antibody. In some embodiments, if the subject has a reduced level of PRO-C3 in the test sample compared to a control sample, the subject is not administered an increased dose of anti-claudin-1 antibody. In some embodiments, if the subject does not have a reduced level of PRO-C3 in the test sample compared to a control sample, the subject is administered an increased dose of anti-claudin-1 antibody.

[0045] In some embodiments, this specification provides a method for designing a personalized therapy for a subject with cancer, comprising: (a) quantifying the level of PRO-C3 in a test sample from the subject; (b) comparing the level of PRO-C3 in the test sample obtained in step (a) with the level of PRO-C3 in a control sample; (c) determining that the subject has an undecreased level of PRO-C3 compared to the level of PRO-C3 in the control sample; and (d) administering an increased dose of anti-claudin-1 antibody. In some embodiments, if the level of PRO-C3 in the test sample obtained in step (a) is undecreased compared to the level of PRO-C3 in the control sample, the subject is sensitive to administration of an increased dose of anti-claudin-1 antibody. In some embodiments, the subject has previously been administered a certain dose of anti-claudin-1 antibody.

[0046] In some embodiments, this specification provides a method for classifying subjects with cancer into a cohort and treating said subjects, comprising: (a) quantifying the level of PRO-C3 in a test sample from the subject; (b) comparing the level of PRO-C3 in the test sample obtained in step (a) with the level of PRO-C3 in a control sample; (c) determining that the subject has an undecreased level of PRO-C3 compared to the level of PRO-C3 in a control sample; (d) classifying the subject into a cohort based on the level of PRO-C3 in the test sample compared to a control sample; and (e) administering an increased dose of anti-claudin-1 antibody. In some embodiments, the subject has previously been administered a certain dose of anti-claudin-1 antibody. In some embodiments, if the level of PRO-C3 in the test sample obtained in step (a) is undecreased compared to the level of PRO-C3 in a control sample, the subject is sensitive to administration of an increased dose of anti-claudin-1 antibody.

[0047] In some embodiments, the Specified provides a method for treating cancer, comprising: (a) administering an anti-claudin-1 antibody to a subject; (b) determining the level of C4G in a test sample from the subject; and (c) comparing the level of C4G in the test sample to the level of C4G in a control sample. In some embodiments, if the level of C4G in the test sample is not increased compared to the level of C4G in the control sample, the dose of anti-claudin-1 antibody administered to the subject is increased. In some embodiments, if the level of C4G in the test sample is increased compared to the control sample, the dose of anti-claudin-1 antibody administered to the subject is not increased.

[0048] In some embodiments, the Specified Method for Treating Cancer in a Subject Determined to Have C4G Levels in a Test Sample from a Subject Whose C4G Levels Have Not Increased Compared to C4G Levels in a Control Sample. In some embodiments, the Subject Has Previously Received an Anti-Claudin-1 Therapeutic Agent, and the Method comprises Administering an Increased Dose of the Anti-Claudin-1 Therapeutic Agent to the Subject.

[0049] In some embodiments, the Specified Publicly Provided is a method for monitoring cancer progression in a subject, comprising: (a) quantifying the level of C4G in a test sample from the subject, indicating that the subject has previously been administered an anti-claudin-1 therapeutic agent; and (b) comparing the level of C4G in the test sample to the level of C4G in a control sample. In some embodiments, a level of C4G in the test sample that has not increased compared to the level of C4G in the control sample indicates that the subject is sensitive to an increased dose of the anti-claudin-1 therapeutic agent.

[0050] In some embodiments, the Specified Public Service provides a method for monitoring cancer progression in cancer, comprising: (a) determining the level of C4G in a test sample from a subject; and (b) comparing the level of C4G in the test sample to the level of C4G in a control sample. In some embodiments, the subject has previously been administered an anti-claudin-1 antibody. In some embodiments, a level of C4G in the test sample that has not increased compared to the level of C4G in the control sample indicates that the subject is sensitive to an increased dose of anti-claudin-1 antibody.

[0051] In some embodiments, the Specified Provision provides a method for treating a subject having cancer, comprising the step of determining whether the subject has an elevated level of C4G by obtaining a test sample from the subject and comparing the level of C4G in the test sample with that of a control sample. In some embodiments, the subject has previously been administered a certain dose of anti-claudin-1 antibody. In some embodiments, if the subject has an elevated level of C4G in the test sample compared to a control sample, the subject is not administered an elevated dose of anti-claudin-1 antibody. In some embodiments, if the subject does not have an elevated level of C4G in the test sample compared to a control sample, the subject is administered an elevated dose of anti-claudin-1 antibody.

[0052] In some embodiments, the Specified provides a method for designing an individualized therapy for a subject with cancer, comprising: (a) quantifying the level of C4G in a test sample from the subject; (b) comparing the level of C4G in the test sample obtained in step (a) with the level of C4G in a control sample; (c) determining that the subject has an unincreased level of C4G compared to the level of C4G in the control sample; and (d) administering an increased dose of anti-claudin-1 antibody. In some embodiments, the subject has previously been administered a certain dose of anti-claudin-1 antibody. In some embodiments, if the level of C4G in the test sample obtained in step (a) is unincreased compared to the level of C4G in the control sample, the subject is sensitive to administration of an increased dose of anti-claudin-1 antibody.

[0053] In some embodiments, this specification provides a method for classifying subjects with cancer into a cohort and treating said subjects, comprising: (a) quantifying the level of C4G in a test sample from the subject; (b) comparing the level of C4G in the test sample obtained in step (a) with the level of C4G in a control sample; (c) determining that the subject has an elevated level of C4G compared to the level of C4G in a control sample; (d) classifying the subject into a cohort based on the level of C4G in the test sample compared to a control sample; and (e) administering an increased dose of anti-claudin-1 antibody. In some embodiments, the subject has previously been administered a certain dose of anti-claudin-1 antibody. In some embodiments, if the level of C4G in the test sample obtained in step (a) is not elevated compared to the level of C4G in a control sample, the subject is sensitive to administration of an increased dose of anti-claudin-1 antibody.

[0054] definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art in which this disclosure pertains. In case of any conflict, the application containing the definitions shall prevail. Unless otherwise required by context, singular terms shall include plural forms, and plural terms shall include singular forms. All publications, patents, and other references mentioned herein are incorporated together by reference for all purposes, as is specifically and individually indicated, that each individual publication or patent application is incorporated by reference.

[0055] While similar or equivalent methods and materials described herein may be used in the practice or testing of this disclosure, suitable methods and materials are described below. The materials, methods, and examples are illustrative and not intended to limit the scope. Other features and advantages of this disclosure will become apparent from the modes for carrying out the invention and the claims.

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

[0057] Unless otherwise explicitly stated by the context, the singular forms “a,” “an,” and “the” refer to multiple objects. The terms “a” (or “an”), “one or more,” and “at least one” are used interchangeably herein. In certain aspects, the terms “a” or “an” mean “single.” In other aspects, the terms “a” or “an” include “two or more” or “multiple.”

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

[0059] Throughout this disclosure, various aspects of this disclosure are presented in the form of scopes. Naturally, the use of scopes is for convenience and conciseness and should not be interpreted as an inflexible limitation on the scope of this disclosure. Therefore, a scope description should be considered to have not only the individual numerical values ​​within that scope, but also all possible partial scopes that are specifically disclosed. For example, a scope description such as 1 to 6 should be considered to have not only the individual numerical values ​​within that scope, e.g., 1, 2, 3, 4, 5, and 6, but also the specifically disclosed partial scopes, e.g., 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc. This applies regardless of the width of the scope. A numerical range described includes the boundary values ​​of the numerical values ​​that define the scope, and includes each integer within the defined scope.

[0060] Units, prefixes, and symbols are shown in the format approved by the International System of Units (SI). Numerical ranges include the numbers that define the range. Where ranges of values ​​are enumerated, it should be understood that each intervening integer value between the enumerated upper and lower limits of that range, and any portion thereof, is also specifically disclosed along with each partial range between such values. The upper and lower limits of any range may or may not be independently included within that range, and each range that includes either one of the limits, does not include any limits, or includes both of these limits is also included in this application. Therefore, it should be understood that the ranges enumerated herein are abbreviated representations of all values ​​within that range (including the enumerated ends). For example, the range 1 to 10 is understood to include any number, combination of numbers, or partial range from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10.

[0061] Where values ​​are explicitly enumerated, it is understood that values ​​that are approximately the same quantity or amount as the enumerated values ​​are also within the scope of this disclosure. Where combinations are disclosed, each partial combination of the elements of that combination is also specifically disclosed and is within the scope of this disclosure. Conversely, where different elements or groups of elements are disclosed individually, their combinations are also disclosed. Where any element of this disclosure is disclosed as having multiple substitutes, examples of such disclosures are also disclosed herein, where each substitute is excluded individually or in any combination with other substitutes, and multiple elements of this disclosure may have such exclusions, and all combinations of elements having such exclusions are disclosed herein.

[0062] As used herein, the term “and / or” should be interpreted as the specific disclosure of each of two identified features or components, with or without the other. Accordingly, as used herein in phrases such as “A and / or B,” the term “and / or” is intended to include “A and B,” “A or B,” “A” (alone), and “B” (alone). Similarly, as used in phrases such as “A, B, and / or C,” the term “and / or” is intended to include each of the following embodiments: 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] The terms “polypeptide,” “peptide,” and “protein” are used herein interchangeably to refer to polymers of amino acids of any length. The polymers may be linear or branched, may contain modified amino acids, or may be interrupted by non-amino acids. These terms also encompass amino acid polymers modified naturally or by intervention, e.g., disulfide bond formation, glycosylation, lipidization, acetylation, phosphorylation, or any other operation or modification, e.g., conjugation with a labeling component. Also included in this definition are polypeptides containing one or more analogues of amino acids (including, for example, non-natural amino acids), and other modifications known in the art. Since the polypeptides of this disclosure are antibody-based, it will be understood that in certain embodiments, such polypeptides may exist as single-chain or conjugated chains.

[0064] As used herein, “administer,” “dosage,” and “administer” refer to the physical introduction of a composition containing a therapeutic agent (e.g., a combination of anti-claudin-1 antibodies) into a target by any of the various methods and delivery systems known to those skilled in the art. Routes of administration include, for example, intravenous, intramuscular, subcutaneous, intraperitoneal, spinal, or other parenteral routes, such as injection or infusion. As used herein, the term "parenteral administration" refers to administration methods other than enteral and topical administration, usually by injection, and includes, but is not limited to, intravenous injection and infusion, intramuscular injection and infusion, intra-arterial injection and infusion, intrathecal injection and infusion, intralymphatic injection and infusion, intrafocal injection and infusion, intracapsular injection and infusion, intraorbital injection and infusion, intracardiac injection and infusion, intradermal injection and infusion, intraperitoneal injection and infusion, transtracheal injection and infusion, subcutaneous injection and infusion, subepidermal injection and infusion, intra-articular injection and infusion, subcapsular injection and infusion, subarachnoid injection and infusion, intrathecal injection and infusion, epidural injection and infusion, intrasternal injection and infusion, and in vivo electroporation. Other parenteral routes include topical, dermal, or mucosal administration routes, such as intranasal, intravaginal, rectal, sublingual, or local. Administration can also be performed, for example, once, multiple times, and / or over one or more extended periods.

[0065] As used herein, the terms “to treat,” “to cure,” or “to treat” mean, for example, reducing the severity of a disease or condition, reducing the duration of the disease course, improving or eliminating one or more symptoms associated with a disease or condition, or providing a beneficial effect to a subject having a disease or condition without necessarily curing the disease or condition. In one embodiment, the terms “to treat” or “to cure” mean treating cancer (for example, head and neck cancer (e.g., head and neck squamous cell carcinoma), lung cancer, breast cancer, melanoma, colorectal cancer, pancreatic cancer, esophageal cancer, cholangiocarcinoma, and hepatocellular carcinoma).

[0066] The terms “subject,” “patient,” “individual,” and “host,” and their variations, are used interchangeably herein and refer to any mammalian subject, including humans, livestock (e.g., including dogs and cats), livestock (e.g., cattle, sheep, pigs, and horses), and laboratory animals (e.g., monkeys, rats, mice, rabbits, and guinea pigs), particularly humans, for which diagnosis, treatment, or therapy is desired. The methods described herein are applicable to both human therapeutic and veterinary uses. Where used herein, the phrase “subject in need of it” includes subjects who would benefit from the administration of a therapeutic agent, e.g., an anti-claudin-1 antibody, e.g., mammalian subjects.

[0067] As used herein, “anti-claudin-1 therapeutic agent” is intended to mean a therapy that targets claudin-1, such as therapy with an anti-claudin-1 antibody or a claudin-1 targeting CAR as described herein. In some embodiments, the anti-claudin-1 therapeutic agent is an anti-claudin-1 antibody disclosed herein.

[0068] Where an aspect is described herein using the word "comprising," it should be understood that similar aspects are also provided, except that they are always described using the terms "consisting of" and / or "consisting essentially of."

[0069] As used herein, the terms “derived” or “derivative” refer to components isolated from or created using information from a particular molecule (e.g., nucleic acid sequences). For example, a polynucleotide sequence derived from another polynucleotide sequence may include polynucleotide sequences that are identical or substantially similar to the polynucleotide sequence from which it is derived. In the case of polynucleotides, derived species can be obtained, for example, by naturally occurring mutagenesis, artificially directed mutagenesis, or artificially random mutagenesis. The mutagenesis used to induce polynucleotides may be intentionally directed, intentionally random, or a mixture thereof. Mutagenesis of a polynucleotide to create a different polynucleotide derived from a first polynucleotide may be a random event (e.g., caused by polymerase infidelity), and the identification of the derived polynucleotide can be carried out by appropriate screening methods known in the art.In some embodiments, the polynucleotide sequences derived from the first polynucleotide sequence are, respectively, at least about 50%, at least about 51%, at least about 52%, at least about 53%, at least about 54%, at least about 55%, at least about 56%, at least about 57%, at least about 58%, at least about 59%, at least about 60%, at least about 61%, at least about 62%, at least about 63%, at least about 64%, at least about 65%, at least about 66%, at least about 67%, at least about 68%, at least about 69%, at least about 70%, at least about 71%, at least about 72%, at least about 73%, and at least about 7 Having 4%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identity, where the derived polynucleotide sequence retains the biological activity of the original polynucleotide.

[0070] The term "human claudin-1 (or CLDN1)" refers to a protein having the sequence shown in NCBI accession number NP_066924.1, or any naturally occurring variant commonly found in HCV-tolerant human populations.

[0071] As used herein, the term “antibody” refers to any immunoglobulin containing an antigen-binding site that binds immunospecifically to an antigen. Therefore, the term “antibody” encompasses not only the entire antibody molecule but also antibody fragments, and derivatives and variants (including derivatives) of antibodies and antibody fragments, insofar as the fragments maintain their specific binding ability. This term encompasses monoclonal and polyclonal antibodies. The term also encompasses any protein having a binding domain that is homologous or largely homologous to the immunoglobulin-binding domain. These proteins may be of natural origin or produced partially or entirely synthetically. When used in relation to antibodies, the term “specific binding” refers to an antibody that binds to a given antigen. Typically, an antibody has a binding capacity of at least 1 × 10⁻⁶. 7 It binds with an affinity of M1 and binds to a given antigen with an affinity at least twice as great as its affinity for binding to nonspecific antigens (e.g., BSA, casein).

