WO2025224337A1 - Anti-cldn1 antibody-drug conjugates comprising exatecan and methods of use thereof - Google Patents

Abstract

The disclosure relates to antibody-drug conjugates comprising an anti-Claudin-1 antibody or antigen-binding fragment thereof and an exatecan moiety, connected by a linker, as well as methods of making and using the same.

Description

ANTI-CLDNI ANTIBODY-DRUG CONJUGATES COMPRISING EXATECAN AND METHODS OF

USE THEREOF

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims the benefit of U.S. Provisional Patent Application No. 63/639,375 filed April 26, 2024, which is incorporated herein by reference in its entirety.

INCORPORATION BY REFERENCE OF SEQUENCE LISTING

[0002] The contents of the text file named “ ALNT-017_001WO_SeqList.xml”, which was created on April 11, 2025 and is 15,336 bytes in size, are hereby incorporated by reference in their entirety.

Background

[0003] Anti-Claudin-1 antibodies have been used to treat an array of diseases, such as hepatocellular carcinoma (e.g., U.S. Pat. No. 10,815,298), non-alcoholic fatty liver disease (e.g., U.S. Pat. No. 10,927,170), and fibrotic diseases such as renal fibrosis, pulmonary fibrosis, cholangial carcinoma and skin fibrosis (e.g., WO 2021/094469 Al).

[0004] An antibody-drug conjugate (ADC) is typically composed of an antibody covalently attached to a cytotoxic drug via a chemical linker. ADCs may be used to deliver highly potent drugs directly to cancer cells by targeting specific antigens present on tumor cells (see Fu et al., Signal Transduct Target Ther. 2022 Mar 22;7( 1 ): 93). The antibody portion of the ADC binds to a surface protein specific to the cancer cells (e.g., tumor-specific antigens or tumor- associated antigens). However, not any antibody is suitable for use in antibody-drug conjugates. Provided herein are antibody-drug conjugates comprising anti-Claudin-1 antibodies.

SUMMARY OF THE INVENTION

In one aspect, provided herein is an anti-Claudin-1 antibody-drug conjugate of Formula (I): A-(L¹-L²-D)_n

(I) or a pharmaceutically acceptable salt or solvate thereof, wherein:

A is an anti-Claudin-1 antibody comprising (i) a heavy chain variable region (VH) comprising a heavy chain complementarity determining region (HCDR)l comprising the amino acid sequence of SEQ ID NO: 5, a HCDR2 comprising the amino acid sequence of SEQ ID NO: 6 and a HCDR3 comprising the amino acid sequence of SEQ ID NO: 7; and (ii) a light chain variable region comprising a light chain complementarity determining region (LCDR)l comprising the amino acid sequence of SEQ ID NO: 8, a LCDR2 comprising the amino acid sequence of GAS, and a LCDR3 comprising the amino acid sequence of SEQ ID NO: 10;

L¹ is a divalent moiety comprising a functional group attached to the anti-Claudin-1 antibody;

L² is a divalent peptide linker comprising at least two amino acids;

D is an exatecan moiety; and n is an integer between about 2 and about 10.

[0005] In embodiments, the anti-Claudin-1 antibody comprises a VH comprising a sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 3 or 13. In embodiments, the anti-Claudin-1 antibody comprises a VL comprising a sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 4 or 14. In embodiments, the anti-Claudin-1 antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 3 and a light chain comprising the amino acid sequence of SEQ ID NO: 4. In embodiments, the anti-Claudin-1 antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 13 and a light chain comprising the amino acid sequence of SEQ ID NO: 14. In embodiments, the anti-Claudin-1 antibody is a monoclonal antibody. In embodiments, the anti-Claudin-1 antibody is a rabbit, mouse, chimeric, humanized or fully human monoclonal antibody. In embodiments, the anti-Claudin-1 antibody is an IgG isotype. In embodiments, the anti-Claudin-1 antibody is an IgGl isotype.

In embodiments, , wherein denotes attachment to A and denotes attachment to D . In embodiments, D is an exatecan moiety. In embodiments, wherein denotes attachment to L².

[0006] In another aspect, provided herein is a method of treating cancer in a subject in need thereof, comprising administering to the subject an antibody-drug conjugate described herein. In embodiments, the cancer is urothelial cancer, esophageal cancer, head and neck cancer, lung cancer, colorectal cancer, liver cancer, or breast cancer.

BRIEF DESCRIPTION OF THE FIGURES

[0007] FIGs. 1 A-1I show Claudin-1 (CLDN1) protein expression in 9 major tumor types. Scoring was done as follows: Negative: H-Score 0-10; Low: H-Score 11-100; Medium: Fl- Score 101-200; High: H-Score 201-300. HNSCC: Head and neck squamous cell carcinoma; Sq.: Squamous; NSCLC: Non-small cell lung cancer; CRC: colorectal carcinoma; HCC: hepatocellular carcinoma; iCCA: intrahepatic cholangiocarcinoma; TNBC: triple negative breast cancer.

[0008] FIG. 2 shows showing binding of ADC2, or ADC2 naked antibody control (Anti- Claudin antibody but no exatecan conjugation) to CLDN1 in Huh7 HCC cancer cells as measured by FACS.

[0009] FIGs. 3 A-3C show the in vitro characterization of ADC2. FIG. 3 A shows binding of ADC2, ADC2 isotype control (IgGl conjugated to exatecan) or ADC2 naked antibody control to Claudin-1 -expressing NCI-H1975 cells. FIG. 3B shows internalization of ADC2, ADC2 isotype control or ADC2 naked antibody control into Claudin-1 -expressing NCL H1975 cells. FIG. 3C shows killing of Claudin-1 -expressing NCI-H1975 cells by ADC2, ADC2 isotype control or free exatecan.

[0010] FIGs. 4A-4C show Immunogenic cell death markers in CLDN1 overexpressing NCI- H1975 NCSCLC cancer cells treated with ADC2. FIG. 4A is a Western Blot showing expression of ER stress-related proteins, apoptosis-related proteins and DNA damage-related proteins after treatment with IgGl naked antibody control, ADC2 isotype control and ADC2, FIGs. 4B and 4C show ATP release and HMGB1 release as measured by FACS after treatment with ADC2 isotype control, ADC2 or exatecan. [0011] FIG. 5 A shows images of a cell population comprising Claudin-1 -positive cells and Claudin-1 -negative cells after treatment with ADC2 isotype control, ADC2 or exatecan. FIG. 5B shows killing of Claudin-1 expressing cells and Claudin-1 -deficient cells by ADC2 isotype control, ADC2 or exatecan.

[0012] FIGs. 6A-6D show tumor regression in an in vivo CDX model of lung cancer (NCI- 111975 overexpressing CLDN1 cells) with medium Claudin-1 expression after administration of ADC2. FIG. 6A shows expression of Claudin-1 measured by IHC and FIG. 6B shows the change in tumor volume after administration of ADC2 isotype control, ADC2 or datopotamab deruxtecan (Dato-DxD). Arrow indicates treatment administration. FIGs. 6C and 6D show responses of individual animals to 10 mg/kg body weight of ADC2 and 10 mg/kg body weight of Dato-DxD, respectively.

[0013] FIGs. 7A and 7B show tumor regression in an in vivo CDX model of liver cancer (Huh7 cells) with low Claudin-1 expression after administration of ADC2. FIG. 7A shows expression of Claudin-1 measured by IHC and FIG. 7B shows the change in tumor volume after administration of ADC2 isotype control or ADC2. Arrows indicate treatment administration.

[0014] FIGs. 8A and 8B show tumor regression in an in vivo patient-derived model of nasopharyngeal cancer with low Claudin-1 expression after administration of ADC2. FIG. 8 A shows the change in tumor volume after administration of ADC2 isotype control or ADC2. Arrow indicates treatment administration. FIG. 8B shows responses of individual animals to 10 mg/kg body weight ADC2.

[0015] FIGs. 9A-9C show anti-CLDNl antibody binding to CLDN1 expressed in NCI- H1975 cells. FIG. 9A shows FACS binding of anti-CLDNl mouse antibody clones. FIG. 9B shows FACS binding of anti-CLDNl rat antibody clone. FIG. 9C shows FACS binding of anti-CLDNl rabbit antibody clone.

[0016] FIGs. 10A-10C show imaging of anti-CLDNl antibody binding to CLDN1 overexpressed in NCI-H1975 cells. FIG. 10A shows representative images of anti-CLDNl antibody binding. FIGs. 10B and 10C show quantification of fluorescence intensity in each picture either represented as the total area of staining per cell (FIG. 10B; total area / cell count) or total fluorescence intensity per cell count (FIG. 10C; total intensity/cell count). [0017] FIGs. 11A and 11B show anti-CLDNl antibody internalization in CLDN1 overexpressing NCI-H1975. FIG. 11 A shows a representative image of internalized pHrodo- conjugated anti-CLDNl antibody at 6h post antibody addition. FIG. 1 IB shows quantification of the total area of dots per cell as a readout of antibody internalization (sum area/cell count).

[0018] FIGs. 12A and 12B show internalization of pHrodo-conjugated anti-CLDNl antibody in CLDN1 overexpressing NCI-H1975. FIG. 12A shows kinetics of internalization represented by the MFI of pHrodo-conjugated anti-CLDNl antibody. FIG. 12B shows internalization of each anti-CLDNl antibody at 6h (MFI).

[0019] FIGs. 13A-13E show CLDN1 staining and internalization of HILI and H3L3 in CLDNl-transduced NCI-H1975 cells. CLDN1 was detected at the cell surface of CLDN1- transduced NCI-H1975 cells using either pHrodo-conjugated HILI or H3L3 (FIG. 13 A). FIG. 13B and 13C show comparisons of HILI and H3L3 internalization in CLDN1- transduced NCI-H1975 using a protocol without washout step. FIG. 13B shows representative pictures of HILI and H3L3 internalization at 2h, 4h and 6h. FIG. 13C shows corresponding quantification of the pHrodo-positive total area per cell. FIG. 13D and 13E show a comparison of HILI and H3L3 internalization in CLDN1 -transduced NCI-H1975 using a protocol with a washout step. FIG. 13D shows representative pictures of HILI and H3L3 internalization at 2h, 4h and 6h. FIG. 13E shows corresponding quantification of the pHrodo-positive total area per cell.

