Antibodies to CLDN4 and methods of use thereof

Abstract

The present invention includes human antibodies that bind to claudin 4 domain (CLDN4) proteins, which can be used to treat cancer. TIFF2025527491000017.tif88170

Description

[Technical Field]

[0001] This application is an international application claiming priority to U.S. Provisional Patent Application No. 63 / 398,075, filed August 15, 2022, the entire contents of which are incorporated herein by reference.

[0002] All patents, patent applications, and publications cited herein are incorporated by reference in their entirety. The disclosures of these publications in their entireties are incorporated by reference into this application in order to more fully describe the state of the art known to those skilled in the art as of the date of the invention described and claimed herein.

[0003] This patent disclosure contains material that is subject to copyright protection. The copyright owner has no objection to the facsimile reproduction by anyone of the patent document or the patent disclosure, as it appears in the U.S. Patent and Trademark Office patent file or records, but otherwise reserves all copyright rights whatsoever.

[0004] Sequence Listing This application has been submitted electronically in ASCII format and contains a Sequence Listing which is incorporated herein by reference in its entirety. The ASCII copy made in [ ] has the name [ ] and is [ ] bytes in size.

[0005] FIELD OF THE INVENTION The present invention includes antibodies against claudin 4 (CLDN4) and methods of using the same. [Background technology]

[0006] background Claudins are four-transmembrane proteins of tight junctions. Claudin 4 protein is encoded by the CLDN4 gene. Summary of the Invention

[0007] An embodiment of the present invention is directed to an isolated monoclonal antibody or antigen-binding fragment thereof that binds to a claudin 4 (CLDN4) protein or a fragment thereof, including a heavy chain, a light chain, or both a heavy chain and a light chain.

[0008] In embodiments, the heavy chain comprises a CDR1 comprising GFTFNNYA (SEQ ID NO: 9), GFTFGGYA (SEQ ID NO: 12), GGTFSSYA (SEQ ID NO: 15), or GGTFNNYA (SEQ ID NO: 18), a CDR2 comprising IRDSGGST (SEQ ID NO: 10), LSNSGSNA (SEQ ID NO: 13), or IIPIVDIA (SEQ ID NO: 16), a CDR3 comprising ARRGYSSSWYGDGYYYGMDV (SEQ ID NO: 11), ARAVMSSWYMRRYYYYYMDV (SEQ ID NO: 14), or ARGGSQGAYYMDV (SEQ ID NO: 17), or a combination of these CDRs. and a combination thereof, wherein the light chain comprises a CDR1 comprising SGSIASSF (SEQ ID NO: 19), RSNIGSNT (SEQ ID NO: 22), SGSIASNY (SEQ ID NO: 25), or QSVSNY (SEQ ID NO: 28), a CDR2 comprising ENN (SEQ ID NO: 20), SNN (SEQ ID NO: 23), EDN (SEQ ID NO: 26), or GAS (SEQ ID NO: 29), and a CDR3 comprising QSYDSTSHV (SEQ ID NO: 21), AAWDDSLNGLYV (SEQ ID NO: 24), QSYDDSNRVV (SEQ ID NO: 27), or HQYGSLPQT (SEQ ID NO: 30), or a combination of these CDRs. In embodiments, the antibody sequence is determined according to the IMGT numbering scheme.

[0009] In embodiments, the claudin 4 protein is a human claudin 4 protein.

[0010] In embodiments, the antibodies are fully human or humanized.

[0011] In embodiments, the antibodies are monospecific, bispecific, or multispecific.

[0012] In embodiments, the antibody is an IgG, for example, the antibody is an IgG1, IgG2, IgG3, or IgG4 antibody.

[0013] In embodiments, the antibody is a single chain antibody.

[0014] In embodiments, the antibody is at least 1.0 x 10 -9 It has a binding affinity of M.

[0015] In embodiments, the antibody or fragment comprises a heavy chain constant region, a light chain constant region, an Fc region, and an Fc variant, or a combination thereof.

[0016] In embodiments, the antibody comprises Gly1-2-F4, Gly1-4-G3, Gly1-1-H9, or Gly1-1-B2.

[0017] In embodiments, the antibody competes with the binding of Gly1-2-F4, Gly1-4-G3, Gly1-1-H9, or Gly1-1-B2.

[0018] In embodiments, the antibody or fragment is linked to a therapeutic agent.

[0019] In embodiments, the antibody is a single chain fragment.

[0020] Embodiments of the present invention are also directed to isolated antibodies or fragments thereof that bind to human claudin-4 protein.

[0021] In embodiments, the antibody comprises: (a) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 9, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 10, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 11, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 19, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 20, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 21; or (b) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 12, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 13, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 14, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 22, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 23, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 24; or (c) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 15, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 16, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 17, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 25, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 26. or (d) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 18, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 16, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 17, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 28, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 29, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 30. In embodiments, the antibody sequences are determined according to the IMGT numbering scheme.

[0022] Embodiments of the present invention are also directed to isolated scFv antibodies that bind to human claudin-4 protein, comprising: (a) a VH CDR1 comprising the amino acid sequence of SEQ ID NO:9, a VH CDR2 comprising the amino acid sequence of SEQ ID NO:10, a VH CDR3 comprising the amino acid sequence of SEQ ID NO:11, a VL CDR1 comprising the amino acid sequence of SEQ ID NO:19, a VL CDR2 comprising the amino acid sequence of SEQ ID NO:20, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO:21; or (b) a VH CDR1 comprising the amino acid sequence of SEQ ID NO:12, a VH CDR2 comprising the amino acid sequence of SEQ ID NO:13, a VH CDR3 comprising the amino acid sequence of SEQ ID NO:14, a VL CDR1 comprising the amino acid sequence of SEQ ID NO:22, a VL CDR2 comprising the amino acid sequence of SEQ ID NO:23, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO:24; or (c) a VH CDR1 comprising the amino acid sequence of SEQ ID NO:15, a VH CDR2 comprising the amino acid sequence of SEQ ID NO:16, a VH CDR3 comprising the amino acid sequence of SEQ ID NO:17, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO:25. or (d) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 18, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 16, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 17, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 28, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 29, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 30. In embodiments, the antibody sequences are determined according to the IMGT numbering scheme.

[0023] An embodiment of the present invention is directed to an isolated antibody or fragment thereof that binds to human claudin-4 protein, comprising a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 3, 5, and 7, or a sequence at least 90% identical thereto.

[0024] An embodiment of the present invention is directed to an isolated antibody or fragment thereof that binds to human claudin-4 protein, comprising a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 4, 6, and 8, or a sequence at least 90% identical thereto.

[0025] An embodiment of the present invention is directed to an isolated antibody or fragment thereof that binds to human claudin-4 protein, comprising a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 3, 5, and 7, or a sequence at least 90% identical thereto, and a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 4, 6, and 8, or a sequence at least 90% identical thereto.

[0026] An embodiment of the present invention is directed to an isolated scFv that binds to human claudin-4 protein, comprising a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 3, 5, and 7, or a sequence at least 90% identical thereto.

[0027] An embodiment of the present invention is directed to an isolated scFv that binds to human claudin-4 protein, comprising a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 4, 6, and 8, or a sequence at least 90% identical thereto.

[0028] An embodiment of the present invention is directed to an isolated scFv that binds to human claudin-4 protein, comprising a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 3, 5, and 7, or a sequence at least 90% identical thereto, and a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 4, 6, and 8, or a sequence at least 90% identical thereto.

[0029] An embodiment of the present invention includes an isolated monoclonal antibody or antigen-binding fragment thereof that binds to human claudin-4 protein, comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence that is about 95% identical to SEQ ID NO:1 and the light chain comprises an amino acid sequence that is about 95% identical to SEQ ID NO:2.

[0030] An embodiment of the present invention includes an isolated scFv that binds to human claudin-4 protein, comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence that is about 95% identical to SEQ ID NO:1 and the light chain comprises an amino acid sequence that is about 95% identical to SEQ ID NO:2.

[0031] An embodiment of the present invention includes an isolated monoclonal antibody or antigen-binding fragment thereof that binds to human claudin-4 protein, comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence that is about 95% identical to SEQ ID NO:3 and the light chain comprises an amino acid sequence that is about 95% identical to SEQ ID NO:4.

[0032] An embodiment of the present invention includes an isolated scFv antibody that binds to human claudin-4 protein, comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence about 95% identical to SEQ ID NO:3 and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO:4.

[0033] An embodiment of the present invention includes an isolated monoclonal antibody or antigen-binding fragment thereof that binds to human claudin-4 protein, comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence that is about 95% identical to SEQ ID NO:5, and the light chain comprises an amino acid sequence that is about 95% identical to SEQ ID NO:6.

[0034] An embodiment of the present invention includes an isolated scFv that binds to human claudin-4 protein, comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence that is about 95% identical to SEQ ID NO:5 and the light chain comprises an amino acid sequence that is about 95% identical to SEQ ID NO:6.

[0035] An embodiment of the present invention includes an isolated monoclonal antibody or antigen-binding fragment thereof that binds to human claudin-4 protein, comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence about 95% identical to SEQ ID NO:7 and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO:8.

[0036] An embodiment of the present invention includes an isolated scFv that binds to human claudin-4 protein, comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence that is about 95% identical to SEQ ID NO:7 and the light chain comprises an amino acid sequence that is about 95% identical to SEQ ID NO:8.

[0037] Aspects of the present invention are directed to isolated bispecific antibodies comprising an antibody fragment described herein and a second antigen-binding fragment having specificity for a molecule on an immune cell. In embodiments, the molecule is selected from the group consisting of CCR4, CXCR4, B7H3, B7H4, CD27, CD28, CD40, CD40L, CD47, CD122, CTLA-4, GITR, GITRL, ICOS, ICOSL, LAG-3, LIGHT, OX-40, OX40L, PD-1, TIM3, 4-1BB, TIGIT, VISTA, HEVM, BTLA, and KIR.

[0038] In embodiments, the fragment and the second fragment are each independently selected from a Fab fragment, a single chain variable fragment (scFv), or a single domain antibody.

[0039] Embodiments of the present invention are further directed to bispecific T cell engagers (BiTEs) that bind to human claudin-4 protein. In embodiments, a BiTE comprises: (a) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 9, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 10, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 11, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 19, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 20, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 21; or (b) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 12, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 13, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 14, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 22, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 23, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 24; or (c) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 15, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 16, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 17, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 25, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 26. or (d) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 18, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 16, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 17, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 28, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 29, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 30. In embodiments, the antibody sequences are determined according to the IMGT numbering scheme.

[0040] Embodiments of the present invention further include a bispecific T cell engager (BiTE) that binds to human claudin-4 protein, comprising a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 3, 5, and 7, or a sequence at least 90% identical thereto.

[0041] Embodiments of the present invention further include a bispecific T cell engager (BiTE) that binds to human claudin-4 protein, comprising a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 4, 6, and 8, or a sequence at least 90% identical thereto.

[0042] Embodiments of the present invention further include a bispecific T cell engager (BiTE) that binds to human claudin-4 protein, comprising a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 3, 5, and 7, or a sequence at least 90% identical thereto, and a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 4, 6, and 8, or a sequence at least 90% identical thereto.

[0043] The embodiments described herein may further comprise an Fc fragment.

[0044] Aspects of the invention are further directed to nucleic acids encoding the antibodies or fragments described herein.

[0045] Additionally, aspects of the present invention are also directed to pharmaceutical compositions comprising an antibody or fragment thereof described herein and a pharmaceutically acceptable carrier or excipient.

[0046] In embodiments, the pharmaceutical composition may further comprise at least one additional therapeutic agent. For example, the therapeutic agent may be a toxin, a radiolabel, an siRNA, a small molecule, or a cytokine.

[0047] Aspects of the present invention are directed to isolated cells comprising one or more polynucleotides encoding the antibodies or fragments described herein.

[0048] Aspects of the invention are also directed to vectors comprising the nucleic acids described herein.

[0049] Further aspects of the present invention are directed to cells containing the vectors described herein.

[0050] Aspects of the present invention are directed to engineered cells containing chimeric antigen receptors. In embodiments, the chimeric antigen receptor comprises an extracellular ligand-binding domain specific for an antigen on the surface of a cancer cell, the antigen comprising claudin 4, and the extracellular ligand-binding domain comprises an antibody or fragment thereof, wherein the antibody or fragment thereof comprises: (a) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 9, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 10, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 11, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 19, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 20, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 21; or (b) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 12, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 13, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 14, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 22, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 23, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 24; or (c) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 15, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 16, or (d) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 18, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 16, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 17, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 25, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 26, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 27, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 28, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 29, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 30. In embodiments, the antibody sequences are determined according to the IMGT numbering scheme.

[0051] In embodiments, the antibody or fragment thereof comprises a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 3, 5, and 7, or a sequence at least 90% identical thereto.

[0052] In embodiments, the antibody or fragment thereof comprises a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 4, 6, and 8, or a sequence at least 90% identical thereto.

[0053] In embodiments, the antibody or fragment thereof comprises a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 3, 5, and 7, or a sequence at least 90% identical thereto, and a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 4, 6, and 8, or a sequence at least 90% identical thereto.

[0054] In embodiments, the cells include T cells, NK cells, NKT cells, iPS cells, iPS-derived cells, cell lines, or B cells. For example, the cells include CD4+, CD8+, CD3+ pan T cells, or any combination thereof.

[0055] Aspects of the present invention are also directed to kits, which in embodiments include at least one pharmaceutical composition according to claim 36 or 39, a syringe, needle, or applicator for administering at least one antibody to a subject, and instructions for use.

[0056] Further aspects of the present invention are directed to methods for detecting the presence of claudin 4 in a sample. In embodiments, the method comprises contacting the sample with an isolated monoclonal antibody or fragment thereof described herein and detecting the presence or absence of an antibody-antigen complex, thereby detecting the presence of claudin 4 in the sample. In embodiments, the contacting comprises immunohistochemistry. For example, immunohistochemistry includes precipitation, immunofluorescence, Western blot, or ELISA. In embodiments, the sample is whole blood, a blood component, a body fluid, a biopsy, a tissue, serum, or one or more cells.

[0057] In embodiments, the sample includes a normal sample or a cancerous sample. For example, the cancer expresses Claudin 4. For example, the cancer includes biliary tract cancer, breast cancer, uterine cancer, colon cancer, esophageal cancer, intestinal cancer, lung cancer, pancreatic cancer, prostate cancer, kidney cancer, rectal cancer, stomach cancer, thyroid cancer, and uterine cancer.

[0058] In embodiments, the one or more cells comprise an in vitro culture.

[0059] In embodiments, the one or more cells comprise a claudin 4-expressing cell.

[0060] In an embodiment, the sample is an in vitro sample.

[0061] Embodiments may further include obtaining a sample from a subject.

[0062] Aspects of the present invention are further directed to methods for treating cancer in a subject by administering to the subject a pharmaceutical composition described herein. For example, the cancer expresses claudin 4. For example, the cancer includes biliary tract cancer, breast cancer, uterine cancer, colon cancer, esophageal cancer, intestinal cancer, lung cancer, pancreatic cancer, prostate cancer, kidney cancer, rectal cancer, gastric cancer, thyroid cancer, and uterine cancer. In embodiments, the antibodies or fragments described herein bind to claudin 3 with a binding affinity that is at least 5, 10, 25, 50, 100, or 1000 times lower than the antibody, fragment, or bispecific antibody binds to claudin 4.

[0063] Other objects and advantages of the present invention will become readily apparent from the description which follows. [Brief explanation of the drawings]

[0064] [Figure 1] Non-limiting exemplary data are presented validating cytopathic effect flow cytometry.

[0065] [Figure 2]FIG. 1 shows a non-limiting, exemplary schematic of panning against claudin-4.

[0066] [Figure 3] 1 shows a chart of non-limiting exemplary data of scFvs targeting claudin-4 by phage panning using soluble claudin-4 expressed in insect cells.

[0067] [Figure 4] 1 shows a graph of non-limiting exemplary data for EK01 (see WO 2019 / 178359) which specifically binds to soluble claudin 4.

[0068] [Figure 5] 1 shows a graph of non-limiting exemplary data of F4 binding to claudin-4.

[0069] [Figure 6] 1 shows non-limiting exemplary histograms of flow cytometry data.

[0070] [Figure 7] 1 shows non-limiting exemplary Western blots from protein purifications.

[0071] [Figure 8]

[0023] Figure 1 shows non-limiting exemplary data for EK01 (see WO 2019 / 178356) minibody binding data.

[0072] [Figure 9]

[0023] Figure 1 shows a non-limiting, exemplary schematic diagram of paramagnetic proteoliposome (PMPL) panning. In an embodiment, three rounds of panning were performed on soluble claudin-4 and screened with claudin-4. After ELISA and dilution series confirmation of soluble claudin-4, one hit was initially identified. Panning with PMPL initially failed. New PMPLs were then generated.

[0073] [Figure 10] Non-limiting exemplary data for PMPL production is presented.

[0074] [Figure 11] 1 shows a non-limiting, exemplary schematic diagram of panning.

[0075] [Figure 12] Figure 11 shows non-limiting exemplary results of 6 plates from a re-rescue panning.

[0076] [Figure 13] Non-limiting exemplary results of clones and frequencies from rounds 1 and 2 of panning are shown in FIG.

[0077] [Figure 14] Figure 11 shows non-limiting exemplary data from round 1 and round 2 next generation sequencing (NGS).

[0078] [Figure 15] Non-limiting exemplary data from a phage-binding soluble claudin-4 ELISA is shown.

[0079] [Figure 16] Non-limiting exemplary data of phage binding in claudin-4 positive cells and untransfected cells are shown.

[0080] [Figure 17] Non-limiting exemplary data is presented for phage binding on untransduced cells.

[0081] [Figure 18] Non-limiting exemplary ELISA data is shown.

[0082] [Figure 19] Non-limiting exemplary minibody binding data is shown.

[0083] [Figure 20] Non-limiting exemplary minibody binding data is shown.

[0084] [Figure 21] FIG. 1 shows a non-limiting, exemplary schematic of the claudin-4 whole-cell panning pathway.

[0085] [Figure 22] A non-limiting exemplary purified phage binding curve (CDLN-4) is shown.

[0086] [Figure 23] Non-limiting exemplary CLDN-4 minibody binding of MB231 CLDN-4 is shown. In this example, KM is an anti-CLDN4 antibody synthesized from U.S. Patent No. 8,076,458 and cloned into our scFv-Fc vector as a control. Miltenyi and R&D were purchased and used as is.

[0087] [Figure 24]

[0023] Figure 1 shows a non-limiting, exemplary schematic diagram of CLDN protein structure. In embodiments, the structure and expression of CLDN-3 and CLDN-4 may be similar. In embodiments, the expression levels may also be similar in cancer tissues.

[0088] [Figure 25]

[0023] Figure 1 shows non-limiting, exemplary schematic diagrams of the extracellular domains of human CLDN-3 and CLDN-4. In some embodiments, homology in the extracellular domains can be used to discover CLDN-4-specific antibodies. Homology: ECL1 98%, ECL2 79%, both 93%.

[0089] [Figure 26] An example of the stability of a commercially available antibody is shown.

[0090] [Figure 27] 1 shows non-limiting exemplary quantitative data for cell line staining.

[0091] [Figure 28] 1 shows non-limiting exemplary data of CLDN-3 binding of aCLDN-4 minibody.

