Antibodies to CLDN4 and methods of use thereof

Abstract

The present invention comprises human antibodies that bind to sealin 4 domain (CLDN4) protein. These antibodies can be used to treat cancer.

Description

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

[0002] All patents, patent applications, and publications cited herein are incorporated herein by reference in their entirety. The disclosures of these publications are hereby incorporated by reference in their entireties into this application to more fully describe the prior art known to those skilled in the art as of the date of the present 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 any and all copyright rights whatsoever. Sequence Listing

[0004] This application contains a sequence listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on [], is named [] and is [] bytes in size. Technical Field

[0005] The present invention includes antibodies against claudin 4 (CLDN4) and methods of using the same. background

[0006] Claudin is a four-span transmembrane protein of tight junctions. The claudin 4 protein is encoded by the CLDN4 gene. Summary of the invention

[0007] Aspects of the invention relate to isolated monoclonal antibodies or antigen-binding fragments thereof that bind to a claudin 4 (CLDN4) protein or fragment thereof, comprising 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), ARAVMSSSWYMRRYYYYYMDV (SEQ ID NO: 14), or ARGGSQGAYYMDV (SEQ ID NO: 17), or a combination of their CDRs; and 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: 26). ID NO: 28), a CDR1 containing ENN (SEQ ID NO: 20), SNN (SEQ ID NO: 23), EDN (SEQ ID NO: 26) or GAS (SEQ ID NO: 29), a CDR3 containing 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 their CDRs. In the embodiments, the antibody sequences have been identified 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 an embodiment, 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 has a -9 The 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 for binding with 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] Aspects of the invention also relate 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 VL CDR2 comprising the amino acid sequence of SEQ ID NO: 17, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 18. NO: 16, a VH CDR2 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. In an embodiment, the antibody sequence has been determined according to the IMGT numbering scheme.

[0022] Aspects of the present invention also relate to an isolated scFv antibody that binds to a human claudin 4 protein, the isolated scFv antibody 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. In an embodiment, the antibody sequence has been determined according to the IMGT numbering scheme.

[0023] Aspects of the present invention relate to an isolated antibody or fragment thereof that binds to human claudin 4 protein, the isolated antibody or fragment thereof comprising a heavy chain variable region comprising an amino acid sequence selected from the group consisting of: SEQ ID NO: 1, 3, 5 and 7, or a sequence at least 90% identical thereto.

[0024] Aspects of the present invention relate to an isolated antibody or fragment thereof that binds to human claudin 4 protein, the isolated antibody or fragment thereof comprising a light chain variable region comprising an amino acid sequence selected from the group consisting of: SEQ ID NO: 2, 4, 6 and 8, or a sequence at least 90% identical thereto.

[0025] Aspects of the present invention relate to an isolated antibody or fragment thereof that binds to human claudin 4 protein, the isolated antibody or fragment thereof comprising a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 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 NO: 2, 4, 6 and 8, or a sequence at least 90% identical thereto.

[0026] Aspects of the invention relate to an isolated scFv that binds to human claudin 4 protein, the isolated scFv comprising a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1, 3, 5 and 7, or a sequence at least 90% identical thereto.

[0027] Aspects of the invention relate to an isolated scFv that binds to human claudin 4 protein, the isolated scFv comprising a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 2, 4, 6 and 8, or a sequence at least 90% identical thereto.

[0028] Aspects of the invention relate to an isolated scFv that binds to human claudin 4 protein, the isolated scFv comprising a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 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 NO: 2, 4, 6 and 8, or a sequence at least 90% identical thereto.

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

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

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

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

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

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

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

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

[0037] Aspects of the invention relate to separated bispecific antibodies comprising an antibody fragment as described herein and a second antigen binding fragment specific for a molecule on an immune cell. In an embodiment, 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 an embodiment, 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] Aspects of the invention further relate to bispecific T cell engagers (BiTEs) that bind to human claudin 4 protein. In an embodiment, the 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 CDR2 comprising the amino acid sequence of SEQ ID NO: 18, a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 19 NO: 16, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 17, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 25, a VLCDR2 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. In an embodiment, the antibody sequence has been identified according to the IMGT numbering scheme.

[0040] Aspects of the invention further include a bispecific T cell engager (BiTE) that binds to a human claudin 4 protein, the BiTE comprising a heavy chain variable region comprising an amino acid sequence selected from the group consisting of: SEQ ID NO: 1, 3, 5 and 7, or a sequence at least 90% identical thereto.

[0041] Aspects of the invention further include a bispecific T cell engager (BiTE) that binds to a human claudin 4 protein, the BiTE comprising a light chain variable region comprising an amino acid sequence selected from the group consisting of: SEQ ID NO: 2, 4, 6 and 8, or a sequence that is at least 90% identical thereto.