DETAILED DESCRIPTION

[0020] Provided herein are antibody-drug conjugates comprising anti-Claudin-1 antibodies and one or more exatecan moiety. The anti-Claudin-1 antibody and each exatecan moiety are connected by a linker. Also provided are methods of making the antibody-drug conjugates and methods of treating a disease or disorder comprising administering a therapeutically effective amount of the antibody-drug conjugates provided herein.

Definitions

[0021] The chemical names provided for the intermediate compounds and/or the compounds of this disclosure described herein may refer to any one of the tautomeric representations of such compounds (in some instances, such alternate names are provided with the experimental). It is to be understood that any reference to a named compound (an intermediate compound or a compound of the disclosure) or a structurally depicted compound (an intermediate compound or a compound of the disclosure) is intended to encompass all tautomeric forms including zwitterionic forms of such compounds and any mixture thereof. [0022] The term “about”, as used herein, generally means ±10% of the value stated. In embo diments, “about” or “approximately” generally means ±9%, ±8%, ±7%, ±6%, ±5%, ±4%, ±3 %, ±2%, or ±1% of the stated value.

[0023] Recitation of ranges of values are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. A range used herein, unless otherwise specified, includes the two limits of the range. In embodiments, the expressions “x being an integer between 1 and 6” and “x being an integer of 1 to 6” both mean “x being 1, 2, 3, 4, 5, or 6”, i.e., the terms “between X and Y” and “range from X to Y, are inclusive of X and Y and the integers there between.

[0024] As utilized in accordance with the present disclosure, the following terms, unless otherwise indicated, shall be understood to have the following meanings:

[0025] The term "alkylene", as used herein, represents a divalent saturated, straight or branched hydrocarbon group having the specified number of carbon atoms. The term “Ci-Ce alkylene” or "Ci-6 alkylene" refers to a methylene moiety or a straight or branched alkylene moiety comprising from 2 to 6 carbon atoms.

[0026] Exemplary alkylenes include, but are not limited to methylene, ethylene, w-propylene, isopropylene, ^-butylene, isobutylene, -butylene, /-butylene, pentylene, and hexylene. [0027] The terms "conjugate(s) of the disclosure" or "conjugate(s) of the present disclosure", as used herein, mean a conjugate as defined herein, in any form, i.e., any tautomeric form, any isomeric form, any salt or non-salt form (e.g., as a free acid or base form, or as a salt, particularly a pharmaceutically acceptable salt thereof) and any physical form thereof (e.g., including non-solid forms (e.g., liquid or semi-solid forms), and solid forms (e.g., amorphous or crystalline forms, specific polymorphic forms, solvate forms, including hydrate forms (e.g., mono-, di- and hemi- hydrates)), and mixtures of various forms.

[0028] Accordingly, included within the present disclosure are the conjugates as disclosure herein, in any salt or non-salt form and any physical form thereof, and mixtures of various forms. While such are included within the present disclosure, it will be understood that the conjugates of the present disclosure, in any salt or non-salt form, and in any physical form thereof, may have varying levels of activity, different bioavailabilities and different handling properties for formulation purposes.

[0029] It is understood that, throughout the description, where compositions are described as having, including, or comprising specific components, it is contemplated that compositions also consist essentially of, or consist of, the recited components. Similarly, where methods or processes are described as having, including, or comprising specific process steps, the processes also consist essentially of, or consist of, the recited processing steps. Further, it should be understood that the order of steps or order for performing certain actions is immaterial so long as the invention remains operable. Moreover, two or more steps or actions can be conducted simultaneously.

[0030] All percentages and ratios used herein, unless otherwise indicated, are by weight. Other features and advantages of the present disclosure are apparent from the different examples. The provided examples illustrate different components and methodology useful in practicing the present disclosure. The examples do not limit the claimed disclosure. Based on the present disclosure the skilled artisan can identify and employ other components and methodology useful for practicing the present disclosure.

[0031] All publications and patent documents cited herein are incorporated herein by reference as if each such publication or document was specifically and individually indicated to be incorporated herein by reference. Citation of publications and patent documents is not intended as an admission that any is pertinent prior art, nor does it constitute any admission as to the contents or date of the same. The invention having now been described by way of written description, those of skill in the art will recognize that the invention can be practiced in a variety of embodiments and that the foregoing description and examples below are for purposes of illustration and not limitation of the claims that follow.

Anti-Claudin-1 Antibody-Drug Conjugates

[0032] In aspects, the present disclosure provides an anti-Claudin-1 antibody-drug conjugate. In embodiments, the anti-Claudin-1 antibody-drug conjugate is biodegradable and biocompatible, and/or exhibits high drug load and strong binding to a target antigen.

[0033] In embodiments, the present disclosure provides an anti-Claudin-1 antibody-drug conjugate, comprising an anti-Claudin-1 antibody and one or more Linker-Drug moi eties, wherein the anti-Claudin-1 antibody is covalently linked to the one or more Linker-Drug moieties.

[0034] In embodiments, the anti-Claudin-1 antibody is an antibody, a cysteine engineered antibody, or a modified antibody.

[0035] In embodiments, the present disclosure provides an anti-Claudin-1 antibody-drug conjugate of Formula (I):

A-(L^J-L²-D)_n (I) or a pharmaceutically acceptable salt or solvate thereof, wherein:

A is an anti-Claudin-1 antibody or antigen binding fragment thereof;

L¹ is a divalent moiety comprising a functional group attached to the anti-Claudin-1 antibody or antigen binding fragment thereof;

L² is a divalent peptide linker comprising at least two amino acids;

D is an exatecan moiety; and n is an integer between about 2 and about 10.

[0036] In embodiments, A is an anti-Claudin-1 antibody or antigen binding fragment thereof.

In embodiments, A is an anti-Claudin-1 antibody. In embodiments, A is an anti-Claudin-1 antigen binding fragment.

[0037] In embodiments, LI has a corresponding monovalent moiety L¹’ comprising a functional group capable of forming a covalent bond with the anti-Claudin-1 antibody. In embodiments, L¹ has a corresponding monovalent moiety L¹’ comprising a functional group capable of forming a covalent bond with the anti-Claudin-1 antigen binding fragment.

[0038] In embodiments, L comprises , , wherein ^? denotes attachment to A. alkylene) ³ —

[0039] In embodiments, , wherein denotes attachment alkylene) -i- to L². In embodiments, , wherein ⁵ denotes attachment denotes attachment to L². # J

[0040] In embodiments, L¹ is , wherein denotes attachment to

L². In embodiments, L¹ is , wherein denotes attachment to A # , and < denotes attachment to L². [0041] In embodiments, L² comprises between two to four amino acids. In embodiments, L² comprises between two to five amino acids. In embodiments, L² comprises between two to six amino acids. In embodiments, L² comprises between two to seven amino acids. In embodiments, L² comprises between two to eight amino acids. In embodiments, L² comprises between two to nine amino acids. In embodiments, L² comprises between two and ten amino acids.

[0042] In embodiments, L² comprises two amino acids. In embodiments, L² comprises three amino acids. In embodiments, L² comprises four amino acids. In embodiments, L² comprises five amino acids. In embodiments, L² comprises six amino acids. In embodiments, L² comprises seven amino acids. In embodiments, L² comprises eight amino acids. In embodiments, L² comprises nine amino acids. In embodiments, L² comprises ten amino acids. In embodiments, L² comprises at least ten amino acids.

[0043] In embodiments, L² comprises amino acids that are each independently selected from glycine and phenylalanine. In embodiments, L² comprises glycine. In embodiments, L² comprises phenylalanine.

[0044] In embodiments, L² is a divalent peptide linker comprising glycine and phenylalanine. In embodiments, L² is a divalent peptide linker consisting of about three glycine and about one phenylalanine. In embodiments, L² is a divalent peptide linker consisting of three glycine and one phenylalanine.

[0045] In embodiments, , denotes

## s attachment to L¹ and denotes attachment to D. In embodiments, L² is w ere n denotes attachment to L¹ and denotes attachment to D.

[0046] In embodiments, wherein denotes attachment to A and denotes attachment to D.

[0047] In embodiments, D comprises an exatecan moiety.

[0048] In embodiments, D is an exatecan moiety.

[0049]

[0050] In embodiments, wherein denotes attachment to L².

[0051] In embodiments, wherein denotes attachment L². [0053] In embodiments, -L^J-L²-D is

[0054] In embodiments, n is an integer between about 2 and about 9. In embodiments, n is an integer between about 2 and about 8. In embodiments, n is an integer between about 2 and about 7. In embodiments, n is an integer between about 2 and about 6. In embodiments, n is an integer between about 2 and about 5. In embodiments, n is an integer between about 2 and about 4.

[0055] In embodiments, n is an integer between about 3 and about 10. In embodiments, n is an integer between about 4 and about 10. In embodiments, n is an integer between about 5 and about 10. In embodiments, n is an integer between about 6 and about 10. In embodiments, n is an integer between about 7 and about 10. In embodiments, n is an integer between about 8 and about 10.

[0056] In embodiments, n is an integer between about 3 and about 9. In embodiments, n is an integer between about 4 and about 8. In embodiments, n is an integer between about 5 and about 7.

[0057] In embodiments, n is an integer between about 2 and about 5. In embodiments, n is an integer between about 2 and about 4. In embodiments, n is an integer between about 2 and about 3.

[0058] In embodiments, n is an integer between about 3 and about 6. In embodiments, n is an integer between about 4 and about 6. In embodiments, n is an integer between about 5 and about 6.