[0072] The term “monoclonal antibody” or its antigen-binding fragment refers to a homogeneous population of antibodies or antigen-binding fragments involved in the highly specific recognition and binding of a single antigenic determinant, i.e., an epitope. This is in contrast to polyclonal antibodies, which typically contain different antibodies against different antigenic determinants. The term “monoclonal antibody” or its antigen-binding fragment encompasses intact, full-length monoclonal antibodies as well as antibody fragments (e.g., Fab, Fab', F(ab')2, Fv), single-chain (scFv) variants, fusion proteins containing antibody moieties, and any other immunoglobulin molecules containing antigen recognition sites. Furthermore, “monoclonal antibody” or its antigen-binding fragment refers to antibodies and their antigen-binding fragments produced by any method, including but not limited to hybridomas, phage selection, recombinant expression, and transgenic animals.

[0073] As used herein, the term “humanized antibody” refers to a chimeric antibody comprising amino acid residues derived from a non-human hypervariable region and amino acid residues derived from a human framework (FR). In particular, a humanized antibody comprises all or substantially all of at least one, typically two, variable domains, and all or substantially all of the complementarity-determining regions (CDRs) are from a human antibody. A humanized antibody may optionally also comprise at least a portion of the antibody constant region derived from a human antibody. The “humanized form” of an antibody, e.g., a non-human antibody, refers to an antibody that has undergone humanization.

[0074] The term "effective dose" refers to the amount of drug that produces a desired biological, therapeutic, and / or prophylactic outcome. This outcome may be a reduction, remission, mitigation, decrease, delay, and / or reduction of signs, symptoms, or etiologies of a disease, or any other desirable change in a biological system (e.g., cancer). An effective dose may be administered in one or more doses.

[0075] The term "PRO-C3" refers to the N-terminal propeptide of type III collagen. The cleavage of the N-terminal propeptide of type III collagen can be mediated by N-proteases. In some embodiments, PRO-C3 is derived from the human COL3A1 homotrimer (e.g., NP_000081.2).

[0076] The term "C4G" refers to granzyme B-degraded type IV collagen product.

[0077] Anti-claudin-1 antibody Antibodies against human claudin-1 have been previously described as being able to treat hepatitis C virus infection, hepatocellular carcinoma, and certain fibrotic diseases such as pulmonary fibrosis (see WO2010 / 034812, WO2016 / 146809, and WO2021 / 094469, each of which is incorporated herein by reference in whole). Anti-claudin-1 antibodies that may be used in carrying out the methods disclosed herein include any antibodies produced against claudin-1. Examples are disclosed in WO2010 / 034812 and WO2017 / 162678, each of which is incorporated herein by reference in whole as examples of anti-claudin-1 antibodies that may be used in the methods disclosed herein.

[0078] Other examples of suitable anti-claudin-1 antibodies include those disclosed in European Patent No. EP1167389, U.S. Patent No. 6,627,439, international patent applications published under WO2014 / 132307, WO2015 / 014659 and WO2015 / 014357, and those disclosed in Yamashita et al., J. Pharmacol. Ther., 2015, 353(1):112-118, each of which is incorporated herein by reference as an example of an anti-claudin-1 antibody that may be used in the methods described herein.

[0079] Suitable anti-claudin-1 antibodies for use in the methods disclosed herein may be polyclonal or monoclonal antibodies.

[0080] Anti-claudin-1 antibodies suitable for use in this disclosure may also be “humanized.” Sequence differences between rodent antibodies and human sequences can be minimized by replacing residues different from those in the human sequence by site-directed mutagenesis of individual residues, by grafting of entire regions, or by chemosynthesis. Humanized antibodies can also be produced using recombinant methods. In the humanized form of an antibody, some, most, or all of the amino acids outside the CDR region are replaced with amino acids derived from human immunoglobulin molecules, 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 acceptable as long as they do not significantly alter the biological activity of the resulting antibody. Suitable human “substitution” immunoglobulin molecules include IgG1, IgG2, IgG2a, IgG2b, IgG3, IgG4, IgA, IgM, IgD, or IgE molecules and their fragments.

[0081] Similarly, conservative amino acid substitutions can be introduced into antibody sequences disclosed herein, provided that they do not significantly alter the biological activity of the resulting antibody. As used herein, “conservative amino acid substitution” means a substitution in which one amino acid residue is replaced by another amino acid residue, thereby changing one amino acid to a different amino acid having similar biochemical properties (e.g., charge, hydrophobicity, and size). For example, lysine, arginine, and histidine have similar properties in that they have basic side chains, and aspartic acid and glutamic acid have similar properties in that they have acidic side chains. Furthermore, glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, and tryptophan have similar properties in that they have non-charged side chains, and alanine, valine, leucine, threonine, isoleucine, proline, phenylalanine, and methionine have similar properties in that they have nonpolar side chains. Also, tyrosine, phenylalanine, tryptophan, and histidine have similar properties in that they have aromatic side chains. Therefore, as will be apparent to those skilled in the art, substitution of amino acid residues in the group exhibiting similar properties as described above does not result in any particular change in properties. Polypeptide variants also include additions and deletions to the polypeptide sequences disclosed herein. Furthermore, variant nucleotide sequences include their analogues and derivatives. Binding protein variants disclosed herein include proteins that bind to the same antigen or epitope as the binding protein.

[0082] In some embodiments, the humanized anti-claudin-1 antibody for use in accordance with this disclosure is one previously described in WO2017 / 162678. Exemplary sequences of the antibody or antigen-binding fragments provided herein are listed in Table 1. [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4]

[0083] In some embodiments, the anti-claudin-1 antibody comprises a complementation-determining region (CDR) H1 containing the amino acid sequence described in SEQ ID NO: 5, a CDR H2 containing the amino acid sequence described in SEQ ID NO: 6, and a CDR H3 containing the amino acid sequence described in SEQ ID NO: 7.

[0084] In some embodiments, the anti-claudin-1 antibody comprises a complementation-determining region (CDR) L1 containing the amino acid sequence described in SEQ ID NO: 8, a CDR L2 containing the amino acid sequence "Gly Ala", and a CDR L3 containing the amino acid sequence described in SEQ ID NO: 10.

[0085] In some embodiments, the Complementarity Determination Region (CDR) disclosed herein is defined in accordance with IMGT™. However, it should be understood that other methods of defining CDR in the art may also be used.

[0086] In some embodiments, the anti-claudin-1 antibody comprises a VH having the amino acid sequence described in SEQ ID NO: 3 or SEQ ID NO: 13.

[0087] In some embodiments, the anti-claudin-1 antibody comprises a VH having an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 3. In some embodiments, the anti-claudin-1 antibody comprises a VH having an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 13.

[0088] In some embodiments, the anti-claudin-1 antibody comprises a VL having the amino acid sequence described in SEQ ID NO: 4 or SEQ ID NO: 14.

[0089] In some embodiments, the anti-claudin-1 antibody comprises a VL containing an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 4. In some embodiments, the anti-claudin-1 antibody comprises a VL containing an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 14.

[0090] In some embodiments, the anti-claudin-1 antibody comprises VH containing the amino acid sequence described in SEQ ID NO: 3, and VL containing the amino acid sequence described in SEQ ID NO: 4.

[0091] In some embodiments, the anti-claudin-1 antibody comprises VH, which contains the amino acid sequence described in SEQ ID NO: 13, and VL, which contains the amino acid sequence described in SEQ ID NO: 14.

[0092] In some embodiments, the anti-claudin-1 antibody comprises a heavy chain having the amino acid sequence described in SEQ ID NO: 1, SEQ ID NO: 11, or SEQ ID NO: 21.

[0093] In some embodiments, the anti-claudin-1 antibody comprises a heavy chain having an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1. In some embodiments, the anti-claudin-1 antibody comprises a heavy chain having an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 11. In some embodiments, the anti-claudin-1 antibody comprises a heavy chain having an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 21.

[0094] In some embodiments, the anti-claudin-1 antibody comprises a light chain having the amino acid sequence described in SEQ ID NO: 2 or SEQ ID NO: 12.

[0095] In some embodiments, the anti-claudin-1 antibody comprises a light chain having an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 2.

[0096] In some embodiments, the anti-claudin-1 antibody comprises a light chain having an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 12.

[0097] In some embodiments, the anti-claudin-1 antibody comprises a heavy chain containing the amino acid sequence described in SEQ ID NO: 1 and a light chain containing the amino acid sequence described in SEQ ID NO: 2.

[0098] In some embodiments, the anti-claudin-1 antibody comprises a heavy chain containing the amino acid sequence described in SEQ ID NO: 21 and a light chain containing the amino acid sequence described in SEQ ID NO: 2.

[0099] In some embodiments, the anti-claudin-1 antibody comprises a heavy chain containing the amino acid sequence described in SEQ ID NO: 11 and a light chain containing the amino acid sequence described in SEQ ID NO: 12.

[0100] In some embodiments, the anti-claudin-1 antibody comprises a complementarity-determining region (CDR) H1 containing the amino acid sequence described in SEQ ID NO: 5, a CDR H2 containing the amino acid sequence described in SEQ ID NO: 6, a CDR H3 containing the amino acid sequence described in SEQ ID NO: 7, and VH containing the amino acid sequence described in SEQ ID NO: 3. In some embodiments, the anti-claudin-1 antibody comprises a complementarity-determining region (CDR) H1 containing the amino acid sequence described in SEQ ID NO: 5, a CDR H2 containing the amino acid sequence described in SEQ ID NO: 6, a CDR H3 containing the amino acid sequence described in SEQ ID NO: 7, and VL containing the amino acid sequence described in SEQ ID NO: 4. In some embodiments, the anti-claudin-1 antibody comprises a complementarity-determining region (CDR) H1 containing the amino acid sequence described in SEQ ID NO: 5, a CDR H2 containing the amino acid sequence described in SEQ ID NO: 6, a CDR H3 containing the amino acid sequence described in SEQ ID NO: 7, and VH containing the amino acid sequence described in SEQ ID NO: 3 and VL containing the amino acid sequence described in SEQ ID NO: 4.

[0101] In some embodiments, the anti-claudin-1 antibody comprises a complementarity-determining region (CDR) H1 containing the amino acid sequence described in SEQ ID NO: 5, a CDR H2 containing the amino acid sequence described in SEQ ID NO: 6, a CDR H3 containing the amino acid sequence described in SEQ ID NO: 7, and VH containing the amino acid sequence described in SEQ ID NO: 13. In some embodiments, the anti-claudin-1 antibody comprises a complementarity-determining region (CDR) H1 containing the amino acid sequence described in SEQ ID NO: 5, a CDR H2 containing the amino acid sequence described in SEQ ID NO: 6, a CDR H3 containing the amino acid sequence described in SEQ ID NO: 7, and VL containing the amino acid sequence described in SEQ ID NO: 14. In some embodiments, the anti-claudin-1 antibody comprises a complementarity-determining region (CDR) H1 containing the amino acid sequence described in SEQ ID NO: 5, a CDR H2 containing the amino acid sequence described in SEQ ID NO: 6, a CDR H3 containing the amino acid sequence described in SEQ ID NO: 7, and VH containing the amino acid sequence described in SEQ ID NO: 13 and VL containing the amino acid sequence described in SEQ ID NO: 14.

[0102] In some aspects, the six complementarity-determining regions (CDRs) of the anti-claudin-1 antibody are the same as those of the anti-claudin-1 monoclonal antibody secreted by a hybridoma cell line deposited in DSMZ on July 29, 2008, under accession number DSM ACC2938.

[0103] In some embodiments, the heavy chain variable region ("VH") and light chain variable region ("VL") of the anti-claudin-1 antibody are identical to those of the anti-claudin-1 monoclonal antibody secreted by a hybridoma cell line deposited in DSMZ on July 29, 2008, under accession number DSM ACC2938.

[0104] In some embodiments, the heavy and light chains of the anti-claudin-1 antibody are identical to those of the anti-claudin-1 monoclonal antibody secreted by a hybridoma cell line deposited in DSMZ on July 29, 2008, under accession number DSM ACC2938.

[0105] A humanized anti-claudin-1 antibody may be a complete monoclonal antibody having isotopes 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, such as a variable fragment (Fv), an antigen-binding fragment (Fab), a bivalent antibody fragment (F(ab')2), a Fab prime fragment (Fab'), a disulfide-stabilized Fv fragment (dsFv), a single-chain variable fragment (scFv), or a tandem single-chain variable fragment (sc(Fv)2). In some embodiments, the humanized anti-claudin-1 antibody may be a bispecific antibody, such as a diabody.

[0106] An anti-claudin-1 antibody (or its biologically active variant or fragment) suitable for use according to this disclosure may be functionally conjugated to one or more other molecular entities (e.g., by chemical coupling, gene fusion, non-covalent bonding, or other methods). Methods for preparing such modified antibodies (or conjugated antibodies) are known in the art (see, for example, “Affinity Techniques. Enzyme Purification: Part B”, Methods in Enzymol., 1974, Vol.34, Jakoby and Wilneck (Eds.), Academic Press: New York, NY, and Wilchek and Bayer, Anal. Biochem., 1988, 171:1-32). Preferably, the molecular entities are conjugated at a position on the antibody molecule that does not interfere with the binding properties of the resulting conjugate, for example, at a position that is not involved in the specific binding of the antibody to its target.

[0107] Antibody molecules and molecular entities may be directly covalently bonded to each other. Alternatively, antibody molecules and molecular entities may be covalently bonded to each other via linker groups. This can be achieved by using one of the wide variety of stable bifunctional agents known in the art, including homofunctional and heterofunctional linkers.

[0108] In some embodiments, the anti-claudin-1 antibody (or a biologically active fragment thereof) for use in accordance with this disclosure is conjugated to a detectable agent. Any of the wide variety of detectable agents available may be used, including, but are not limited to, various ligands, radionuclides (e.g., 3H, 125I, 131I, etc.), fluorescent dyes (e.g., fluorescein isothiocyanate, rhodamine, phycoerytherin, phycocyanin, allophycocyanin, o-phthalaldehyde, and fluorescamine), chemiluminescent agents (e.g., luciferin, luciferase, and aequorin), microparticles (e.g., quantum dots, nanocrystals, phosphors, etc.), enzymes (e.g., those used in ELISA, i.e., horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase), colorimetric labels, magnetic labels, and biotin, dioxygenin, or other haptens and proteins for which antiserum or monoclonal antibodies are available.

[0109] Other molecular entities that may be conjugated to the anti-claudin-1 antibody (or its biologically active fragment) of this disclosure include, but are not limited to, linear or branched hydrophilic polymer groups, fatty acid groups, or fatty acid ester groups.

[0110] Accordingly, in the course of this disclosure, anti-claudin-1 antibodies may be used in the form of molecules such as full-length antibodies, their biologically active variants or fragments, chimeric antibodies, humanized antibodies, and antibody-derived molecules comprising at least one complementarity-determining region (CDR) from the heavy-chain or light-chain variable region of an anti-claudin-1 antibody, e.g., Fab fragments, F(ab')2 fragments, Fd fragments, Fabc fragments, Sc antibodies (single-chain antibodies), diabodies, individual antibody light chains, nanobodies, individual antibody heavy chains, antibodies consisting only of heavy chains, chimeric fusions of antibody chains with other molecules, and antibody conjugates, e.g., antibodies conjugated to therapeutic agents or detectable drugs. Preferably, the anti-claudin-1 antibody-related molecules according to this disclosure retain the ability of the antibody to bind to its antigen, particularly the extracellular domain of claudin-1.