[0092] [Figure 29] Non-limiting exemplary data for the specificity of claudin antibodies are shown. Similar results were observed at 0.1 mg / ml.

[0093] [Figure 30] 1 shows non-limiting exemplary data of MB231 staining by aCXCR4 for KO.

[0094] [Figure 31] Figure 1 shows a non-limiting, exemplary graph of CD8+ anti-CLDN-4 CART killing of CLDN-4-expressing MB231 cells. Positive controls KM3900 and CPE nonspecifically killed tumors. aC4-G3 and aC4-F4 specifically recognized and killed CLDN+ tumors (9-fold higher). Negative controls had low background killing (less than 10%). Our anti-CLDN-4 CART kills tumor cells that specifically express high levels of CLDN-4.

[0095] [Figure 32] 1 shows a graph of non-limiting exemplary data of the kinetics of CD8+ anti-CLDN-4 CART killing at an E:T ratio of 2:1. DETAILED DESCRIPTION OF THE INVENTION

[0096] Detailed Description Abbreviations and Definitions

[0097] Detailed descriptions of one or more embodiments are provided herein. However, it should be understood that the present invention can be embodied in various forms. Therefore, the specific details disclosed herein should not be construed as limiting, but as a basis for the claims and as a representative basis for teaching one skilled in the art how to use the present invention in any suitable manner.

[0098] The singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. The use of the words "a" or "an" when used in conjunction with the word "comprising" in the claims and / or specification can mean "one," but is also consistent with the meanings of "one or more," "at least one," and "one or more."

[0099] Whenever any of the phrases "for example," "such as," "including," etc. are used herein, unless expressly stated otherwise, it is understood that the phrase "without limitation" is also involved. Similarly, "one example," "exemplary," etc. are understood to be non-limiting.

[0100] The term "substantially" permits deviations from the descriptor that do not adversely affect the intended purpose. It is understood that a descriptor is modified by the term "substantially" even if the word "substantially" is not explicitly recited.

[0101] Terms such as "comprising," "including," "having," and "involving" (and similarly, "comprises," "includes," "has," and "involves") are used interchangeably and have the same meaning. Specifically, each term is defined consistent with the general U.S. patent law definition of "comprising" and, therefore, is construed as an open term meaning "at least the following" and not excluding additional features, limitations, aspects, etc. Thus, for example, "a process comprising steps a, b, and c" means that the process includes at least steps a, b, and c. Whenever the terms "a" or "an" are used, they are understood to mean "one or more," unless such interpretation is inessential from the context.

[0102] As used herein, the term "about" is used herein to mean approximately, roughly, around, or within a range thereof. When the term "about" is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the stated numerical values. In general, the term "about" is used herein to modify numerical values ​​above and below the stated value by a variance above or below 20 percent (higher or lower).

[0103] Unique recombinant claudin 4 (CLDN4) antibodies are described herein. "Recombinant," as it relates to a polypeptide (such as an antibody) or polynucleotide, can refer to a form of the polypeptide or polynucleotide that does not occur in nature, a non-limiting example of which is one that can be made by combining polynucleotides or polypeptides that do not normally occur together.

[0104] In addition to exemplary wild-type IgG constant regions useful in combination with the VH and VL sequences provided herein (see Tables 1-4), amino acid sequences of monoclonal CLDN4 antibodies are provided herein. The amino acid sequences of the heavy and light chain complementarity determining regions (CDRs) of the CLDN4 antibodies are shown below as underlined ( CDR1 ), Underlined and bold ( CDR2 ), or Underlined, italicized, and bold ( CDR3 ) is shown. Table 1: Gly1-2-F4_PelB-F_2019-12-08_C03 Ab variable region amino acid sequence TIFF2025527491000002.tif42140 Table 2: Gly1-4-G3_PelB-F_2019-12-08_H03 Ab variable region amino acid sequence TIFF2025527491000003.tif42140 Table 3: Gly1-1-H9_PelB-F_2019-12-08_B03 Ab variable region amino acid sequence TIFF2025527491000004.tif38140 Table 4: Gly1-1-B2_PelB-F_2019-12-08_A03 Ab variable region amino acid sequence TIFF2025527491000005.tif38140

[0105] The amino acid sequences of the complementarity determining regions of the heavy and light chains of the CLDN4 antibody are shown in Tables 5A and 5B below. Table 5A. Heavy chain (V) of CLDN4 antibody H ) Complementarity-Determining Region (CDR) TIFF2025527491000006.tif61128 Table 5B. Light chain (V) of CLDN4 antibody L ) Complementarity-Determining Region (CDR) TIFF2025527491000007.tif60128

[0106] The amino acid sequences of the framework regions of the heavy and light chains of the CLDN4 antibody are shown in Tables 6A and 6B below. Table 6A: Heavy chain (V) of CLDN4 antibody H ) Framework region (FR) TIFF2025527491000008.tif106134 (Table 6B) Light chain (V) of CLDN4 antibody L ) Framework region (FR) TIFF2025527491000009.tif98133

[0107] The nucleic acid sequences of monoclonal CLDN4 antibodies are provided herein, and the nucleic acid sequences of the heavy and light chain complementarity determining regions (CDRs) of the CLDN4 antibodies are as follows: Underlined (CDR1), Underlined and bold (CDR2), or Underlined, italicized, and bold (CDR3).

[0108] Table 7A: Heavy chain (V) of CLDN4 antibody H ) Complementarity-Determining Region (CDR) TIFF2025527491000010.tif81139

[0109] Table 7B: Heavy chain (V) of CLDN4 antibody H ) Complementarity-Determining Region (CDR) TIFF2025527491000011.tif77139

[0110] Table 8A. VDJ regions of CLDN4 antibodies TIFF2025527491000012.tif148140

[0111] Table 8B. VJ regions of CLDN4 antibodies TIFF2025527491000013.tif136139

[0112] The CLDN4 antibodies described herein bind to CLDN4. In one embodiment, the CLDN4 antibody has high affinity and high specificity for CLDN4. Some embodiments also feature antibodies that share a certain percentage of identity or similarity with the amino acid or nucleotide sequence of an anti-CLDN4 antibody described herein. For example, "homology" or "identity" or "similarity" refers to the sequence similarity between two peptides or two nucleic acid molecules. Homology can be determined by comparing positions in each sequence that can be aligned for comparison purposes. If a position in the compared sequences is occupied by the same base or amino acid, the molecules are homologous at that position. The degree of homology between sequences is a function of the number of matching or homologous positions shared by the sequences. For example, an antibody can have 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more amino acid sequence identity when compared to a specific region or the full length of any one of the anti-CLDN4 antibodies described herein. For example, an antibody can have 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more nucleic acid sequence identity when compared to a specific region or the full length of any one of the anti-CLDN4 antibodies described herein. Sequence identity or similarity for the nucleic acids and proteins of the invention can be determined by sequence comparison and / or alignment by methods known in the art, for example, using software programs known in the art, such as those described in Ausubel et al. eds. (2007) Current Protocols in Molecular Biology. For example, sequence comparison algorithms (i.e., BLAST or BLAST 2.0), manual alignment, or visual inspection can be used to determine the percent sequence identity or similarity for the nucleic acids and proteins of the invention.

[0113] As used herein, "polypeptide" can encompass a single "polypeptide" as well as multiple "polypeptides" and can refer to a molecule composed of monomers (amino acids) linked in a linear chain by amide bonds (also known as peptide bonds). The term "polypeptide" can refer to any chain of two or more amino acids and does not refer to a specific length of the product. Thus, peptide, dipeptide, tripeptide, oligopeptide, "protein," "amino acid chain," or any other term that can be used to refer to a chain of two or more amino acids can refer to a "polypeptide" herein, and the term "polypeptide" can be used in place of or interchangeably with any of these terms. "Polypeptide" can also refer to post-expression modified products of a polypeptide, such as, but not limited to, glycosylation, acetylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, or modification with non-naturally occurring amino acids. A polypeptide can be derived from a natural biological source or produced by recombinant technology and is not necessarily translated from a specific nucleic acid sequence. It can be generated by any method, including chemical synthesis. With respect to amino acid sequences, those skilled in the art will readily recognize that individual substitutions, deletions, or additions to nucleic acid, peptide, polypeptide, or protein sequences that alter, add, delete, or substitute a single amino acid or a small percentage of amino acids in the encoded sequence are collectively referred to herein as "conservatively modified variants." In some embodiments, the alteration results in the replacement of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. Such conservatively modified variants of the anti-CLDN4 antibodies disclosed herein can exhibit increased cross-reactivity with CLDN4 compared to unmodified CLDN4 antibodies.

[0114] For example, a "conservative amino acid substitution" is one in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art as follows: basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, a non-essential amino acid residue in an immunoglobulin polypeptide is replaced with another amino acid residue from the same side chain family. In another embodiment, the amino acid chain can be replaced with a structurally similar chain that differs in the order and / or composition of the side chain family members.

[0115] antibody

[0116] As used herein, "antibody" or "antigen-binding polypeptide" can refer to a polypeptide or polypeptide complex that specifically recognizes and binds to an antigen. An antibody can be a full-length antibody or any antigen-binding fragment, or single chain thereof. For example, an "antibody" can include any protein- or peptide-containing molecule containing at least a portion of an immunoglobulin molecule that has the biological activity of binding to an antigen. Non-limiting examples include the complementarity-determining regions (CDRs) of a heavy or light chain or a ligand-binding portion thereof, the variable region of a heavy or light chain, the constant region of a heavy or light chain, the framework (FR) region, or any portion thereof, or at least a portion of a binding protein. As used herein, the term "antibody" can refer to immunoglobulin molecules and immunologically active portions of immunoglobulin (Ig) molecules, i.e., molecules that contain an antigen-binding site that specifically binds (immunoreacts with) an antigen. "Specifically binds" or "immunoreacts" means that the antibody reacts with one or more antigenic determinants of an antigen and not with other polypeptides.

[0117] As used herein, the term "antibody fragment" or "antigen-binding fragment" refers to an antibody fragment of F (ab’)2 , F (ab)2 , F ab ', F ab, Fv, scFv, etc. Antibody fragments, regardless of structure, bind to the same antigen recognized by the intact antibody. The term "antibody fragment" can encompass aptamers (such as spiegelmers), minibodies, and diabodies. The term "antibody fragment" can also encompass any synthetic or genetically engineered protein that acts like an antibody by binding to a specific antigen to form a complex. Antibodies, antigen-binding polypeptides, variants, or derivatives described herein include, but are not limited to, polyclonal, monoclonal, multispecific, human, humanized, or chimeric antibodies, single-chain antibodies, epitope-binding fragments such as Fab, Fab', F(ab'), Fd, Fv, single-chain Fv (scFv), single-chain antibodies, dAb (domain antibodies), minibodies, disulfide-linked Fv (sdFv), fragments comprising either the VL or VH domain, fragments produced by a Fab expression library, and anti-idiotypic (anti-Id) antibodies.

[0118] A "single-chain variable fragment" or "scFv" is a fragment of an immunoglobulin heavy chain (V H ) and light chain (V L (scFv) refers to a fusion protein of the variable regions of a single-chain Fv ("scFv") polypeptide molecule. A single-chain Fv ("scFv") polypeptide molecule is a covalently linked VH:VL heterodimer, which can be expressed from a gene fusion comprising genes encoding VH and VL linked by a peptide-encoding linker. (See Huston et al. (1988) Proc Nat Acad Sci USA 85(16):5879-5883.) In some embodiments, the regions are linked by a short linker peptide of 10 to about 25 amino acids. The linker can be rich in glycine for flexibility and serine or threonine for solubility, and the V H N-terminus of V Lor vice versa. This protein retains the specificity of the original immunoglobulin despite the removal of the constant region and the introduction of the linker. Many methods have been described for identifying chemical structures for converting naturally aggregated but chemically separated light and heavy polypeptide chains from antibody V regions into scFv molecules that fold into a three-dimensional structure substantially similar to that of an antigen-binding site. See, e.g., U.S. Pat. Nos. 5,091,513, 5,892,019, 5,132,405, and 4,946,778, each of which is incorporated herein by reference in its entirety.

[0119] Very large naive human scFv libraries have been and can be generated to provide a large source of rearranged antibody genes against a large number of target molecules. Smaller libraries can be constructed from individuals with infectious diseases to isolate disease-specific antibodies. (See Barbas et al., Proc. Natl. Acad. Sci. USA 89:9339-43 (1992); Zebedee et al., Proc. Natl. Acad. Sci. USA 89:3 175-79 (1992)).

[0120] Antibody molecules obtained from humans are classified into five classes of immunoglobulins: IgG, IgM, IgA, IgE, and IgD, which differ from each other in the nature of the heavy chains present in the molecule. Those skilled in the art will understand that heavy chains are classified as gamma, mu, alpha, delta, or epsilon (γ, μ, α, δ, ε), with several subclasses within each (e.g., γ1-γ4). Certain classes also have subclasses, such as IgG1, IgG2, IgG3, and IgG4. Immunoglobulin subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, and IgG5, are well characterized and are known to confer functional differentiation. With regard to IgG, a typical immunoglobulin molecule contains two identical light chain polypeptides with a molecular weight of approximately 23,000 daltons and two identical heavy chain polypeptides with a molecular weight of 53,000-70,000. The four chains are joined by disulfide bonds in a "Y" configuration, with the light chains flanking the heavy chains, which begin at the mouth of the "Y" and continue through the variable region. The immunoglobulin or antibody molecules described herein can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2) or subclass of immunoglobulin molecule.

[0121] Light chains are classified as kappa or lambda (κ, λ). Each heavy chain class can be associated with either a kappa or lambda light chain. Generally, light and heavy chains are covalently linked to each other, and when immunoglobulins are produced by hybridomas, B cells, or genetically engineered host cells, the "tails" of the two heavy chains are linked to each other by covalent disulfide bonds or non-covalent bonds. In the heavy chains, the amino acid sequence extends from the N-terminus at the forked end of the Y-shape to the C-terminus at the bottom of each chain.

[0122] Both light and heavy chains are divided into regions of structural and functional homology. The terms "constant" and "variable" are used functionally. The variable domains (VL and VH) of both the light and heavy chains determine antigen recognition and specificity. Conversely, the constant domains (CL and CH1, CH2, or CH3) of the light and heavy chains confer important biological properties, such as secretion, transplacental mobility, Fc receptor binding, and complement binding. The term "antigen-binding site" or "binding portion" can refer to the portion of an immunoglobulin molecule involved in antigen binding. The antigen-binding site is formed by amino acid residues in the N-terminal variable ("V") regions of the heavy ("H") and light ("L") chains. Three highly divergent sections within the V regions of the heavy and light chains, called "hypervariable regions," are interspersed between more conserved adjacent sections known as "framework regions" or "FRs." Thus, the term "FR" can refer to the amino acid sequences naturally found between and adjacent to the hypervariable regions of immunoglobulins. In an antibody molecule, the three hypervariable regions of the light chain and the three hypervariable regions of the heavy chain are arranged relative to each other in three-dimensional space to form an antigen-binding surface. The antigen-binding surface is complementary to the three-dimensional surface of a bound antigen, and the three hypervariable regions of each of the heavy and light chains are called "complementarity-determining regions" or "CDRs." The VH and VL regions containing the CDRs and frameworks (FRs) of the CLDN4 antibody are shown in Tables 1 to 4.

[0123] The six CDRs present in each antigen-binding domain are short, noncontiguous sequences of amino acids that are specifically arranged to form the antigen-binding domain when the antibody assumes its three-dimensional configuration in an aqueous environment. The remaining amino acids of the antigen-binding domain, the FR regions, show little inter-molecular variability. The framework regions primarily adopt a beta-sheet conformation, and the CDRs form loops that connect them and, in some cases, form part of the beta-sheet structure. The framework regions function to form a scaffold that orients the CDRs through non-covalent interactions between the chains. The antigen-binding domain formed by the arranged CDRs provides a surface complementary to the epitope on the antigen in an immune response, promoting non-covalent binding of the antibody to its cognate epitope. The amino acids comprising the CDRs and framework regions, respectively, have been previously identified for heavy or light chain variable regions and can be readily identified by one of skill in the art (see "Sequences of Proteins of Immunological Interest," Kabat, E., et al., USDapartment of Health and Human Services, (1983); and Chothia and Lesk, J. Mol. Biol., 196:901-917 (1987)).

[0124] Where there are more than one definition for a term used and / or accepted in the art, the definition of the term used herein is intended to encompass such meanings unless specifically and explicitly stated to the contrary. A specific example is the use of the term "complementarity-determining region" ("CDR") to describe the non-contiguous antigen-binding sites found within the variable regions of both heavy and light chain polypeptides. This particular region is described in Kabat et al., U.S. Dept. of Health and Human Services, "Sequences of Proteins of Immunological Interest" (1983) and Chothia et al., Mol. Biol. 196:901-917 (1987), which are incorporated herein by reference in their entireties. The definitions of CDRs by Kabat and Chothia include overlapping or subsets of amino acid residues when compared with each other. Nevertheless, application of the definitions to refer to CDRs of an antibody or variants thereof is intended to be within the scope of the term as defined and used herein. For comparison, the appropriate amino acid residues encompassing the CDRs defined by each of the above cited references are set forth in the following table. The exact residue numbers which encompass a CDR will vary depending on the sequence and size of the CDR, and one of skill in the art can routinely determine which residues constitute a CDR, given the variable region amino acid sequence of an antibody. TIFF2025527491000014.tif44128

[0125] Kabat et al. defined a numbering system for variable domain sequences that is applicable to any antibody. One of skill in the art can unambiguously assign this "Kabat numbering" system to any variable domain sequence, without reliance on other experimental data on the sequence itself. As used herein, "Kabat numbering" refers to the numbering system described in Kabat et al., U.S. Department of Health and Human Services, "Sequence of Proteins of Immunological Interest" (1983).

[0126] In addition to the above table, the Kabat numbering system describes the CDR regions as follows: CDR-H1 begins at about amino acid 31 (i.e., about 9 residues after the first cysteine ​​residue), includes about 5-7 amino acids, and ends at the next tryptophan residue. CDR-H2 begins at the 15th residue after the end of CDR-H1, includes about 16-19 amino acids, and ends at the next arginine or lysine residue. CDR-H3 begins at about the 33rd amino acid residue after the end of CDR-H2; includes 3-25 amino acids and ends with the sequence WGXG (X is any amino acid). CDR-L1 begins at about residue 24 (i.e., after the cysteine ​​residue), includes about 10-17 residues, and ends at the next tryptophan residue. CDR-L2 begins at about the 16th residue after the end of CDR-L1 and includes about 7 residues. CDR-L3 begins approximately 33 residues after the end of CDR-L2 (i.e., after the cysteine ​​residue), includes about 7-11 residues, and ends with the sequence F or WGXG (where X is any amino acid).