[0059] In embodiments, n is an integer between about 3 and about 5.

[0060] In embodiments, n is an integer between 2 and 9. In embodiments, n is an integer between 2 and 8. In embodiments, n is an integer between 2 and 7. In embodiments, n is an integer between 2 and 6. In embodiments, n is an integer between 2 and 5. In embodiments, n is an integer between 2 and 4. [0061] In embodiments, n is an integer between 3 and 10. In embodiments, n is an integer between 4 and 10. In embodiments, n is an integer between 5 and 10. In embodiments, n is an integer between 6 and 10. In embodiments, n is an integer between 7 and 10. In embodiments, n is an integer between 8 and 10.

[0062] In embodiments, n is an integer between 3 and 9. In embodiments, n is an integer between 4 and 8. In embodiments, n is an integer between 5 and 7.

[0063] In embodiments, n is an integer between 2 and 5. In embodiments, n is an integer between 2 and 4. In embodiments, n is an integer between 2 and 3.

[0064] In embodiments, n is an integer between 3 and 6. In embodiments, n is an integer between 4 and 6. In embodiments, n is an integer between 5 and 6.

[0065] In embodiments, n is an integer between 3 and 5.

[0066] In embodiments, n is about 2. In embodiments, n is about 3. In embodiments, n is about 4. In embodiments, n is about 5. In embodiments, n is about 6. In embodiments, n is about 7. In embodiments, n is about 8. In embodiments, n is about 9. In embodiments, n is about 10.

[0067] In embodiments, n is 2. In embodiments, n is 3. In embodiments, n is 4. In embodiments, n is 5. In embodiments, n is 6. In embodiments, n is 7. In embodiments, n is 8. In embodiments, n is 9. In embodiments, n is 10.

[0068] In embodiments, the conjugate is of Formula (I), or a pharmaceutically acceptable salt or solvate thereof, wherein:

A is an anti-Claudin-1 antibody; , denotes attachment to A and denotes attachment to L²;

L² is a divalent peptide linker comprising glycine and phenylalanine;

D is an exatecan moiety; and n is an integer about 4.

[0069] In embodiments, the conjugate is of Formula (II),

or a pharmaceutically acceptable salt or solvate thereof, wherein A and n are as described herein.

[0070] In embodiments, the conjugate is of Formula (II), or a pharmaceutically acceptable salt or solvate thereof, and A is an anti-Claudin-1 antibody.

[0071] In embodiments, the conjugate is of Formula (IF),

[0072] or a pharmaceutically acceptable salt or solvate thereof, wherein A and n are as described herein. .In embodiments, the conjugate is of Formula (IF), or a pharmaceutically acceptable salt or solvate thereof, and A is an anti-Claudin-1 antibody.

[0073] In embodiments, -L^J-L²-D, prior to attachment to A, is

[0074] In embodiments, -L^J-L²-D, prior to attachment to A, is

[0075] In embodiments, conjugates of the disclosure comprise two or more occurrences of D, wherein the two or more occurrences of D may be the same or different from each other. In embodiments, conjugates of the disclosure comprise two or more occurrences of L¹, wherein the two or more occurrences of L¹ may be the same or different from each other. In embodiments, conjugates of the disclosure comprise two or more occurrences of L², wherein the two or more occurrences of L² may be the same or different from each other.

[0076] In embodiments, at least one instance of a -L^J-L²-D moiety is attached to A via a thioether bond (e.g., via wherein denotes attachment to the rest of

_ + +

A, and denotes attachment to the rest of the -L¹-L²-D moiety).

[0077] In embodiments, the L¹, L², and D moieties described herein can be assembled into the conjugate of the disclosure, for example according to the disclosed techniques and methods. Therapeutic and targeting conjugates of the disclosure, and methods for producing them, are described below by way of non-limiting example.

[0078] In embodiments, each instance of a -L^J-L²-D moiety is attached to A via a thioether bond. [0079] In embodiments, the conjugate of the disclosure comprises one or more

[0080] Conjugates disclosed herein can be purified (e.g., removal of any starting materials) by extensive diafiltration. If necessary, additional purification by size exclusion chromatography can be conducted to remove any aggregated conjugates. In general, the conjugates as purified typically contain less than 5% (e.g., <2% w/w) aggregated conjugates as determined by SEC; less than 0.5% (e.g., <0.1% w/w) free (unconjugated) drug as determined by RP-HPLC; less than 1% of the unattached L^J-L²-D as determined by SEC and less than about 10% (e.g., about 5% w/w) unconjugated anti-Claudin-1 antibody as determined by HIC-HPLC.

Antibodies

[0081] Examples of anti-Claudin-1 antibodies that may be used in the antibody-drug conjugates described herein are disclosed in WO 2010/034812 and WO 2017/162678, each of which is incorporated by reference herein in its entirety. Claudin-1 may also be referred to as “CLDN1 ”

[0082] Other examples of suitable anti-Claudin-1 antibodies include those disclosed in European Patent No. EP 1 167 389, in U.S. Patent No. 6,627,439, in international patent application published under No. WO 2014/132307, in international patent applications published under No. WO 2015/014659 and No. WO 2015/014357, and in Yamashita et al., J. Pharmacol. Exp. Then, 2015, 353(1): 112-118, each of which is incorporated by reference herein in its entirety.

[0083] Anti-Claudin-1 antibodies or antigen binding fragments thereof suitable for use in the present invention may be polyclonal antibodies or monoclonal antibodies.

[0084] Anti-Claudin-1 antibodies or antigen binding fragments thereof suitable for use according to the present invention may also be "humanized", (e.g., sequence differences between rodent antibodies and human sequences can be minimized by replacing residues which differ from those in the human sequences by site-directed mutagenesis of individual residues or by grafting of entire regions or by chemical synthesis). Humanized antibodies can also be produced using recombinant methods. In the humanized form of the antibody, some, most or all of the amino acids outside the CDR regions are replaced with amino acids from human immunoglobulin molecules, while some, most or all amino acids within one or more CDR regions are unchanged. Small additions, deletions, insertions, substitutions or modifications of amino acids are permissible as long as they do not significantly modify the biological activity of the resulting antibody. Suitable human "replacement" immunoglobulin molecules include IgGl, IgG2, IgG2a, IgG2b, IgG3, IgG4, IgA, IgM, IgD or IgE molecules, and fragments thereof.

[0085] In embodiments, an anti-Claudin-1 antibody or antigen-binding fragment thereof is a fully human antibody or antigen-binding fragment thereof.

[0086] In aspects, a humanized anti-Claudin-1 antibody for use according to the present invention includes those described in WO 2017/162678, which is incorporated by reference in its entirety. Exemplary sequences for the anti-Claudin-1 antibody or antigen binding fragment used in the methods of the disclosure are described in Table 1.

Table 1: Exemplary anti-Claudin-1 antibody sequences

[0087] In aspects, the “HILI” anti-Claudin-1 antibody or antigen binding fragment thereof comprises a complementarity determining region (CDR) Hl comprising the amino acid sequence set forth in SEQ ID NO: 5, a CDR H2 comprising the amino acid sequence set forth in SEQ ID NO: 6, and a CDR H3 comprising the amino acid sequence set forth in SEQ ID NO: 7.

[0088] In aspects, the “HILI” anti-Claudin-1 antibody or antigen binding fragment thereof comprises a complementarity determining region (CDR) LI comprising the amino acid sequence set forth in SEQ ID NO: 8, a CDR L2 comprising the amino acid sequence "Gly Ala Ser", and a CDR L3 comprising the amino acid sequence set forth in SEQ ID NO: 10. [0089] In aspects, the “HILI” anti-Claudin-1 antibody or antigen binding fragment thereof comprises a complementarity determining region (CDR) Hl comprising the amino acid sequence set forth in SEQ ID NO: 5, a CDR H2 comprising the amino acid sequence set forth in SEQ ID NO: 6, a CDR H3 comprising the amino acid sequence set forth in SEQ ID NO: 7, a complementarity determining region (CDR) LI comprising the amino acid sequence set forth in SEQ ID NO: 8, a CDR L2 comprising the amino acid sequence "Gly Ala Ser", and a CDR L3 comprising the amino acid sequence set forth in SEQ ID NO: 10.

[0090] In aspects, the complementarity determining regions (CDRs) disclosed herein are defined according to IMGT®. However, it is appreciated that other methods of defining the CDRs in the art can also be used.

[0091] In aspects, the “HILI” anti-Claudin-1 antibody or antigen fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 3.

[0092] In aspects, the anti-Claudin-1 antibody or antigen fragment thereof comprises a VH comprising the amino acid sequence set forth in SEQ ID NO: 3 or SEQ ID NO: 13.

[0093] In aspects, the anti-Claudin-1 antibody or antigen fragment thereof comprises a VH comprising an amino acid sequence 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 aspects, the anti- Claudin-1 antibody or antigen fragment thereof comprises a VH comprising an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 13.

[0094] In aspects, the “HILI” anti-Claudin-1 antibody or antigen fragment thereof comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 4.

[0095] In aspects, the anti-Claudin-1 antibody or antigen fragment thereof comprises a VL comprising the amino acid sequence set forth in SEQ ID NO: 4 or SEQ ID NO: 14.

[0096] In aspects, the anti-Claudin-1 antibody or antigen fragment thereof comprises a VL comprising an amino acid sequence 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 aspects, the anti- Claudin-1 antibody or antigen fragment thereof comprises a VL comprising an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 14.

[0097] In aspects, the anti-Claudin-1 antibody or antigen fragment thereof 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. In aspects, the “HILI” anti-Claudin-1 antibody or antigen fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 3 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 4.

[0098] In aspects, the anti-Claudin-1 antibody or antigen fragment thereof 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.