[0111] Chimeric antigen receptor Chimeric antigen receptor (CAR) T-cell therapy, or CAR T-cell therapy, is a cancer treatment based on the use of T cells that have been genetically engineered to express synthetic receptors that bind to tumor antigens. The engineered CAR T cells are grown in vitro and injected into the patient's body to attack and destroy chemotherapy-resistant cancers.

[0112] The term "chimeric antigen receptor" (CAR) refers to a molecule that combines a binding domain to a component present on a target cell, such as antibody-based specificity against a desired antigen (e.g., a tumor antigen such as CLDN1), with a T cell receptor-activating intracellular domain to produce a chimeric protein that exhibits specific anti-target cell immune activity.

[0113] The "signal transducing domain" or "signaling domain" of a CAR is responsible for intracellular signaling after the extracellular ligand-binding domain binds to a target, leading to the activation of immune cells and an immune response. In other words, the signaling domain is responsible for activating at least one of the normal effector functions of the immune cells on which the CAR is expressed. For example, the effector function of a T cell may be helper activity, including cytolytic activity or cytokine secretion. Therefore, the term "signaling domain" refers to the part of a protein that transduces effector function signals and instructs cells to perform specialized functions. Examples of signaling domains for use in CARs include cytoplasmic sequences of T cell receptors and co-receptors that act simultaneously and initiate signaling after antigen receptor binding, 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 includes two distinct classes of cytoplasmic signaling sequences: those that initiate antigen-dependent primary activation, and those that act antigen-independently to provide secondary or co-stimulatory signals. Primary cytoplasmic signaling sequences may include signaling motifs known as the immunoreceptor-activating tyrosine motif of ITAMs. ITAMs are well-defined signaling motifs found in the cytoplasmic tails of various receptors that function as binding sites for syk / zap70 class tyrosine kinases. Exemplary ITAMs include those derived from TCRζ, FcRγ, FcRβ, FcRε, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, and CD66d. In some embodiments, the signaling domain of the CAR may include a CD3ζ signaling domain (SEQ ID NO: 15).

[0114] Generally, CARs are synthetic receptors consisting of a targeting moiety that associates with one or more signaling domains in a single fusion molecule. Typically, the binding moiety of a CAR consists of the antigen-binding domain of a monoclonal antibody (scFv) containing light and heavy chain variable fragments of a monoclonal antibody conjugated by a flexible linker. This molecule typically binds to an intracellular signaling molecule containing one or more intracellular signaling domains that mediate T cell activation. The signaling domains of first-generation CARs generally derive from the cytoplasmic region of the CD3ζ or Fc receptor gamma chain (or the intracellular signaling domain of another immunoreceptor-activating tyrosine motif [ITAM]-containing protein). First-generation CARs have been shown to successfully redirect the cytotoxicity of T cells. However, they have not been able to provide long-term proliferation and antitumor activity in vivo. The addition of signaling domains from costimulatory molecules, as well as transmembrane and hinge domains, forms second, third, and fourth-generation CARs. Second-generation chimeric receptors generally incorporate a costimulatory endodomain (e.g., 4-1BB / CD3ζ). Third-generation CARs generally contain multiple co-stimulatory signaling modules. Fourth-generation CARs are generated by adding IL-12 to the base of second-generation constructs and are known as T cells redirected to universal cytokine-mediated killing (TRUCKs). TRUCKs enhance T cell activation and activate and induce innate immune cells to eliminate antigen-negative cancer cells in target lesions. Human therapeutic trials using CAR T cell therapy have shown some success. For example, CAR-redirected T cells specific to the B cell differentiation antigen CD19 have shown dramatic efficacy in treating B cell malignancies, and TCR-redirected T cells have shown benefits in patients with solid tumors. Stauss et al. have described strategies for modifying therapeutic CARs and TCRs for use in cancer treatment, for example, to enhance antigen-specific effector function and limit the toxicity of engineered T cells (Current Opinion in Pharmacology 2015, 24:113-118).

[0115] In some embodiments, this specification provides CARs specific to claudin-1 expressed on the surface of cancer cells. In some embodiments of this disclosure, the CAR comprises an extracellular target-specific binding domain, a transmembrane domain, an intracellular signaling domain (e.g., a signaling domain derived from CD3ζ or FcRγ), and / or one or more costimulatory signaling domains derived from a costimulatory molecule, e.g., 4-1BB, but not limited to these. In some embodiments, the CAR comprises a hinge or spacer region between the extracellular binding domain and the transmembrane domain, e.g., a CD8α hinge. In some embodiments, the CAR is an anti-claudin single-chain antibody (scFv) comprising an extracellular target-specific binding domain, which may be mouse, human, or humanized scFv. The single-chain antibody may be cloned from a V-region gene of a hybridoma specific to the desired target. Techniques that can be used for cloning variable region heavy chains (VH) and variable region light chains (VL) are described, for example, in Orlandi et al., PNAS 86:3833-3837 (1989). Therefore, in some embodiments, the binding domain may include an antibody-derived binding domain, but it may also be a non-antibody-derived binding domain. The antibody-derived binding domain may be a fragment of an antibody, or a genetically modified product of one or more fragments of an antibody, which is involved in binding to the antigen.

[0116] In some embodiments, the CARs of this disclosure may include linkers between various domains added for proper spacing and conformation of the molecule. For example, in some embodiments, a linker which may be 1 to 10 amino acid lengths may be present between binding domains VH or VL. In some embodiments, a linker between any of the domains of the chimeric antigen receptor may be 1 to 20 or 20 amino acid lengths. In this regard, the linker may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid lengths. In some embodiments, the linker may be 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acid lengths. A range including the numbers described herein, for example, linkers of 10 to 30 amino acid lengths, is also included herein.

[0117] In some embodiments, the linker suitable for use in the CAR described herein is a flexible linker. The suitable linker can be easily selected and may be any of several suitable lengths, such as 1 amino acid (e.g., Gly) to 20 amino acids, 2 amino acids to 15 amino acids, 3 amino acids to 12 amino acids, 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 may be 1, 2, 3, 4, 5, 6, or 7 amino acids.

[0118] Exemplary flexible linkers include glycine polymer (G)n, glycine-serine polymer (where n is an integer of at least 1), glycine-alanine polymer, alanine-serine polymer, and other flexible linkers known in the art. Glycine and glycine-serine polymers are relatively structurally indeterminate and can therefore function as neutral tethers between domains of fusion proteins such as CARs described herein. Glycine has significantly more access to the phi-pse space than alanine and is less restrictive than residues with longer side chains (see Scheraga, Rev. Computational Chem. 11173-142 (1992)). Those skilled in the art will recognize that the design of a CAR may include all or part flexible linkers, thereby the linker may include a flexible linker and one or more parts that confer less flexible structures in order to provide the desired CAR structure. Certain linkers include the (G4S)n linker, where n = 1 to 3. In some embodiments, the linker includes the amino acid sequence of SEQ ID NO: 16 or SEQ ID NO: 17.

[0119] The binding domain of a CAR may be followed by a “spacer” or “hinge,” which refers to a region that moves the antigen-binding domain away from the effector cell surface, enabling proper cell / cell contact, antigen binding, and activation (Patel et al., Gene Therapy 6:412-419 (1999)). The hinge region of a CAR is generally located between the transmembrane (TM) domain and the binding domain. In some embodiments, the hinge region is an immunoglobulin hinge region, which may be a wild-type immunoglobulin hinge region or a modified wild-type immunoglobulin hinge region. Other exemplary hinge regions used in CARs described herein include hinge regions derived from the extracellular regions of type 1 membrane proteins such as CD8α, CD4, CD28, and CD7, which may be wild-type hinge regions derived from these molecules or modified. In some embodiments, the hinge region includes a CD8α hinge (SEQ ID NO: 18).

[0120] The "transmembrane" region or domain is a portion of the CAR that fixes the extracellular binding portion to the plasma membrane of an immunoeffector cell, facilitating the binding of the target antigen-binding domain. The transmembrane domain may be the CD3ζ transmembrane domain; however, other transmembrane domains that may be used include those derived from CD8α, CD4, CD28, CD45, CD9, CD16, CD22, CD33, CD64, CD80, CD86, CD134, CD137, and CD154. In some embodiments, the transmembrane domain is the transmembrane domain of CD137. In some embodiments, the transmembrane domain contains the amino acid sequence of SEQ ID NO: 19. In some embodiments, the transmembrane domain is synthetic, in which case it mainly contains hydrophobic residues such as leucine and valine.

[0121] As described above, “intracellular signaling domain” or “signaling domain” refers to a portion of a chimeric antigen receptor protein that transduces an effective CAR binding message to a target antigen into the interior of an immune effector cell, thereby inducing effector cell function, such as activation, cytokine production, proliferation, or cytotoxic activity, such as the release of cytotoxic factors to CAR-bound target cells, or other cellular responses induced by antigen binding to an extracellular CAR domain. The term “effector function” refers to the cell’s specialization function. The effector function of a T cell may be, for example, cytolytic activity or helper activity including cytokine secretion. Therefore, the terms “intracellular signaling domain” or “signaling domain” are used interchangeably herein and refer to a portion of a protein that transmits effector function signals and instructs cells to perform specialization functions. Usually, the entire intracellular signaling domain may be used, but in many cases, it is not necessary to use the entire domain. A truncated portion of the intracellular signaling domain may be used to the extent that such a truncated portion can be used in place of the entire domain, as long as it transmits effector function signals. The term intracellular signaling domain refers to any truncated portion of an intracellular signaling domain sufficient to transmit effector functional signals. Intracellular signaling domains are also known as “signaling domains” and are typically derived from portions of the human CD3 or FcRγ chain.

[0122] It is known in the art that signals generated solely via T cell receptors are insufficient for complete T cell activation, and that secondary or co-stimulatory signals are also necessary. Therefore, it can be said that T cell activation is mediated by two distinct classes of cytoplasmic signaling sequences: those that initiate antigen-dependent primary activation via T cell receptors (primary cytoplasmic signaling sequences) and those that act antigen-independently to provide secondary or co-stimulatory signals (secondary cytoplasmic signaling sequences). Co-stimulatory cytoplasmic signaling sequences may include signaling motifs known as immunoreceptor-activated tyrosine motifs or ITAMs.

[0123] Examples of ITAM-containing primary cytoplasmic signaling sequences particularly useful in the CARs disclosed herein include those derived from TCRζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, and CD66d. In one particular embodiment, the intracellular signaling domain of the anti-BCMA CAR described herein is derived from CD3ζ. In one particular embodiment, the signaling domain comprises the amino acid sequence of SEQ ID NO: 15.

[0124] As used herein, the terms “costimulatory signaling domain” or “costimulatory domain” refer to the portion of a CAR containing the intracellular domain of a costimulatory molecule. A costimulatory molecule is a cell surface molecule other than an antigen receptor or Fc receptor that provides a secondary signal necessary for the efficient activation and function of T lymphocytes upon binding to an antigen. Examples of such costimulatory molecules include ligands that specifically bind to CD27, CD28, 4-1BB (CD137), OX40 (CD134), CD30, CD40, PD-1, ICOS (CD278), LFA-1, CD2, CD7, LIGHT, NKD2C, B7-H2, and CD83. Therefore, this disclosure provides exemplary sequences of costimulatory domains derived from CD3ζ and 4-1BB, but other costimulatory domains are intended for use with the CARs described herein. Inclusion of one or more costimulatory signaling domains may enhance the efficacy and proliferation of T cells expressing CAR receptors. The intracellular signaling and co-stimulatory signaling domains may be tandem-linked to the carboxyl terminus of the transmembrane domain in any order. In some embodiments, the co-stimulatory domain includes the amino acid sequence of SEQ ID NO: 20.

[0125] In some embodiments, the anti-claudin-1 CAR of the present disclosure includes any of the elements in Table 2. [Table 2]

[0126] While scFv-based CARs engineered to include signaling domains derived from CD3 or FcRγ have been shown to deliver potent signals for T cell activation and effector function, they are typically insufficient to induce signals that promote T cell survival and proliferation in the absence of accompanying co-stimulatory signals. Other CARs containing one or more co-stimulatory signaling domains (e.g., intracellular co-stimulatory domains derived from CD28, CD137, CD134, and CD278) along with binding domains, hinges, transmembrane domains, and signaling domains derived from CD3ζ or FcRγ may more effectively induce antitumor activity, as well as increased cytokine secretion, lytic activity, survival, and proliferation in CAR-expressing T cells in vitro, as well as in animal models and cancer patients (Milone et al., Molecular Therapy 17:1453-1464 (2009), Zhong et al., Molecular Therapy 18:413-420 (2010), Carpenito et al., PNAS 106:3360-3365 (2009)).

[0127] In some embodiments, the anti-claudin-1 CAR of the present disclosure comprises (a) an anti-claudin-1 binding domain (e.g., an scFv having a binding region (e.g., a CDR or variable domain) from one or more of the sequences specified 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, e.g., 4-1BB.

[0128] The anti-claudin-1 binding domain may include either the VH sequence or the VL sequence described herein. For example, the anti-claudin-1 binding domain may include VH containing the sequence described in SEQ ID NO: 3 or 13, and VL containing the sequence described in SEQ ID NO: 4 or 14. In some embodiments, the anti-claudin-1 binding domain includes CDR H1 containing the amino acid sequence described in SEQ ID NO: 5, CDR H2 containing the amino acid sequence described in SEQ ID NO: 6, and CDR H3 containing the amino acid sequence described in SEQ ID NO: 7. In some embodiments, the anti-claudin-1 binding domain includes complementarity-determining region (CDR) L1 containing the amino acid sequence described in SEQ ID NO: 8, CDR L2 containing the amino acid sequence "Gly Ala", and CDR L3 containing the amino acid sequence described in SEQ ID NO: 10. In some embodiments, the anti-claudin-1 binding domain includes a VH having an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 3. In some aspects, the anti-claudin-1 binding domain includes a VH having an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 13. In some embodiments, the anti-claudin-1 binding domain includes a VL containing an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 4. In some embodiments, the anti-claudin-1 binding domain includes a VL containing an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 14. In some embodiments, the anti-claudin-1 binding domain includes a VH containing the amino acid sequence described in SEQ ID NO: 3 and a VL containing the amino acid sequence described in SEQ ID NO: 4. In some embodiments, the anti-claudin-1 binding domain includes a VH containing the amino acid sequence described in SEQ ID NO: 13 and a VL containing the amino acid sequence described in SEQ ID NO: 14.

[0129] In some embodiments, different protein domains are arranged in the following order from the amino terminus to the carboxyl terminus, and from the N terminus to the C terminus: an anti-claudin-1 binding domain, a hinge region, and a transmembrane domain. Intracellular signaling domains and optionally selected co-stimulatory signaling domains are tandem-linked to the transmembrane carboxyl terminus in any order to form a single-chain chimeric polypeptide.