[0127] In certain embodiments, the CDRs of an antibody can be determined according to the IMGT numbering system, which is unique to IMGT and is designed to compare variable domains regardless of antigen receptor, chain type, or species [Lefranc M.-P., Immunology Today 18, 509 (1997) / Lefranc M.-P., The Immunologist, 7, 132-136 (1999) / Lefranc, M.-P., Pommie, C., Ruiz, M., Giudicelli, V., Foulquier, E., Truong, L., Thouvenin-Contet, V., and Lefranc, Dev. Comp. Immunol., 27, 55-77 (2003)]. In the IMGT-specific numbering, conserved amino acids always have the same positions, e.g., cysteine ​​23 (1st-CYS), tryptophan 41 (CONSERVED-TRP), hydrophobic amino acid 89, cysteine ​​104 (2nd-CYS), phenylalanine or tryptophan 118 (J-PHE or J-TRP). The IMGT-specific numbering provides standardized boundaries for the framework regions (FR1-IMGT: positions 1-26, FR2-IMGT: positions 39-55, FR3-IMGT: positions 66-104, and FR4-IMGT: positions 118-128) and the complementarity-determining regions: CDR1-IMGT: positions 27-38, CDR2-IMGT: positions 56-65, and CDR3-IMGT: positions 105-117. Since gaps represent unoccupied positions, the length of the CDR-IMGT (shown between brackets and separated by dots, e.g., [8.8.13]) is important information.The IMGT-specific numbering is used in the 2D graphic representations called IMGT Colliers de Perles [Ruiz, M. and Lefranc, M.-P., Immunogenetics, 53, 857-883 (2002) / Kaas, Q. and Lefranc, M.-P., Current Bioinformatics, 2, 21-30 (2007)] and in the 3D structures of the IMGT / 3Dstructure-DB [Kaas, Q., Ruiz, M. and Lefranc, M.-P., T cell receptor and MHC structural data. Nucl. Acids. 32, D208-D210 (2004)].

[0128] As used herein, the term "epitope" may include any protein determinant capable of specific binding to an immunoglobulin, scFv, or T-cell receptor. The variable region enables an antibody to selectively recognize and specifically bind to an epitope on an antigen. For example, the VL and VH domains of an antibody, or a subset of complementarity-determining regions (CDRs), combine to form variable regions that define a three-dimensional antigen-binding site. This quaternary antibody structure forms the antigen-binding site present at the end of each arm of a Y. Epitope determinants may consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and have specific three-dimensional structural and charge characteristics. For example, antibodies can be raised against N- or C-terminal peptides of a polypeptide. More specifically, the antigen-binding site is defined by three CDRs (i.e., CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3) on each of the VH and VL chains. In one embodiment, an antibody has the amino acid sequence: It may be for claudin 4 (CLDN4) with UniProtKB ID: O14493 CLD4_HUMAN (209 amino acid residues long), including TIFF2025527491000015.tif33128.

[0129] As used herein, the terms "immunological binding" and "immunological binding properties" can refer to the type of non-covalent interactions that occur between an immunoglobulin molecule and an antigen for which the immunoglobulin is specific. The strength, or affinity, of an immunological binding interaction is determined by the dissociation constant (K d ) and K d A smaller K represents a greater affinity. The immunological binding properties of a selected polypeptide can be quantified using methods well known in the art. One such method involves measuring the rates of antigen-binding site / antigen complex formation and dissociation, which depend on the concentrations of the complex partners, the affinity of the interaction, and geometric parameters that affect the rates in both directions equally. Thus, both "on rate constants" (K on ) and "off rate constant" (K off ) can be determined by calculation of the concentration and the actual rates of association and dissociation. (See Nature 361:186-87 (1993)). K off / K on The ratio of α to β allows for the elimination of parameters not related to affinity, and the equilibrium binding constant K D (See Davies et al. (1990) Annual Rev Biochem 59:439-473.) The antibodies of the present invention may have an equilibrium binding constant (K) as measured by a kinetic assay, e.g., a radioligand binding assay or similar assay known to those skilled in the art, e.g., BIAcore or Octet (BLI). D ) is ≦1 μM, ≦10 μM, ≦10 nM, ≦10 pM, or ≦100 pM to about 1 pM, the antibody can specifically bind to the CLDN4 epitope. For example, in some embodiments, D is about 1E-12M and about 1E-11M. D In some embodiments, K D is about 1E-11M and about 1E-10M. D In some embodiments, K D is about 1E-10M and about 1E-9M. D In some embodiments, K Dis about 1E-9M and about 1E-8M D In some embodiments, K D is about 1E-8M and about 1E-7M D In some embodiments, K D is about 1E-7M and about 1E-6M. D For example, in some embodiments, K D is about 1E-12M, and in other embodiments, K D is about 1E-11M. In some embodiments, K D is about 1E-10M, and in other embodiments, K D is about 1E-9M. In some embodiments, K D is about 1E-8M, and in other embodiments, K D is about 1E-7M. In some embodiments, K D is about 1E-6M, and in other embodiments, K D is about 1E-5M. In some embodiments, for example, K D is about 3E-11M, and in other embodiments, K D is about 3E-12M. In some embodiments, K D is approximately 6E-11M. "Specifically binds" or "having specificity" can refer to an antibody that binds to an epitope via the antibody's antigen-binding domain, where the binding involves some complementarity between the antigen-binding domain and the epitope. For example, an antibody is said to "specifically bind" to an epitope if it binds to that epitope via its antigen-binding domain more readily than it binds to a random, unrelated epitope.

[0130] For example, CLDN4 antibodies can be monovalent or bivalent and can comprise single or double chains. Functionally, the binding affinity of CLDN4 antibodies can be greater than or equal to 10 -5 M~10 -12 For example, the binding affinity of a CLDN4 antibody is in the range of 10 -6 M~10 -12 M, 10 -7 M~10 -12 M, 10 -8M~10 -12 M、10 -9 M~10 -12 M、10 -5 M~10 -11 M、10 -6 M~10 -11 M、10 -7 M~10 -11 M、10 -8 M~10 -11 M、10 -9 M~10 -11 M、10 -10 M~10 -11 M、10 -5 M~10 -10 M、10 -6 M~10 -10 M、10 -7 M~10 -10 M、10 -8 M~10 -10 M、10 -9 M~10 -10 M、10 -5 M~10 -9 M、10 -6 M~10 -9 M、10 -7 M~10 -9 M、10 -8 M~10 -9 M、10 -5 M~10 -8 M、10 -6 M~10 -8 M、10 -7 M~10 -8 M、10 -5 M~10 -7 M、10 -6 M~10 -7 M、10 -5 M~10 -6 Mである。

[0131] CLDN4 protein, or a derivative, fragment, analog, homolog, or ortholog thereof, can be used as an immunogen in the generation of antibodies that immunologically specifically bind to these protein components. CLDN4 protein, or a derivative, fragment, analog, homolog, or ortholog thereof, coupled to a proteoliposome, can be used as an immunogen in the generation of antibodies that immunologically specifically bind to these protein components.

[0132] Those skilled in the art will recognize that, without undue experimentation, one can determine whether a human monoclonal antibody has the same specificity as a human monoclonal antibody of the present invention by determining whether the former prevents the latter from binding to CLDN4. For example, if the human monoclonal antibody being tested exhibits reduced binding by and competes with a human monoclonal antibody of the present invention, then these two monoclonal antibodies bind to the same or closely related epitopes.

[0133] Another method for determining whether a human monoclonal antibody has the specificity of the human monoclonal antibody of the present invention is to preincubate the human monoclonal antibody of the present invention with the CLDN4 protein with which it is normally reactive, then add the human monoclonal antibody to be tested and determine whether the human monoclonal antibody to be tested is inhibited in its ability to bind to CLDN4. If the human monoclonal antibody to be tested is inhibited, it may have the same or functionally equivalent epitope specificity as the monoclonal antibody of the present invention. Screening of the human monoclonal antibodies of the present invention can also be carried out by using CLDN4 to determine whether the monoclonal antibody to be tested can neutralize CLDN4.

[0134] Various procedures known in the art can be used for the production of polyclonal or monoclonal antibodies directed against the proteins of the invention or against their derivatives, fragments, analogs, homologs, or orthologs (see, e.g., Antibodies: A Laboratory Manual, Harlow E, and Lane D, 1988, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, incorporated herein by reference).

[0135] Antibodies can be purified by well-known techniques, such as affinity chromatography using protein A or protein G, which primarily provide the IgG fraction of immune serum. Subsequently, or alternatively, the specific antigen that is the target of the desired immunoglobulin, or an epitope thereof, can be immobilized on a column, and immune-specific antibodies can be purified by immunoaffinity chromatography. Immunoglobulin purification is discussed, for example, by D. Wilkinson (The Scientist, published by The Scientist, Inc., Philadelphia, PA, Vol. 14, No. 8 (April 17, 2000), pp. 25-28).

[0136] As used herein, the term "monoclonal antibody" or "mAb" or "Mab" or "monoclonal antibody composition" can refer to a population of antibody molecules containing only one antibody molecule species consisting of a unique light chain gene product and a unique heavy chain gene product. The complementarity-determining regions (CDRs) of a monoclonal antibody are identical among the molecules of the population. MAbs contain an antigen-binding site capable of immunoreacting with an epitope of an antigen characterized by a unique binding affinity for it.

[0137] Monoclonal antibodies can be prepared using hybridoma methods, such as those described by Kohler and Milstein, Nature, 256:495 (1975). In the hybridoma method, a mouse, hamster, or other suitable host animal is immunized with an immunizing agent to induce lymphocytes that produce, or are capable of producing, antibodies that will specifically bind to the immunizing agent. Alternatively, lymphocytes can be immunized in vitro.

[0138] The immunizing agent can comprise a protein antigen, a fragment thereof, or a fusion protein thereof. For example, peripheral blood lymphocytes can be used if cells of human origin are preferred, or spleen cells or lymph node cells can be used if a non-human mammalian source is preferred. The lymphocytes are then fused with an immortalized cell line using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell (see Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, (1986) pp. 59-103). The immortalized cell line can be transformed mammalian cells, such as myeloma cells of rodent, bovine, and human origin. For example, rat or mouse myeloma cell lines can be used. The hybridoma cells can be cultured in a suitable medium containing one or more substances that inhibit the growth or survival of the unfused, immortalized cells. For example, if the parent cells lack the enzyme hypoxanthine guanine phosphoribosyltransferase (HGPRT or HPRT), the culture medium for the hybridoma contains hypoxanthine, aminopterin, and thymidine ("HAT medium"), which substances prevent growth of the HGPRT-deficient cells.

[0139] Useful immortalized cell lines are those that fuse efficiently, maintain stable high-level expression of antibody by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium. Immortalized cell lines are, for example, mouse myeloma lines available from the Salk Institute Cell Distribution Center (San Diego, California) and the American Type Culture Collection (Manassas, Virginia). Human myeloma and mouse-human heteromyeloma cell lines have also been described for the production of human monoclonal antibodies. (See Kozbor, J. Immunol., 133:3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, Marcel Dekker, Inc., New York, (1987) pp. 51-63.)

[0140] The culture medium in which the hybridoma cells are cultured can then be assayed for the presence of monoclonal antibodies against the antigen. For example, the binding specificity of monoclonal antibodies produced by hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA). Such techniques and assays are known in the art. The binding affinity of a monoclonal antibody can be determined, for example, by the Scatchard analysis of Munson and Pollard, Anal. Biochem., 107:220 (1980). Furthermore, in therapeutic applications of monoclonal antibodies, it is important to identify antibodies that have a high degree of specificity and high binding affinity for the target antigen.

[0141] After hybridoma cells are identified, the clones can be subcloned by limiting dilution procedures and grown by standard methods. (See Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, (1986) pp. 59-103.) Suitable culture media for this purpose include, for example, Dulbecco's modified Eagle's medium and RPMI-1640 medium. Alternatively, the hybridoma cells can be grown in vivo as ascites in a mammal.

[0142] The monoclonal antibodies secreted by the subclones can be isolated or purified from the culture medium or ascites fluid by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

[0143] Monoclonal antibodies can also be made by recombinant DNA methods, such as those described in U.S. Pat. No. 4,816,567, which is incorporated herein by reference in its entirety. DNA encoding the monoclonal antibodies of the invention can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes capable of binding specifically to genes encoding the heavy and light chains of murine antibodies). The hybridoma cells of the invention serve as a source of such DNA. Once isolated, the DNA can be placed into an expression vector, which is then transfected into host cells such as monkey COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of the monoclonal antibody in the recombinant host cells. The DNA can also be modified, for example, by substituting the coding sequence for human heavy and light chain constant domains for the homologous murine sequences (see U.S. Pat. No. 4,816,567; Morrison, Nature 368, 812-13 (1994)), or by covalently linking all or part of the coding sequence for a non-immunoglobulin polypeptide to the immunoglobulin coding sequence. Such a non-immunoglobulin polypeptide can be substituted for the constant domains of an antibody of the invention and / or for the variable domains of one antigen-binding site of an antibody of the invention to create a chimeric bivalent antibody.

[0144] A fully human antibody is an antibody molecule in which the entire sequence of both the light and heavy chains, including, for example, the CDRs, arises from human genes. Such antibodies are referred to herein as "humanized antibodies" or "fully human antibodies." Human monoclonal antibodies, such as fully human antibodies and humanized antibodies, can be prepared by using trioma technology, human B-cell hybridoma technology (see Kozbor, et al., 1983 Immunol Today 4:72), and EBV hybridoma technology to produce human monoclonal antibodies (see Cole, et al., 1985 In:MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp.77-96). Human monoclonal antibodies are available and can be produced by using human hybridomas (see Cote, et al, 1983. Proc Natl Acad Sci USA 80:2026-2030) or by transforming human B cells with Epstein-Barr virus in vitro (see Cole, et al., 1985 In: MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp. 77-96).

[0145] A "humanized antibody" may be an antibody derived from a non-human species (such as a mouse) whose amino acid sequence (e.g., in the CDR regions) has been modified to increase similarity to antibody variants produced in humans. Antibodies can be humanized by methods known in the art, such as CDR grafting. See also Safdari et al., (2013) Biotechnol Genet Eng Rev.; 29:175-86. Furthermore, humanized antibodies can be produced in transgenic plants as an inexpensive alternative to existing mammalian systems. For example, transgenic plants can be tobacco plants, i.e., Nicotiania benthamiana and Nicotiana tabaccum. Antibodies are purified from plant leaves. Stable transformation of plants can be achieved using Agrobacterium tumefaciens or particle bombardment. For example, a nucleic acid expression vector containing at least the heavy and light chain sequences can be expressed via transformation in a bacterial culture, i.e., A. tumefaciens strain BLA4404. Infiltration of plants can be achieved by injection. Soluble leaf extracts can be prepared by crushing leaf tissue in a mortar and centrifugation. Isolation and purification of antibodies can be carried out by many methods known to those skilled in the art. Other methods for antibody production in plants are described, for example, in Fischer et al., Vaccine, 2003, 21:820-5, and Ko et al., Current Topics in Microbiology and Immunology, Vol. 332, 2009, pp. 55-78. Thus, the present invention further provides any cell or plant containing a vector encoding or producing an antibody of the present invention.

[0146] Antibodies can be modified by, for example, CDR grafting (EP 239,400; WO 91 / 09967; U.S. Pat. Nos. 5,225,539; 5,530,101; and 5,585,089), veneering, or resurfacing (EP 592,106; EP 519,596; Padlan, Molecular Immunology 28(4 / 5):489-498 (1991); Studnicka et al., Protein Engineering 7(6):805-814 (1994); Roguska et al., Proc. Natl. Sci. USA 91:969-973 (1994)) and chain shuffling (U.S. Pat. No. 5,565,332, the entirety of which is incorporated by reference). "Humanization" (also called reshaping or CDR grafting) is a well-established technique understood by those skilled in the art to reduce the immunogenicity of monoclonal antibodies (mAbs) derived from heterologous sources (e.g., rodents) and improve activation of the human immune system (see, e.g., Hou S, Li B, Wang L, Qian W, Zhang D, Hong X, Wang H, Guo Y (July 2008). "Humanization of an anti-CD34 monoclonal antibody by complementarity-determining region grafting based on computer-assisted molecular modeling," J. Biochem. 144(1):115-20).

[0147] In addition, antibodies (such as human antibodies) can also be produced using other techniques, including phage display libraries. (See Hoogenboom and Winter, J. Mol. Biol, 227:381 (1991); Marks et al., J. Mol. Biol, 222:581 (1991)). Similarly, human antibodies can be made by introducing human immunoglobulin loci into transgenic animals, e.g., mice in which the endogenous immunoglobulin genes have been partially or completely inactivated. Following challenge, human antibody production is observed, which closely resembles that seen in humans in all respects, including gene rearrangement, assembly, and antibody repertoire. This approach is described, for example, in U.S. Pat. Nos. 5,545,807, 5,545,806, 5,569,825, 5,625,126, 5,633,425, and 5,661,016, as well as Marks et al., Bio / Technology 10, 779-783 (1992), Lonberg et al., Nature 368, 856-859 (1994), Morrison, Nature 368, 812-13 (1994), Fishwild et al., Nature Biotechnology 14, 845-51 (1996), Neuberger, Nature Biotechnology 14, 826 (1996), and Lonberg and Huszar, Intern. Rev. Immunol. 13:65-93 (1995).

[0148] Human antibodies can also be produced using transgenic non-human animals that are modified to produce fully human antibodies in response to antigen challenge rather than the animal's endogenous antibodies (see WO 94 / 02602 and U.S. Pat. No. 6,673,986). Endogenous genes encoding heavy and light immunoglobulin chains in the non-human host are disabled, and active loci encoding human heavy and light immunoglobulin chains are inserted into the host's genome. Human genes are incorporated, for example, using yeast artificial chromosomes containing the necessary human DNA segments. Animals providing the desired modifications are then obtained as progeny by breeding intermediate transgenic animals containing less than the full complement of modifications. A non-realistic example of such a non-human animal is a mouse, referred to as the Xenomouse™, as disclosed in WO 96 / 33735 and WO 96 / 34096. This animal produces B cells that secrete fully human immunoglobulins. Antibodies can be obtained, for example, as polyclonal antibody preparations, directly from animals after immunization with an immunogen of interest, or alternatively, from immortalized B cells derived from animals, such as hybridomas, that produce monoclonal antibodies. Additionally, genes encoding immunoglobulins with human variable regions can be recovered and expressed to obtain antibodies directly or further modified to obtain antibody analogs, such as, for example, single-chain Fv (scFv) molecules.

[0149] Thus, using such a technique, it is possible to produce therapeutically useful IgG, IgA, IgM, and IgE antibodies. For an overview of this technology for producing human antibodies, see Lonberg and Huszar Int. Rev. Immunol. 73:65-93 (1995). For a detailed discussion of this technology for producing human antibodies and human monoclonal antibodies and protocols for producing such antibodies, see, e.g., WO 98 / 24893, WO 96 / 34096, WO 96 / 33735, U.S. Patent Nos. 5,413,923, 5,625,126, 5,633,425, 5,569,825, 5,661,016, 5,545,806, 5,814,318, and 5,939,598, which are incorporated by reference in their entireties. Additionally, companies such as Creative BioLabs (Shirley, NY) offer services to provide human antibodies directed against a selected antigen using technology similar to that described above.

[0150] An example of a method for producing a non-human host, exemplified as a mouse, lacking expression of endogenous immunoglobulin heavy chains is disclosed in U.S. Patent No. 5,939,598. This can be obtained by a method comprising deleting a J segment gene from at least one endogenous heavy chain locus in embryonic stem cells to prevent rearrangement of the locus and the formation of transcripts of the rearranged immunoglobulin heavy chain locus, the deletion being carried out by a targeting vector containing a gene encoding a selectable marker, and producing a transgenic mouse from the embryonic stem cells, the somatic and germ cells of which contain the gene encoding the selectable marker.