[0099] In aspects, the anti-Claudin-1 antibody or antigen fragment thereof comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 1, SEQ ID NO: 11, or SEQ ID NO: 15. In aspects the “HILI” anti-Claudin-1 antibody or antigen fragment thereof comprises the heavy chain comprising the amino acid sequence of SEQ ID NO: 1.

[0100] In aspects, the anti-Claudin-1 antibody or antigen fragment thereof comprises a Heavy Chain comprising an amino acid sequence 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 aspects, the anti-Claudin-1 antibody or antigen fragment thereof comprises a Heavy Chain comprising an amino acid sequence 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 aspects, the anti-Claudin-1 antibody or antigen fragment thereof comprises a Heavy Chain comprising an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 15.

[0101] In aspects, the anti-Claudin-1 antibody or antigen fragment thereof comprises a light chain comprising the amino acid sequence set forth in SEQ ID NO: 2 or SEQ ID NO: 12. In aspects, the “HILI” anti-Claudin-1 antibody or antigen fragment thereof comprises the light chain comprising the amino acid sequence of SEQ ID NO: 2.

[0102] In aspects, the anti-Claudin-1 antibody or antigen fragment thereof comprises a light chain comprising an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 2.

[0103] In aspects, the anti-Claudin-1 antibody or antigen fragment thereof comprises a light chain comprising an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 12. [0104] The term “identity” or “homology” refers to a relationship between the sequences of two or more polypeptides, as determined by comparing the sequences. The term "identity" also means the degree of sequence relatedness between the polypeptides, as determined by the number of matches between strings of two or more amino acid residues. The percent “identity” between the two sequences is a function of the number of identical positions shared by the sequences (i.e., percent identity equals number of identical positions/total number of positions x 100), taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences. The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters. Additionally, or alternatively, the amino acid sequences disclosed herein can further be used as a “query sequence” to perform a search against public databases to, for example, identify related sequences. For example, such searches can be performed using the BLAST program of Altschul et al. (J. Mol. Biol. 215:403-10, 1990). Optionally, the identity exists over a region that is at least about 10 amino acids in length, or over a region that is about 20, 50, 200 or more amino acids in length. Preferably, the sequence identity is determined over the entire length of the sequences.

[0105] In aspects, the anti-Claudin-1 antibody or antigen fragment thereof comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 1; and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 2. In aspects, the “HILI” anti- Claudin-1 antibody or antigen fragment thereof comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 1; and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 2.

[0106] In aspects, the anti-Claudin-1 antibody or antigen fragment thereof comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 15; and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 2. [0107] In aspects, the anti-Claudin-1 antibody or antigen fragment thereof comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 11; and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 12.

[0108] In aspects, the six complementarity determining regions (CDRs) of the anti-Claudin-1 antibody or antigen fragment thereof are the same as those in the anti-Claudin-1 monoclonal antibody or antigen fragment thereof secreted by a hybridoma cell line deposited at the DSMZ on July 29, 2008 under an Accession Number DSM ACC2938.

[0109] In aspects, the heavy chain variable region ("VH") and the light chain variable region ("VL") of the anti-Claudin-1 antibody or antigen fragment thereof are the same as those in the anti-Claudin-1 monoclonal antibody or antigen fragment thereof secreted by a hybridoma cell line deposited at the DSMZ on July 29, 2008 under an Accession Number DSM ACC2938.

[0110] In aspects, the heavy chain and light chain of the anti-Claudin-1 antibody or antigen fragment thereof are the same as those in the anti-Claudin-1 monoclonal antibody or antigen fragment thereof secreted by a hybridoma cell line deposited at the DSMZ on July 29, 2008 under an Accession Number DSM ACC2938.

[OHl] The humanized anti-Claudin-1 antibody or antigen fragment thereof may be a full monoclonal antibody or antigen fragment thereof having an isotope selected from the group consisting of IgGl, IgG2, IgG3 and IgG4. Alternatively, the humanized anti-Claudin-1 antibody or antigen fragment thereof may be a fragment of a monoclonal antibody or antigen fragment thereof selected from the group consisting of Fv, Fab, F(ab')2, Fab', dsFv, scFv, sc(Fv)2 and diabodies.

[0112] Anti-Claudin-1 antibodies (or biologically active variants or fragments thereof) suitable for use according to the present invention may be functionally linked (e.g., by chemical coupling, genetic fusion, non-covalent association or otherwise) to one or more other molecular entities. Methods for the preparation of 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, molecular entities are attached at positions on the antibody molecule that do not interfere with the binding properties of the resulting conjugate, e.g., positions that do not participate in the specific binding of the antibody to its target.

[0113] The antibody molecule and molecular entity may be covalently, directly linked to each other. Or, alternatively, the antibody molecule and molecular entity may be covalently linked to each other through a linker group. This can be accomplished by using any of a wide variety of stable bifunctional agents well known in the art, including homofunctional and heterofunctional linkers.

[0114] In aspects, an anti-Claudin-1 antibody (or a biologically active fragment thereof) for use according to the present invention is conjugated to a detectable agent. Any of a wide variety of detectable agents can be used, including, without limitation, various ligands, radionuclides (e.g., 3H, 1251, 1311, and the like), fluorescent dyes (e.g., fluorescein isothiocyanate, rhodamine, phycoerytherin, phycocyanin, allophycocyanin, o-phthalaldehyde and fluorescamine), chemiluminescent agents (e.g., luciferin, luciferase and aequorin), microparticles (such as, for example, quantum dots, nanocrystals, phosphors and the like), enzymes (such as, for example, those used in an ELISA, i.e., horseradish peroxidase, betagalactosidase, luciferase, alkaline phosphatase), colorimetric labels, magnetic labels, and biotin, dioxigenin or other haptens and proteins for which antisera or monoclonal antibodies are available.

[0115] Other molecular entities that can be conjugated to an anti-Claudin-1 antibody of the present invention (or a biologically active fragment thereof) include, but are not limited to, linear or branched hydrophilic polymeric groups, fatty acid groups, or fatty ester groups. [0116] Thus, in the practice of the present invention, anti-Claudin-1 antibodies can be used under the form of full length antibodies, biologically active variants or fragments thereof, chimeric antibodies, humanized antibodies, and antibody-derived molecules comprising at least one complementarity determining region (CDR) from either a heavy chain or light chain variable region of an anti-Claudin-1 antibody, including molecules such as Fab fragments, F(ab')2 fragments, Fd fragments, Fabc fragments, Sc antibodies (single chain antibodies), diabodies, individual antibody light single chains, individual antibody heavy chains, chimeric fusions between antibody chains and other molecules, and antibody conjugates, such as antibodies conjugated to a therapeutic agent or a detectable agent. Preferably, anti-Claudin-1 antibody-related molecules according to the present invention retain the antibody's ability to bind its antigen, in particular the extracellular domain of Claudin-1.

[0117] Anti-Claudin-1 antibodies that are internalized by the target cell are especially suitable for use in antibody-drug conjugates. This, in embodiments, the anti-Claudin-1 antibody used in the antibody-drug conjugates describes herein is an antibody that is internalized by the target cell upon binding to Claudin-1. Methods of Use

[0118] In another aspect, provided herein are methods of treating a disease or disorder in a subject in need thereof, the method comprising administering to the subject an antibody-drug conjugate described herein.

[0119] As a general proposition, the therapeutically effective amount of the antibody-drug conjugate administered to human subject will be in the range of about 0.5 to about 50 mg/kg of patient body weight whether by one or more administrations. In embodiments, the therapeutically effective amount is in the range of about 0.5 to about 5 mg/kg of patient body weight. In embodiments, the therapeutically effective amount is in the range of about 5 to about 10 mg/kg of patient body weight. In embodiments, the therapeutically effective amount is in the range of about 10 to about 15 mg/kg of patient body weight. In embodiments, the therapeutically effective amount is in the range of about 15 to about 20 mg/kg of patient body weight. In embodiments, the therapeutically effective amount is in the range of about 20 to about 25 mg/kg of patient body weight. In embodiments, the therapeutically effective amount is in the range of about 25 to about 30 mg/kg of patient body weight. In embodiments, the therapeutically effective amount is in the range of about 30 to about 35 mg/kg of patient body weight. In embodiments, the therapeutically effective amount is in the range of about 35 to about 40 mg/kg of patient body weight. In embodiments, the therapeutically effective amount is in the range of about 40 to about 45 mg/kg of patient body weight. In embodiments, the therapeutically effective amount is in the range of about 45 to about 50 mg/kg of patient body weight.

[0120] In embodiments, the therapeutically effective amount is about 3 mg/kg of patient body weight. In embodiments, the therapeutically effective amount is about 4 mg/kg of patient body weight. In embodiments, the therapeutically effective amount is about 5 mg/kg of patient body weight. In embodiments, the therapeutically effective amount is about 6 mg/kg of patient body weight. In embodiments, the therapeutically effective amount is about 7 mg/kg of patient body weight. In embodiments, the therapeutically effective amount is about 8 mg/kg of patient body weight. In embodiments, the therapeutically effective amount is about 9 mg/kg of patient body weight. In embodiments, the therapeutically effective amount is about 10 mg/kg of patient body weight. In embodiments, the therapeutically effective amount is about 15 mg/kg of patient body weight. In embodiments, the therapeutically effective amount is about 20 mg/kg of patient body weight. In embodiments, the therapeutically effective amount is about 25 mg/kg of patient body weight. In embodiments, the therapeutically effective amount is about 30 mg/kg of patient body weight. In embodiments, the therapeutically effective amount is about 35 mg/kg of patient body weight. In embodiments, the therapeutically effective amount is about 40 mg/kg of patient body weight. In embodiments, the therapeutically effective amount is about 45 mg/kg of patient body weight. In embodiments, the therapeutically effective amount is about 40 mg/kg of patient body weight. In embodiments, the therapeutically effective amount is about 50 mg/kg of patient body weight.