[0130] Polynucleotides and vectors In some embodiments, polynucleotides encoding CARs as described herein are provided. In some embodiments, the nucleic acid construct encoding anti-claudin-1 CAR is a chimeric nucleic acid molecule comprising a nucleic acid molecule comprising different coding sequences, e.g., anti-claudin-1 scFv (from 5' to 3'), human CD8α hinge, human CD8α transmembrane domain, and CD3ζ intracellular signaling domain coding sequences. In some embodiments, the nucleic acid construct encoding anti-claudin-1 CAR is a chimeric nucleic acid molecule comprising a nucleic acid molecule comprising different coding sequences, e.g., anti-claudin-1 scFv (from 5' to 3'), human CD8α hinge, human CD8α transmembrane domain, 4-1BB costimulatory domain, and CD3ζ costimulatory domain coding sequences.

[0131] In some embodiments, the polynucleotide encoding the CAR described herein is inserted into a vector. For the expression of anti-claudin-1 CAR, the vector may be introduced into a host cell to enable the expression of the polypeptide within the host cell. The expression vector may include, but is not limited to, a promoter sequence, a transcription start sequence, an enhancer sequence, a selectable marker, and a signal sequence, to control expression. These elements may be appropriately selected by those skilled in the art, as described above. For example, the promoter sequence may be selected to promote the transcription of the polynucleotide in the vector. Suitable promoter sequences include, but are not limited to, the T7 promoter, T3 promoter, SP6 promoter, β-actin promoter, EF1a promoter, CMV promoter, and SV40 promoter. The enhancer sequence may be selected to enhance the transcription of the polynucleotide. The selectable marker may be selected to enable the selection of a host cell into which the vector has been inserted from one into which it has not; for example, the selectable marker may be a gene that confers antibiotic resistance. The signal sequence may be selected to enable the expressed polypeptide to be transported outside the host cell.

[0132] In some embodiments, the vector is a plasmid, such as plasmid DNA, phage DNA, bacterial plasmid, or phage DNA. In some embodiments, the vector is a viral vector, such as a retrovirus, adenovirus, vaccinia virus, or baculovirus.

[0133] Modified cells The CARs of this disclosure may be introduced into host cells using transfection and / or transduction techniques known in the art. As used herein, the terms “transfection” and “transduction” refer to the process by which an exogenous nucleic acid sequence is introduced into a host cell. The nucleic acid may be incorporated into the host cell DNA or maintained outside the chromosome. The nucleic acid may be transiently maintained or a stable introduced product. Transfection may 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 bioristics. Transduction refers to the delivery of a gene(s) using a virus or retroviral vector by viral infection, rather than by transfection.

[0134] As used herein, the terms “genetically engineered” or “genetically modified” in relation to cells refer to the addition of additional genetic material in the form of DNA or RNA to the total genetic material within a cell. The terms “genetically modified cell,” “modified cell,” and “redirected cell” are used interchangeably.

[0135] In some embodiments, the CARs of this disclosure are introduced and expressed in immunoeffector cells to redirect their specificity to a target antigen of interest, such as claudin-1.

[0136] This disclosure provides methods for producing immunoeffector cells expressing CARs as described herein. In some embodiments, the method involves transfecting or transfecting immunoeffector cells isolated from a subject, such as a subject having claudin-1 expressing tumor cells, so that the immunoeffector cells express one or more CARs as described herein. In some embodiments, the immunoeffector cells are isolated from an organism and genetically modified without further in vitro manipulation. Such cells can then be directly re-administered to an organism. In some embodiments, the immunoeffector cells are first activated and stimulated and proliferated in vitro before being genetically modified to express a CAR. In this regard, the immunoeffector cells can be cultured before or after genetic modification (i.e., transfected or transfected to express a CAR as described herein).

[0137] Prior to the in vitro manipulation or genetic modification of the immunoeffector cells described herein, source cells may be obtained from the subject. In some embodiments, the immunoeffector cells for use with the CARs described herein include T cells. T cells may be obtained from several sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymic tissue, tissue from infection sites, ascites, pleural fluid, splenic tissue, and tumors. The cells may be autologous or allogeneic. In some embodiments, T cells may be obtained from blood units collected from the subject using any number of techniques known to those skilled in the art, such as Ficoll® isolation. In some embodiments, cells derived from the circulating blood of an individual are obtained by apheresis. Apheresis products typically contain lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated leukocytes, erythrocytes, and platelets. In some embodiments, cells collected by apheresis may be washed to remove the plasma fraction, and the cells may be placed in a buffer or medium appropriate for subsequent processing. In some embodiments, the cells are washed with PBS. In some embodiments, the washing solution may be calcium-deficient, magnesium-deficient, or many but not all divalent cations. As will be understood by those skilled in the art, the washing step may be achieved by methods known to those skilled in the art, for example, by using semi-automatic flow-through centrifugation. After washing, the cells may be resuspended in various biocompatible buffers or other saline solutions (with or without buffers). In some embodiments, undesirable components of the apheresis sample may be removed by transferring the cells directly to the culture medium in which they are resuspended.

[0138] In some embodiments, T cells are isolated from peripheral blood mononuclear cells by lysing erythrocytes and depleting monocytes, for example, by centrifugation using a PERCOLL® gradient. Specific subpopulations of T cells, such as CD28+, CD4+, CD8+, CD45RA+, and CD45RO+ T cells, may be further isolated by positive or negative selection techniques. For example, enrichment of a T cell population by negative selection may be achieved using a combination of antibodies directed to surface markers specific to negatively selected cells. One method for use herein is cell sorting and / or selection by negative magnetic immunoadhesion or flow cytometry using a cocktail of monoclonal antibodies directed to cell surface markers present on negatively selected cells. For example, to enrich CD4+ cells by negative selection, a cocktail of monoclonal antibodies typically includes antibodies against CD14, CD20, CD1b, CD16, HLA-DR, and CD8. Flow cytometry and cell sorting may also be used to isolate the desired cell population for use according to this disclosure.

[0139] PBMCs can be used directly for CAR-mediated genetic modification using the methods described herein. In some embodiments, after isolation of PBMCs, T lymphocytes are further isolated, and in some embodiments, both cytotoxic T lymphocytes and helper T lymphocytes can be classified into naive T cell subpopulations, memory T cell subpopulations, and effector T cell subpopulations either before or after genetic modification and / or proliferation. CD8+ cells can be obtained using standard methods. In some embodiments, CD8+ cells are further classified into naive cells, central memory cells, and effector cells by identifying the cell surface antigens associated with each of their types of CD8+ cells. In some embodiments, memory T cells are present in both the CD62L+ subset and the CD62L- subset of CD8+ peripheral blood lymphocytes. After staining with anti-CD8 and anti-CD62L antibodies, PBMCs are classified into CD62L-CD8+ and CD62L+CD8+ fractions. In some embodiments, 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 embodiments, central memory T cells are CD45RO+, CD62L+, CD8+ T cells. In some embodiments, effector T cells are negative for CD62L, CCR7, CD28, and CD127, and positive for granzyme B and perforin. In some embodiments, naive CD8+ T lymphocytes are characterized by the expression of naive T cell phenotypic markers including CD62L, CCR7, CD28, CD3, CD127, and CD45RA.

[0140] In some embodiments, CD4+ T cells are further classified into subpopulations. For example, CD4+ T helper cells can be classified into naive cells, central memory cells, and effector cells by identifying cell populations with cell surface antigens. CD4+ lymphocytes can be obtained by standard methods. In some embodiments, naive CD4+ T lymphocytes are CD45RO-, CD45RA+, and CD62L+ CD4+ T cells. In some embodiments, central memory CD4+ cells are CD62L-positive and CD45RO-positive. In some embodiments, effector CD4+ cells are CD62L-negative and CD45RO-negative.

[0141] Immune effector cells, such as T cells, can be genetically modified after isolation using known methods, or immune effector cells can be activated and proliferated (or differentiated in the case of progenitor cells) in vitro before genetic modification. In some embodiments, immune effector cells, such as T cells, are genetically modified with chimeric antigen receptors as described herein (e.g., transduced with a viral vector containing nucleic acids encoding CARs), and then activated and proliferated in vitro. Methods for activating and proliferating T cells are known in the art and are described, for example, in U.S. Patents 6,905,874, 6,867,041, 6,797,514, and WO2012079000. Generally, such methods involve contacting PBMCs or isolated T cells with stimulatory and co-stimulatory factors, such as anti-CD3 and anti-CD28 antibodies, generally bound to beads or other surfaces, in a medium containing a suitable cytokine, such as IL-2. The anti-CD3 and anti-CD28 antibodies bound to the same beads function as "surrogate" antigen-presenting cells (APCs). In some embodiments, T cells are activated and stimulated and proliferated using feeder cells and appropriate antibodies and cytokines, such as in the methods described in U.S. Patent No. 6,040,177, No. 5,827,642, and WO2012129514.

[0142] biomarkers This specification provides biomarkers that can be used to identify patients suitable for treatment with anti-claudin-1 antibodies and to guide treatment strategies. In some embodiments, the biomarker is PRO-C3. In some embodiments, the biomarker is C4G. In some embodiments, the biomarker is a combination of PRO-C3 and C4G.

[0143] PRO-C3 Type III collagen, along with type I collagen, constitutes a major structural protein in the human body, and is essential for type I collagen fiber formation, except in bone, which is composed almost entirely of type I collagen (Bao, X., et al., Journal of Genetics and Genomics 34(3):223-228 (2014), Jensen, LT, and Host, NB, Cardiovascular Research 33(3):535-539 (1997)). During fiber assembly, the N-terminal propeptide of type III collagen is cleaved by a specific N-protease before mature collagen is incorporated into the extracellular matrix (ECM), thus being released into the ECM and entering circulation. The propeptide molecule consists of three identical α-chains with a total molecular weight of 42 kDa. Propeptide removal is sometimes incomplete, leaving propeptides bound to the molecule, resulting in thin fibrils with abnormal crosslinking, which facilitates rapid metabolic turnover (Niemelae, O., et al., The Biochemical Journal 232(1):145-150 (1985), Wang, W., et al., The Biochemical Journal 398(3):515-519 (2006)). Therefore, PIIINP can be a marker for both formation and degradation. Enzyme-linked immunosorbent assays (ELISAs) against the N-terminal propeptide of type III collagen ("PRO-C3") have been developed to assess true formation through the development of monoclonal antibodies against the N-protease-producing neoepitope of the N-terminal propeptide of type III collagen (Nielsen, M., et al., American Journal of Translational Research 5(3):303-315 (19 Apr. 2013)).

[0144] In some embodiments, PRO-C3 can be used as a biomarker to measure the binding of anti-CLDN1 antibody targets in cancer.

[0145] In some embodiments, cancer is selected from the group consisting of head and neck cancer (e.g., head and neck squamous cell carcinoma), lung cancer, breast cancer, melanoma, colorectal cancer, pancreatic cancer, esophageal cancer, bile duct cancer, and hepatocellular carcinoma.

[0146] In some embodiments, PRO-C3 levels are measured by enzyme-linked immunosorbent assay (ELISA). In some embodiments, PRO-C3 levels are measured by mass spectrometry.

[0147] C4G Granzyme B-degraded type IV collagen product (C4G) is a biomarker used to measure granzyme B-degraded type IV collagen product (C4G) in serum (Jensen, C., et al., Cancers 12(10):2786(2020)). The discovery of C4G is based on the research of Prakash et al., which showed that granzyme B promotes the migration of cytotoxic lymphocytes across the basement membrane by degrading components such as type IV collagen (Prakash, M., et al., Immunity 41(6):960-972(2014)).

[0148] In some embodiments, C4G levels are measured by enzyme-linked immunosorbent assay (ELISA). In some embodiments, C4G levels are measured by mass spectrometry.

[0149] Detection method In a particular embodiment, identifying patients suitable for anti-claudin-1 antibody therapy for the present method involves measuring or evaluating the expression of PRO-C3 and / or C4G in a sample, e.g., a cancer test tissue sample. The method for measuring or evaluating the expression of PRO-C3 and / or C4G can be achieved by any applicable method.

[0150] To evaluate the expression of PRO-C3 and / or C4G, test tissue samples can be obtained from patients in need of therapy. Test samples include, but are not limited to, any clinically appropriate tissue sample, such as tumor biopsy, core biopsy tissue sample, fine-needle aspiration, or body fluid sample, such as blood, plasma, serum, lymph, ascites, cystic fluid, or urine. In some embodiments, the test tissue sample is from a primary tumor. In some embodiments, the test tissue sample is from metastases. In some embodiments, the test tissue sample is collected from the subject at multiple time points, for example, before, during, and / or after treatment. In some embodiments, the test tissue sample is collected from different locations in the subject, for example, a sample from a primary tumor and a sample from metastases at a distant location. In some embodiments, the test tissue sample is from cancer tissue.

[0151] In some embodiments, the test tissue sample is a paraffin-embedded and fixed tissue sample. In some embodiments, the test tissue sample is a formalin-fixed, paraffin-embedded (FFPE) tissue sample. In some embodiments, the test tissue sample is a fresh tissue (e.g., tumor) sample. In some embodiments, the test tissue sample is a frozen tissue sample. In some embodiments, the test tissue sample is a fresh-frozen (FF) tissue (e.g., tumor) sample. In some embodiments, the test tissue sample is cells isolated from a liquid. In some embodiments, the test tissue sample contains circulating tumor cells (CTCs). In some embodiments, the test tissue sample contains tumor-infiltrating lymphocytes (TILs). In some embodiments, the test tissue sample contains tumor cells and tumor-infiltrating lymphocytes (TILs). In some embodiments, the test tissue sample contains circulating lymphocytes. In some embodiments, the test tissue sample is a preserved tissue sample. In some embodiments, the test tissue sample is a preserved tissue sample with a known history of diagnosis, treatment, and / or outcome. In some embodiments, the sample is a block of tissue. In some embodiments, the test tissue sample is dispersed cells. In some embodiments, the sample size is approximately 1 cell to approximately 1 × 10⁶ cells or more. In some embodiments, the sample size is approximately 1 cell to approximately 1 × 10⁵ cells. In some embodiments, the sample size is approximately 10,000 cells. In some embodiments, the sample size is approximately 1 cell to approximately 1,000 cells. In some embodiments, the sample size is approximately 1 cell to approximately 100 cells. In some embodiments, the sample size is approximately 1 cell to approximately 10 cells. In some embodiments, the sample size is a single cell.

[0152] In some embodiments, the test tissue sample is obtained from a liquid biopsy. In some embodiments, the test tissue sample is urine, plasma, or serum. In some embodiments, the test tissue sample is obtained by a nasal brush. In some embodiments, the test tissue sample is obtained by a hair biopsy or scalp biopsy. In some embodiments, the test tissue sample is obtained by bronchoalveolar lavage.

[0153] In some embodiments, the evaluation of PRO-C3 and / or C4G expression can be achieved without obtaining test tissue samples.

[0154] In some embodiments, selecting a suitable patient includes (i) preparing a test tissue sample obtained from a patient having tissue cancer, wherein the test tissue sample contains cancer cells, and (ii) evaluating the percentage of cells in the test tissue sample that express PRO-C3 and / or C4G on the cell surface, based on the evaluation that the percentage of cells in the test tissue sample that express PRO-C3 and / or C4G on the cell surface is higher than a predetermined threshold level.