[0042] Aspects of the invention further include a bispecific T cell engager (BiTE) that binds to human claudin 4 protein, the BiTE comprising a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 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 NO: 2, 4, 6 and 8, or a sequence at least 90% identical thereto.

[0043] Embodiments as described herein may further comprise an Fc fragment.

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

[0045] Still further, aspects of the present invention relate to pharmaceutical compositions comprising an antibody or fragment thereof as described herein, and a pharmaceutically acceptable carrier or excipient.

[0046] In an embodiment, 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 invention relate to isolated cells comprising one or more polynucleotides encoding an antibody or fragment as described herein.

[0048] Furthermore, aspects of the invention relate to vectors comprising a nucleic acid as described herein.

[0049] Still further, aspects of the invention relate to cells comprising a vector as described herein.

[0050] Aspects of the invention relate to engineered cells comprising a chimeric antigen receptor. In an embodiment, the chimeric antigen receptor comprises an extracellular ligand binding domain that is specific for an antigen on the surface of a cancer cell, wherein the antigen comprises claudin 4, wherein the extracellular ligand binding domain comprises an antibody or a 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 VLCDR1 comprising the amino acid sequence of SEQ ID NO: 22, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 23 CDR2, 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. In the Examples, antibody sequences have been identified 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 NO: 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 NO: 2, 4, 6 and 8, or a sequence at least 90% identical thereto.

[0053] In an embodiment, 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 an embodiment, the cell comprises a T cell, a NK cell, a NKT cell, an iPS cell, an iPS-derived cell, a cell line, or a B cell. For example, the cell comprises a CD4+, CD8+, CD3+ pan T cell, or any combination thereof.

[0055] Aspects of the invention further relate to kits. In an embodiment, the kit comprises at least one pharmaceutical composition of 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 relate to methods for detecting the presence of claudin 4 in a sample. In an embodiment, the method comprises contacting the sample with an isolated monoclonal antibody or fragment thereof as described herein, and detecting the presence or absence of the antibody-antigen complex, thereby detecting the presence of claudin 4 in the sample. In an embodiment, contacting comprises immunohistochemistry. For example, immunohistochemistry comprises precipitation, immunofluorescence, protein blot or ELISA. In an embodiment, the sample is whole blood, a blood component, a body fluid, a biopsy, a tissue, serum or one or more cells.

[0057] In an embodiment, the sample comprises a normal sample or a cancerous sample. For example, the cancer expresses claudin 4. For example, the cancer comprises gallbladder cancer, breast cancer, cervical cancer, colorectal cancer, esophageal cancer, intestinal cancer, lung cancer, pancreatic cancer, prostate cancer, kidney cancer, rectal cancer, stomach cancer, thyroid cancer or uterine cancer.

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

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

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

[0061] Embodiments may further comprise the step of obtaining a sample from a subject.

[0062] Aspects of the invention further relate to a method of treating cancer in a subject by administering to the subject a pharmaceutical composition as described herein. For example, the cancer expresses claudin 4. For example, the cancer includes gallbladder cancer, breast cancer, cervical cancer, colorectal cancer, esophageal cancer, intestinal cancer, lung cancer, pancreatic cancer, prostate cancer, kidney cancer, rectal cancer, gastric cancer, thyroid cancer or uterine cancer. In embodiments, the binding affinity of the antibody or fragment as described herein to claudin 3 is at least 5, 10, 25, 50, 100 or 1000 times lower than the binding affinity of the antibody, fragment or bispecific antibody to claudin 4.

[0063] Other objects and advantages of the present invention will become apparent from the ensuing description. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0065] Figure 2 A non-limiting exemplary schematic diagram of panning against claudin-4 is shown.

[0066] Figure 3 A graph showing non-limiting exemplary data of scFvs targeting claudin 4 based on phage panning using lysed claudin 4 expressed in insect cells.

[0067] Figure 4 A graph showing non-limiting exemplary data of EK01 (see, WO 2019 / 178359) phage specifically binding to soluble claudin-4.

[0068] Figure 5 A graph showing non-limiting exemplary data of F4 binding to claudin-4 is shown.

[0069] Figure 6 Histograms of non-limiting exemplary flow cytometry data are shown.

[0070] Figure 7 Non-limiting exemplary Western blots from protein purification are shown.

[0071] Figure 8 Non-limiting exemplary data of EK01 minibody binding data are shown (see, WO 2019 / 178356).

[0072] Fig. 9A non-limiting exemplary schematic diagram of paramagnetic proteoliposome (PMPL) panning is shown. In an embodiment, three rounds of panning were performed on soluble sealin-4 and screened with sealin-4. After ELISA confirmation and a dilution series of soluble sealin 4, a hit was initially identified. The panning initially failed on PMPL. A new PMPL was then prepared.

[0073] Fig.10 Non-limiting exemplary data for PMPL production are shown.

[0074] Fig.11 A non-limiting exemplary schematic diagram of panning is shown.