[0121] The dose may be administered as a single dose or as multiple doses (e.g., 2 or 3 doses), such as infusions. For repeated administrations over several days or longer, depending on the condition, the treatment would generally be sustained until a desired suppression of disease symptoms occurs. An exemplary dose would be in the range from about 0.3 mg/kg to about 20 mg/kg. Thus, one or more doses of about 0.3 mg/kg, 3 mg/kg, 10 mg/kg, 15 mg/kg or 20 mg/kg (or any combination thereof) may be administered to the patient. Such doses may be administered intermittently, for example, every week or every three weeks (e.g., such that the patient receives from about two to about twenty, or, for example, about three doses of the antibody-drug conjugate). An initial higher loading dose, followed by one or more lower doses may be administered. The progress of this therapy is easily monitored by the methods described herein or conventional techniques and assays.

[0122] In aspects, the antibody-drug conjugate is administered intratumorally, intravenously, intraperitoneally, intramuscularly, intrathecally or subcutaneously, n embodiments, the antibody drug conjugate is administered intravenously. In embodiments, the antibody-drug conjugate is administered subcutaneously.

[0123] Without wishing to be bound by theory, it is believed that any disease or disorder that responds to anti-Claudin-1 antibodies may be treated with an antibody-drug conjugate disclosed herein.

[0124] In embodiments, the disease or disorder treated in accordance with a method described herein is cancer. In embodiments, the cancer is urothelial cancer. In embodiments, the cancer is esophageal cancer. In embodiments, the cancer is head and neck cancer. In embodiments, the cancer is lung cancer. In embodiments, the cancer is non-small cell lung cancer. In embodiments, the cancer is a squamous non-small cell lung cancer. In embodiments, the cancer is colorectal cancer. In embodiments, the cancer is cervical cancer. In embodiments, the cancer is a squamous cervical cancer. In embodiments, the cancer is liver cancer. In embodiments, the cancer is hepatocellular carcinoma. In embodiments, the cancer in intrahepatic cholangiocarcinoma. In embodiments, the cancer is breast cancer. In embodiments, the cancer is triple-negative breast cancer. [0125] In embodiments, a method of treatment described herein results in a decrease in tumor size. The decrease in tumor size may be measured using any suitable method known in the art or described herein, for example, using calipers or bioluminescence imaging in animal models, or using imaging such as computer tomography (CT) scanning, magnetic resonance imagining (MRI), positron emission tomography (PET), x-ray, or physical examination in human subjects.

[0126] In embodiments, a method of treatment described herein results in a decrease in tumor size of at least 5% compared to the tumor size before the first administration of the antibodydrug conjugate. In embodiments, a method of treatment described herein results in a decrease in tumor size of at least 10% compared to the tumor size before the first administration of the antibody-drug conjugate. In embodiments, a method of treatment described herein results in a decrease in tumor size of at least 20% compared to the tumor size before the first administration of the antibody-drug conjugate. In embodiments, a method of treatment described herein results in a decrease in tumor size of at least 30% compared to the tumor size before the first administration of the antibody-drug conjugate. In embodiments, a method of treatment described herein results in a decrease in tumor size of at least 40% compared to the tumor size before the first administration of the antibody-drug conjugate. In embodiments, a method of treatment described herein results in a decrease in tumor size of at least 50% compared to the tumor size before the first administration of the antibody-drug conjugate.

[0127] The decrease in tumor size may be measured at any suitable time point after administration of the antibody-drug conjugate, for example, the decrease in tumor size may be determined about 1 week to about 2 weeks, about 2 weeks to about 3 weeks, about 3 weeks to about 4 weeks, about 4 weeks to about 5 weeks, about 5 weeks to about 6 weeks, about 6 weeks to about 7 weeks, about 7 weeks to about 8 weeks, about 2 month to about 3 month, about 3 months to about 4 months, about 4 months to about 5 months, or about 5 months to about 6 months, after the first administration of the antibody-drug conjugate. Alternatively, the decrease in tumor size may be determined about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 7 weeks, about 2 month, about 3 months, about 4 months, about 5 months, or about 6 months, after the first administration of the antibody-drug conjugate.

[0128] In embodiments, a method of treatment described herein results in a decrease Extracellular Matrix (ECM) remodeling as measured by collagen fragments levels in the plasma. Collagen fragments that may be monitored include collagen 1 (C1M), collagen 3 (C3M) and collagen 4 (C4M and C4M12). Collagen fragment levels may be measured by any suitable method known in the art or described herein, e.g., by liquid chromatography-mass spectrometry (LC-MS/MS).

[0129] In embodiments, a method of treatment described herein results in a decrease in the levels of a collagen fragment in the plasma of at least 10% compared to levels before the first administration of the antibody-drug conjugate. In embodiments, a method of treatment described herein results in a decrease in the levels of a collagen fragment in the plasma of at least 20% compared to levels before the first administration of the antibody-drug conjugate. In embodiments, a method of treatment described herein results in a decrease in the levels of a collagen fragment in the plasma of at least 30% compared to levels before the first administration of the antibody-drug conjugate. In embodiments, a method of treatment described herein results in a decrease in the levels of a collagen fragment in the plasma of at least 40% compared to levels before the first administration of the antibody-drug conjugate. In embodiments, a method of treatment described herein results in a decrease in the levels of a collagen fragment in the plasma of at least 50% compared to levels before the first administration of the antibody-drug conjugate.

[0130] The levels of a collagen fragment in the plasma may be measured at any suitable time point after administration of the antibody-drug conjugate, for example, the decrease in tumor size may be determined about 1 week to about 2 weeks, about 2 weeks to about 3 weeks, about 3 weeks to about 4 weeks, about 4 weeks to about 5 weeks, about 5 weeks to about 6 weeks, about 6 weeks to about 7 weeks, about 7 weeks to about 8 weeks, about 2 month to about 3 month, about 3 months to about 4 months, about 4 months to about 5 months, or about 5 months to about 6 months, after the first administration of the antibody-drug conjugate. Alternatively, the decrease in levels of a collagen fragment may be determined about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 7 weeks, about 2 month, about 3 months, about 4 months, about 5 months, or about 6 months, after the first administration of the antibody-drug conjugate.

[0131] The antibody-drug conjugates described herein may be administered in combination with other therapeutic agents. Thus, in aspects, provided herein are methods of treating a disease or disorder in a subject in need thereof, the method comprising administering to the subject an antibody-drug conjugate described herein and one or more additional therapeutic agent. In embodiments, the additional therapeutic agent is a checkpoint inhibitor. In embodiments, the checkpoint inhibitor is an antibody or an antibody fragment. [0132] In embodiments, the checkpoint inhibitor is an anti -Cytotoxic T-Lymphocyte Antigen-4 (CTLA-4) antibody. In embodiments the antibody or antigen-binding portion thereof binds specifically to CTLA-4 and inhibits CTLA-4 activity. In embodiments, the anti- CTLA-4 antibody is a human CTLA-4-blocking antibody. In embodiments, the anti-CTLA-4 antibody is ipilimumab or tremelimumab.

[0133] In embodiments, the anti-CTLA-4 antibody is ipilimumab. In embodiments, the ipilimumab is administered to the subject in an amount of about 0.5 mg/kg to about 10 mg/kg. In embodiments, the ipilimumab is administered to the subject in an amount of about 1 mg/kg every six weeks. In embodiments, the ipilimumab is administered to the subject intravenously.

[0134] In embodiments, the anti-CTLA-4 antibody is tremelimumab. In embodiments, the tremelimumab is administered to the subject in an amount of about 1 mg/kg to about 50 mg/kg. In embodiments, the tremelimumab is administered to the subject in an amount of about 15 mg/kg every 90 days. In embodiments, the tremelimumab is administered to the subject intravenously.

[0135] In embodiments, the checkpoint inhibitor is an anti -Programmed Death Ligand- 1 (PD-L1) antibody. In embodiments, the anti-PD-Ll antibody is a human PD-L1 -blocking antibody. In embodiments, the anti-PD-Ll antibody is atezolizumab, durvalumab, or avelumab.

[0136] In embodiments, the anti-PD-Ll antibody is atezolizumab. In embodiments, the atezolizumab is administered to the subject in an amount of about 500 mg to about 2000 mg. In embodiments, the atezolizumab is administered to the subject in an amount of about 840 mg every 2 weeks. In embodiments, the atezolizumab is administered to the subject in an amount of about 1200 mg every 3 weeks. In embodiments, the atezolizumab is administered to the subject in an amount of about 1680 mg every 4 weeks. In embodiments, the atezolizumab is administered to the subject for up to one year. In embodiments, the atezolizumab is administered to the subject intravenously. In embodiments, the atezolizumab is administered to the subject intravenously over 60 minutes. In embodiments, the atezolizumab is administered to the subject intravenously over 60 minutes, and subsequently, intravenously over 30 minutes.

[0137] In embodiments, the anti-PD-Ll antibody is durvalumab. In embodiments, the durvalumab is administered to the subject in an amount of about 5 mg/kg to about 20 mg/kg. In embodiments, the durvalumab is administered to the subject in an amount of about 10 mg/kg every two weeks. In embodiments, the durvalumab is administered to the subject in an amount of about 10 mg/kg every two weeks where the subject weighs less than 30 kg. In embodiments, the durvalumab is administered to the subject in an amount of about 1000 mg to about 2000 mg. In embodiments, the durvalumab is administered to the subject in an amount of about 1500 mg every 4 weeks. In embodiments, the durvalumab is administered to the subject in an amount of about 1500 mg every 4 weeks where the subject weighs 30 kg or more. In embodiments, the durvalumab is administered to the subject intravenously. In embodiments, the durvalumab is administered intravenously to the subject over 60 minutes. [0138] In embodiments, the anti-PD-Ll antibody is avelumab. In embodiments, the avelumab is administered to the subject in an amount of about 500 mg to about 1500 mg. In embodiments, the avelumab is administered to the subject in an amount of about 800 mg every 2 weeks. In embodiments, the avelumab is administered to the subject intravenously. In embodiments, the avelumab is administered to the subject intravenously over 60 minutes. [0139] In embodiments, the checkpoint inhibitor is an anti -Programmed Death receptor- 1 (PD-1) antibody. In embodiments, the antibody or antigen-binding portion thereof binds specifically to a Programmed Death-1 (PD-1) receptor and inhibits PD-1 activity. In embodiments, the anti-PD-1 antibody is a human PD-l-blocking antibody. In embodiments, the anti-PD-1 antibody is nivolumab, pembrolizumab, cemiplimab, or dostarlimab.