[0155] However, it should be understood that in any method involving the measurement of PRO-C3 and / or C4G expression in a test tissue sample, the step of preparing a test tissue sample obtained from a patient is an optional step. That is, in some embodiments, the method includes this step, while in other embodiments, this step is not included in the method. It should also be understood that in certain embodiments, the “measurement” or “evaluation” step for identifying cells in a test tissue sample that express PRO-C3 and / or C4G, or determining their number or proportion, may be performed by an alternative method for assaying PRO-C3 and / or C4G expression, for example, by performing an IHC assay. In some other embodiments, no alternative step is involved, and PRO-C3 and / or C4G expression is evaluated, for example, by reviewing the test results reported from a laboratory. In some embodiments, PRO-C3 and / or C4G expression is evaluated by reviewing the results of an immunohistochemical assay from a laboratory. In some embodiments, the steps of a method including the evaluation of PRO-C3 and / or C4G expression provide an interim result, which may be provided to a physician or other healthcare provider for use in selecting a suitable candidate for claudin-1 antibody therapy. In some embodiments, the steps of a method including the evaluation of PRO-C3 and / or C4G expression provide an interim result, which may be provided to a physician or other healthcare provider for use in selecting a suitable candidate for treatment. In certain embodiments, the steps of a method including the evaluation of PRO-C3 and / or C4G expression provide an interim result, which may be provided to a physician or other healthcare provider for use in selecting a suitable candidate for claudin-1 antibody therapy. In some embodiments, the step of providing an interim result is performed by a physician or a person acting under the direction of a physician. In other embodiments, these steps are performed by an independent laboratory or by an independent person, such as a laboratory technician.

[0156] In other embodiments, the amount of PRO-C3 and / or C4G is assessed by performing an assay to detect the presence of PRO-C3 and / or C4G polypeptides. In further embodiments, the presence of PRO-C3 and / or C4G polypeptides is detected by IHC, enzyme-linked immunosorbent assay (ELISA), in vivo imaging, or flow cytometry. In some embodiments, the expression of PRO-C3 and / or C4G is assayed by IHC. In all other embodiments of these methods, the cell surface expression of PRO-C3 and / or C4G is assayed using, for example, IHC or in vivo imaging.

[0157] In some embodiments of this method, the percentage of cells expressing PRO-C3 and / or C4G in the test tissue sample is assessed by performing an assay to detect the presence of PRO-C3 and / or C4G polypeptides. In some embodiments, the presence of PRO-C3 and / or C4G polypeptides is detected by an immunohistochemical assay. In some embodiments, the test tissue sample is a tumor biopsy. In some embodiments, the test tissue sample is a formalin-fixed paraffin-embedded (FFPE) sample.

[0158] In some embodiments, the immunohistochemical assay is a monoplex assay. In some embodiments, the immunohistochemical assay is a multiplex assay.

[0159] In some embodiments of this method, an automated IHC method is used to assay the expression of PRO-C3 and / or C4G in FFPE tissue samples. This disclosure provides a method for detecting the presence of human PRO-C3 and / or C4G antigen in a test tissue sample, or a method for quantifying the level of human PRO-C3 and / or C4G antigen or the percentage of cells in the sample expressing said antigen, the method comprising contacting a test sample and a negative control sample with an antibody that specifically binds to human PRO-C3 and / or C4G under conditions that allow for the formation of a complex between the antibody or a portion thereof and human PRO-C3 and / or C4G. In some embodiments, the test tissue sample and the control tissue sample are FFPE samples. Complex formation is then detected, where the difference in complex formation between the test sample and the negative control sample indicates the presence of human PRO-C3 and / or C4G antigen in the sample. Various methods are used to quantify the expression of PRO-C3 and / or C4G.

[0160] In some embodiments, the automated IHC method includes (a) deparaffinizing and rehydrating tissue sections mounted in an automated staining device; (b) retrieving antigens in an automated staining device; (c) setting reagents on the automated staining device; and (d) operating the automated staining device to neutralize endogenous peroxidase in the specimen, block nonspecific protein binding sites on the slide, incubate the slide with a primary antibody, incubate with a post-primary blocking agent, incubate with a post-primary antibody detection agent such as another antibody which may or may not be conjugated to the detection enzyme, incubate with a high molecular weight enzyme detection reagent, add a chromogenic substrate to develop color, and counterstain with hematoxylin. In some embodiments, antigen retrieval includes using any heat-based antigen retrieval device.

[0161] Treatment method In some embodiments, this specification provides a method for treating cancer, comprising: (a) administering an anti-claudin-1 antibody to a subject; (b) determining the level of PRO-C3 in a test sample from the subject; and (c) comparing the level of PRO-C3 in the test sample to the level of PRO-C3 in a control sample. In some embodiments, if the level of PRO-C3 in the test sample is not decreased compared to the level of PRO-C3 in the control sample, the dose of anti-claudin-1 antibody administered to the subject is increased. In some embodiments, if the level of PRO-C3 in the test sample is decreased compared to the control sample, the dose of anti-claudin-1 antibody administered to the subject is not increased. In some embodiments, if the level of PRO-C3 in the test sample is decreased compared to the control sample, the dose of anti-claudin-1 antibody administered to the subject is not increased, but the subject is monitored. Monitoring of the subject may include, for example, periodic examinations, further measurement of PRO-C3 levels, or observation of tumors using imaging such as PET or CT scans. In some embodiments, this specification provides a method for treating cancer in a subject. In some embodiments, the subject is determined to have a level of PRO-C3 in the test sample from the subject that has not decreased compared to the level of PRO-C3 in the control sample. In some embodiments, the subject has previously been administered an anti-claudin-1 therapeutic agent (e.g., an anti-claudin-1 antibody or a claudin-1 targeting CAR disclosed herein), and the method comprises administering an increased dose of the anti-claudin-1 therapeutic agent to the subject.

[0162] In some embodiments, this specification provides a method for monitoring cancer progression in a subject, comprising: (a) quantifying the level of PRO-C3 in a test sample from the subject, indicating that the subject has previously been administered an anti-claudin-1 therapeutic agent; and (b) comparing the level of PRO-C3 in the test sample to the level of PRO-C3 in a control sample. In some embodiments, a level of PRO-C3 in the test sample that is not decreased compared to the level of PRO-C3 in the control sample indicates that the subject is sensitive to an increased dose of the anti-claudin-1 therapeutic agent. In some embodiments, if the subject is not sensitive to an increased dose of the anti-claudin-1 antibody, the subject is monitored. Monitoring of the subject may include, for example, regular examinations, further measurements of PRO-C3 levels, or observation of tumors using imaging such as PET or CT scans.

[0163] In some embodiments, this specification provides a method for monitoring cancer progression in a subject, comprising: (a) determining the level of PRO-C3 in a test sample from the subject; and (b) comparing the level of PRO-C3 in the test sample to the level of PRO-C3 in a control sample. In some embodiments, the subject has previously been administered an anti-claudin-1 antibody. In some embodiments, a level of PRO-C3 in the test sample that is not decreased compared to the level of PRO-C3 in the control sample indicates that the subject is sensitive to an increased dose of anti-claudin-1 antibody. In some embodiments, if the subject is not sensitive to an increased dose of anti-claudin-1 antibody, the subject is monitored. Monitoring of the subject may include, for example, regular examinations, further measurements of PRO-C3 levels, or observation of tumors using imaging such as PET or CT scans.

[0164] In some embodiments, the Specified Provision provides a method for treating a subject having cancer, comprising the step of obtaining a test sample from the subject and determining whether the subject has a reduced level of PRO-C3 by comparing the level of PRO-C3 in the test sample with that of a control sample. In some embodiments, the subject has previously been administered a certain dose of anti-claudin-1 antibody. In some embodiments, if the subject has a reduced level of PRO-C3 in the test sample compared to a control sample, the subject is not administered an increased dose of anti-claudin-1 antibody. In some embodiments, if the subject does not have a reduced level of PRO-C3 in the test sample compared to a control sample, the subject is administered an increased dose of anti-claudin-1 antibody. In some embodiments, if the subject has a reduced level of PRO-C3 in the test sample compared to a control sample, the subject is not administered an increased dose of anti-claudin-1 antibody, but the subject is monitored. Monitoring of the subject may include, for example, periodic examinations, further measurements of PRO-C3 levels, or observation of tumors using imaging such as PET or CT scans.

[0165] In some embodiments, this specification provides a method for designing an individualized therapy for a subject with cancer, comprising: (a) quantifying the level of PRO-C3 in a test sample from the subject; (b) comparing the level of PRO-C3 in the test sample obtained in step (a) with the level of PRO-C3 in a control sample; (c) determining that the subject has an undecreased level of PRO-C3 compared to the level of PRO-C3 in the control sample; and (d) administering an increased dose of anti-claudin-1 antibody. In some embodiments, the subject has previously been administered a certain dose of anti-claudin-1 antibody. In some embodiments, if the level of PRO-C3 in the test sample obtained in step (a) is undecreased compared to the level of PRO-C3 in the control sample, the subject is suitable for administration of an increased dose of anti-claudin-1 antibody. In some embodiments, if the subject is not suitable for administration of an increased dose of anti-claudin-1 antibody, the subject is monitored. Monitoring of the target may include, for example, regular examinations, further measurement of PRO-C3 levels, or observation of tumors using imaging such as PET or CT scans.

[0166] In some embodiments, this specification provides a method for classifying subjects with cancer into a cohort and treating said subjects, comprising: (a) quantifying the level of PRO-C3 in a test sample from the subject; (b) comparing the level of PRO-C3 in the test sample obtained in step (a) with the level of PRO-C3 in a control sample; (c) determining that the subject has an undecreased level of PRO-C3 compared to the level of PRO-C3 in a control sample; (d) classifying the subject into a cohort based on the level of PRO-C3 in the test sample compared to a control sample; and (e) administering an increased dose of anti-claudin-1 antibody. In some embodiments, the subject has previously been administered a certain dose of anti-claudin-1 antibody. In some embodiments, if the level of PRO-C3 in the test sample obtained in step (a) is undecreased compared to the level of PRO-C3 in a control sample, the subject is suitable for administration of an increased dose of anti-claudin-1 antibody. In some embodiments, if the subject is not suitable for administration of increased doses of anti-claudin-1 antibody, the subject is monitored. Monitoring of the subject may include, for example, regular examinations, further measurement of PRO-C3 levels, or observation of tumors using imaging such as PET or CT scans.

[0167] In some embodiments, the Specified Provision provides a method for treating cancer, comprising: (a) administering an anti-claudin-1 antibody to a subject; (b) determining the level of C4G in a test sample from the subject; and (c) comparing the level of C4G in the test sample to the level of C4G in a control sample. In some embodiments, if the level of C4G in the test sample is not increased compared to the level of C4G in the control sample, the dose of anti-claudin-1 antibody administered to the subject is increased. In some embodiments, if the level of C4G in the test sample is increased compared to the control sample, the dose of anti-claudin-1 antibody administered to the subject is not increased. In some embodiments, if the level of C4G in the test sample is increased compared to the control sample, the dose of anti-claudin-1 antibody administered to the subject is not increased, but the subject is monitored. Monitoring of the subject may include, for example, periodic examinations, further measurements of C4G levels, or observation of tumors using imaging such as PET or CT scans.

[0168] In some embodiments, the Specified provides a method for treating cancer in a subject that has been determined to have a level of C4G in a test sample from a subject that has not increased compared to the level of C4G in a control sample. In some embodiments, the subject has previously been administered an anti-claudin-1 therapeutic agent (e.g., an anti-claudin-1 antibody or a claudin-1 targeting CAR disclosed herein). In some embodiments, the method comprises administering an increased dose of the anti-claudin-1 therapeutic agent to the subject.

[0169] In some embodiments, the Specified Public Service provides a method for monitoring cancer progression in cancer, comprising (a) quantifying the level of C4G in a test sample from a subject, and (b) comparing the level of C4G in the test sample to the level of C4G in a control sample. In some embodiments, the subject has previously been administered an anti-claudin-1 therapeutic agent. In some embodiments, a level of C4G in the test sample that has not increased compared to the level of C4G in the control sample indicates that the subject is sensitive to an increased dose of the anti-claudin-1 therapeutic agent. In some embodiments, if the subject is not sensitive to an increased dose of the anti-claudin-1 therapeutic agent, the subject is monitored. Monitoring of the subject may include, for example, regular examinations, further measurements of C4G levels, or observation of the tumor using imaging such as PET or CT scans.

[0170] In some embodiments, this specification provides a method for monitoring cancer progression in cancer, comprising: (a) determining the level of C4G in a test sample from a subject; and (b) comparing the level of C4G in the test sample to the level of C4G in a control sample. In some embodiments, the subject has previously been administered an anti-claudin-1 antibody. In some embodiments, a level of C4G in the test sample that is not elevated compared to the level of C4G in the control sample indicates that the subject is sensitive to an increased dose of anti-claudin-1 antibody. In some embodiments, if the subject is not sensitive to an increased dose of anti-claudin-1 antibody, the subject is monitored. Monitoring of the subject may include, for example, regular examinations, further measurements of C4G levels, or observation of the tumor using imaging such as PET or CT scans.

[0171] In some embodiments, the Specified Method is provided for treating a subject having cancer, comprising the step of determining whether the subject has an elevated level of C4G by obtaining a test sample from the subject and comparing the level of C4G in the test sample with that of a control sample. In some embodiments, the subject has previously been administered a certain dose of anti-claudin-1 antibody. In some embodiments, if the subject has an elevated level of C4G in the test sample compared to a control sample, the subject is not administered an elevated dose of anti-claudin-1 antibody. In some embodiments, if the subject does not have an elevated level of C4G in the test sample compared to a control sample, the subject is administered an elevated dose of anti-claudin-1 antibody. In some embodiments, if the subject has an elevated level of C4G in the test sample compared to a control sample, the subject is not administered an elevated dose of anti-claudin-1 antibody, but the subject is monitored. Monitoring of the subject may include, for example, periodic examinations, further measurement of PRO-C3 levels, or observation of tumors using imaging such as PET or CT scans.

[0172] In some embodiments, this specification provides a method for designing an individualized therapy for a subject with cancer, comprising: (a) quantifying the level of C4G in a test sample from the subject; (b) comparing the level of C4G in the test sample obtained in step (a) with the level of C4G in a control sample; (c) determining that the subject has an unincreased level of C4G compared to the level of C4G in the control sample; and (d) administering an increased dose of anti-claudin-1 antibody. In some embodiments, the subject has previously been administered a certain dose of anti-claudin-1 antibody. In some embodiments, if the level of C4G in the test sample obtained in step (a) is unincreased compared to the level of C4G in the control sample, the subject is suitable for administration of an increased dose of anti-claudin-1 antibody. In some embodiments, if the subject is not suitable for administration of an increased dose of anti-claudin-1 antibody, the subject is monitored. Monitoring of the target may include, for example, regular examinations, further measurement of C4G levels, or observation of tumors using imaging such as PET or CT scans.