[0151] One method for producing a desired antibody, such as a human antibody, is disclosed in U.S. Patent No. 5,916,771. This method involves introducing an expression vector containing a nucleotide sequence encoding the heavy chain into one mammalian host cell in culture, introducing an expression vector containing a nucleotide sequence encoding the light chain into another mammalian host cell, and fusing the two cells to form a hybrid cell. The hybrid cell expresses an antibody containing the heavy and light chains.

[0152] In a further improvement of this procedure, methods for identifying clinically relevant epitopes on immunogens and correlative methods for selecting antibodies that immunospecifically bind with high affinity to the relevant epitopes are disclosed in WO 99 / 53049.

[0153] The antibody of interest can also be expressed by a vector containing a DNA segment encoding the single-chain antibody described herein. Vectors include, but are not limited to, chemical conjugates such as those described in WO 93 / 64701, which have a targeting moiety (e.g., a ligand for a cell surface receptor) and a nucleic acid-binding moiety (e.g., polylysine), viral vectors (e.g., DNA or RNA viral vectors), fusion proteins such as those described in PCT / US95 / 02140 (WO 95 / 22618), which are fusion proteins containing a targeting moiety (e.g., an antibody specific for a target cell) and a nucleic acid-binding moiety (e.g., protamine), plasmids, phages, viral vectors, and the like. Vectors can be chromosomal, non-chromosomal, or synthetic. Retroviral vectors can also be used, including Moloney murine leukemia virus. DNA viral vectors can also be used, including pox vectors such as orthopox or avipox vectors, herpes virus vectors such as herpes simplex virus (HSV) vectors (see Geller, AI et al, J. Neurochem, 64:487 (1995); Lim, F., et al, DNA Cloning: Mammalian Systems, D. Glover, Ed. (Oxford Univ. Press, Oxford England) (1995); Geller, AI et al, Proc Natl. Acad. Sci.: USA 90:7603 (1993); Geller, AI, et al, Proc Natl. Acad. Sci USA 87:1149 (1990)), and adenovirus vectors (LeGal LaSalle et al, Science, 259:988 (1993); Davidson, et al, Nat. Genet. 3:219 (1993); Yang, et al., J. Virol. 69:2004 (1995)) and adeno-associated virus vectors (Kaplitt, MGet al., Nat. Genet. 8:148 (1994)).

[0154] Poxvirus vectors transfer genes into the cytoplasm of cells. Avipoxvirus vectors result in only short-term expression of nucleic acids. Adenovirus vectors, adeno-associated virus vectors, and herpes simplex virus (HSV) vectors can be used to transfer nucleic acids into neural cells. Adenovirus vectors result in shorter-term expression (approximately 2 months) than adeno-associated virus (approximately 4 months), which in turn is shorter than HSV vectors. The vector selected will depend on the target cell and the condition being treated. Introduction can be by standard techniques (e.g., infection, transfection, transduction, or transformation). Examples of modes of gene transfer include naked DNA, CaP04 precipitation, DEAE-dextran, electroporation, protoplast fusion, lipofection, cell microinjection, and viral vectors.

[0155] Vectors can be used to target essentially any target cell. For example, stereotactic injection can be used to direct vectors (e.g., adenovirus, HSV) to a desired location. In addition, particles can be delivered by intracerebroventricular (icv) injection using a minipump infusion system such as the SynchroMed Infusion System. A method based on bulk flow, called convection, has also proven effective in delivering large molecules to extended areas of the brain and may be useful for delivering vectors to target cells. (See Bobo et al., Proc. Natl. Acad. Sci. USA 91:2076-2080 (1994); Morrison et al., Am. J. Physiol. 266:292-305 (1994)). Other methods that can be used include catheter, intravenous, parenteral, intraperitoneal, and subcutaneous injection, as well as oral or other known administration routes.

[0156] These vectors can be used to express large amounts of antibodies that can be used in a variety of ways, for example, to detect the presence of CLDN4 in a sample. The antibodies can also be used to attempt to bind and destroy CLDN4 activity.

[0157] In one embodiment, the antibodies described herein may be full-length antibodies, including those that contain an Fc region similar to a wild-type Fc region that binds to an Fc receptor.

[0158] Techniques can be adapted for the production of single chain antibodies specific to an antigenic protein of the invention (see, e.g., U.S. Pat. No. 4,946,778). In addition, methods can be used to generate monoclonal antibodies with a preferred specificity for a protein or a derivative, fragment, analog, or homolog thereof. ab To allow for rapid and effective identification of fragments, F ab Methods can be adapted for the construction of expression libraries (see, e.g., Huse, et al., 1989 Science 246:1275-1281). Antibody fragments containing the idiotype to a protein antigen can be produced by techniques known in the art, including, but not limited to: (i) F produced by pepsin digestion of antibody molecules. (ab’)2 Fragment, (ii)F (ab’)2 F generated by reducing the disulfide bridges of the fragment ab (iii) F fragments produced by treating antibody molecules with papain and a reducing agent. ab fragments, and (iv) F v piece.

[0159] Heteroconjugate antibodies are also within the scope of the present invention. Heteroconjugate antibodies are composed of two covalently linked antibodies. Such antibodies can, for example, target immune system cells to unwanted cells (see U.S. Pat. No. 4,676,980) or be used to treat HIV infection (see WO 91 / 00360 and WO 92 / 20373). Antibodies can be prepared in vitro using known methods in synthetic protein chemistry, including those involving crosslinking agents. For example, immunotoxins can be constructed using a disulfide exchange reaction or by forming a thioether bond. Examples of suitable reagents for this purpose include iminothiolate and methyl-4-mercaptobutyrimidate, as well as those disclosed, for example, in U.S. Pat. No. 4,676,980.

[0160] The antibodies of the present invention can be modified with respect to effector function, for example, to enhance the effectiveness of the antibody in treating cancer. For example, cysteine ​​residues can be introduced into the Fc region, thereby allowing interchain disulfide bond formation in this region. The homodimeric antibody thus generated may have improved internalization capability and / or increased complement-mediated cell killing and antibody-dependent cellular cytotoxicity (ADCC). (See Caron et al., J. Exp Med., 176:1191-1195 (1992) and Shopes, J. Immunol., 148:2918-2922 (1992)). Alternatively, antibodies can be engineered with dual Fc regions, thereby enabling enhanced complement lysis and ADCC capabilities. (See Stevenson et al., Anti-Cancer Drug Design, 3:219-230 (1989)). In one embodiment, an antibody of the present invention has a modified Fc region such that the Fc region does not bind to Fc receptors. For example, the Fc receptor is an Fcγ receptor. Antibodies with modified Fc regions such that the Fc region does not bind Fcγ but still binds to neonatal Fc receptors are useful as described herein.

[0161] In embodiments, antibodies of the invention may comprise Fc variants. See, for example, WO 2018 / 145075 and WO 2019 / 183362, which provide Fc variant compositions for enhancing antibody-mediated receptor signaling. In embodiments, Fc variants may comprise amino acid substitutions that alter the antigen-independent effector function of the antibody, such as the circulating half-life of the antibody. Such antibodies exhibit increased or decreased binding to FcRn and therefore have increased or decreased serum half-lives, respectively, when compared to antibodies lacking these substitutions. Fc variants with improved affinity for FcRn are expected to have longer serum half-lives, and such molecules have useful applications in methods of treating mammals where a longer half-life of the administered antibody is preferred, for example, to treat a chronic disease or disorder. In contrast, Fc variants with reduced FcRn-binding affinity have a shorter resting period, and such molecules may also be useful, for example, for administration to mammals where a shortened circulation time may be advantageous, such as in in vivo imaging, or in situations where the starting antibody has toxic side effects if present in the circulation for an extended period of time. Fc variants with reduced FcRn-binding affinity are also less likely to cross the placenta and are therefore useful in treating diseases or disorders in pregnant women. In addition, other applications in which reduced FcRn-binding affinity may be preferable include applications in which localization to the brain, kidney, and / or liver is preferred. In one embodiment, Fc variant-containing antibodies may exhibit reduced transport from the vasculature across the epithelium of renal glomeruli. In another embodiment, Fc variant-containing antibodies may exhibit reduced transport from the brain across the blood-brain barrier (BBB) ​​into the vascular space. In one embodiment, an antibody with altered FcRn binding comprises an Fc domain with one or more amino acid substitutions within the "FcRn-binding loop" of the Fc domain. The FcRn-binding loop is composed of amino acid residues 280 to 299 (EU numbering). Exemplary amino acid substitutions that alter FcRn-binding activity are disclosed in WO 05 / 047327, which is incorporated herein by reference.In certain exemplary embodiments, an antibody of the invention, or a fragment thereof, comprises an Fc domain with one or more of the following substitutions: V284E, H285E, N286D, K290E, and S304D (EU numbering).

[0162] In some embodiments, mutations are introduced into the constant region of the mAb to alter the antibody-dependent cell-mediated cytotoxicity (ADCC) activity of the mAb. For example, the mutation is a LALA mutation in the CH2 domain. In one embodiment, the antibody (e.g., a human mAb or a bispecific Ab) contains a mutation on one scFv unit of the heterodimeric mAb that reduces ADCC activity. In another embodiment, the mAb contains mutations on both chains of the heterodimeric mAb that completely eliminate ADCC activity. For example, the mutation introduced into one or both scFv units of the mAb is a LALA mutation in the CH2 domain. These mAbs with variable ADCC activity can be optimized so that the mAb exhibits maximal selective killing toward cells expressing one antigen recognized by the mAb, but minimal killing toward a second antigen recognized by the mAb.

[0163] In other embodiments, antibodies of the invention for use in the diagnostic and treatment methods described herein have a constant region, e.g., an IgG1 or IgG4 heavy chain constant region, which may be altered to reduce or eliminate glycosylation. For example, antibodies of the invention may also include Fc variants containing amino acid substitutions that alter the glycosylation of the antibody. For example, Fc variants may have reduced glycosylation (e.g., N-linked or O-linked glycosylation). In some embodiments, Fc variants have reduced glycosylation of the N-linked glycan normally found at amino acid position 297 (EU numbering). In another embodiment, the antibody has an amino acid substitution near or within a glycosylation motif, e.g., an N-linked glycosylation motif comprising the amino acid sequence NXT or NXS. In embodiments, the antibody comprises an Fc variant with an amino acid substitution at amino acid position 228 or 299 (EU numbering). In another embodiment, the antibody comprises an IgG1 or IgG4 constant region comprising S228P and T299A mutations (EU numbering).

[0164] Exemplary amino acid substitutions that reduce or alter glycosylation are disclosed in WO 05 / 018572, incorporated herein by reference. In some embodiments, antibodies of the invention or fragments thereof are modified to eliminate glycosylation. Such antibodies or fragments thereof may be referred to as "agly" antibodies or fragments thereof (e.g., "agly" antibodies). Without wishing to be bound by theory, "agly" antibodies or fragments thereof may have an improved safety and stability profile in vivo. An exemplary agly antibody or fragment thereof comprises a deglycosylated Fc region of an IgG4 antibody that lacks Fc effector function, thereby eliminating the potential for Fc-mediated toxicity to normal living tissues and cells that express CLDN4. In yet other embodiments, antibodies of the invention or fragments thereof comprise an altered glycan. For example, the antibody may have a reduced number of fucose residues on the N-glycan at Asn297 of the Fc region, i.e., be defucosylated. In another embodiment, the antibody can have an altered number of sialic acid residues on the N-glycan at Asn297 of the Fc region.

[0165] The present invention is also directed to immunoconjugates comprising antibodies conjugated to cytotoxic agents such as toxins (e.g., enzymatically active toxins of bacterial, fungal, plant, or animal origin, or fragments thereof), or to radioisotopes (the latter being radioconjugates).

[0166] Enzymatically active toxins and fragments thereof that can be used include diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii protein, dianthin protein, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogenin, restrictocin, phenomycin, enomycin, and the trichothecenes. A variety of radionuclides are available for the production of radioconjugated antibodies. Non-limiting examples include: 212 B.I., 131 I, 131 In, 90 Y, and 186 Re is an example.

[0167] Conjugates of antibodies and cytotoxic agents have been prepared using a variety of bifunctional protein-linking agents, such as N-succinimidyl-3-(2-pyridyldithiol)propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (e.g., dimethyl adipimidate HCl), active esters (e.g., disuccinimidyl suberate), aldehydes (e.g., glutaraldehyde), bis-azido compounds (e.g., bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (e.g., bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (e.g., triene 2,6-diisocyanate), and bis-active fluorine compounds (e.g., 1,5-difluoro-2,4-dinitrobenzene). For example, ricin immunotoxins can be prepared as described in Vitetta et al., Science 238:1098 (1987). Carbon-14 labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugating radionucleotides to antibodies (see WO 94 / 11026 and U.S. Pat. No. 5,736,137).

[0168] Those skilled in the art will recognize that a wide variety of possible moieties can be attached to a given antibody or other molecule of the invention (see, e.g., "Conjugate Vaccines," Contributions to Microbiology and Immunology, JM Cruse and RE Lewis, Jr (eds), Carger Press, New York, (1989), incorporated herein by reference in its entirety).

[0169] Conjugation can be achieved by any chemical reaction that will link two molecules, so long as the antibody and other moiety retain their respective activities. This conjugation can involve many chemical mechanisms, such as covalent bonding, affinity binding, intercalation, coordinate bonding, and complex formation. In one embodiment, the conjugation is a covalent bond. Covalent bonding can be achieved by direct condensation of existing side chains or by incorporation of an external crosslinking molecule. Many bivalent or polyvalent linking agents are useful for linking protein molecules, such as the antibodies of the present invention, to other molecules. For example, representative linking agents can include organic compounds such as thioesters, carbodiimides, succinimide esters, diisocyanates, glutaraldehyde, diazobenzene, and hexamethylenediamine. This list is not intended to be exhaustive of the various classes of linking agents known in the art, but rather is illustrative of more common linking agents. (See Killen and Lindstrom, Jour. Immun. 133:1335-2549 (1984); Jansen et al., Immunological Reviews 62:185-216 (1982); and Vitetta et al., Science 238:1098 (1987)). Non-limiting examples of linkers are described in the literature. (See, for example, Ramakrishnan, S. et al., Cancer Res. 44:201-208 (1984), which describes the use of MBS (M-maleimidobenzoyl-N-hydroxysuccinimide ester).) See also U.S. Pat. No. 5,030,719, which describes the use of halogenated acetylhydrazide derivatives attached to antibodies via oligopeptide linkers.Non-limiting examples of useful linkers that can be used with the antibodies of the invention include: (i) EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride); (ii) SMPT (4-succinimidyloxycarbonyl-α-methyl-α-(2-pyridyldithio)-toluene) (Pierce Chem. Co., catalog (21558G); (iii) SPDP (succinimidyl-6[3-(2-pyridyldithio)propionamido]hexanoate) (Pierce Chem. Co., catalog number 21651G); (iv) sulfo-LC-SPDP (sulfosuccinimidyl-6[3-(2-pyridyldithio)-propianamido]hexanoate (Pierce Chem. Co. catalog number 2165-G); and (v) sulfo-NHS (-hydroxysulfo-succinimide) conjugated to EDC (Pierce Chem. Co., catalog number 2165-G). Chem. Co., Cat. No. 24510).

[0170] The linkers described herein contain components with different attributes, thereby resulting in conjugates with different physicochemical properties. For example, sulfo-NHS esters of alkyl carboxylates are more stable than sulfo-NHS esters of aromatic carboxylates. NHS-ester-containing linkers are less soluble than sulfo-NHS esters. Furthermore, the linker SMPT contains a sterically hindered disulfide bond, allowing for the formation of conjugates with improved stability. Disulfide bonds are generally less stable than other bonds because they are cleaved in vitro, resulting in fewer available conjugates. Sulfo-NHS can enhance the stability of carbodiimide bonds. Carbodiimide bonds (such as EDC) when used in combination with sulfo-NHS form esters that are more resistant to hydrolysis than the carbodiimide bond reaction alone.

[0171] The antibodies disclosed herein can also be formulated as immunoliposomes. Liposomes containing antibodies can be prepared by methods known in the art, such as those described in Epstein et al., Proc. Natl. Acad. Sci. USA, 82:3688 (1985); Hwang et al., Proc. Natl. Acad. Sci. USA, 77:4030 (1980); and U.S. Patent Nos. 4,485,045 and 4,544,545. Liposomes with enhanced circulation time are disclosed in U.S. Patent No. 5,013,556.

[0172] Non-limiting examples of useful liposomes can be generated by the reverse-phase evaporation method with a lipid composition comprising phosphatidylcholine, cholesterol, and PEG-derivatized phosphatidylethanolamine (PEG-PE). Liposomes are extruded through filters of defined pore size to yield liposomes with the desired diameter. Fab' fragments of the antibody of the present invention can be conjugated to liposomes via a disulfide exchange reaction as described in Martin et al., J. Biol. Chem., 257:286-288 (1982).

[0173] Multispecific antibodies (bispecific and trispecific)

[0174] The embodiments described herein may include monospecific or multispecific antibodies.

[0175] Monospecific antibodies are antibodies that have one or more binding sites that specifically bind to a single antigen.