[0140] In embodiments, the anti-PD-1 antibody is nivolumab. In embodiments, the nivolumab is administered to the subject in an amount of about 200 mg to about 500 mg. In embodiments, the nivolumab is administered to the subject in an amount of about 240 mg every 2 weeks. In embodiments, the nivolumab is administered to the subject in an amount of about 360 mg every 3 weeks. In embodiments, the nivolumab is administered to the subject in an amount of about 480 mg every 4 weeks. In embodiments, the nivolumab is administered to the subject intravenously. In embodiments, nivolumab is administered to the subject intravenously over 30 minutes

[0141] In embodiments, the anti-PD-1 antibody is pembrolizumab. In embodiments, the pembrolizumab is administered to the subject in an amount of about 100 mg to about 500 mg. In embodiments, the pembrolizumab is administered to the subject in an amount of about 200 mg every 3 weeks. In embodiments, the pembrolizumab is administered to the subject in an amount of about 400 mg every 6 weeks. In embodiments, the pembrolizumab is administered to the subject in an amount of about 1 mg/kg to about 5 mg/kg. In embodiments, the pembrolizumab is administered to the subject in an amount of about 2 mg/kg every 3 weeks. In embodiments, the pembrolizumab is administered to the subject intravenously. In embodiments, the pembrolizumab is administered to the subject intravenously over 30 minutes.

[0142] In embodiments, the anti-PD-1 antibody is cemiplimab. In embodiments, the cemiplimab is administered to the subject in an amount of about 200 mg to about 500 mg. In embodiments, the cemiplimab is administered to the subject in an amount of about 350 mg every 3 weeks. In embodiments, the cemiplimab is administered to the subject intravenously. In embodiments, the cemiplimab is administered to the subject intravenously over 30 minutes.

[0143] In embodiments, the anti-PD-1 antibody is dostarlimab. In embodiments, the dostarlimab is administered to the subject in an amount of about 100 mg to about 2000 mg. In embodiments, the dostarlimab is administered to the subject at a dose of about 500 mg every three weeks for 12 weeks (or four doses), followed by subsequent doses of about 1000 mg every 6 weeks, beginning three weeks after the 12 weeks (i.e., after the 4th dose). In embodiments, the dostarlimab is administered to the subject intravenously over 30 minutes. [0144] The additional therapeutic agent may be administrated before, after or concurrently with the antibody-drug conjugate described herein.

EXAMPLES

[0145] The following working examples are illustrative of the linkers, drug molecules and antibodies or antibody fragments, and methods for preparing same. These are not intended to be limiting and it will be readily understood by one of skill in the art that other reagents or methods may be utilized.

Example 1: Expression Levels of Claudin-1 in Cancer

[0146] In order to determine expression of Claudin-1 (CLND1), tissues microarrays (TMAs) from different indications were stained with a Roche Discovery Ultra autostainer. Slides were baked at 60 °C for 1 hour followed by deparaffinization using standard autostainer protocol. Heat-Induced Epitope Retrieval (HIER) was performed using Roche CC1 (high pH) for 48 minutes at 95 °C. Peroxidase inhibitor was applied. The primary antibody (anti-CLDNl, Sigma-Aldrich HPA048319 at 1 :50) was incubated for 24 minutes at 37 °C. The secondary antibody Roche anti-Rabbit HQ was incubated for 8 minutes at 37 °C followed by Roche anti -HQ HRP for 8 minutes at 37 °C. DAB was applied using the Roche ChromoMap DAB kit, followed by Roche Hematoxylin II. [0147] Based on CLDN1 staining, the H-score was determined by the formula: 3 x percentage of strongly staining nuclei + 2 x percentage of moderately staining nuclei + percentage of weakly staining nuclei, giving a range of 0 to 300. Expression was categorized as follows: Negative: H-Score 0-10 ; Low: H-Score 11-100 ; Medium: H-Score 101-200 ; High: H-Score 201-300

[0148] Results are shown in FIGs. 1 A-1I and indicate that Claudin-1 is frequently overexpressed in different indications.

Example 2: ADC2 Binding to CLDN1

[0149] Binding of ADC2 (exatecan conjugated to an anti-Claudin-1 antibody and an ADC2 naked antibody control (anti-Claudin antibody but no exatecan conjugation) was measured in Huh7 hepatocellular carcinoma cancer cells using Fluorescence-activated cell sorting (FACS). Results are shown in FIG. 2 and indicate that exatecan conjugation did not interfere with ADC2 binding to CLDN1, IgGl naked antibody control (IgGl but no exatecan conjugation) did not bind CLDN1 (data not shown).

Example 3: ADC2 in vitro Profiling

[0150] Binding of ADC2 to CLDN1 overexpressing NS CLC NCI-H1975 cancer cells was analyzed by FACS. ADC2 naked antibody control and ADC2 isotype control (IgGl conjugated to exatecan) were used as controls. Results are shown in FIG. 3 A and indicate that ADC2 bound to Claudin-1 expressed on the cells.

[0151] Internalization of ADC2 into Claudin-1 -expressing NCLH1975 cells was analyzed using pHrodo dye. ADC2 naked antibody control and ADC2 isotype control were included and exatecan was used as negative control. Results are shown in FIG. 3B and indicate that ADC2 was efficiently internalized into Claudin-1 overexpressing NSCLC NCI-H1975 cells. [0152] Killing of CLND1 -overexpressing NCI-H1975 cells by ADC2 was analyzed by CellTiter-Glo assay. Cells were seeded in 96 well plates and cultured in complete cell culture medium at 37 °C. On day 0, cells were treated with either ADC2, ADC2 isotype control or free exatecan at different concentrations. On day 5, cell viability was determined by CellTiter-Glo (CTG).

[0153] Data are shown in FIG. 3C and indicate that ADC2 mediated cancer cell killing in vitro. Example 4: ADC2 Induces Immunogenic Cell Death in Cancer Cell

[0154] Three immunogenic cell death (ICD) markers were assessed in vitro in CLDN1 overexpressing NCI-H1975 NCSCLC cancer cells. ER stress was measured by JNK phosphorylation as measured by Western Blotting. ATP and HMGB1 release from cells were measure by ELISA in the culture medium. DNA damage induced by ADC2 was assessed by measuring H2A phosphorylation using Western Blotting.

[0155] Results are shown in FIGs. 4A-4C and indicate that ADC2 induces immunogenic cell death in cancer cells.

Example 5: ADC2 Bystander Effect in NCI-H1975 Isogenic Cell System

[0156] A CLDN1 Isogenic cancer cell system was generated in NCI-H1975 NSCLC cancer cells. The systems comprises parental NCI-H1975 CLDN1 -negative cells and CLDN1 overexpressing NCI-H1975 cells. CLND1 -deficient and CLDN1 -positive NCI-H1975 cells were mixed with a 1 : 1 ratio and treated with ADC2, ADC2 isotype control or exatecan. Cell killing was monitored over time by life cell imaging using IncuCyte. Illustrative images are shown in FIG. 5 A and the cell killing is quantified in FIG. 5B. These results show that ADC2 directly kills CLDN1+ cells via internalization and release of payload and that CLDN1- cell are killed by the diffusion of the released payload from CLDN1+ cell into the tumor microenvironment (bystander effect).

Example 6: ADC2 drives Tumor Regression in Cell Line Derived Models In Vivo.

[0157] NCI-H1975 non-small cell lung (NSCLC) cancer cells overexpressing human CLDN1 or Huh7 hepatocellular carcinoma (HCC) cancer cells were subcutaneously engrafted in NOD SCID female mice (17-21 g). When tumors reached 150 mm³ in size in the NSCLC model or 180 mm³ in the HCC model, treatment was initiated (this is Day 0 ; arrow in the graph).

[0158] For the NSCLC model (NCLH1975 cells overexpressing CLDN1), ADC2, or ADC2 isotype control or dato-DxD (an anti-TROP2 ADC that has shown efficacy in preclinical studies) were administered as single dose at 10 mg/kg body weight intravenously. For the HCC model (Huh7), 10 mg/kg body weight of ADC2 or 10 mg/kg body weight of ADC2 isotype control were administered intravenously on Days and 7.

[0159] FIGs. 6A and 7A show expression of CLDN1 in the tumor models. FIGs. 6B and 7B show tumor growth in the tumor models. For both models, no body weight loss was observed. These data indicate that ADC2 drove complete tumor regression in cell line derived cancer models vivo.

Example 7: ADC2 Drives Complete Tumor Regression in a Patient-Derived Nasopharyngeal Cancer Model In Vivo

[0160] Patient derived material from nasopharyngeal cancer was subcutaneously engrafted in NOD SCID female mice (17-21 g). When tumors reached 200 mm³ in size, treatment with either 10 mg/kg body weight of ADC2 or 10 mg/kg body weight with ADC2 isotype control was initiated (this is Day 0; see arrow in the graph). Both single doses of ADC2 and ADC2 isotype control were administered intravenously. On day 24 the experiment was terminated. All mice treated with ADC2 were in regression. No body weight loss was observed.

[0161] Results are shown in FIGs. 8A and 8B and indicate that ADC2 was able to drive complete tumor regression.