[0173] In some embodiments, this specification provides a method for classifying subjects with cancer into a cohort and treating said subjects, comprising: (a) quantifying the level of C4G in a test sample from the subject; (b) comparing the level of C4G in the test sample obtained in step (a) with the level of C4G in a control sample; (c) determining that the subject has a level of C4G that is not elevated compared to the level of C4G in a control sample; (d) classifying the subject into a cohort based on the level of C4G in the test sample compared to a control sample; and (e) administering an increased dose of anti-claudin-1 antibody. In some embodiments, the subject has previously been administered a certain dose of anti-claudin-1 antibody. In some embodiments, if the level of C4G in the test sample obtained in step (a) is not elevated compared to the level of C4G in a control sample, the subject is suitable for administration of an increased dose of anti-claudin-1 antibody. In some embodiments, if the subject is not suitable for administration of increased doses of anti-claudin-1 antibody, the subject is monitored. Monitoring of the subject may include, for example, periodic examinations, further measurement of C4G levels, or observation of tumors using imaging such as PET or CT scans.

[0174] In some embodiments, the Specified Provision provides a method for treating cancer, comprising (a) determining the level of PRO-C3 in a test sample from a subject, and (b) comparing the level of PRO-C3 in the test sample to the level of PRO-C3 in a control sample. In some embodiments, if the level of PRO-C3 in the test sample is not reduced compared to the level of PRO-C3 in the control sample, the subject is administered a certain dose of anti-claudin-1 antibody. In some embodiments, if the level of PRO-C3 in the test sample is reduced compared to the control sample, the subject is not administered a certain dose of anti-claudin-1 antibody. In some embodiments, if the level of PRO-C3 in the test sample is reduced compared to the control sample, the subject is not administered a certain dose of anti-claudin-1 antibody, but the subject is monitored. Monitoring of the subject may include, for example, periodic examinations, further measurements of PRO-C3 levels, or observation of tumors using imaging such as PET or CT scans.

[0175] In some embodiments, methods for treating cancer in a subject are provided herein. In some embodiments, the subject is determined to have a level of PRO-C3 in a test sample from the subject that has not decreased compared to the level of PRO-C3 in a control sample. In some embodiments, the method comprises administering an increased dose of an anti-claudin-1 therapeutic agent (e.g., an anti-claudin-1 antibody or a claudin-1 targeting CAR disclosed herein) to the subject.

[0176] In some embodiments, the Specified Public Service provides a method for monitoring cancer progression in a subject, comprising: (a) quantifying the level of PRO-C3 in a test sample from the subject; and (b) comparing the level of PRO-C3 in the test sample to the level of PRO-C3 in a control sample. In some embodiments, a level of PRO-C3 in the test sample that has not decreased compared to the level of PRO-C3 in the control sample indicates that the subject is suitable for administration of a certain dose of an anti-claudin-1 therapeutic agent. In some embodiments, if the subject is not suitable for administration of a certain dose of an anti-claudin-1 therapeutic agent, the subject is monitored. Monitoring of the subject may include, for example, periodic examinations, further measurements of PRO-C3 levels, or observation of tumors using imaging such as PET or CT scans.

[0177] In some embodiments, the Specified Public provides a method for monitoring cancer progression in a subject, comprising: (a) determining the level of PRO-C3 in a test sample from the subject; and (b) comparing the level of PRO-C3 in the test sample to the level of PRO-C3 in a control sample. In some embodiments, a level of PRO-C3 in the test sample that is not decreased compared to the level of PRO-C3 in the control sample indicates that the subject is suitable for administration of a certain dose of anti-claudin-1 antibody. In some embodiments, if the subject is not suitable for administration of a certain dose of anti-claudin-1 antibody, the subject is monitored. Monitoring of the subject may include, for example, periodic examinations, further measurements of PRO-C3 levels, or observation of tumors using imaging such as PET or CT scans.

[0178] In some embodiments, the Specified Provision provides a method for treating a subject with cancer, comprising the step of obtaining a test sample from the subject and determining whether the subject has a reduced level of PRO-C3 by comparing the level of PRO-C3 in the test sample with that of a control sample. In some embodiments, if the subject has a reduced level of PRO-C3 in the test sample compared with that of a control sample, the subject is not administered an increased dose of anti-claudin-1 antibody. In some embodiments, if the subject does not have a reduced level of PRO-C3 in the test sample compared with that of a control sample, the subject is administered a certain dose of anti-claudin-1 antibody. In some embodiments, if the subject has a reduced level of PRO-C3 in the test sample compared with that of a control sample, the subject is not administered an increased dose of anti-claudin-1 antibody, but the subject is monitored. Monitoring of the subject may include, for example, periodic examinations, further measurements of PRO-C3 levels, or observation of tumors using imaging such as PET or CT scans.

[0179] In some embodiments, this specification provides a method for designing a personalized therapy for a subject with cancer, comprising: (a) quantifying the level of PRO-3 in a test sample from the subject; (b) comparing the level of PRO-C3 in the test sample obtained in step (a) with the level of PRO-C3 in a control sample; (c) determining that the subject has an undecreased level of PRO-C3 compared to the level of PRO-C3 in the control sample; and (d) administering a certain dose of anti-claudin-1 antibody. In some embodiments, if the level of PRO-C3 in the test sample obtained in step (a) is undecreased compared to the level of PRO-C3 in the control sample, the subject is suitable for administration of a certain dose of anti-claudin-1 antibody. In some embodiments, if the subject is not suitable for administration of a certain dose of anti-claudin-1 antibody, the subject is monitored. Monitoring of the subject may include, for example, periodic examinations, further measurements of PRO-C3 levels, or observation of tumors using imaging such as PET or CT scans.

[0180] In some embodiments, this specification provides a method for classifying subjects with cancer into a cohort and treating the subjects, comprising: (a) quantifying the level of PRO-C3 in a test sample from the subject; (b) comparing the level of PRO-C3 in the test sample obtained in step (a) with the level of PRO-C3 in a control sample; (c) determining that the subject has an undecreased level of PRO-C3 compared to the level of PRO-C3 in a control sample; (d) classifying the subject into a cohort based on the level of PRO-C3 in the test sample compared to a control sample; and (e) administering a certain dose of anti-claudin-1 antibody. In some embodiments, if the level of PRO-C3 in the test sample obtained in step (a) is undecreased compared to the level of PRO-C3 in a control sample, the subject is suitable for administration of a certain dose of anti-claudin-1 antibody. In some embodiments, if the subject is not suitable for administration of an increased dose of anti-claudin-1 antibody, the subject is monitored. Monitoring of the target may include, for example, regular examinations, further measurement of PRO-C3 levels, or observation of tumors using imaging such as PET or CT scans.

[0181] In some embodiments, the Specified Provision provides a method for treating cancer, comprising (a) determining the level of C4G in a test sample from a subject, and (b) comparing the level of C4G in the test sample to the level of C4G in a control sample. In some embodiments, if the level of C4G in the test sample is not elevated compared to the level of C4G in the control sample, the subject is administered a certain dose of anti-claudin-1 antibody. In some embodiments, if the level of C4G in the test sample is elevated compared to the control sample, the subject is not administered anti-claudin-1 antibody. In some embodiments, if the level of C4G in the test sample is elevated compared to the control sample, the subject is not administered anti-claudin-1 antibody, but the subject is monitored. Monitoring of the subject may include, for example, periodic examinations, further measurements of C4G levels, or observation of tumors using imaging such as PET or CT scans.

[0182] In some embodiments, the Specified provides a method for treating cancer in a subject that has been determined to have a level of C4G in a test sample that has not increased compared to the level of C4G in a control sample. In some embodiments, the method comprises administering a certain dose of an anti-claudin-1 therapeutic agent (e.g., an anti-claudin-1 antibody or a claudin-1 targeting CAR disclosed herein) to the subject.

[0183] In some embodiments, the Specified Public Service provides a method for monitoring cancer progression in cancer, comprising (a) quantifying the level of C4G in a test sample from a subject, and (b) comparing the level of C4G in the test sample to the level of C4G in a control sample. In some embodiments, a level of C4G in the test sample that has not increased compared to the level of C4G in the control sample indicates that the subject is sensitive to a certain dose of an anti-claudin-1 therapeutic agent. In some embodiments, if the subject is not sensitive to a certain dose of an anti-claudin-1 therapeutic agent, the subject is monitored. Monitoring of the subject may include, for example, regular examinations, further measurements of C4G levels, or observation of the tumor using imaging such as PET or CT scans.

[0184] In some embodiments, the Specified Provision provides a method for monitoring cancer progression in cancer, comprising: (a) determining the level of C4G in a test sample from a subject; and (b) comparing the level of C4G in the test sample to the level of C4G in a control sample. In some embodiments, a level of C4G in the test sample that is not elevated compared to the level of C4G in the control sample indicates that the subject is sensitive to a certain dose of anti-claudin-1 antibody. In some embodiments, if the subject is not sensitive to a certain dose of anti-claudin-1 antibody, the subject is monitored. Monitoring of the subject may include, for example, regular examinations, further measurements of C4G levels, or observation of the tumor using imaging such as PET or CT scans.

[0185] In some embodiments, the Specified Provision provides a method for treating a subject having cancer, comprising the step of determining whether the subject has an elevated level of C4G by obtaining a test sample from the subject and comparing the level of C4G in the test sample with that of a control sample. In some embodiments, if the subject has an elevated level of C4G in the test sample compared to that of a control sample, the subject is not administered a certain dose of anti-claudin-1 antibody. In some embodiments, if the subject does not have an elevated level of C4G in the test sample compared to that of a control sample, the subject is administered a certain dose of anti-claudin-1 antibody. In some embodiments, if the subject has an elevated level of C4G in the test sample compared to that of a control sample, the subject is not administered a certain dose of anti-claudin-1 antibody, but the subject is monitored. Monitoring of the subject may include, for example, periodic examinations, further measurements of C4G levels, or observation of tumors using imaging such as PET or CT scans.

[0186] In some embodiments, this specification provides a method for designing a personalized therapy for a subject with cancer, comprising: (a) quantifying the level of C4G in a test sample from the subject; (b) comparing the level of C4G in the test sample obtained in step (a) with the level of C4G in a control sample; (c) determining that the subject has an unincreased level of C4G compared to the level of C4G in the control sample; and (d) administering a certain dose of anti-claudin-1 antibody. In some embodiments, if the level of C4G in the test sample obtained in step (a) is unincreased compared to the level of C4G in the control sample, the subject is suitable for administration of a certain dose of anti-claudin-1 antibody. In some embodiments, if the subject is not suitable for administration of a certain dose of anti-claudin-1 antibody, the subject is monitored. Monitoring of the subject may include, for example, periodic examinations, further measurements of C4G levels, or observation of tumors using imaging such as PET or CT scans.

[0187] In some embodiments, this specification provides a method for classifying subjects with cancer into a cohort and treating the subjects, comprising: (a) quantifying the level of C4G in a test sample from the subject; (b) comparing the level of C4G in the test sample obtained in step (a) with the level of C4G in a control sample; (c) determining that the subject has a level of C4G that is not elevated compared to the level of C4G in a control sample; (d) classifying the subject into a cohort based on the level of C4G in the test sample compared to a control sample; and (e) administering a certain dose of anti-claudin-1 antibody. In some embodiments, if the level of C4G in the test sample obtained in step (a) is not elevated compared to the level of C4G in a control sample, the subject is suitable for administration of a certain dose of anti-claudin-1 antibody. In some embodiments, if the subject is not suitable for administration of a certain dose of anti-claudin-1 antibody, the subject is monitored. Monitoring of the target may include, for example, regular examinations, further measurement of C4G levels, or observation of tumors using imaging such as PET or CT scans.

[0188] In some embodiments, this specification provides a method for treating cancer, comprising: (a) administering an anti-claudin-1 antibody to a subject; (b) determining the levels of PRO-C3 and C4G in a test sample from the subject; and (c) comparing the levels of PRO-C3 and C4G in the test sample to the levels of PRO-C3 and C4G in a control sample. In some embodiments, if the levels of PRO-C3 and C4G in the test sample are not decreased compared to the levels of PRO-C3 and C4G in the control sample, the dose of anti-claudin-1 antibody administered to the subject is increased. In some embodiments, if the levels of PRO-C3 and C4G in the test sample are not decreased compared to the levels of PRO-C3 and C4G in the control sample, the dose of anti-claudin-1 antibody administered to the subject is not increased. In some embodiments, if the levels of PRO-C3 and C4G in the test sample are not decreased compared to the levels of PRO-C3 and C4G in the control sample, the dose of anti-claudin-1 antibody administered to the subject is not increased, but the subject is monitored. Monitoring of the target may include, for example, regular examinations, further measurement of PRO-C3 and C4G levels, or observation of tumors using imaging such as PET or CT scans.

[0189] In some embodiments, methods for treating cancer in a subject are provided herein. In some embodiments, the subject is determined to have PRO-C3 and C4G levels in a test sample from the subject that have not decreased compared to the levels of PRO-C3 and C4G in a control sample. In some embodiments, the subject has previously been administered an anti-claudin-1 therapeutic agent (e.g., an anti-claudin-1 antibody or a claudin-1 targeting CAR disclosed herein), and the method comprises administering an increased dose of the anti-claudin-1 therapeutic agent to the subject.

[0190] In some embodiments, this specification provides a method for monitoring cancer progression in a subject, comprising: (a) quantifying the levels of PRO-C3 and C4G in a test sample from the subject, indicating that the subject has previously been administered an anti-claudin-1 therapeutic agent; and (b) comparing the levels of PRO-C3 and C4G in the test sample to the levels of PRO-C3 and C4G in a control sample. In some embodiments, levels of PRO-C3 and C4G in the test sample that are not decreased compared to the levels of PRO-C3 and C4G in the control sample indicate that the subject is sensitive to an increased dose of the anti-claudin-1 therapeutic agent. In some embodiments, if the subject is not sensitive to an increased dose of the anti-claudin-1 antibody, the subject is monitored. Monitoring of the subject may include, for example, regular examinations, further measurements of PRO-C3 and C4G levels, or observation of tumors using imaging such as PET or CT scans.

[0191] In some embodiments, the Specified Public provides a method for monitoring cancer progression in a subject, comprising: (a) determining the levels of PRO-C3 and C4G in a test sample from the subject; and (b) comparing the levels of PRO-C3 and C4G in the test sample to the levels of PRO-C3 and C4G in a control sample. In some embodiments, the subject has previously been administered an anti-claudin-1 antibody. In some embodiments, levels of PRO-C3 and C4G in the test sample that are not decreased compared to the levels of PRO-C3 and C4G in the control sample indicate that the subject is sensitive to an increased dose of anti-claudin-1 antibody. In some embodiments, if the subject is not sensitive to an increased dose of anti-claudin-1 antibody, the subject is monitored. Monitoring of the subject may include, for example, regular examinations, further measurements of PRO-C3 and C4G levels, or observation of tumors using imaging such as PET or CT scans.