[0176] A multispecific antibody is an antibody that can recognize two or more different antigens. For example, a bispecific antibody (bsAb) is an antibody that contains two variable domains or scFv units such that the resulting antibody recognizes two different antigens. For example, a trispecific antibody (tsAb) is an antibody that contains two variable domains or scFv units such that the resulting antibody recognizes three different antigens. The present invention provides multispecific antibodies, such as bispecific and trispecific antibodies, that recognize CLDN4 and a second and / or third antigen. In one embodiment, the multispecific antibody (e.g., bispecific and trispecific antibodies) can comprise a CLDN4-specific fusion protein comprising an antibody described herein. Exemplary second and / or third antigens include tumor-associated antigens (e.g., LINGO1), cytokines (e.g., IL-12 (IL-12A (p35 subunit) protein sequence having NCBI Reference No. NP_000873.2); IL-12B (p40 subunit) protein sequence having NCBI Reference No. NP_002178.2); IL-18 (protein sequence having NCBI Reference No. NP_001553.1); IL-15 (protein sequence having NCBI Reference No. NP_000576.1); IL-7 (protein sequence having NCBI Reference No. NP_000871.1); IL-2 (protein sequence having NCBI Reference No. NP_000577.2) and IL-21 (protein sequence having NCBI reference number NP_068575.1), cytokine cognate receptors (e.g., IL-12R), and cell surface receptors. Non-limiting examples of second and / or third antigens include CTLA-4, CXCR4, LAG-3, CD28, CD122, 4-1BB, TIM3, OX-40, OX40L, CD40, CD40L, LIGHT, ICOS, ICOSL, GITR, GITRL, TIGIT, CD27, VISTA, B7H3, B7H4, HEVM (or BTLA), CD47, and CD73. In one embodiment, the bispecific and trispecific antibodies comprise a CLDN4 fusion protein.For example, the fusion protein can include an antibody comprising a variable domain or scFv unit described herein and a ligand or antigen and / or a third ligand or antigen, such that the resulting antibody recognizes the antigen and binds to a ligand-specific receptor. Exemplary antibody compositions (e.g., VH and / or VL sequences or fragments thereof) useful in designing the CLDN4 fusion proteins described herein include the anti-CAIX antibodies described in PCT / US2006 / 046350 and PCT / US2015 / 067178; the anti-CXCR4 antibodies described in PCT / US20006 / 005691 and PCT / US2019 / 022272; the anti-CCR4 antibodies described in PCT / US2008 / 088435, PCT / US2013 / 039744, and PCT / US2015 / 054202; Examples of suitable antibodies include, but are not limited to, the anti-PD-L1 antibodies described in PCT Patent Applications PCT / US2008 / 088435 and PCT / US2020 / 062815; the anti-PD-1 antibodies described in PCT Patent Applications PCT / US2020 / 037791 and PCT / US2020 / 037781; the anti-GITR antibodies described in PCT / US2017 / 043504; the anti-claudin-4 antibodies described in PCT / US2019 / 022272; and the anti-MUC1 antibodies described in PCT / US2020 / 037783 (each of which is incorporated by reference in its entirety). In one embodiment, the fusion protein further comprises a constant region and / or a linker as described herein. Different formats of multispecific antibodies (e.g., bispecific and trispecific antibodies, such as fusion proteins comprising an antibody and a ligand that recognize CLDN4) are described herein.Ligands can be tumor-associated antigens (e.g., LINGO1, ErbB2 (HER2 / neu), carcinoembryonic antigen (CEA), epithelial cell adhesion molecule (EpCAM), epidermal growth factor receptor (EGFR), MUC1, MSLN, CD19, CD20, CD30, CD40, CD22, RAGE-1, MN-CA IX, RET1, RET2 (AS), prostate-specific antigen (PSA), TAG-72, PAP, p53, Ras, prostein, PSMA, survivin, 9D7, prostate cancer tumor antigen-1 (PCTA-1), GAGE, MAGE, mesothelin, β-catenin, TGF-βRII, BRCA1 / 2, SAP-1, HPV-E6, HPV-E7 (see PCT / US2015 / 067225, incorporated by reference in its entirety, for additional tumor-associated surface antigens). and PCT / US2019 / 022272); cytokines (e.g., IL-12 (IL-12A (p35 subunit) protein sequence having NCBI Reference No. NP_000873.2; IL-12B (p40 subunit) protein sequence having NCBI Reference No. NP_002178.2; IL-18 (protein sequence having NCBI Reference No. NP_001553.1); IL-15 (protein sequence having NCBI Reference No. NP_0 00576.1); IL-7 (protein sequence with NCBI reference number NP_000871.1); IL-2 (protein sequence with NCBI reference number NP_000577.2); and IL-21 (protein sequence with NCBI reference number NP_068575.1)); CTLA-4, LAG-3, CD28, CD122, 4-1BB, TIM3, OX-40, OX40L, CD40, CD40L, LIGHT, ICOS, IC The antigen-specific fragment may be OSL, GITR, GITRL, TIGIT, CD27, VISTA, B7H3, B7H4, HEVM (or BTLA), CD47, or CD73. Bispecific or trispecific antibodies in various formats are also provided herein. In some embodiments, the anti-CLDN4 fragment and the second antigen-specific fragment and / or the third antigen-specific fragment are each independently selected from a Fab fragment, a single-chain variable fragment (scFv), or a single-domain antibody.In some embodiments, the bispecific or trispecific antibody further comprises an Fc fragment (e.g., as described in PCT / US2015 / 021529 and PCT / US2019 / 023382, each of which is incorporated by reference in its entirety). The bispecific or trispecific antibody of the invention can comprise a heavy and light chain combination or scFv of a CLDN4 antibody described herein.

[0177] Multispecific antibodies (e.g., bispecific and trispecific antibodies) of the present invention (e.g., anti-CLDN4-scFv fusion proteins) can be constructed using methods known in the art. In some embodiments, a bispecific antibody is a single polypeptide in which two scFv fragments are joined by a long linker polypeptide of sufficient length to allow intramolecular interactions between the two scFv units to form the antibody. In other embodiments, a bispecific antibody is two or more polypeptides linked by covalent or non-covalent bonds. In some embodiments, the amino acid linker shown herein (GGGGSGGGGS; "(G4S)2") can be generated using a longer G4S linker to improve flexibility. For example, the linker can also be "(G4S)3" (e.g., GGGSGGGGSGGGGS); "(G4S)4" (e.g., GGGGSGGGGSGGGGSGGGGS); "(G4S)5" (e.g., GGGGSGGGGSGGGGSGGGGSGGGGS); "(G4S)6" (e.g., GGGGSGGGGSGGGGSGGGGSGGGGSGGGGS); For example, the use of a (G4S)5 linker can provide greater flexibility to the ligands described herein and can improve expression. In some embodiments, the linker can also be a (GS) n , (GGS) n , (GGGS) n , (GGSG)n , (GGSGG) n , or (GGGGS) n where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. Non-limiting examples of linkers known to those of skill in the art that can be used to construct the fusions described herein can be found in U.S. Pat. No. 9,708,412, U.S. Patent Application Publication Nos. 20180134789 and 20200148771, and WO 2019051122 (each of which is incorporated by reference in its entirety).

[0178] In another embodiment, multispecific antibodies (e.g., bispecific and trispecific antibodies, such as anti-CLDN4-scFv fusions) can be constructed using the "knob-into-hole" method (Ridgway et al., Protein Eng 7:617-621 (1996)). In this method, Ig heavy chains of two different variable domains are reduced to selectively cleave heavy chain pairing while preserving the heavy-light chain pairing. Two heavy-light chain heterodimers that recognize two or three different antigens / ligands are mixed to promote heterologous ligand binding pairing mediated through the engineered "knob-in-hole" of the CH3 domain.

[0179] In another embodiment, multispecific antibodies (e.g., bispecific and trispecific antibodies such as anti-CLDN4-scFv fusions) can be constructed by exchanging heavy-light chain dimers from two or more different antibodies to generate hybrid antibodies, where a first heavy-light chain dimer recognizes CLDN4 and a second heavy-light chain dimer recognizes a second and / or third antigen. The mechanism of heavy-light chain dimerization is similar to the formation of human IgG4, which also functions as a bispecific molecule. Dimerization of IgG heavy chains is promoted by intramolecular forces such as the pairing of the CH3 domains of each heavy chain with disulfide bridges. The presence of a specific amino acid (R409) in the CH3 domain has been shown to promote dimer exchange and the assembly of IgG4 molecules. Heavy chain pairing is also further stabilized by inter-heavy chain disulfide bridges in the hinge region of the antibody. Specifically, in IgG4, the hinge region contains the amino acid sequence Cys-Pro-Ser-Cys (compared to the stable IgG1 hinge region, which contains the sequence Cys-Pro-Pro-Cys) at amino acids 226 to 230. This difference in the sequence of serine at position 229 has been linked to the propensity of IgG4 to form intrachain disulfides in the hinge region (Van der Neut Kolfschoten, M. et al., 2007, Science 317:1554-1557 and Labrijn, A. F. et al., 2011, Journal of Immunol 187:3238-3246).

[0180] Multispecific antibodies of the present invention (e.g., bispecific and trispecific antibodies such as anti-CLDN4-scFv fusions) can be generated by introducing the R409 residue in the CH3 domain and a Cys-Pro-Ser-Cys sequence in the hinge region of an antibody that recognizes CLDN4 or a second and / or third antigen, such that the heavy-light chain dimers are swapped to produce an antibody molecule having one heavy-light chain dimer that recognizes CLDN4 and a second heavy-light chain dimer that recognizes a second and / or third antigen, where the second and / or third antigen (or ligand) is any antigen (or ligand) disclosed herein. Known IgG4 molecules can also be modified so that the heavy and light chains recognize CLDN4 or a second and / or third antigen, as disclosed herein. The use of this method to construct multispecific antibodies of the present invention (e.g., bispecific and trispecific antibodies such as anti-CLDN4-scFv fusions) can be advantageous due to the unique characteristics of the IgG4 molecule, whose Fc region differs from other IgG subtypes in that it interacts poorly with effector systems of the immune response, such as complement and Fc receptors expressed by certain leukocytes. This particular property makes these IgG4-based multispecific antibodies (e.g., bispecific and trispecific antibodies such as anti-CLDN4-scFv fusions) attractive for therapeutic applications, where the antibody is required to bind to a target and functionally modify a signaling pathway associated with the target, but does not induce effector activity.

[0181] The multispecific antibodies described herein (e.g., bispecific and trispecific antibodies such as anti-CLDN4-scFv fusions) can be engineered with a non-depleting heavy chain isotype, such as IgG1-LALA or stabilized IgG4 or one of the other non-depleting variants. In some embodiments, mutations are introduced into the constant region of the bsAb such that the antibody-dependent cell-mediated cytotoxicity (ADCC) activity of the bsAb is altered. For example, the mutation is a LALA mutation in the CH2 domain. In one aspect, multispecific antibodies (e.g., bispecific and trispecific antibodies such as anti-CLDN4-scFv fusions) contain a mutation on one scFv unit of the heterodimeric multispecific antibody that reduces ADCC activity. In another aspect, multispecific antibodies (e.g., bispecific and trispecific antibodies such as anti-CLDN4-scFv fusions) contain mutations on both chains of the heterodimeric multispecific antibody that completely eliminate ADCC activity. For example, a mutation introduced into one or both scFv units of a multispecific antibody (e.g., bispecific antibodies such as anti-CLDN4-scFv fusions and trispecific antibodies) is a LALA mutation in the CH2 domain. These multispecific antibodies (e.g., bispecific antibodies such as anti-CLDN4-scFv fusions and trispecific antibodies) with variable ADCC activity can be optimized so that the multispecific antibody exhibits maximum selective killing toward cells expressing one antigen recognized by the multispecific antibody, but minimal killing toward the second antigen recognized by the multispecific antibody.

[0182] The multispecific antibodies (e.g., bispecific antibodies) described herein can be engineered as modular tetrameric bispecific antibodies (tBsAbs). See, e.g., WO 2018 / 071913, incorporated herein by reference in its entirety. For example, a tetravalent antibody can be a dimer of bispecific scFv fragments comprising a first binding site for a first antigen and a second binding site for a second antigen. In embodiments, the anti-CLDN4 antibody can be the first binding site for the first antigen. In embodiments, the anti-CLDN4 antibody can be the second binding site for the second antigen. The two binding sites can be linked together via a linker domain. In embodiments, the scFv fragment is a tandem scFv, and the linker domain comprises an immunoglobulin hinge region (e.g., an IgG1, IgG2, IgG3, or IgG4 hinge region) amino acid sequence. In embodiments, the immunoglobulin hinge region amino acid sequence can be, for example, the linker amino acid sequence (GGGS). x1-6 , (GGGGS) x1-6 , or GSAGSAAGSGEF. In embodiments, the linker domain comprises at least a portion of an immunoglobulin Fc domain, e.g., an IgG1, IgG2, IgG3, or IgG4 Fc domain. In embodiments, at least a portion of the immunoglobulin Fc domain does not comprise a CH2 domain. In embodiments, at least a portion of the immunoglobulin Fc domain can be a CH2 domain. An exemplary CH2 domain amino acid sequence comprises APELLGGPDVFLF (SEQ ID NO: 95). The Fc domain can be linked to the C-terminus of an immunoglobulin hinge region (e.g., an IgG1, IgG2, IgG3, or IgG4 hinge region) amino acid sequence. The linker domain can be flanked at one or both ends by a flexible linker amino acid sequence (e.g., (GGGS) x1-6 , (GGGGS) x1-6 , or GSAGSAAGSGEF).

[0183] In embodiments, the tBsAb is specific for CLDN4 and may also be specific for a target selected from the group consisting of B7H3, B7H4, CD27, CD28, CD40, CD40L, CD47, CD122, CCR4, CXCR4, CTLA-4, GITR, GITRL, ICOS, ICOSL, LAG-3, LIGHT, OX-40, OX40L, PD-L1, PD-1, TIM3, 4-1BB, TIGIT, VISTA, HEVM, BTLA, and KIR.

[0184] In embodiments, the multispecific antibody may be a bispecific T cell engager (BiTE). The term "BiTE" (bispecific T cell engager) refers to a single polypeptide chain molecule having two antigen-binding domains, one of which binds to a T cell antigen. For example, a BiTE may comprise a CLDN4 antibody or functional fragment thereof disclosed herein and an antibody or fragment thereof that binds to a T cell antigen. For example, the antibody or fragment thereof that binds to the T cell antigen may be specific for CD3.

[0185] In embodiments, the multispecific antibody may be a trispecific T cell engager (TriTE). The term "TriTE" (trispecific T cell engager) may refer to a single polypeptide chain molecule having three antigen-binding domains, one or more of which bind to a T cell antigen. For example, a TriTE may comprise a CLDN4 antibody or functional fragment thereof disclosed herein and an antibody or fragment thereof that binds to a T cell antigen. For example, the antibody or fragment thereof that binds to a T cell antigen may be specific for CD3, CD28, or both.

[0186] The multispecific antibodies disclosed herein (e.g., bispecific and trispecific antibodies such as anti-CLDN4-scFv fusions) may be useful in the treatment of chronic infections, diseases, or medical conditions, such as cancer.

[0187] fusion proteins

[0188] The present invention provides fusion proteins containing the CLDN4 antibody disclosed herein or a functional fragment thereof operably linked to a second protein. The second protein can be, for example, a cytokine or growth factor. In embodiments, the cytokine is IL-2 or TGF-beta and variants thereof. In some other embodiments, the second protein can be a therapeutic agent such as a toxin, a detectable moiety such as a fluorescent protein for detection, or a biological agent such as an agent that stimulates T cells (i.e., CD3). In some embodiments, the CLDN4 antibody of the present invention can be operably linked to multiple additional proteins or peptides, for example, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additional protein or peptide sequences.

[0189] In some embodiments, the CLDN4 antibody or functional fragment thereof disclosed herein is directly linked to the second protein. In other embodiments, the CLDN4 antibody or functional fragment thereof is linked to the second protein via a linker, such as a flexible polypeptide chain. The linker can be any suitable linker of any length, but can be at least 1, 2, 3, 4, 5, 10, 15, 20, 25, or 30 amino acids in length. In one embodiment, the linker is an amino acid sequence that naturally occurs in a host immunoglobulin molecule, and the presence of the linker cannot result in an immune response by the mammal against the linker sequence. Fusion proteins of the present invention comprising multiple additional proteins directed against a CLDN4 antibody can have multiple linker sequences linking each additional protein or peptide sequence.

[0190] The fusion proteins of the present invention can be constructed by recombinant methods known to those skilled in the art. For example, an expression vector containing a nucleic acid sequence encoding a CLDN4 antibody of the present invention can be operably linked to a nucleic acid sequence encoding a second protein and introduced into an expression system for translation and production of the fusion protein. Alternatively, those skilled in the art can readily utilize novel protein synthesis techniques to produce the fusion proteins described herein.

[0191] Use of antibodies against CLDN4

[0192] Antibodies of the present invention that specifically bind to the CLDN4 protein or a fragment thereof can be administered to treat CLDN4-associated diseases or disorders. "CLDN4-associated diseases or disorders" include disease states and / or symptoms associated with disease states in which elevated levels of CLDN4 and / or activation of cell signaling pathways involving CLDN4 are observed. Exemplary CLDN4-associated diseases or disorders include, but are not limited to, T cell-suppressed diseases, such as cancer and infectious diseases. In some embodiments, the cancer may be lung cancer, kidney cancer, ovarian cancer, prostate cancer, colon cancer, breast cancer, cervical cancer, uterine cancer, brain cancer, skin cancer, liver cancer, pancreatic cancer, or gastric cancer. In embodiments, the cancer may be a CLDN4-associated cancer. In embodiments, the CLDN4-associated cancer may be triple-negative breast cancer.

[0193] The antibodies of the present invention, including bispecific, polyclonal, monoclonal, humanized, and fully human antibodies, can be used as therapeutic agents. Such agents can be used to treat cancer in a subject, improve vaccine efficacy, or enhance natural immune responses. An antibody preparation, e.g., one with high specificity and high affinity for its target antigen, is administered to a subject to exert an effect resulting from binding to the target. Administration of the antibody can neutralize, inhibit, or interfere with the activity of the CLDN4 protein.

[0194] Pharmaceutical Composition

[0195] The antibody of the present invention that specifically binds to the CLDN4 protein or a fragment thereof can be administered for cancer treatment in the form of a pharmaceutical composition. Principles and considerations for preparing therapeutic pharmaceutical compositions containing antibodies, as well as guidance in selecting components, are provided, for example, in Remington: The Science and Practice of Pharmacy, 20th ed. (Alfonso R. Gennaro, et al., editors), Mack Pub. Co., Easton, Pa., 2000; Drug Absorption Enhancement: Concepts, Possibilities, Limitations, and Trends, Harwood Academic Publishers, Langhorne, Pa., 1994; and Peptide and Protein Drug Delivery (Advances in Parenteral Sciences, Vol. 4), 1991, M. Dekker, New York.

[0196] The specific dosage and treatment regimen for a patient will depend on various factors, such as the specific antibody, variant, or derivative thereof used, the patient's age, weight, general health, sex, and diet, as well as the time of administration, frequency of excretion, concomitant drug use, and the severity of the disease being treated. Assessment of such factors by a medical professional is within the skill of one of ordinary skill in the art. The amount will also depend on the individual patient being treated, the route of administration, the type of formulation, the characteristics of the compound used, the severity of the disease, and the desired effect. The amount used can be determined by pharmaceutical and pharmacokinetic principles well known in the art.

[0197] A therapeutically effective amount of an antibody of the present invention can be the amount necessary to achieve a therapeutic goal. As noted above, this can be a binding interaction between the antibody and its target antigen, which in certain cases interferes with the function of the target. The amount that needs to be administered further depends on the binding affinity of the antibody for its specific antigen and also on the rate at which the administered antibody is depleted from the free volume of the other subject to which it is administered. The dosage of an antigen-binding polypeptide described herein administered to a subject (e.g., a patient) is approximately 0.1 mg / kg to 100 mg / kg of patient body weight, 0.1 mg / kg to 20 mg / kg of patient body weight, or 1 mg / kg to 10 mg / kg of patient body weight. Human antibodies have a longer half-life in the human body than antibodies from other species due to the immune response to foreign polypeptides. Therefore, lower dosages and less frequent administration of human antibodies are often possible. Furthermore, the dosage and frequency of administration of the antibodies of the present disclosure can be reduced by enhancing antibody uptake and tissue (e.g., brain) penetration through modifications such as lipidation. A typical range for therapeutically effective administration of an antibody or antibody fragment of the invention can be, by way of non-limiting example, about 0.1 mg / kg body weight to about 50 mg / kg body weight, and a typical administration frequency can range, for example, from twice daily to once weekly.

[0198] When antibody fragments are used, the smallest inhibitory fragment that specifically binds to the binding domain of the target protein is preferred. For example, peptide molecules that retain the ability to bind to the target protein sequence can be designed based on the variable region sequence of the antibody. Such peptides can be chemically synthesized and / or produced by recombinant DNA technology. (See, e.g., Marasco et al., Proc. Natl. Acad. Sci. USA, 90:7889-7893 (1993)). The formulation may also contain two or more active compounds necessary for the indication being treated, e.g., those with complementary activities that do not adversely affect each other. Alternatively, or in addition, the composition may contain an agent that enhances its function (e.g., a cytotoxic agent, a cytokine (e.g., IL-15), a chemotherapeutic agent, or a growth-inhibitory agent, etc.). Such molecules are preferably present in combination in amounts effective for the intended purpose.