Example 9: Characterization of Antibodies to Determine Suitability for use in ADCs [0162] In this example, several anti-CLDNl antibodies targeting the extracellular domain of CLDN1 were compared based on the pivotal parameters for an antibody to be suitable for an ADC strategy, including efficient binding and subsequent internalization into cancer cells expressing CLDN1. Ideally, for use in an ADC, the target antigen should efficiently internalize once bound by the antibody, allowing the payload to enter the cell and exert its cytotoxic effect.

Methods

Cell lines and culture condition

[0163] At confluence, cell culture was split with a sub-cultivation ratio of 1 :3 to 1 :5. [0164] All cell cultures were grown at 37°C and 5% CO2 with saturating humidity unless otherwise specified.

Test articles used in the study

[0165] Commercial anti-CLDNl antibodies were evaluated for their ability to bind extracellular domain of CLDN1 present on the CLDN1 -transduced non-small cell lung cancer NCI-H1975 cell line (Clones 7A5, 6F6 and 421203). Additional antibodies that target CLDN1 with an unknown epitope were also evaluated (2E2-H5 and 9C3P1). All tested antibodies are listed in Table 2. Table 2: Antibodies used in Example 9

[0166] To facilitate the comparison with commercially available antibodies, a m!gG2a version of HILI was used in the study.

Antibody binding to CLDN1 by FACS

[0167] Cells were seeded at 50.10³ cells per well of a 96 well plate in 100 pL of complete growth medium and kept on culture for 24h. Cell medium was then removed, and cells were washed with 100 pL of PBS. Cells were incubated in 100 pL of cell dissociation buffer until they detached. Detached cells were collected and transferred into a V-bottom plate, and the plate was spun for 3 min at 350 g. The supernatants were discarded and cells incubated in 100 pL of anti-CLDNl antibody solution for Ih at 4°C (all antibodies were used in a dose-range from 0.0032 pg/mL (0.00213 nM) to 50 pg/mL (333.5 nM) in 5-fold steps). Cells were washed twice in cold PBS and incubated in 100 pL of secondary antibody solution (dilution 1 :400, Table 3.1) containing Zombie Violet Live/Dead cell marker (1 :500 dilution) for 30 min at 4°C. Cells were washed twice with 100 pL of FACS buffer, and resuspended in 45 pL of FACS buffer, before flow cytometry analysis on a CytoFLEX benchtop flow cytometer (Beckman Coulter) for Zombie Violet negative and Alexa Fluor® 647 positive cells. Antibody reference is listed in Table 2.

Antibody binding to CLDN1 by microscopy

[0168] 12xl0³ CLDN1-H1975 cells were seeded in a flat bottom 96-well plate in 100 pL of cell culture medium and incubated for 18h. Then the medium was removed and replaced by 100 pl of Hoechst 33342 solution (4pg/mL in assay medium) and cells incubated for 15 min at 37°C. The Hoechst solution was then removed, washed twice with 100 pL of assay medium and replaced with a mix of 50 pL of fresh media and 50 pL of Zenon pHrodo labeled complex (anti-CLDNl antibody). Cells were incubated 30 min and unbound antibodies were washed off with three washing steps in 100 pL of assay medium. Then cells were incubated in 100 pL of assay medium pH=3 to allow pHrodo to fluoresce and image for anti-CLDNl staining. Images were acquired with Cytation CIO microscope (Agilent) using DAPI and Cy5 channels, 20x objective.

Antibody labeling with Zenon pHrodo reagent

[0169] Zenon pHrodo IgG labelling reagent was used to labelled anti-CLDNl antibodies and isotype controls. A 4x working solution of Zenon™ pHrodo™ iFL IgG Labelling Reagent Deep Red was prepared and mixed with a 4x antibody solution (40 pg/mL) for 5 min at room temperature to allow labelling complexes to form between the Fab fragments from the Zenon pHrodo reagent and the Fc portion of IgG antibodies.

Internalization assay by FACS

[0170] 50xl0³ CLDN1-H1975 cells were seeded in a flat bottom 96-well plate in 100 pL of cell culture medium and incubated for 18h. Then the medium was removed, cells washed with 100 pL of FACS buffer (D-PBS without calcium and magnesium, 1 mM EDTA, 10% FBS) and then incubated at room temperature in 100 pL of FACS buffer until the cells detached. The cells were collected and transferred to a V-bottom plate. The plate was spun at 350g for 3 min, the supernatant removed and cell pellet resuspended in 50 pL of assay medium (RPMH640, 1% FBS, 1% Penicillin/Streptomycin) and transferred into a flat-bottom plate. 50 pL of the Zenon pHrodo labeled complex (anti-CLDNl antibody) was then added and the plate was incubated 30 min at 37°C. After 30 min, cells were transferred into a V- bottom plate and unbound antibodies were washed out by two washing steps with 100 pL of assay medium. Cells were resuspended in 100 pL of assay medium, transferred into a flatbottom plate and incubated under standard cell culture conditions for 2h, 4h and 6h to allow the antibodies to internalize. pHrodo dye is pH sensitive, and therefore shows little to no fluorescent signal at neutral pH and fluoresces brightly in acidic environments like lysosomal vesicles. When incubation was completed, the medium was removed, and the cells were detached in 100 pL of FACS buffer. Cells were washed twice with cold PBS and incubated in 50 pL of Zombie Violet Live/Dead cell marker (1 :500 dilution) for 15 min at 4°C. After a final washing step with FACS buffer, cells were resuspended in 40 pL of FACS buffer and analyzed by flow cytometry on a CytoFLEX benchtop flow cytometer (Beckman Coulter) for Zombie Violet negative and pHrodo-positive cells (excitation at 560 nm, emission at 585 nm).

Internalization by microscopy

[0171] 12xl0³ CLDN1-H1975 cells were seeded in a flat bottom 96-well plate in 100 pL of cell culture medium and incubated for 18h. Then the medium was removed and replaced by 100 pl of Hoechst 33342 solution (4pg/mL in assay medium) and cells incubated for 15 min at 37°C. The Hoechst solution was then removed, washed twice with 100 pL of assay medium and replaced with a mix of 50 pL of fresh media and 50 pL of Zenon pHrodo labeled complex (anti-CLDNl antibody). Cells were incubated 30 min and unbound antibodies were washed off with three washing steps in 100 pL of assay medium. Then cells were incubated 2h, 4h and 6 h in 100 pL of assay medium to allow antibodies to internalize. When incubation was completed, the medium was removed, and the cells were washed with PBS before a fixation step. Images were acquired with Cytation CIO microscope (Agilent) using DAPI and Cy5 channels, 20x objective.

Data analysis

[0172] Data are expressed as mean ± S.D. Statistical analyses were calculated by using GraphPad Prism software (GraphPad Software, La Jolla, CA).

Results

Anti-CLDN 1 Antibody binding to CLDN1

[0173] First, all selected commercially available anti-CLDNl antibodies were tested for their ability to bind CLDN1 by FACS on detached cells. When cells are detached, cell-cell interaction are disrupted and CLDN1 disengages from CLDN1-CLDN1 interaction, making the epitopes of the extracellular part of CLDN1 more accessible.

[0174] Under these condition, HILI and anti-CLDNl antibodies 7A5, 6F6 and 421203 were shown to bind CLDN1 expressed in NCI-H1975 with Km values varying from 1.45 nM for 7A5 to 28.9 nM for 6F6 (FIGs. 9A and 9B and Table 3).

Table 3: Summary table with Km and CI values for each Antibody

[0175] No binding was observed for antibodies 2E2-H5 and 9C3P1 (FIGs. 9A-9C). As the epitopes for these two antibodies were not defined, the most likely reason for lack of binding was that these antibodies are not able to bind extracellular domains of CLDN1.

[0176] Then, anti-CLDNl antibodies found to bind CLDN1 by FACS were further tested by imaging for their ability to bind CLDN1 on adherent cells. Only HILI and 6F6 were observed to bind CLDN1 by immunofluorescence (FIGs. 10A and 10B). Antibody 7A5 was showed to bind very weakly to CLDN1 while antibody 421203 was not able to interact with CLDN1 on adherent cells (FIG. 10A and 10B), suggesting that the CLDN1 epitopes bound by 7A5 and 421203 were masked or buried when cells were grown on plastic, making it likely that these epitopes would also not be accessible in cells organized in tissues.

[0177] The quantification of HILI and 6F6 staining showed that the binding of HILI is stronger than the binding of 6F6 on both total fluorescence per cell or on the total area stained per cell (FIGs. 10A and 10B).

Anti-CLDN 1 Antibody Internalization

[0178] The anti-CLDNl antibodies identified as CLDN1 binders were tested for their ability to internalize into CLDN1 -expressing NCLH1975. To do so, pHrodo Deep Red dye was used. Since pHrodo dye is pH sensitive, little to no fluorescent signal is detected at neutral pH while it becomes highly fluorescent in acidic internalization and lysosomal vesicles.

[0179] First, all anti-CLDNl antibodies were conjugated with pHrodo Deep Red dye and tested in an internalization assay on adherent cells by imaging. As shown in FIG. 11 A and 1 IB, only antibodies HILI, 7A5 and 6F6 were internalized. HILI was the most internalized antibody, 7A5 the least internalized and 6F6 showed an intermediate internalization ability. The total area of pHrodo-positive pixels per cell was quantified at 16.63, 8.89 and 4.29 for HILI, 6F6 and 7A5 respectively (FIG. 1 IB).

[0180] Then, the internalization of anti-CLDNl antibodies was assessed by FACS on adherent cells. Under these conditions, HILI showed the highest internalization rate with a MFI value at 6h of 40386, while this MFI value was 14388 and 10736 for 6F6 and 7A5, respectively (FIGs. 12A and 12B).

[0181] Altogether, both internalization studies (by imaging and by FACS) demonstrated a much stronger ability of HILI to internalize compared to the other tested anti-CLDNl antibodies.

Conclusion and Discussion

[0182] Five commercially available anti-CLDNl antibodies were compared to HILI for their ability to bind to CLDN1 and to undergo a subsequent internalization, since efficient binding and internalization into cancer cells is a desirable characteristic of an antibody that is to be used in an ADC.