[0192] In some embodiments, the Specified Provision provides a method for treating a subject having cancer, comprising the step of obtaining a test sample from the subject and determining whether the subject has reduced levels of PRO-C3 and C4G by comparing the levels of PRO-C3 and C4G in the test sample with those of a control sample. In some embodiments, the subject has previously been administered a certain dose of anti-claudin-1 antibody. In some embodiments, if the subject has reduced levels of PRO-C3 and C4G in the test sample compared to a control sample, the subject is not administered an increased dose of anti-claudin-1 antibody. In some embodiments, if the subject does not have reduced levels of PRO-C3 and C4G in the test sample compared to a control sample, the subject is administered an increased dose of anti-claudin-1 antibody. In some embodiments, if the subject has reduced levels of PRO-C3 and C4G in the test sample compared to a control sample, the subject is not administered an increased dose of anti-claudin-1 antibody, but the subject is monitored. Monitoring of the target may include, for example, regular examinations, further measurement of PRO-C3 and C4G levels, or observation of tumors using imaging such as PET or CT scans.

[0193] In some embodiments, this specification provides a method for designing an individualized therapy for a subject with cancer, comprising: (a) quantifying the levels of PRO-C3 and C4G in a test sample from the subject; (b) comparing the levels of PRO-C3 and C4G in the test sample obtained in step (a) with the levels of PRO-C3 and C4G in a control sample; (c) determining that the subject has undecreased levels of PRO-C3 and C4G compared to the levels of PRO-C3 and C4G in the control sample; and (d) administering an increased dose of anti-claudin-1 antibody. In some embodiments, the subject has previously been administered a certain dose of anti-claudin-1 antibody. In some embodiments, if the levels of PRO-C3 and C4G in the test sample obtained in step (a) are undecreased compared to the levels of PRO-C3 and C4G in the control sample, the subject is suitable for administration of an increased dose of anti-claudin-1 antibody. In some embodiments, if the subject is not suitable for administration of increased doses of anti-claudin-1 antibody, the subject is monitored. Monitoring of the subject may include, for example, periodic examinations, further measurement of PRO-C3 and C4G levels, or observation of tumors using imaging such as PET or CT scans.

[0194] In some embodiments, the Specified provides a method for classifying subjects with cancer into a cohort and treating said subjects, comprising: (a) quantifying the levels of PRO-C3 and C4G in a test sample from the subject; (b) comparing the levels of PRO-C3 and C4G in the test sample obtained in step (a) with the levels of PRO-C3 and C4G in a control sample; (c) determining that the subject does not have reduced levels of PRO-C3 and C4G compared to the levels of PRO-C3 and C4G in the control sample; (d) classifying the subject into a cohort based on the levels of PRO-C3 and C4G in the test sample compared with the control sample; and (e) administering an increased dose of anti-claudin-1 antibody. In some embodiments, the subject has previously been administered a certain dose of anti-claudin-1 antibody. In some embodiments, if the levels of PRO-C3 and C4G in the test sample obtained in step (a) are not decreased compared to the levels of PRO-C3 and C4G in the control sample, the subject is suitable for administration of an increased dose of anti-claudin-1 antibody. In some embodiments, if the subject is not suitable for administration of an increased dose of anti-claudin-1 antibody, the subject is monitored. Monitoring of the subject may include, for example, periodic examinations, further measurement of PRO-C3 and C4G levels, or observation of tumors using imaging such as PET or CT scans.

[0195] In some embodiments, the Specified Provision provides a method for treating cancer, comprising (a) determining the levels of PRO-C3 and C4G in a test sample from a subject, and (b) comparing the levels of PRO-C3 and C4G in the test sample to the levels of PRO-C3 and C4G in a control sample. In some embodiments, if the levels of PRO-C3 and C4G in the test sample are not reduced compared to the levels of PRO-C3 and C4G in the control sample, the subject is administered a certain dose of anti-claudin-1 antibody. In some embodiments, if the levels of PRO-C3 and C4G in the test sample are reduced compared to the control sample, the subject is not administered a certain dose of anti-claudin-1 antibody. In some embodiments, if the levels of PRO-C3 and C4G in the test sample are reduced compared to the control sample, the subject is not administered a certain dose of anti-claudin-1 antibody, but the subject is monitored. Monitoring of the subject may include, for example, periodic examinations, further measurements of PRO-C3 and C4G levels, or observation of tumors using imaging such as PET or CT scans.

[0196] In some embodiments, methods for treating cancer in a subject are provided herein. In some embodiments, the subject is determined to have PRO-C3 and C4G levels in a test sample from the subject that have not decreased compared to the levels of PRO-C3 and C4G in a control sample. In some embodiments, the method comprises administering an increased dose of an anti-claudin-1 therapeutic agent (e.g., an anti-claudin-1 antibody or a claudin-1 targeting CAR disclosed herein) to the subject.

[0197] In some embodiments, this specification provides a method for monitoring cancer progression in a subject, comprising (a) quantifying the levels of PRO-C3 and C4G in a test sample from the subject, and (b) comparing the levels of PRO-C3 and C4G in the test sample with the levels of PRO-C3 and C4G in a control sample. In some embodiments, levels of PRO-C3 and C4G in the test sample that have not decreased compared to the levels of PRO-C3 and C4G in the control sample indicate that the subject is suitable for administration of a certain dose of an anti-claudin-1 therapeutic agent. In some embodiments, if the subject is not suitable for administration of a certain dose of an anti-claudin-1 therapeutic agent, the subject is monitored. Monitoring of the subject may include, for example, periodic examinations, further measurements of PRO-C3 and C4G levels, or observation of tumors using imaging such as PET or CT scans.

[0198] In some embodiments, this specification provides a method for monitoring cancer progression in a subject, comprising: (a) determining the levels of PRO-C3 and C4G in a test sample from the subject; and (b) comparing the levels of PRO-C3 and C4G in the test sample with the levels of PRO-C3 and C4G in a control sample. In some embodiments, levels of PRO-C3 and C4G in the test sample that are not decreased compared to the levels of PRO-C3 and C4G in the control sample indicate that the subject is suitable for administration of a certain dose of anti-claudin-1 antibody. In some embodiments, if the subject is not suitable for administration of a certain dose of anti-claudin-1 antibody, the subject is monitored. Monitoring of the subject may include, for example, periodic examinations, further measurements of PRO-C3 and C4G levels, or observation of tumors using imaging such as PET or CT scans.

[0199] In some embodiments, this specification provides a method for treating a subject with cancer, comprising the step of obtaining a test sample from the subject and determining whether the subject has reduced levels of PRO-C3 and C4G by comparing the levels of PRO-C3 and C4G in the test sample with those of a control sample. In some embodiments, if the subject has reduced levels of PRO-C3 and C4G in the test sample compared with that of a control sample, the subject is not administered an increased dose of anti-claudin-1 antibody. In some embodiments, if the subject does not have reduced levels of PRO-C3 and C4G in the test sample compared with that of a control sample, the subject is administered a certain dose of anti-claudin-1 antibody. In some embodiments, if the subject has reduced levels of PRO-C3 and C4G in the test sample compared with that of a control sample, the subject is not administered an increased dose of anti-claudin-1 antibody, but the subject is monitored. Monitoring of the subject may include, for example, periodic examinations, further measurements of PRO-C3 and C4G levels, or observation of tumors using imaging such as PET or CT scans.

[0200] In some embodiments, this specification provides a method for designing an individualized therapy for a subject with cancer, comprising: (a) quantifying the levels of PRO-C3 and C4G in a test sample from the subject; (b) comparing the levels of PRO-C3 and C4G in the test sample obtained in step (a) with the levels of PRO-C3 and C4G in a control sample; (c) determining that the subject has undecreased levels of PRO-C3 and C4G compared to the levels of PRO-C3 and C4G in the control sample; and (d) administering a certain dose of anti-claudin-1 antibody. In some embodiments, if the levels of PRO-C3 and C4G in the test sample obtained in step (a) are undecreased compared to the levels of PRO-C3 and C4G in the control sample, the subject is suitable for administration of a certain dose of anti-claudin-1 antibody. In some embodiments, if the subject is not suitable for administration of a certain dose of anti-claudin-1 antibody, the subject is monitored. Monitoring of the target may include, for example, regular examinations, further measurement of PRO-C3 and C4G levels, or observation of tumors using imaging such as PET or CT scans.

[0201] In some embodiments, this specification provides a method for classifying subjects with cancer into a cohort and treating said subjects, comprising: (a) quantifying the levels of PRO-C3 and C4G in a test sample from the subject; (b) comparing the levels of PRO-C3 and C4G in the test sample obtained in step (a) with the levels of PRO-C3 and C4G in a control sample; (c) determining that the subject does not have reduced levels of PRO-C3 and C4G compared to the levels of PRO-C3 and C4G in a control sample; (d) classifying the subject into a cohort based on the levels of PRO-C3 and C4G in the test sample compared with a control sample; and (e) administering a certain dose of anti-claudin-1 antibody. In some embodiments, if the levels of PRO-C3 and C4G in the test sample obtained in step (a) are not reduced compared to the levels of PRO-C3 and C4G in a control sample, the subject is suitable for administration of a certain dose of anti-claudin-1 antibody. In some embodiments, if the subject is not suitable for administration of increased doses of anti-claudin-1 antibody, the subject is monitored. Monitoring of the subject may include, for example, periodic examinations, further measurement of PRO-C3 and C4G levels, or observation of tumors using imaging such as PET or CT scans.

[0202] In some embodiments, if the subject is sensitive to an increased dose of anti-claudin-1 antibody, the anti-claudin-1 antibody is administered to the subject.

[0203] In some embodiments, the anti-claudin-1 therapeutic agent is an anti-claudin-1 antibody.

[0204] In some embodiments, anti-claudin-1 antibodies are administered intratumorally, intravenously, intraperitoneally, intramuscularly, intrathecally, or subcutaneously.

[0205] In some embodiments, cancer is selected from the group consisting of head and neck cancer (e.g., head and neck squamous cell carcinoma), lung cancer, breast cancer, melanoma, colorectal cancer, pancreatic cancer, esophageal cancer, bile duct cancer, and hepatocellular carcinoma.

[0206] In some embodiments, the control sample is a sample from a subject before administration of an anti-claudin-1 antibody or anti-claudin-1 therapeutic agent.

[0207] In some embodiments, the level of PRO-C3 in the control sample is approximately 15 g / mL. In some embodiments, the level of PRO-C3 in the control sample is approximately 10 ng / mL to approximately 30 ng / mL. In some embodiments, the level of PRO-C3 in the control sample is approximately 10 ng / mL to approximately 25 ng / mL. In some embodiments, the level of PRO-C3 in the control sample is approximately 10 ng / mL to approximately 20 ng / mL. In some embodiments, the level of PRO-C3 in the control sample is approximately 10 ng / mL to approximately 15 ng / mL. In some embodiments, the level of PRO-C3 in the control sample is approximately 15 ng / mL to approximately 30 ng / mL. In some embodiments, the level of PRO-C3 in the control sample is approximately 20 ng / mL to approximately 30 ng / mL. In some embodiments, the level of PRO-C3 in the control sample is approximately 25 ng / mL to approximately 30 ng / mL.

[0208] In some embodiments, the PRO-C3 level in the control sample is 15 g / mL. In some embodiments, the PRO-C3 level in the control sample is 10 ng / mL to 30 ng / mL. In some embodiments, the PRO-C3 level in the control sample is 10 ng / mL to 25 ng / mL. In some embodiments, the PRO-C3 level in the control sample is 10 ng / mL to 20 ng / mL. In some embodiments, the PRO-C3 level in the control sample is 10 ng / mL to 15 ng / mL. In some embodiments, the PRO-C3 level in the control sample is 15 ng / mL to 30 ng / mL. In some embodiments, the PRO-C3 level in the control sample is 20 ng / mL to 30 ng / mL. In some embodiments, the PRO-C3 level in the control sample is 25 ng / mL to 30 ng / mL.

[0209] In some embodiments, the C4G level in the control sample is approximately 25 ng / mL. In some embodiments, the C4G level in the control sample is approximately 20 ng / mL to approximately 35 ng / mL. In some embodiments, the C4G level in the control sample is approximately 25 ng / mL to approximately 35 ng / mL. In some embodiments, the C4G level in the control sample is approximately 30 ng / mL to approximately 35 ng / mL. In some embodiments, the C4G level in the control sample is approximately 25 ng / mL to approximately 35 ng / mL. In some embodiments, the C4G level in the control sample is approximately 30 ng / mL to approximately 35 ng / mL. In some embodiments, the C4G level in the control sample is approximately 25 ng / mL to approximately 30 ng / mL.

[0210] In some embodiments, the C4G level in the control sample is 25 ng / mL. In some embodiments, the C4G level in the control sample is 20 ng / mL to 35 ng / mL. In some embodiments, the C4G level in the control sample is 25 ng / mL to 35 ng / mL. In some embodiments, the C4G level in the control sample is 30 ng / mL to 35 ng / mL. In some embodiments, the C4G level in the control sample is 25 ng / mL to 35 ng / mL. In some embodiments, the C4G level in the control sample is 30 ng / mL to 35 ng / mL. In some embodiments, the C4G level in the control sample is 25 ng / mL to 30 ng / mL.

[0211] In some embodiments, the level of PRO-C3 in the control sample is approximately 15 g / mL, and the level of C4G in the control sample is approximately 25 ng / mL.

[0212] In some embodiments, the PRO-C3 level in the control sample is approximately 10 ng / mL to approximately 30 ng / mL, and the C4G level in the control sample is approximately 20 ng / mL to approximately 35 ng / mL. In some embodiments, the PRO-C3 level in the control sample is approximately 10 ng / mL to approximately 25 ng / mL, and the C4G level in the control sample is approximately 25 ng / mL to approximately 35 ng / mL. In some embodiments, the PRO-C3 level in the control sample is approximately 10 ng / mL to approximately 20 ng / mL, and the C4G level in the control sample is approximately 30 ng / mL to approximately 35 ng / mL. In some embodiments, the PRO-C3 level in the control sample is approximately 10 ng / mL to approximately 15 ng / mL, and the C4G level in the control sample is approximately 25 ng / mL to approximately 35 ng / mL. In some embodiments, the PRO-C3 level in the control sample is approximately 15 ng / mL to approximately 30 ng / mL, and the C4G level in the control sample is approximately 30 ng / mL to approximately 35 ng / mL. In some embodiments, the PRO-C3 level in the control sample is approximately 20 ng / mL to approximately 30 ng / mL, and the C4G level in the control sample is approximately 25 ng / mL to approximately 30 ng / mL. In some embodiments, the PRO-C3 level in the control sample is approximately 25 ng / mL to approximately 30 ng / mL, and the C4G level in the control sample is approximately 25 ng / mL to approximately 30 ng / mL.

[0213] In some embodiments, the PRO-C3 level in the control sample is 15 g / mL and the C4G level in the control sample is 25 ng / mL. In some embodiments, the PRO-C3 level in the control sample is 10 ng / mL to 30 ng / mL and the C4G level in the control sample is 20 ng / mL to 35 ng / mL. In some embodiments, the PRO-C3 level in the control sample is 10 ng / mL to 25 ng / mL and the C4G level in the control sample is 25 ng / mL to 35 ng / mL. In some embodiments, the PRO-C3 level in the control sample is 10 ng / mL to 20 ng / mL and the C4G level in the control sample is 30 ng / mL to 35 ng / mL. In some embodiments, the PRO-C3 level in the control sample is 10 ng / mL to 15 ng / mL and the C4G level in the control sample is 25 ng / mL to 35 ng / mL. In some embodiments, the PRO-C3 level in the control sample is 15 ng / mL to 30 ng / mL, and the C4G level in the control sample is 30 ng / mL to 35 ng / mL. In some embodiments, the PRO-C3 level in the control sample is 20 ng / mL to 30 ng / mL, and the C4G level in the control sample is 25 ng / mL to 30 ng / mL. In some embodiments, the PRO-C3 level in the control sample is 25 ng / mL to 30 ng / mL, and the C4G level in the control sample is 25 ng / mL to 30 ng / mL.