[0199] The active ingredient can also be encapsulated in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, e.g., hydroxymethylcellulose or gelatin-microcapsules and poly-(methyl methacrylate) microcapsules, in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules), or in macroemulsions, respectively.

[0200] Formulations to be used for in vivo administration must be sterile, which is readily accomplished by filtration through sterile filtration membranes.

[0201] Sustained-release preparations can be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g., films, or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (e.g., poly(2-hydroxyethyl-methacrylate) or poly(vinyl alcohol)), polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and γ-ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers, such as LUPRON DEPOT™ (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D-(-)-3-hydroxybutyric acid. While polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid enable release of molecules for over 100 days, certain hydrogels release proteins for shorter periods of time.

[0202] The antibodies or agents of the present invention (also referred to herein as "active compounds"), as well as their derivatives, fragments, analogs, and homologs, can be incorporated into pharmaceutical compositions suitable for administration. Such compositions typically comprise the antibody or agent and a pharmaceutically acceptable carrier. As used herein, "pharmaceutically acceptable carrier" can include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Suitable carriers are described in the most recent edition of Remington's Pharmaceutical Sciences, a standard reference text in the field, incorporated herein by reference. Preferred examples of such carriers or diluents include, but are not limited to, water, saline, Ringer's solution, dextrose solution, and 5% human serum albumin. Non-aqueous vehicles, such as liposomes and fixed oils, can also be used. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, its use in the compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions.

[0203] The pharmaceutical composition of the present invention is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (i.e., topical), transmucosal, and rectal administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application may contain the following components: a sterile diluent such as water for injection, saline, fixed oils, polyethylene glycol, glycerin, propylene glycol, or other synthetic solvents; an antibacterial agent such as benzyl alcohol or methylparaben; an antioxidant such as ascorbic acid or sodium bisulfite; a chelating agent such as ethylenediaminetetraacetic acid (EDTA); a buffer such as acetate, citrate, or phosphate, and an agent for adjusting tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases such as hydrochloric acid or sodium hydroxide. Parenteral preparations can be enclosed in ampoules, disposable syringes, or multiple-dose vials made of glass or plastic.

[0204] Pharmaceutical compositions suitable for injectable use may include sterile aqueous solutions (where water soluble) or dispersions, and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, NJ), or phosphate-buffered saline (PBS). In embodiments, the composition is sterile and fluid to the extent that easy syringability exists. It may be stable under the conditions of manufacture and storage and may be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc. In many cases, isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride, can be included in the composition. Prolonged absorption of injectable compositions can be achieved by including in the composition an agent that delays absorption, for example, aluminum monostearate and gelatin.

[0205] Sterile injectable solution can be prepared by incorporating the required amount of active compound into a suitable solvent with one or a combination of the ingredients listed above as needed, and then sterilize by filtration.For example, dispersion is prepared by incorporating active compound into a sterile vehicle that contains basic dispersion medium and the other ingredients listed above as needed.For the preparation of sterile injectable solution, the method of preparation is vacuum drying and freeze-drying, which produces a powder of active compound and any additional ingredients from the solution that has been previously sterile filtered.

[0206] Oral compositions include inert diluents or edible carriers. They can be enclosed in gelatin capsules or compressed into tablets. For oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules. Oral compositions can also be prepared using fluid carriers for use as mouthwashes, in which the compound in the fluid carrier is orally applied, swirled in the mouth, and expectorated or swallowed. Pharmaceutically compatible binders and / or adjuvant materials can be included as part of the composition. The tablets, pills, capsules, troches and the like may contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.

[0207] For administration by inhalation, the compounds can be delivered in the form of an aerosol spray from pressured container or dispenser which contains a suitable propellant, eg, a gas such as carbon dioxide, or a nebulizer.

[0208] Systemic administration can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are known in the art and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be achieved through the use of nasal sprays or suppositories. For transdermal administration, the active compound is formulated into ointments, salves, gels, or creams known in the art.

[0209] The compounds can also be prepared in the form of suppositories (eg, with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.

[0210] In one embodiment, the active compound is prepared with a carrier that will protect the compound against rapid elimination from the body, such as a controlled-release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. Methods for preparing such formulations will be apparent to those skilled in the art. Materials are also commercially available from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies against viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Pat. No. 4,522,811.

[0211] For the sake of ease of administration and uniformity of dosage, oral or parenteral compositions can be formulated in dosage unit form.As used herein, dosage unit refers to a physically separate unit suitable as a unit dose for the subject to be treated, and each unit contains a predetermined amount of active compound calculated to produce a desired therapeutic effect in association with required pharmaceutical carrier.The specification of dosage unit form of the present invention is determined by and can directly depend on the specific characteristics of active compound and the therapeutic effect to be achieved, and the inherent limitations of the technology of compounding such active compound for individual treatment.

[0212] The pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration.

[0213] diagnosis

[0214] The antibody according to the present invention can be used as an agent for detecting the presence of CLDN4 (or a protein fragment thereof) in a sample. For example, the sample can be a cancer sample or a sample from a subject at risk of having cancer. For example, the cancer can be lung cancer, kidney cancer, ovarian cancer, prostate cancer, colon cancer, breast cancer, cervical cancer, uterine cancer, brain cancer, skin cancer, liver cancer, pancreatic cancer, or stomach cancer. For example, the antibody can comprise a detectable label. The antibody can be polyclonal or monoclonal. An intact antibody, or a fragment thereof (e.g., F ab , scFv, or F (ab)2 ) can be used. With respect to a probe or antibody, the term "labeled" can encompass direct labeling of the probe or antibody by conjugating (i.e., physically linking) a detectable substance to the probe or antibody, as well as indirect labeling of the probe or antibody by reactivity with another reagent that is directly labeled. Examples of indirect labeling include detection of a primary antibody using a fluorescently labeled secondary antibody and end-labeling a DNA probe with biotin so that it can be detected with fluorescently labeled streptavidin. The term "biological sample" can include tissues, cells, and biological fluids isolated from a subject, as well as tissues, cells, and biological fluids present within a subject. Thus, the use of the term "biological sample" includes blood and fractions or components of blood, including serum, plasma, or lymph. That is, the detection methods of the present invention can be used to detect analyte mRNA, protein, or genomic DNA in biological samples in vitro and in vivo. For example, in vitro techniques for detecting analyte mRNA include Northern hybridization and in situ hybridization. In vitro techniques for detection of an analyte protein include enzyme linked immunosorbent assays (ELISAs), Western blots, immunoprecipitations, and immunofluorescence. In vitro techniques for detection of analyte genomic DNA include Southern hybridizations.

[0215] Procedures for performing immunoassays are described, for example, in "ELISA: Theory and Practice: Methods in Molecular Biology", Vol. 42, J.R.Crowther (Ed.) Human Press, Totowa, NJ, 1995; "Immunoassay", E. Diamandis and T. Christopoulus, Academic Press, Inc., San Diego, CA, 1996; and "Practice and Theory of Enzyme Immunoassays", P. Tijssen, Elsevier Science Publishers, Amsterdam, 1985. Additionally, in vivo techniques for detecting an analyte protein include introducing into a subject a labeled anti-analyte protein antibody. For example, the antibody can be labeled with a radioactive marker whose presence and location in a subject can be detected by standard imaging techniques.

[0216] Antibodies to the CLDN4 protein (or fragments thereof) can be used in methods known in the art related to localizing and / or quantitating the CLDN4 protein (e.g., for use in measuring the level of CLDN4 protein in an appropriate physiological sample, for use in diagnostic methods, for use in protein imaging). In certain embodiments, antibodies specific for the CLDN4 protein, or derivatives, fragments, analogs, or homologs thereof, containing an antigen-binding domain derived from the antibody, are utilized as pharmaceutically active compounds (hereinafter referred to as "therapeutic agents").

[0217] Antibodies specific to the CLDN4 protein of the present invention can be used to isolate CLDN4 polypeptides by standard techniques such as immunoaffinity, chromatography, or immunoprecipitation. Antibodies to the CLDN4 protein (or fragments thereof) can be used diagnostically to monitor protein levels in tissues as part of a clinical testing procedure, for example, to determine the effectiveness of a given treatment regimen.

[0218] Detection can be facilitated by conjugating (i.e., physically linking) the antibody to a detectable substance. Examples of detectable substances include, but are not limited to, various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials. Non-limiting examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin / biotin and avidin / biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride, or phycoerythrin; examples of luminescent materials include luminol; examples of bioluminescent materials include luciferase, luciferin, and aequorin; and examples of suitable radioactive materials include 125 I, 131 I, 35 S, 32 P, or 3 H is one example.

[0219] Screening Methods

[0220] The present invention provides methods (also referred to herein as "screening assays") for identifying modulators, i.e., candidate or test compounds or agents (e.g., peptides, peptidomimetics, small molecules, or other drugs), that regulate or otherwise interfere with CLDN4 activity. Methods for identifying compounds useful for treating cancer are also provided. The present invention also encompasses compounds identified using the screening assays described herein.

[0221] For example, the present invention provides assays for screening candidate or test compounds that modulate the expression and / or activity of CLDN4. Test compounds of the present invention can be obtained using any of a number of approaches in combinatorial library methods known in the art, including, but not limited to, biological libraries; spatially addressable parallel solid-phase or liquid-phase libraries; synthetic library methods requiring deconvolution; "one bead, one compound" library methods; and synthetic library methods using affinity chromatography selection. While the biological library approach is limited to peptide libraries, the other four approaches are applicable to peptide, non-peptide oligomer, or small molecule libraries of compounds. (See, e.g., Lam, 1997, Anticancer Drug Design 12:145.)

[0222] As used herein, "small molecule" may refer to a composition having a molecular weight of less than about 5 kD, most preferably less than about 4 kD. Small molecules may be, for example, nucleic acids, peptides, polypeptides, peptidomimetics, carbohydrates, lipids, or other organic or inorganic molecules. Libraries of chemical and / or biological mixtures, such as fungal, bacterial, or algal extracts, are known in the art and can be screened with any of the assays of the present invention.

[0223] Examples of methods for the synthesis of molecular libraries can be found in the art, for example, DeWitt, et al., 1993. Proc. Natl. Acad. Sci. USA 90:6909; Erb, et al., 1994. Proc. Natl. Acad. Sci. USA 91:11422; Zuckermann, et al., 1994. J. Med. Chem. 37:2678; Cho, et al., 1993. Science 261:1303; Carrell, et al., 1994. Angew. Chem. Int. Ed. Engl. 33:2059; Carell, et al., 1994. Angew. Chem. Int. Ed. Engl. 33:2061; and Gallop, et al., 1994. J. Med. Chem. 37:1233.

[0224] Libraries of compounds can be stored in solution (see, e.g., Houghten, 1992, Biotechniques 13:412-421), on beads (see, e.g., Lam, 1991, Nature 354:82-84), on chips (see, e.g., Fodor, 1993, Nature 364:555-556), bacteria (see, e.g., U.S. Pat. No. 5,223,409), spores (see, e.g., U.S. Pat. No. 5,223,409), plasmids (see, e.g., Cull, et al., 1992, Proc. Natl. Acad. Sci. USA 89:1865-1869), or on phage (see, e.g., Scott and Smith, 1990, Science 249:386-390; Devlin, 1990, Science 249:404-406; Cwirla, et al., 1990, Science 249:406-406). al., 1990. Proc. Natl. Acad. Sci. USA 87:6378-6382; Felici, 1991. J. Mol. Biol. 222:301-310; and U.S. Pat. No. 5,233,409).

[0225] In one embodiment, a candidate compound is introduced to the antibody-antigen complex to determine whether the candidate compound disrupts the antibody-antigen complex, where disruption of the complex indicates that the candidate compound modulates CLDN4 activity.

[0226] In another embodiment, at least one CLDN4 protein is exposed to at least one monoclonal antibody. The formation of an antibody-antigen complex is detected, and one or more candidate compounds are introduced into the complex. If the antibody-antigen complex is destroyed after the introduction of the one or more candidate compounds, the candidate compounds are useful for treating cancer or a proliferative disease or disorder.

[0227] Determining the ability of a test compound to interfere with or disrupt the antibody-antigen complex can be accomplished, for example, by conjugating the test compound with a radioisotope or enzyme label, so that binding of the test compound to the antigen or a biologically active portion thereof can be determined by detecting the labeled compound in the complex. For example, the test compound can be directly or indirectly labeled with 125r, 35S, 14C, or 3H, and the radioisotope can be detected by direct counting of radioemission or by scintillation counting. Alternatively, the test compound can be enzymatically labeled with, for example, horseradish peroxidase, alkaline phosphatase, or luciferase, and detected by measuring the conversion of the enzyme label and an appropriate substrate to product.

[0228] In one embodiment, the assay comprises contacting the antibody-antigen complex with a test compound and determining the ability of the test compound to interact with the antigen or disrupt a pre-existing antibody-antigen complex. In this embodiment, determining the ability of the test compound to interact with the antigen and / or disrupt the antibody-antigen complex comprises determining the ability of the test compound to preferentially bind to the antigen or a biologically active portion thereof compared to the antibody.

[0229] In another embodiment, the assay comprises contacting the antibody-antigen complex with a test compound and determining the ability of the test compound to modulate the antibody-antigen complex. Determining the ability of the test compound to modulate the antibody-antigen complex can be accomplished, for example, by determining the ability of the antigen to bind to or interact with the antibody in the presence of the test compound.

[0230] Those skilled in the art will recognize that in any of the screening methods disclosed herein, the antibody may be a CLDN4 antibody. Further, the antigen may be a CLDN4 protein or a portion thereof.

[0231] The screening methods disclosed herein can be performed as cell-based or cell-free assays. For cell-free assays involving membrane-bound CLDN4 protein, it may be desirable to utilize a solubilizing agent to maintain the membrane-bound form of the protein in solution. Examples of such solubilizing agents include non-ionic detergents such as n-octylglucoside, n-dodecylglucoside, n-dodecylmaltoside, octanoyl-N-methylglucamide, decanoyl-N-methylglucamide, Triton® X-100, Triton® X-114, Thesit®, isotridecyl poly(ethylene glycol ether) n, N-dodecyl-N,N-dimethyl-3-ammonium-1-propanesulfonic acid, 3-(3-cholamidopropyl)dimethylammonium-1-propanesulfonic acid (CHAPS), or 3-(3-cholamidopropyl)dimethylammonium-2-hydroxy-1-propanesulfonic acid (CHAPSO).

[0232] In some embodiments, it may be desirable to immobilize the antibody or antigen to facilitate separation of the complexed form from the uncomplexed form of either or both after introduction of the candidate compound and to adapt the assay to automation. Observation of the antibody-antigen complex in the presence and absence of the candidate compound can be achieved in any vessel suitable for containing the reactants. Examples of such vessels include microtiter plates, test tubes, and microcentrifuge tubes. In one embodiment, fusion proteins can be provided that add a domain that allows one or both of the proteins to be bound to a matrix. For example, a GST-antibody fusion protein or a GST-antigen fusion protein can be adsorbed onto glutathione Sepharose beads (Sigma Chemical, St. Louis, MO) or a glutathione-derivatized microtiter plate, then combined with a test compound, and the mixture can be incubated under conditions that promote complex formation (e.g., physiological conditions of salt and pH). After incubation, the beads or microtiter plate wells are washed to remove unbound components, the matrix is ​​immobilized in the case of beads, and the complex is determined directly or indirectly. Alternatively, the complexes can be dissociated from the matrix, and the level of antibody-antigen complex formation can be determined using standard techniques.

[0233] Other techniques for immobilizing proteins on matrices can also be used in the screening assays of the present invention. For example, antibodies or antigens (e.g., CLDN4 proteins) can be immobilized using conjugation of biotin and streptavidin. Biotinylated antibody or antigen molecules can be prepared from biotin-NHS (N-hydroxysuccinimide) using techniques well known in the art (e.g., biotinylation kit, Pierce Chemicals, Rockford, Ill.) and immobilized to wells of a streptavidin-coated 96-well plate (Pierce Chemical). Alternatively, other antibodies that react with the antibody or antigen of interest but do not interfere with the formation of the antibody-antigen complex of interest can be derivatized to the wells of the plate, and unbound antibody or antigen can be captured in the wells by antibody conjugation. Methods for detecting such complexes include immunodetection of complexes using such other antibodies reactive with the antibody or antigen, in addition to those described herein for GST-immobilized complexes.

[0234] This invention further pertains to novel agents identified by any of the screening assays described herein and uses thereof for treatments described herein.

[0235] Chimeric antigen receptor (CAR) cell therapy

[0236] Also provided herein are cell therapies, such as chimeric antigen receptor (CAR) cell therapies. For example, the cells can be CAR T cells or CAR NK cells.

[0237] CAR cell therapy redirects a patient's T cells and / or NK cells to kill tumor cells, for example, by exogenous expression of a CAR on the T cells or NK cells. CARs can be transmembrane fusion proteins that link the antigen recognition domain of an antibody to the intracellular signaling domain of a T cell receptor and co-receptor or NK cell receptor.

[0238] In one embodiment, a monospecific CAR cell is provided. For example, the anti-CLDN4 antibody described herein can be used as a targeting moiety of the CAR cell. For example, a CLDN4 antibody can have low affinity to its antigen but high avidity. In another example, a CLDN4 antibody can have high affinity to its antigen but low avidity. An antibody with fewer binding sites can have high affinity and low avidity, while an antibody with a larger binding site can have low affinity and high avidity.

[0239] In another embodiment, a bispecific (or dual-target) CAR cell is provided. In another embodiment, the CAR cell is an engineered cell comprising a chimeric antigen receptor, where the chimeric antigen receptor comprises an extracellular ligand-binding domain specific for a first antigen and a second antigen on the surface of a cancer cell, wherein the first antigen comprises an antigen that is not CLDN4 and the second antigen comprises CLDN4.

[0240] In embodiments, the anti-CLDN4 antibodies or CLDN4 fusion proteins described herein can be used as a payload for armored CAR cell therapy. For example, suitable cells capable of secreting the anti-CLDN4 antibodies of the present invention (or alternatively, engineered to express the secreted anti-CLDN4 antibodies described herein) can be used. The secreted anti-CLDN4 "payload" can be, for example, a minibody, scFv, IgG molecule, bispecific fusion molecule, and other antibody fragments described herein. After contacting or engineering, the cells described herein can be introduced into a patient in need of treatment by infusion therapy, as known to those skilled in the art.

[0241] In embodiments, the patient may have a CLDN4-associated disease or disorder, such as a cancer, as described herein. The cells (e.g., T cells) may be, for example, but not limited to, T lymphocytes, CD4+ T cells, CD8+ T cells, or a combination thereof. Exemplary CARs and CAR factories useful in aspects of the present invention include those disclosed in, for example, PCT / US2015 / 067225 and PCT / US2019 / 022272, each of which is incorporated herein by reference in its entirety. In one embodiment, a CLDN4 antibody discussed herein can be used to construct a multispecific antibody or as a payload for CAR-T cells or CAR NK cells. For example, in one embodiment, an anti-CLDN4 antibody discussed herein can be used for targeting a CAR (i.e., as a targeting moiety). In another embodiment, an anti-CLDN4 antibody discussed herein can be used as a targeting moiety, and a different CLDN4 antibody targeting a different epitope can be used as the payload. In another embodiment, the payload can be an immunomodulatory antibody payload.