[0183] Only three of the tested antibodies were able to bind CLDN1 on non-adherent cells (Ab 7A5, 6F6 and 421203) when CLDN1 extracellular epitopes were fully accessible. But when mimicking a tissue organization with adherent cells, antibody 6F6 showed an intermediate binding and antibody 7A5 showed a very low level of binding to CLDN1- positive cells, while HILI was able to strongly bind to CLDN1.

[0184] The antibodies’ ability to be internalized was also assessed on both adherent and nonadherent cells. In both conditions, HILI was internalized much better than the other two antibodies.

[0185] Altogether, these data demonstrate that not any anti-CLDNl Ab is suitable for use in an ADC approach. Some antibodies can bind to CLDN1 and were not internalized (e.g., antibody 421203). Some antibodies bind to CLDN1, but their binding strongly affected by cell-cell interaction limiting their interest for an ADC (e.g., antibody 7A5). Finally, 6F6 was able to bind to CLDN1 and undergo a CLDN1 -dependent internalization, but with a lower efficacy compared to HILI.

[0186] HILI showed good binding and internalization properties to be suitable for an ADC approach. HILI was shown to be superior to all tested antibodies in its ability to be efficiently internalized in NCLH975 overexpressing CLDN1

Example 10: Comparison of Closely Related Antibodies in the Context of ADCs

[0187] This example describes the biological characterization of two variants of anti-human CLDN1 antibodies HILI and H3L3, which have the same CDRs but different VH and VL sequences and different Fc backbones with human IgGl Fc-silent due to the LFLEPS mutation and with a human IgGl wild type sequence, respectively. The two antibodies were compared in an internalization assay in CLDN1 -overexpressing NCI-H1975 lung cancer cells by imaging.

Methods

CLND1 Staining at the Cell Membrane

[0188] CLDN1 -transduced NCI-H1975 cells were seeded at 25xl0³ cells in a flat bottom 96- well plate (collagen-coated PhenoPlate, black, optically clear) in 100 pL of cell culture medium without phenol red and incubated for 18h. Then the medium was removed, cells were washed twice with 200 pL of HBSS. Then, 100 pL of Hoechst solution at 4 pg/mL were added in each well and cells were incubated for 10 min at 37°C. After 3 washing steps with 200 pL of HBSS, 100 pL of pHrodo-labeled HILI, H3L3 or hlgGl control diluted at 10 pg/mL in complete culture medium without phenol red were added and plates were incubated for 30 min at 4°C. to allow antibodies to bind CLDN1 at the cell membrane. Then, the unbound anti-CLDNl antibodies were washed away with 200 pL of HBSS and plates were incubated in an acidic solution of acetic acid (pH 3.5) to allow pH rodo to fluoresce. Images were acquired with Cytation CIO microscope (Agilent) using DAPI and Cy5 channels, 20x objective.

Internalization assay by imaging using pHrodo technology

[0189] 0.5 mg of each antibody were diluted at 2.2 mg/mL in PBS or a similar neutral buffer free of primary amines. 25 pL of a 1 M freshly prepared sodium bicarbonate (pH 8.5) with the kit component C was added to the antibody solution. [0190] The contents of the 100 pg vial of pHrodo™ Deep Red amine-reactive dye was dissolved in 100 pL of water to prepare a 0.77 mM (1 mg/mL) labeling solution.

[0191] 50 pL of the labeling solution were added to the antibody solution and were incubated for 2 hours at room temperature, protected from light.

[0192] The dye/antibody ratio was determined using the method from AAT Bio website (www.aatbio.com/resources/application-notes/how-to-determine-the-degree-of-labeling). These ratio were 4.02, 4.66 and 2.48 for HILI, H3L3 and hlgGl control respectively.

[0193] Internalization assay by imaging (protocol without washout step): CLDN1- transduced NCI-H1975 cells were seeded at 25xl0⁵ cells in a flat bottom 96-well plate (collagen-coated PhenoPlate, black, optically clear) in 100 pL of cell culture medium without phenol red and incubated for 18h. Then the medium was removed, cells were washed twice with 200 pL of HBSS. Then, 100 pL of Hoechst solution at 4 pg/mL were added in each well and cells were incubated for 10 min at 37°C. After 3 washing steps with 200 pL of HBSS, 100 pL of pHrodo-labeled HILI, H3L3 or hlgGl control diluted at 10 pg/mL in complete culture medium without phenol red were added and plates were incubated for 2 h, 4 h and 6 h under standard cell culture conditions to allow antibodies to be internalized (pHrodo dye is pH sensitive, little to no fluorescent signal at neutral pH and fluoresces brightly in acidic environments like lysosomal vesicles). When incubation was completed, the medium was removed, and the cells were washed with PBS before a fixation step. Images were acquired with Cytation CIO microscope (Agilent) using DAPI and Cy5 channels, 20x objective.

[0194] Internalization assay by imaging (protocol with a washout step): A similar assay procedure was applied. 100 pL of pHrodo-labeled HILI, H3L3 or hlgGl control diluted at 10 pg/mL in complete culture medium without phenol red were added to the Hoeachst- stained cells and plates were incubated for 30 min at 4°C to allow antibodies to bind CLDN1 at the cell membrane. Then, the unbound anti-CLDNl antibodies were washed away with 200 pL of HBSS and plates were incubated for 2 h, 4 h and 6 h at 37°C to allow antibodies to be internalized.

Data analysis

[0195] Data are expressed as mean ± S.D. Statistical analyses were calculated by using GraphPad Prism software (GraphPad Software, La Jolla, CA). Results

[0196] HILI and H3L3 anti-CLDNl antibodies were compared for their ability to internalize in CLDN1 -positive cells. In both antibodies, the CDR sequences are identical. Only the sequences outside the CDR differ between those antibodies (see Table 1).

[0197] Both HILI and H3L3 antibodies were shown to similarly detect CLDN1 at the cell surface of CLDNl-transduced NCI-H1975 cells (FIG. 13A).

[0198] The ability of HILI and H3L3 to be internalized in CLDN1 -transduced NCI-H1975 cells was assessed by two different approaches. pHrodo-conjugated anti-CLDNl antibodies were applied on cells. The kinetics of internalization was determined either without washing step between antibody binding and internalization, or a washing step was performed to remove the excess of unbound antibodies. In both cases, HILI was shown to be internalized more efficiently than H3L3. With the assay procedure without washout step HILI internalization is significantly higher at 6h compared to H3L3 (FIGs. 13B and 13C). With the protocol including a washout step following antibody binding and before internalization kinetics, at all time points (2h, 4h and 6h), HILI showed better internalization than H3L3 (FIGs. 13D and 13E).

Conclusion and Discussion

[0199] Identical CDR sequences are shared between HILI and H3L3 anti-CLDNl antibodies. Despite a similar binding to CLDN1 expressed at the cell surface of NCI-H1975 cells, internalization of HILI was shown to be significantly higher than that of H3L3.

[0200] This feature suggests that HILI is a better candidate for an ADC approach, because of its internalization capabilities.

Claims

CLAIMS What is claimed is:

1. An antibody-drug conjugate of Formula (I):

A- L^L^Djn

(I) or a pharmaceutically acceptable salt or solvate thereof, wherein:

A is an anti-Claudin-1 antibody comprising

(i) a heavy chain variable region (VH) comprising a heavy chain complementarity determining region (HCDR)l comprising the amino acid sequence of SEQ ID NO: 5, a HCDR2 comprising the amino acid sequence of SEQ ID NO: 6 and a HCDR3 comprising the amino acid sequence of SEQ ID NO: 7; and

(ii) a light chain variable region comprising a light chain complementarity determining region (LCDR)1 comprising the amino acid sequence of SEQ ID NO: 8, a LCDR2 comprising the amino acid sequence of GAS, and a LCDR3 comprising the amino acid sequence of SEQ ID NO: 10;

L¹ is a divalent moiety comprising a functional group attached to the anti-Claudin-1 antibody;

L² is a divalent peptide linker comprising at least two amino acids;

D is an exatecan moiety; and n is an integer between about 2 and about 10.

2. The antibody-drug conjugate of claim 1, wherein the anti-Claudin-1 antibody comprises a VH comprising a sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 3 or 13.

3. The antibody-drug conjugate of claim 1 or 2, wherein the anti-Claudin-1 antibody comprises a VL comprising a sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 4 or 14.

4. The antibody-drug conjugate of any one of claims 1-3, wherein the anti-Claudin-1 antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 3 and a light chain comprising the amino acid sequence of SEQ ID NO: 4.

5. The antibody-drug conjugate of any one of claims 1-3, wherein the anti-Claudin-1 antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 13 and a light chain comprising the amino acid sequence of SEQ ID NO: 14.

6. The antibody-drug conjugate of any one of claims 1-5, wherein the anti-Claudin-1 antibody is a monoclonal antibody.

7. The antibody-drug conjugate of any one of claims 1-6, wherein the anti-Claudin-1 antibody is a rabbit, mouse, chimeric, humanized or fully human monoclonal antibody.

8. The antibody-drug conjugate of any one of claims 1-7, wherein the anti-Claudin-1 antibody is an IgG isotype.

9. The antibody-drug conjugate of any one of claims 1-8, wherein the anti-Claudin-1 antibody is an IgGl isotype.

10. The antibody-drug conjugate of any one of claims 1-9, wherein -iJ-L²- is eno es a ac men o .

11. The antibody-drug conjugate of any one of claims 1-10, wherein

, wherein denotes attachment to L².

12. A method of treating cancer in a subject in need thereof, comprising administering to the subject the antibody-drug conjugate of any one of claims 1-11.

13. The method of claim 12, wherein the cancer is urothelial cancer, esophageal cancer, head and neck cancer, lung cancer, colorectal cancer, liver cancer, or breast cancer.