[0214] In some embodiments, the anti-claudin-1 antibody is a monoclonal antibody containing six complementarity-determining regions (CDRs) of an anti-claudin-1 monoclonal antibody secreted by a hybridoma cell line deposited in DSMZ on July 29, 2008, under accession number DSM ACC2938.

[0215] In some embodiments, anti-claudin-1 antibodies are humanized.

[0216] In some embodiments, the anti-claudin-1 antibody comprises a VH having the amino acid sequence described in SEQ ID NO: 3 or SEQ ID NO: 13.

[0217] In some embodiments, the anti-claudin-1 antibody comprises a VL having the amino acid sequence described in SEQ ID NO: 4 or SEQ ID NO: 14.

[0218] In some embodiments, the anti-claudin-1 antibody comprises VH containing the amino acid sequence described in SEQ ID NO: 3, and VL containing the amino acid sequence described in SEQ ID NO: 4.

[0219] In some embodiments, the anti-claudin-1 antibody comprises VH, which contains the amino acid sequence described in SEQ ID NO: 13, and VL, which contains the amino acid sequence described in SEQ ID NO: 14.

[0220] In some embodiments, the anti-claudin-1 antibody comprises a complementation-determining region (CDR) H1 containing the amino acid sequence described in SEQ ID NO: 5, a CDR H2 containing the amino acid sequence described in SEQ ID NO: 6, and a CDR H3 containing the amino acid sequence described in SEQ ID NO: 7.

[0221] In some embodiments, the anti-claudin-1 antibody comprises a complementation-determining region (CDR) L1 containing the amino acid sequence described in SEQ ID NO: 8, a CDR L2 containing the amino acid sequence Gly Ala, and a CDR L3 containing the amino acid sequence described in SEQ ID NO: 10.

[0222] kit This disclosure also provides a kit or product comprising an anti-claudin-1 antibody and an ELISA kit. In some embodiments, the ELISA kit measures PRO-C3. In some embodiments, the ELISA kit measures C4G. In some embodiments, the kit further comprises a lancet and a vial for obtaining a blood sample. In some embodiments, the blood sample is a plasma sample.

[0223] Those skilled in the art will readily recognize that the anti-claudin-1 antibody of this disclosure can be readily incorporated into one of the well-established kit formats in the art.

[0224] The following embodiments are illustrative and do not limit the scope of the claimed embodiments. [Examples]

[0225] Example 1. PRO-C3 and C4G are biomarkers for target binding of anti-claudin-1 antibodies. Claudin-1 (CLND1) mRNA expression was measured and shown to be expressed in numerous solid tumors of various cancer types (see Figure 1).

[0226] Anti-CLDN1 antibodies with mutant Fc regions and anti-CLDN1 antibodies with WT IgG1 Fc regions were analyzed. Anti-CLDN1 antibodies with WT IgG1 Fc showed better ADCC values ​​when measured in vitro by relative luminescence (RLU) and better tumor growth inhibition in the Cal27 HNSCC CDX model when measured by tumor volume (mm3) (see Figures 4A-4B).

[0227] The efficacy of anti-CLDN1 antibodies was measured using head and neck squamous cell carcinoma ("HNSCC") cells in a patient-derived xenograft ("PDX") model. Tumor volume in the HNSCC PDX model decreased after treatment with anti-CLDN1 antibodies compared to treatment with an IgG1 control (see Figure 2).

[0228] Next, the extracellular matrix remodeling markers PRO-C3 and C4G were quantified in HNSCC PDX models after treatment with anti-CLDN1 antibody or IgG1 control. After treatment with anti-CLDN1 antibody, PRO-C3 levels decreased and C4G levels increased. Therefore, PRO-C3 and C4G represent viable biomarkers for measuring the effectiveness of anti-CLDN1 antibody treatment (see Figures 3A-3B).

[0229] In implementing this application, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology that are within the scope of the art of those skilled in the art will be used. Such techniques are adequately described in the literature.

[0230] All references cited above, and all references cited herein, are incorporated herein by reference as a whole.

Claims

1. A method of treating cancer, (a) To administer anti-claudin-1 antibodies, (b) Determining the level of the N-terminal propeptide ("PRO-C3") of type III collagen in the test sample from the subject, (c) The method comprising comparing the level of PRO-C3 in the test sample with the level of PRO-C3 in a control sample, wherein if the level of PRO-C3 in the test sample is not decreased compared to the level of PRO-C3 in the control sample, the dose of the anti-claudin-1 antibody administered to the subject is increased, and if the level of PRO-C3 in the test sample is decreased compared to the control sample, the dose of the anti-claudin-1 antibody administered to the subject is not increased.

2. A method for treating cancer in a subject, wherein the subject is determined to have a level of PRO-C3 in a test sample from the subject that is not reduced compared to the level of PRO-C3 in a control sample, the subject has been previously administered an anti-claudin-1 therapeutic agent, and the method comprises administering an increased dose of the anti-claudin-1 therapeutic agent to the subject.

3. A method for monitoring cancer progression in a subject, (a) To quantify the level of PRO-C3 in a test sample from the subject, wherein the subject has previously been administered an anti-claudin-1 drug, (b) Comparing the level of PRO-C3 in the test sample with the level of PRO-C3 in the control sample, The method, wherein the level of PRO-C3 in the test sample, which has not decreased compared to the level of PRO-C3 in the control sample, indicates that the subject is sensitive to an increased dose of the anti-claudin-1 therapeutic agent.

4. A method for monitoring cancer progression in a subject, (a) Determining the level of PRO-C3 in a test sample from the subject, wherein the subject has previously been administered an anti-claudin-1 antibody, (b) Comparing the level of PRO-C3 in the test sample with the level of PRO-C3 in the control sample, The method for indicating that the level of PRO-C3 in the test sample, which has not decreased compared to the level of PRO-C3 in the control sample, is sensitive to an increased dose of the anti-claudin-1 antibody.

5. A method for treating a person suffering from cancer, A step of determining whether the subject has an increased level of PRO-C3 by obtaining a test sample from the subject and comparing the level of PRO-C3 in the test sample with that of a control sample, wherein the subject has previously been administered a certain dose of anti-claudin-1 antibody, If the subject has a reduced level of PRO-C3 in the test sample compared to the control sample, the subject is not administered an increased dose of the anti-claudin-1 antibody. The method wherein, if the subject does not have a reduced level of PRO-C3 in the test sample compared to the control sample, the subject is administered an increased dose of the anti-claudin-1 antibody.

6. A method for designing personalized therapies for individuals with cancer, (a) To quantify the level of PRO-C3 in a test sample from the subject, wherein the subject has previously been administered a certain dose of anti-claudin-1 antibody, (b) Comparing the level of PRO-C3 in the test sample obtained in step (a) with the level of PRO-C3 in the control sample, wherein if the level of PRO-C3 in the test sample obtained in step (a) is not decreased compared with the level of PRO-C3 in the control sample, the subject is sensitive to the administration of an increased dose of the anti-claudin-1 antibody, and the comparison is as follows: (c) Determining that the subject has a level of PRO-C3 that is not reduced compared to the level of PRO-C3 in the control sample, (d) The method comprising administering an increased dose of the anti-claudin-1 antibody.

7. A method for classifying subjects suffering from cancer into cohorts and treating said subjects, (a) To quantify the level of PRO-C3 in a test sample from the subject, wherein the subject has previously been administered a certain dose of anti-claudin-1 antibody, (b) Comparing the level of PRO-C3 in the test sample obtained in step (a) with the level of PRO-C3 in the control sample, wherein if the level of PRO-C3 in the test sample obtained in step (a) is not decreased compared with the level of PRO-C3 in the control sample, the subject is sensitive to the administration of an increased dose of the anti-claudin-1 antibody, and the comparison is as follows: (c) Determining that the subject has a level of PRO-C3 that is not reduced compared to the level of PRO-C3 in the control sample, (d) Classifying the subjects into cohorts based on the PRO-C3 level in the test sample compared to the control sample, (e) The method comprising administering an increased dose of the anti-claudin-1 antibody.

8. A method of treating cancer, (a) To administer anti-claudin-1 antibodies, (b) Determining the level of granzyme B-degrading type IV collagen product ("C4G") in the test sample from the subject, (c) Comparing the level of C4G in the test sample with the level of C4G in the control sample, The method comprising increasing the dose of the anti-claudin-1 antibody administered to the subject if the level of C4G in the test sample is not increased compared to the level of C4G in the control sample, and not increasing the dose of the anti-claudin-1 antibody administered to the subject if the level of C4G in the test sample is increased compared to the control sample.

9. A method for treating cancer in a subject, wherein the subject is determined to have a level of C4G in a test sample from the subject that has not increased compared to the level of C4G in a control sample, the subject has been previously administered an anti-claudin-1 therapeutic agent, and the method comprises administering an increased dose of the anti-claudin-1 therapeutic agent to the subject.

10. A method for monitoring cancer progression in a subject, (a) To quantify the level of C4G in a test sample from the subject, wherein the subject has previously been administered an anti-claudin-1 drug, (b) Comparing the level of C4G in the test sample with the level of C4G in the control sample, The method for indicating that the level of C4G in the test sample, which has not increased compared to the level of C4G in the control sample, is sensitive to an increased dose of the anti-claudin-1 therapeutic agent.

11. A method for monitoring cancer progression in a subject, (a) Determining the level of C4G in a test sample from the subject, wherein the subject has previously been administered an anti-claudin-1 antibody, (b) Comparing the level of C4G in the test sample with the level of C4G in the control sample, The method for indicating that the level of C4G in the test sample, which has not increased compared to the level of C4G in the control sample, is sensitive to an increased dose of the anti-claudin-1 antibody in the subject.

12. A method for treating a person suffering from cancer, A step of determining whether the subject has an increased level of C4G by obtaining a test sample from the subject and comparing the level of C4G in the test sample with that of a control sample, wherein the subject has previously been administered a certain dose of anti-claudin-1 antibody, If the subject has an increased level of C4G in the test sample compared to the control sample, the subject is not administered the increased dose of the anti-claudin-1 antibody. The method wherein, if the subject does not have an increased level of C4G in the test sample compared to the control sample, the subject is administered an increased dose of the anti-claudin-1 antibody.

13. A method for designing personalized therapies for individuals with cancer, (a) To quantify the level of C4G in a test sample from the subject, wherein the subject has previously been administered a certain dose of anti-claudin-1 antibody, (b) Comparing the level of C4G in the test sample obtained in step (a) with the level of C4G in the control sample, wherein if the level of C4G in the test sample obtained in step (a) is not increased compared with the level of C4G in the control sample, the subject is sensitive to the administration of the increased dose of the anti-claudin-1 antibody, and the comparison is as follows: (c) Determining that the subject has a level of C4G that is not increased compared to the level of C4G in the control sample, (d) The method comprising administering an increased dose of the anti-claudin-1 antibody.

14. A method for classifying subjects suffering from cancer into cohorts and treating said subjects, (a) To quantify the level of C4G in a test sample from the subject, wherein the subject has previously been administered a certain dose of anti-claudin-1 antibody, (b) Comparing the level of C4G in the test sample obtained in step (a) with the level of C4G in the control sample, wherein if the level of C4G in the test sample obtained in step (a) is not increased compared with the level of C4G in the control sample, the subject is sensitive to the administration of the increased dose of the anti-claudin-1 antibody, and the comparison is as follows: (c) Determining that the subject has a level of C4G that is not increased compared to the level of C4G in the control sample, (d) Classifying the subjects into a cohort based on the level of C4G in the test sample compared to the control sample, (e) The method comprising administering an increased dose of the anti-claudin-1 antibody.

15. The method according to any one of claims 3, 4, 10, or 11, wherein if the subject is sensitive to an increased dose of the anti-claudin-1 antibody, the anti-claudin-1 antibody is administered to the subject.

16. The method according to any one of claims 2, 3, 9, or 10, wherein the anti-claudin-1 therapeutic agent is an anti-claudin-1 antibody.

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

18. The method according to any one of claims 1 to 17, wherein the cancer is selected from the group consisting of head and neck cancer (e.g., head and neck squamous cell carcinoma), lung cancer, breast cancer, melanoma, colorectal cancer, pancreatic cancer, esophageal cancer, bile duct cancer, and hepatocellular carcinoma.

19. The method according to any one of claims 1 to 18, wherein the control sample is a sample from the subject before administration of an anti-claudin-1 antibody or an anti-claudin-1 therapeutic agent.

20. The method according to any one of claims 1 to 8 or 15 to 19, wherein the level of PRO-C3 in the control sample is about 10 ng / mL to about 30 ng / mL.

21. The method according to any one of claims 9 to 19, wherein the level of C4G in the control sample is about 20 ng / mL to about 35 ng / mL.

22. The method according to any one of claims 1 to 21, wherein the anti-claudin-1 antibody is a monoclonal antibody comprising six complementarity-determining regions (CDRs) of an anti-claudin-1 monoclonal antibody secreted by a hybridoma cell line deposited in DSMZ on July 29, 2008, under accession number DSM ACC2938.

23. The method according to any one of claims 1 to 22, wherein the anti-claudin-1 antibody is humanized.

24. The method according to any one of claims 1 to 23, wherein the anti-claudin-1 antibody comprises a VH having the amino acid sequence described in SEQ ID NO: 3 or SEQ ID NO:

13.

25. The method according to any one of claims 1 to 24, wherein the anti-claudin-1 antibody comprises a VL having the amino acid sequence described in SEQ ID NO: 4 or SEQ ID NO:

14.

26. The method according to any one of claims 1 to 25, wherein the anti-claudin-1 antibody comprises VH having the amino acid sequence described in SEQ ID NO: 3 and VL having the amino acid sequence described in SEQ ID NO:

4.

27. The method according to any one of claims 1 to 26, wherein the anti-claudin-1 antibody comprises VH having the amino acid sequence described in SEQ ID NO: 13 and VL having the amino acid sequence described in SEQ ID NO:

14.

28. The method according to any one of claims 1 to 27, wherein the anti-claudin-1 antibody comprises a complementation-determining region (CDR) H1 containing the amino acid sequence described in SEQ ID NO: 5, a CDR H2 containing the amino acid sequence described in SEQ ID NO: 6, and a CDR H3 containing the amino acid sequence described in SEQ ID NO:

7.

29. The method according to any one of claims 1 to 26, wherein the anti-claudin-1 antibody comprises a complementation-determining region (CDR) L1 containing the amino acid sequence described in SEQ ID NO: 8, a CDR L2 containing the amino acid sequence GA, and a CDR L3 containing the amino acid sequence described in SEQ ID NO:

10.

30. The method according to any one of claims 1 to 29, wherein the anti-claudin-1 antibody comprises a heavy chain having the amino acid sequence described in SEQ ID NO:

1.

31. The method according to any one of claims 1 to 30, wherein the anti-claudin-1 antibody comprises a light chain having the amino acid sequence described in SEQ ID NO: 2.

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