[0242] Solid tumors present unique challenges for CAR-T therapy. Some barriers to CAR-T efficacy in solid tumors include heterogeneous antigen expression, poor tissue homing, activation, persistence, and an immunosuppressive tumor microenvironment. Unlike hematological cancers, tumor-associated target proteins are overexpressed between tumors and healthy tissues, resulting in on-target / off-tumor T cell killing of healthy tissues. Furthermore, immunosuppression in the tumor microenvironment (TME) limits CAR-T cell activation toward tumor killing. After such contact or manipulation, the cells can be introduced into a cancer patient in need of treatment via infusion therapy known to those skilled in the art. The cancer patient may have any of the types of cancer disclosed herein. The cells (e.g., T cells) may be, for example, but not limited to, tumor-infiltrating T lymphocytes, CD4+ T cells, CD8+ T cells, or a combination thereof.

[0243] In embodiments, CAR cells (i.e., CAR T cells or CAR NK cells) can be generated according to methods known in the art using lentiviral systems (via transduction), retroviral systems (via transfection (electroporation)), and transposon systems (via PiggyBac). Useful promoters for payloads that can be used in generating CAR-Ts include, for example, constitutive promoters (where the promoter is the same as that of the CAR-T, e.g., EF1a followed by IRES or 2A); inducible promoters (where the promoter is different from that of the CAR-T, e.g., NFAT, IL-2 promoter); and engineered promoters (such as cytokine CLDN4 locus "knock-ins" and / or promoters under the control of endogenous promoters). In one embodiment, the CLDN4 antibodies or CLDN4 fusion proteins discussed herein can be used in the construction of multispecific antibodies or as payloads for CAR cells. For example, in one embodiment, the anti-CLDN4 antibodies or CLDN4 fusion proteins discussed herein can be used for targeting CARs (i.e., as targeting moieties). In one embodiment, an anti-CLDN4 antibody or CLDN4 fusion protein discussed herein can be used as a payload secreted by CAR cells. In another embodiment, an anti-CLDN4 antibody or CLDN4 fusion protein discussed herein can be used as a targeting moiety, and a different CLDN4 antibody targeting a different epitope can be used as the payload. In another embodiment, the payload can be an immunomodulatory antibody payload. In some embodiments, the CLDN4 antibody or CLDN4 fusion protein described herein for use in a CAR-T composition is not a high-affinity CLDN4 antibody (e.g., so that the antibody does not bind strongly to its CLDN4 target). For example, a CLDN4 antibody or CLDN4 fusion protein described herein can be used as a payload secreted by CAR cells, and two targeting moieties (e.g., tumor-associated surface antigens) are selected for a particular cancer.Non-limiting examples of tumor-associated surface antigens include ErbB2 (HER2 / neu), carcinoembryonic antigen (CEA), epithelial cell adhesion molecule (EpCAM), epidermal growth factor receptor (EGFR), MUC1, MSLN, CD19, CD20, CD30, CD40, CD22, RAGE-1, MN-CAIX, RET1, RET2 (AS), prostate-specific antigen (PSA), TAG-72, PAP, p53, Ras, prostein, These include PSMA, survivin, 9D7, prostate cancer tumor antigen-1 (PCTA-1), GAGE, MAGE, mesothelin, β-catenin, TGF-βRII, BRCA1 / 2, SAP-1, HPV-E6, and HPV-E7 (see also PCT / US2015 / 067225 and PCT / US2019 / 022272, which are incorporated by reference in their entireties, for additional tumor-associated surface antigens). Exemplary armored CAR-T cells are listed in the table below. TIFF2025527491000016.tif122142

[0244] Those skilled in the art will recognize that CAR cells can be generated from cell sources known to those skilled in the art. Non-limiting examples include T cells, NK cells, iPSC-derived cells (e.g., iPSC-derived T cells and / or iPSC-derived NK cells), peripheral blood cells (e.g., peripheral blood mononuclear cells), umbilical cord blood cells, cell lines (e.g., the NK92 cell line), human embryonic stem cells (hESCs), and CD34+ hematopoietic progenitor cells (HPCs). See, for example, Lu, Hui, et al., "From CAR-T cells to CAR-NK cells: a developing immunotherapy method for hematological malignancies." Frontiers in Oncology (2021): 3151.

[0245] Chimeric B cell receptor

[0246] Engineered B cell receptors, called chimeric B cell receptors, including B cell receptors containing antibodies or antibody fragments preselected for high affinity to specific disease-associated antigens, represent a powerful new approach to disease. B cells function as professional antigen-presenting cells, processing and presenting antigens on MHC class II molecules, enhancing tumor recognition by immune cells and assisting in the spread of neoantigens. Chimeric antibody signaling and secreting (CASS) B cells, a key component of immunological memory, simultaneously recruit a wide range of immune cells, providing a robust, lifelong surveillance program that reverses tumor-infiltrating lymphocyte depletion and protects against tumor metastasis and recurrence. In embodiments, B cells may contain chimeric, non-natural, and at least partially artificially engineered receptors. In certain cases, engineered chimeric B cell receptors have one, two, three, four, or more components, and in some embodiments, one or more components facilitate B cell targeting or binding to one or more antigen-bearing cells.

[0247] Aspects of the invention include genetically engineered B cells that have been modified to express and carry a chimeric B cell receptor on their surface. In embodiments, the genetically engineered B cells can comprise a single chimeric B cell receptor that targets one antigen, such as CLDN4, or a single chimeric B cell receptor that targets two or more antigens (e.g., a bispecific or multispecific chimeric B cell receptor). In some embodiments, the cells comprise a split-chimeric B cell receptor, e.g., two different scFvs and different costimulatory domains expressed on the B cell surface. Additionally, some embodiments comprise fine-tuned chimeric B cell receptors.

[0248] In embodiments, the chimeric B cell receptor comprises an extracellular domain, a transmembrane domain, and an intracellular signaling domain, and the polypeptides assemble together to form the chimeric B cell receptor.

[0249] For example, the extracellular ligand-binding domain can be selected to recognize a ligand, such as CLDN4, that acts as a cell surface marker on target cells associated with a disease state. For example, the disease state can be cancer and the target ligand can be a cancer-associated antigen, such as CLDN4.

[0250] In embodiments, the extracellular ligand-binding domain may comprise an antigen-binding or antigen-recognition domain derived from an antibody against a target antigen, such as an anti-CLDN4 antibody described herein, hi embodiments, the extracellular ligand-binding domain may comprise an antibody or fragment thereof described herein.

[0251] In one embodiment, the transmembrane domain comprises a stalk region. The stalk region can be derived from all or a portion of a naturally occurring molecule, such as all or a portion of the extracellular region of CD8, CD4, or CD28, or all or a portion of an antibody constant region (e.g., CH1, CH2, CH3, or both CH2 and CH3 for an IgG antibody, or CH1, CH2, CH3, CH4, or any combination thereof for an IgM antibody). In other embodiments, the stalk region can be a synthetic sequence that corresponds to a naturally occurring stalk sequence, or can be an entirely synthetic stalk sequence. In one embodiment, the stalk region is a portion of the human CD8 alpha chain.

[0252] The signaling domain or intracellular signaling domain of the chimeric B cell receptor of the present invention is involved in intracellular signal transduction following binding of the extracellular ligand-binding domain to a target, resulting in activation of immune cells and immune responses. In other words, the signaling domain is involved in activating at least one of the normal functions of a B cell expressing the chimeric B cell receptor. Thus, the term "signaling domain" can refer to the portion of a protein that induces a cell to perform a specialized function, such as, for example, early activation of Lyn and Syk, and late activation of NFAT and NFκB.

[0253] Chimeric B cell receptors can contain native transmembrane and intracellular domains. In native B cells, B cell receptor binding leads to rapid tyrosine phosphorylation of the intracellular domain and calcium ion polarization, resulting in downstream activation of NFAT and NF-κB. We have engineered an inducible expression system that is activated by antigens associated with pathologies such as cancer, using NFAT / NF-κB response elements to drive expression of our secreted proteins.

[0254] Distinguishing features of suitable transmembrane polypeptides include their ability to be expressed on the surface of immune cells, such as B cells, and to interact together to induce a cellular response of the immune cells against a predefined target cell. Different transmembrane polypeptides of a chimeric B cell receptor, comprising an extracellular ligand-binding domain and / or a signaling domain, interact together to participate in signal transduction after binding to a target ligand and induce an immune response. The transmembrane domain may be derived from natural or synthetic sources. The transmembrane domain may be derived from any membrane-bound or transmembrane protein.

[0255] Treatment method

[0256] As used herein, the term "treat" or "treatment" refers to both therapeutic treatment and prophylactic or preventative measures, the purpose of which is to prevent or slow (alleviate) an undesired physiological change or disorder, such as the progression of cancer. Beneficial or favorable clinical results include, but are not limited to, alleviation of symptoms, reduction in extent of disease, stable (i.e., not worsening) state of disease, slowing or delaying of disease progression, improvement or palliation of disease state, remission (partial or total), whether detectable or not. "Treatment" can also refer to prolonging survival as compared to expected survival if not receiving treatment. Those in need of treatment include those already with the condition or disorder, as well as those prone to suffer from the condition or disorder or those in whom the condition or disorder is to be prevented.

[0257] The present invention provides both prophylactic and therapeutic methods for treating subjects at risk (or susceptible) for cancer (e.g., when an early detection cancer biomarker is identified in such a subject) or other cell proliferation-related diseases or disorders. Such diseases or disorders include, but are not limited to, diseases or disorders associated with aberrant expression of CLDN4 and / or aberrant activation of cell signaling pathways involving CLDN4. Such diseases or disorders are included in CLDN-associated diseases or disorders. For example, the method is used to treat, prevent, or alleviate the symptoms of cancer. In one embodiment, the method is used to treat, prevent, or alleviate the symptoms of solid tumors. Non-limiting examples of other tumors that can be treated with the compositions described herein include lung cancer, kidney cancer, ovarian cancer, prostate cancer, colon cancer, cervical cancer, uterine cancer, brain cancer, skin cancer, liver cancer, pancreatic cancer, or gastric cancer. Additionally, the method of the present invention can be used to treat blood cancers such as leukemia and lymphoma. Alternatively, the method can be used to treat, prevent, or alleviate the symptoms of metastatic cancer. For example, cancers that can be treated or prevented or whose symptoms can be alleviated include B-cell chronic lymphocytic leukemia (CLL), non-small cell lung cancer, melanoma, ovarian cancer, lymphoma, or renal cell carcinoma. Cancers that can be treated or prevented or whose symptoms can be alleviated also include solid tumors with high mutational burden and WBCs in the filtrate.

[0258] Thus, in one aspect, the invention provides a method for preventing, treating, or alleviating symptomatic cancer or cell proliferative disease or disorder in a patient by administering to the patient a monoclonal antibody, scFv antibody, or bispecific antibody of the invention. For example, an anti-CLDN4 antibody can be administered in a therapeutically effective amount.

[0259] Subjects at risk for cancer or cell proliferation-related diseases or disorders can include patients with a family history of cancer or subjects who have been exposed to agents known or suspected to cause cancer. Administration of a prophylactic agent can occur prior to the manifestation of cancer, such that the disease is prevented or, alternatively, its progression is delayed.

[0260] In another embodiment, tumor cell growth is inhibited by contacting the cell with an anti-CLDN4 antibody of the invention. The cell can be any cell that expresses CLDN4.

[0261] The present invention further provides both prophylactic and therapeutic methods for treating subjects at risk (or susceptible to) chronic or acute viral, bacterial, or parasitic infections. The present invention also provides therapeutic methods for both prophylactic and therapeutic treatment of subjects at risk for a disease, disorder, or condition associated with T cell depletion, or at risk of developing T cell depletion. The present invention also provides therapeutic methods for both prophylactic and therapeutic treatment of subjects at risk for a disease, disorder, or condition associated with T cell depletion, or at risk of developing T cell depletion. Such diseases or disorders include, but are not limited to, HIV, AIDS, and chronic or acute bacterial, viral, or parasitic infections. Other such chronic infections include, for example, those caused by hepatitis B virus (HBV), hepatitis C virus (HCV), herpes simplex virus type 1 (HSV-1), Helicobacter pylori, or Toxoplasma gondii. Other acute infections that are included are those caused by microorganisms such as gram-positive bacteria, gram-negative bacteria, protozoa, or fungi, for example, as described herein.

[0262] The present invention also encompasses methods for increasing or enhancing an immune response to an antigen. The immune response is increased or enhanced by administering a monoclonal antibody, scFv antibody, or bispecific antibody of the present invention to a subject. The immune response is enhanced, for example, by enhancing antigen-specific T effector function. The antigen is a viral (e.g., HIV), bacterial, parasitic, or tumor antigen. The immune response is a natural immune response. By natural immune response, we mean an immune response that is the result of an infection. The infection is a chronic infection. An increase or enhancement of the immune response to an antigen can be measured by many methods known in the art. For example, the immune response can be measured by measuring one of the following: T cell activity, T cell proliferation, T cell activation, effector cytokine production, and T cell transcriptional profile. Alternatively, the immune response is a response induced by vaccination.

[0263] Thus, in another aspect, the invention provides a method of increasing vaccine efficacy by administering to a subject a monoclonal or scFv antibody of the invention and a vaccine, wherein the antibody and vaccine are administered sequentially or simultaneously, and the vaccine is a tumor vaccine, a bacterial vaccine, or a viral vaccine.

[0264] Combination Method

[0265] The compositions of the present invention described herein can be administered in combination with chemotherapeutic agents. Chemotherapeutic agents that can be administered with the compositions of the present disclosure include antibiotic derivatives (e.g., doxorubicin, bleomycin, daunorubicin, and dactinomycin); antiestrogens (e.g., tamoxifen); antimetabolites (e.g., fluorouracil, 5-FU, methotrexate, floxuridine, interferon α-2b, glutamic acid, plicamycin, mercaptopurine, and 6-thioguanine); cytotoxic agents (e.g., carmustine, BCNU, lomustine, CCNU, cytosine arabinoside, cyclophosphamide, estramustine, hydroxyurea, procarbazine, mitomycin, busulfan, cisplatin, and vincristine sulfate); hormones (e.g., medroxycycline, cyclosporine, cycloheximide ... These include, but are not limited to, progesterone, estramustine sodium phosphate, ethinyl estradiol, estradiol, megestrol acetate, methyltestosterone, diethylstilbestrol diphosphate, chlorotrianisene, and testolactone; nitrogen mustard derivatives (e.g., mephalen, chorambucil, mechlorethamine (nitrogen mustard), and thiotepa); steroids and combinations (e.g., betamethasone sodium phosphate); and others (e.g., dicarbazine, asparaginase, mitotane, vincristine sulfate, vinblastine sulfate, and etoposide).

[0266] In additional embodiments, the compositions of the invention described herein can be administered in combination with cytokines, including but not limited to IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-10, IL-12, IL-13, IL-15, anti-CD40, CD40L, and TNF-α.

[0267] In additional embodiments, the compositions described herein can be administered in combination with other therapeutic or prophylactic regimens, such as, for example, radiation therapy.

[0268] In some embodiments, the compositions described herein can be administered in combination with other immunotherapeutic agents, including, but not limited to, simtuzumab, abagovomab, adecatumumab, afutuzumab, alemtuzumab, altumomab, amatuximab, anatumomab, arcitumomab, bavituximab, bectumomab, bevacizumab, bivatuzumab, blinatumomab, brentuximab, cantuzumab, catumaxomab, cetuximab, sitatuzumab, cixutumumab, clivatuzumab, conatumumab, daratumumab, drozitumab, dutuzumab, and tuzumab. Rigotumab, dusigitumab, detumomab, dacetuzumab, dalotuzumab, ecromeximab, elotuzumab, ensituximab, ertumaxomab, etaracizumab, farletuzumab, ficlatuzumab, fizitumumab, framvotumab, futuximab, ganitumab, gemtuzumab, girentuximab, glembatumumab, ibritumomab, igovomab, imgatuzumab, indatuximab, inotuzumab, intetumumab, ipilimumab Mab, iratumumab, labetuzumab, lexatumumab, lintuzumab, lorvotuzumab, lucatumumab, mapatumumab, matuzumab, milatuzumab, minletumomab, mitumomab, moxetumomab, narnatumumab, naptumomab, necitumumab, nimotuzumab, nofetumomab, ocaratuzumab, ofatumumab, olaratuzumab, onartuzumab, oportuzumab, oregovomab, panitumumab, palsatuzumab, patritumumab, pemtumomab and 3F8.

[0269] The present invention provides a method for treating cancer in a patient by administering two antibodies that bind to the same epitope on the CLDN4 protein, or alternatively, two different epitopes on the CLDN4 protein. Alternatively, cancer can be treated by administering a first antibody that binds to CLDN4 and a second antibody that binds to a protein other than CLDN4. In another embodiment, cancer can be treated by administering a bispecific antibody that binds to both CLDN4 and a protein other than CLDN4. For example, the protein other than CLDN4 can include, but is not limited to, IL-12, IL-12R, IL-2, IL-2R, IL-15, IL-15R, IL-7, IL-7R, IL-21, or IL-21R. For example, the protein other than CLDN4 can be a tumor-associated antigen, or the protein other than CLDN4 can be a cytokine. Non-limiting examples of proteins other than CLDN4 include CTLA-4, CXCR4, LAG-3, CD28, CD122, 4-1BB, TIM3, OX-40, OX40L, CD40, CD40L, LIGHT, ICOS, ICOSL, GITR, GITRL, TIGIT, CD27, VISTA, B7H3, B7H4, HEVM (or BTLA), CD47, and CD73.

[0270] In some embodiments, the invention provides for the administration of anti-PD-1 antibodies, alone or in combination with an additional antibody that recognizes another protein other than CLDN4, along with cells capable of generating or enhancing an immune response. For example, these cells can be peripheral blood mononuclear cells (PBMCs), or any cell type found in PBMCs, such as cytotoxic T cells, macrophages, and natural killer (NK) cells.

[0271] Additionally, the present invention provides for the administration of antibodies that bind to CLDN4 protein with other therapeutic agents, including anti-neoplastic agents such as small molecules, growth factors, cytokines, or biomolecules such as peptides, peptidomimetics, peptoids, polynucleotides, lipid-derived mediators, small biogenic amines, hormones, neuropeptides, and proteases. Small molecules include, but are not limited to, inorganic molecules and small organic molecules. Suitable growth factors or cytokines include IL-2, GM-CSF, IL-12, and TNF-alpha. Small molecule libraries are known in the art. (See Lam, Anticancer Drug Des., 12:145, 1997.)

[0272] Diagnostic Assays

[0273] Anti-CLDN4 antibodies can be used diagnostically, for example, to monitor the development or progression of cancer, for example, as part of a clinical trial procedure to determine the effectiveness of a given therapeutic and / or prophylactic regimen.

[0274] In some embodiments, for diagnostic purposes, the anti-CLDN4 antibodies of the invention are linked to a detectable moiety, e.g., to provide a method for detecting cancer cells in a subject at risk for or afflicted with cancer.

[0275] The detectable moiety can be conjugated directly to the antibody or fragment, or indirectly, for example, by using a fluorescent secondary antibody. Direct conjugation can be achieved, for example, by standard chemical coupling of a fluorophore to the antibody or antibody fragment, or through genetic engineering. Chimeric or fusion proteins containing an antibody or antibody fragment linked to a fluorescent or bioluminescent protein can be constructed. For example, Casadei, et al. (Proc Natl Acad Sci USA. 1990 Mar;87(6):2047-51) describe a method for creating a vector construct capable of expressing a fusion protein gene between aequorin and an antibody gene in mammalian cells.

[0276] As used herein, the term "labeled" with respect to a probe or antibody can encompass both direct labeling of the probe or antibody by conjugating (i.e., physically linking) a detectable substance to the probe or antibody, and indirect labeling of the probe or antibody by reactivity with another reagent that is directly labeled. Examples of indirect labeling include detection of a primary antibody using a fluorescently labeled secondary antibody and end-labeling a DNA probe with biotin for detection with fluorescently labeled streptavidin. The term "biological sample" is intended to include tissues, cells, and biological fluids isolated from a subject (e.g., biopsy material), as well as tissues, cells, and biological fluids present within a subject. That is, the detection methods of the present invention can be used in vitro and in vivo to detect cells expressing CLDN4 in biological samples. For example, in vitro techniques for detecting CLDN4 include enzyme-linked immunosorbent assay (ELISA), Western blotting, immunoprecipitation, and immunofluorescence. Furthermore, in vivo techniques for detecting CLDN4 involve introducing a labeled anti-CLDN4 antibody into a subject. For example, the antibody can be labeled with a radioactive marker whose presence and location in a subject can be detected by standard imaging techniques.

[0277] In the case of "targeted" conjugates, i.e., conjugates that include a targeting moiety, which is a molecule or feature designed to localize the conjugate to a specific site within a subject or animal, localization can refer to a state in which equilibrium between the bound "localized" entity and the unbound "free" entity within the subject is essentially achieved. The rate at which such equilibrium is achieved depends on the route of administration. For example, a conjugate administered by intravenous injection can achieve localization within minutes of injection, whereas an orally administered conjugate may take several hours to achieve localization. Alternatively, localization can simply refer to the location of an entity within a subject or animal at a selected period of time after the entity is administered. As another example, localization is achieved when the moiety becomes distributed after administration.

[0278] A reasonable estimate of the time required to achieve localization can be made by one skilled in the art. Furthermore, the state of localization as a function of time can be tracked by imaging a detectable moiety (e.g., a luminescent conjugate) according to the method of the present invention, such as with a photodetector device. The "photodetector device" used can be sensitive enough to allow imaging of weak light from within a mammal in a reasonable time and to use the signal from such a device to construct an image.

[0279] If it is possible to use extremely bright light-generating moieties and / or detect light-generating fusion proteins localized near the surface of the object or animal being imaged, then "night vision" goggles or standard sensitive video cameras such as Silicon Intensified Tube (SIT) cameras (e.g., manufactured by Hammamatsu Photonic Systems, Bridgewater, NJ) can be used. However, more sensitive light detection methods are needed.

[0280] At extremely low light levels, the photon flux per unit area becomes so low that the scene being imaged no longer appears continuous. Instead, it is represented by individual photons that differ from each other both temporally and spatially. When viewed on a monitor, such an image appears as sparkling points of light, each representing a single detected photon. By accumulating these detected photons over time in a digital image processor, an image can be acquired and constructed. In contrast to conventional cameras, where the signal at each image point is assigned an intensity value, in photon-counting imaging, the amplitude of the signal is immaterial. The goal is simply to detect the presence of a signal (photon) and count its occurrence relative to its location over time.

[0281] At least two types of photodetector devices described herein can detect individual photons and generate a signal that can be analyzed by an image processor. Noise-reducing photodetector devices achieve sensitivity by reducing the background noise of the photon detector rather than amplifying the photon signal. Noise is primarily reduced by cooling the detector array. Devices include charge-coupled device (CCD) cameras called "back-thinned" cooled CCD cameras. In more sensitive devices, cooling is achieved using, for example, liquid nitrogen, to bring the temperature of the CCD array to approximately -120°C. "Back-thinned" refers to an ultra-thin backplate that shortens the path length photons must travel before being detected, thereby increasing quantum efficiency. A highly sensitive back-thinned cryogenic CCD camera is the "TECH 512" Series 200 camera available from Photometries, Ltd. (Tucson, Arizona).

[0282] "Photon amplification devices" amplify photons before they hit the detection screen. This class includes CCD cameras equipped with intensifier tubes, such as microchannel intensifier tubes. Microchannel intensifier tubes contain a metal array of channels perpendicular to and coextensive with the camera's detection screen. The microchannel array is positioned between the sample, subject, or animal being imaged and the camera. Most of the photons that enter the channels of the array contact the sides of the channels before exiting. A voltage applied across the array results in the emission of many electrons from each photon collision. Electrons from such collisions exit their channels of origin in a "shotgun" pattern and are detected by the camera.

[0283] Even greater sensitivity can be achieved by arranging intensifying microchannel arrays in series, so that electrons generated in the first stage in turn result in an amplified signal of electrons in the second stage. However, the increased sensitivity is achieved at the expense of spatial resolution, which decreases with each additional stage of amplification. An exemplary microchannel intensifier-tube-based single-photon detection device is the C2400 series available from Hamamatsu.

[0284] The image processor processes signals generated by the photon-counting photodetector device to construct an image that can be displayed on a monitor or printed on a video printer, for example. Such image processors are sold as part of systems that include the highly sensitive photon-counting cameras described herein and are therefore available from the same sources. The image processor is connected to a personal computer, such as an IBM-compatible PC or an Apple Macintosh (Apple Computer, Cupertino, Calif.), which may or may not be included as part of a purchased imaging system. Once the images are in the form of digital files, they can be manipulated and printed using a variety of image processing programs (e.g., "ADOBE PHOTOSHOP," Adobe Systems, Adobe Systems, Mt. View, Calif.).

[0285] In one embodiment, the biological sample contains protein molecules from the test subject. One exemplary biological sample is a peripheral blood leukocyte sample isolated by conventional means from a subject.

[0286] The present invention also encompasses kits for detecting the presence of CLDN4- or TIGIT-expressing cells in a biological sample. For example, the kit may include: a labeled compound or agent (e.g., an anti-CLDN4 scFv or monoclonal antibody) capable of detecting cancer or tumor cells in a biological sample, a means for determining the amount of CLDN4 in the sample, and a means for comparing the amount of CLDN4 in the sample with a standard. In some embodiments, the standard is a non-cancerous cell or a cellular extract thereof. The compound or agent can be packaged in a suitable container. The kit may further include instructions for using the kit to detect cancer in a sample.

[0287] Other embodiments

[0288] While the present invention has been described in conjunction with its detailed description, the foregoing description is intended to be illustrative, but not limiting, of the scope of the invention, which is defined by the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

[0289] The invention is further described in the following examples, which do not limit the scope of the invention described in the claims. [Example]

[0290] Examples are provided below to facilitate a more complete understanding of the present invention. The following examples illustrate exemplary modes of making and practicing the invention. However, the scope of the present invention is not limited to the specific embodiments disclosed in these examples, which are for illustrative purposes only, as alternative methods may be used to obtain similar results.

[0291] Example 1 - Claudin 4 as a therapeutic target We identified two anti-claudin-4 scFvs (Gly1-4-G3 and Gly1-2-F4) that were subjected to three rounds of alternating panning on 293T cells stably transduced to express CLDN4 and Cf2Th cells. Our scFvs showed minimal cross-reactivity with claudin-3, a structurally related protein.

[0292] Claudin 4 is a therapeutic target for many cancers, including triple-negative breast cancer, pancreatic cancer, and biliary tract cancer. Our scFv can specifically target CLDN4 and can be used to target CAR T cells.

[0293] These scFvs can be developed as monoclonal antibodies for therapeutic or diagnostic purposes, and they can be used as the targeting moiety of CAR T cells against cancers that overexpress CLND4.

[0294] Example 2 - We transiently transfected 293T cells with CLDN-3 plasmid

[0295] - Low or no background binding of antibodies to 293T that does not contain CLDN-3 or CLDN-4

[0296] - CLDN-3 antibody specifically recognized only CLDN-3 cell lines

[0297] - CDLN-4 antibody weakly bound to CLDN-3 cell line

[0298] - Some 293T CLDN-4 cells may have lost CLDN-4 expression

[0299] - We can perform binding assays of CLDN-4 minibodies to CLDN-3 expressing cell lines.

[0300] equivalent Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, numerous equivalents to the specific substances and procedures described herein which equivalents are considered to be within the scope of this invention and encompassed by the following claims.

Claims

1. An antibody or antigen-binding fragment thereof that binds to a claudin 4 (CLDN4) protein or a fragment thereof, comprising a heavy chain, a light chain, or both a heavy chain and a light chain, the heavy chain HCDR1 comprising GFTFNNYA (SEQ ID NO: 9), GFTFGGYA (SEQ ID NO: 12), GGTFSSYA (SEQ ID NO: 15), or GGTFNNYA (SEQ ID NO: 18); HCDR2 comprising IRDSGGST (SEQ ID NO: 10), LSNSGSNA (SEQ ID NO: 13), or IIPIVDIA (SEQ ID NO: 16); HCDR3 comprising ARRGYSSSWYGDGYYYGMDV (SEQ ID NO: 11), ARAVMSSSSWYMRRYYYYYMDV (SEQ ID NO: 14), or ARGGSQGAYYMDV (SEQ ID NO: 17); or a combination of these CDRs; the light chain LCDR1 comprising SGSIASSF (SEQ ID NO: 19), RSNIGSNT (SEQ ID NO: 22), SGSIASNY (SEQ ID NO: 25), or QSVSNY (SEQ ID NO: 28); LCDR2 comprising ENN (SEQ ID NO: 20), SNN (SEQ ID NO: 23), EDN (SEQ ID NO: 26), or GAS (SEQ ID NO: 29); an LCDR3 comprising QSYDSTSHV (SEQ ID NO: 21), AAWDDSLNGLYV (SEQ ID NO: 24), QSYDDSNRVV (SEQ ID NO: 27), or HQYGSLPQT (SEQ ID NO: 30); or a combination of these CDRs, An antibody or antigen-binding fragment thereof.

2. The antibody of claim 1, comprising a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 3, 5, and 7, or a sequence at least 90% identical thereto, and a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 4, 6, and 8, or a sequence at least 90% identical thereto.

3. An antibody as described herein.

4. A therapeutic antibody that binds to claudin 4 (CLDN4), comprising a variable domain and a constant domain, wherein the constant domain is IgG, and the variable domain comprises framework regions and complementarity determining means for binding to claudin 4.

5. The antibody of claim 4, wherein the constant region is IgG1 or IgG4.

6. An isolated antibody or fragment thereof that binds to human claudin 4 protein, (a) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 9, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 10, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 11, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 19, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 20, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 21; or (b) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 12, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 13, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 14, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 22, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 23, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 24; or (c) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 15, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 16, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 17, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 25, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 26, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 27; or (d) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 18, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 16, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 17, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 28, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 29, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO:

30. An antibody or fragment thereof comprising:

7. An isolated scFv antibody that binds to human claudin 4 protein, (a) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 9, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 10, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 11, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 19, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 20, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 21; or (b) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 12, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 13, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 14, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 22, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 23, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 24; or (c) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 15, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 16, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 17, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 25, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 26, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 27; or (d) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 18, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 16, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 17, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 28, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 29, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO:

30. An scFv antibody comprising:

8. An isolated antibody or fragment thereof that binds to human claudin-4 protein, comprising a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 3, 5, and 7, or a sequence at least 90% identical thereto, and a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 4, 6, and 8, or a sequence at least 90% identical thereto.

9. An isolated scFv that binds to human claudin-4 protein, comprising a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 3, 5, and 7, or a sequence at least 90% identical thereto, and a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 4, 6, and 8, or a sequence at least 90% identical thereto.

10. A F(ab) that binds to claudin-4, (a) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 9, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 10, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 11, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 19, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 20, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 21; or (b) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 12, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 13, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 14, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 22, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 23, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 24; or (c) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 15, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 16, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 17, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 25, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 26, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 27; or (d) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 18, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 16, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 17, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 28, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 29, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO:

30. F(ab).

11. A VhH that binds to claudin 4, (a) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 9, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 10, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 11; or (b) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 12, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 13, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 14; or (c) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 15, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 16, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 17; or (d) VH CDR1 comprising the amino acid sequence of SEQ ID NO: 18, VH CDR2 comprising the amino acid sequence of SEQ ID NO: 16, and VH CDR3 comprising the amino acid sequence of SEQ ID NO:

17. VhH, including

12. 12. An isolated bispecific antibody comprising the fragment of any one of claims 1 to 11 and a second antigen-binding fragment having specificity for a molecule on an immune cell.

13. 13. The bispecific antibody of claim 12, wherein the molecule is selected from the group consisting of CCR4, B7H3, B7H4, CD27, CD28, CD40, CD40L, CD47, CD122, CTLA-4, GITR, GITRL, ICOS, ICOSL, LAG-3, LIGHT, OX-40, OX40L, PD-1, TIM3, 4-1BB, TIGIT, VISTA, HEVM, BTLA, and KIR.

14. 13. The bispecific antibody of claim 12, wherein the fragment and the second fragment are each independently selected from a Fab fragment, a single-chain variable fragment (scFv), or a single-domain antibody.

15. 13. The bispecific antibody of claim 12, further comprising an Fc fragment.

16. A bispecific T cell engager (BiTE) that binds to human claudin 4 protein, (a) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 9, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 10, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 11, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 19, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 20, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 21; or (b) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 12, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 13, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 14, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 22, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 23, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 24; or (c) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 15, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 16, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 17, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 25, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 26, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 27; or (d) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 18, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 16, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 17, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 28, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 29, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO:

30. A bispecific T cell engager (BiTE), comprising:

17. A bispecific T cell engager (BiTE) that binds to human claudin-4 protein, comprising a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 3, 5, and 7, or a sequence at least 90% identical thereto, and a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 4, 6, and 8, or a sequence at least 90% identical thereto.

18. The antibody of any one of claims 1 to 11, having at least 90% sequence identity.

19. the nucleic acid sequence encoding HCDR1 is SEQ ID NO:62, SEQ ID NO:65, SEQ ID NO:68, or SEQ ID NO:71, or a degenerate variant thereof; the nucleic acid sequence encoding HCDR2 is SEQ ID NO:63, SEQ ID NO:66, SEQ ID NO:69, or SEQ ID NO:72, or a degenerate variant thereof; the nucleic acid sequence encoding HCDR3 is SEQ ID NO:64, SEQ ID NO:67, SEQ ID NO:70, or SEQ ID NO:73, or a degenerate variant thereof; the nucleic acid sequence encoding LCDR1 is SEQ ID NO:74, SEQ ID NO:77, SEQ ID NO:80, or SEQ ID NO:83, or a degenerate variant thereof; the nucleic acid sequence encoding LCDR2 is SEQ ID NO:75, SEQ ID NO:78, SEQ ID NO:81, or SEQ ID NO:84, or a degenerate variant thereof; the nucleic acid sequence encoding LCDR3 is SEQ ID NO:76, SEQ ID NO:79, SEQ ID NO:82, or SEQ ID NO:85, or a degenerate variant thereof; A nucleic acid encoding the antibody of any one of claims 1 to 11.

20. the nucleic acid sequence encoding the HCVR is SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, or SEQ ID NO:89, or a degenerate variant thereof; The nucleic acid sequence encoding the LCVR is SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, or SEQ ID NO: 93, or a degenerate variant thereof; A nucleic acid encoding the antibody of any one of claims 1 to 11.

21. A pharmaceutical composition comprising the antibody of any one of claims 1 to 11 and one or more pharmaceutically acceptable carriers, diluents, or excipients.

22. (a) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 9, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 10, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 11, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 19, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 20, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 21; or (b) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 12, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 13, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 14, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 22, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 23, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 24; or (c) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 15, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 16, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 17, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 25, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 26, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 27; or (d) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 18, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 16, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 17, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 28, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 29, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO:

30.

22. The pharmaceutical composition of claim 21, comprising:

23. 22. The pharmaceutical composition of claim 21, wherein the pharmaceutical composition comprises a heavy chain variable region (HCVR) and a light chain variable region (LCVR), wherein the amino acid sequence of the HCVR is SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, or SEQ ID NO: 7, and the amino acid sequence of the LCVR is SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, or SEQ ID NO:

8.

24. A nucleic acid encoding the antibody or fragment of any one of claims 1 to 11.

25. A nucleic acid encoding the bispecific antibody of any one of claims 12 to 15.

26. A nucleic acid encoding the BiTE of any one of claims 16 to 17.

27. The antibody or fragment of any one of claims 1 to 11, the bispecific antibody of any one of claims 12 to 15, or the BiTE of any one of claims 16 to 17, wherein the antibody, fragment, or bispecific antibody binds to claudin 3 with a binding affinity that is at least 5, 10, 25, 50, 100, or 1000 times lower than the antibody, fragment, or bispecific antibody binds to claudin 4.

28. 1. An engineered cell comprising a chimeric antigen receptor, wherein the chimeric antigen receptor comprises an extracellular ligand-binding domain specific for an antigen on the surface of a cancer cell, the antigen comprising claudin 4, the extracellular ligand-binding domain comprising an antibody or fragment thereof, the antibody or fragment thereof comprising: (a) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 9, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 10, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 11, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 19, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 20, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 21; or (b) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 12, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 13, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 14, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 22, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 23, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 24; or (c) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 15, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 16, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 17, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 25, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 26, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 27; or (d) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 18, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 16, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 17, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 28, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 29, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO:

30. engineered cells, including

29. 29. The engineered cell of claim 28, wherein the antibody or fragment thereof comprises a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 3, 5, and 7, or a sequence at least 90% identical thereto, and a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 4, 6, and 8, or a sequence at least 90% identical thereto.

30. 29. The engineered cell of claim 28, comprising a T cell, an NK cell, an NKT cell, an iPS cell, an iPS-derived cell, a cell line, or a B cell.

31. 29. The engineered cells of claim 28, comprising CD4+, CD8+, CD3+ pan T cells, or any combination thereof.

32. 22. A method for treating cancer, comprising administering to a subject in need thereof an effective amount of (a) the antibody of any one of claims 1 to 11, or (b) the pharmaceutical composition of claim 21.

33. A method for inhibiting claudin 4 in a subject, comprising administering to the subject an effective amount of (a) an antibody described in any one of claims 1 to 11, or (b) a pharmaceutical composition described in claim 21.

34. 34. The method of claim 33, comprising a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 3, 5, and 7, or a sequence at least 90% identical thereto, and a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 4, 6, and 8, or a sequence at least 90% identical thereto.

35. Use of (a) an antibody according to any one of claims 1 to 11, or (b) a pharmaceutical composition according to claim 21, for treating cancer.

36. An antibody according to any one of claims 1 to 11, or (b) a pharmaceutical composition according to claim 21, for use in therapy.

37. (b) an antibody according to any one of claims 1 to 11, or (b) a pharmaceutical composition according to claim 21, for use in the treatment of cancer.

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