[0075] Fig.12 Shows the Fig.11 Non-limiting exemplary results of 6 plates of re-rescue from panning in .

[0076] Fig.13 Shows the Fig.11 Non-limiting exemplary results of clones and frequencies in rounds 1 and 2 of panning.

[0077] Fig.14 Shows the Fig.11 Non-limiting exemplary data for round 1 and round 2 of next generation sequencing (NGS).

[0078] Fig.15 Non-limiting exemplary data of a phage binding lytic claudin-4 ELISA are shown.

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

[0080] Fig.17 Non-limiting exemplary data of phage binding to non-transduced cells are shown.

[0081] Fig.18 Non-limiting exemplary ELISA data are shown.

[0082] Fig.19 Non-limiting exemplary minibody binding data are shown.

[0083] Fig. 20 Non-limiting exemplary minibody binding data are shown.

[0084] Fig.21 A non-limiting exemplary schematic of a claudin-4 whole cell panning pathway is shown.

[0085] Fig. 22Non-limiting exemplary purified phage binding curves are shown (CLDN-4).

[0086] Fig.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 US 8,076,458 and cloned into our scFv-Fc vector as a control. Reagents were purchased from Miltenyi and R&D and used as received.

[0087] Fig.24 A non-limiting exemplary schematic diagram of the structure of CLDN proteins is shown. In embodiments, the structure and expression of CLDN-3 and CLDN-4 may be similar. In embodiments, the expression levels in cancer tissues may also be similar.

[0088] Fig.25 Non-limiting exemplary schematic diagrams of human CLDN-3 and CLDN-4 extracellular domains are shown. In some embodiments, the homology of the extracellular domains can be used to discover CLDN-4 specific antibodies. Homology: ECL1 98%, ECL2 79%, both 93%.

[0089] Fig.26 Examples of commercial antibody stability are shown.

[0090] Fig. 27 Non-limiting exemplary quantitative data of cell line staining are shown.

[0091] Fig.28 Non-limiting exemplary data for CLDN-3 binding of aCLDN-4 minibodies are shown.

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

[0093] Fig.30 Non-limiting exemplary data of MB231 staining with aCXCR4 compared to KO are shown.

[0094] Fig.31 A non-limiting exemplary diagram of CD8+ anti-CLDN-4 CART killing MB231 cells expressing CLDN-4 is shown. Positive controls KM3900 and CPE non-specifically kill tumors, and aC4-G3 and aC4-F4 do specifically recognize and kill CLDN+ tumors (9 times higher). Negative control background killing is low (<10%). Our anti-CLDN-4 CART specifically kills tumor cells expressing high levels of CLDN-4.

[0095] Fig.32 A graph showing non-limiting exemplary data of the kinetics of CD8+ anti-CLDN-4 CART killing at an E:T ratio of 2:1. DETAILED DESCRIPTION

[0096] Abbreviations and definitions

[0097] A detailed description of one or more embodiments is provided herein. However, it should be understood that the present invention can be implemented in various forms. Therefore, the specific details disclosed herein should not be interpreted as being restrictive, but should be interpreted as a basis for the claims and a representative basis for teaching those skilled in the art to adopt the present invention in any appropriate manner.

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

[0099] Whenever any of the phrases "for example," "such as," "including," etc. are used herein, unless expressly stated otherwise, they should be understood to be followed by the phrase "without limitation." Similarly, "example," "exemplary," etc. should be understood to be non-limiting.

[0100] The term "substantially" allows deviations from the descriptive terms that do not adversely affect the intended purpose. The descriptive terms should be understood as being modified by the term "substantially" even if the word "substantially" is not explicitly recited.

[0101] The terms "comprising" and "including," as well as "having" and "involving" (and similarly "comprises," "includes," "has," and "involves") and the like are used interchangeably and have the same meaning. In particular, the definition of each of these terms is consistent with the common definition of "comprising" under U.S. patent law and is therefore interpreted as an open term meaning "at least the following," and is also interpreted as not excluding additional features, limitations, aspects, etc. Thus, for example, "a process involving steps a, b, and c" means that the process includes at least steps a, b, and c. Whenever the term "a" or "an" is used, it should be understood to mean "one or more," unless such an interpretation is meaningless in the context.

[0102] The term "about" is used herein to mean approximately, roughly, roughly, or within the range of. When the term "about" is used in conjunction with a numerical range, it modifies the range by extending the upper and lower boundaries of the numerical values ​​set forth above. Typically, the term "about" is used herein to modify a numerical value above and below the stated value by a variation of 20 percent upward or downward (higher or lower).

[0103] Described herein are unique recombinant monoclonal claudin 4 (CLDN4) antibodies. "Recombinant" when referring to a polypeptide (e.g., an antibody) or polynucleotide may refer to a form of the polypeptide or polynucleotide that does not exist in nature, a non-limiting example of which may be created by combining polynucleotides or polypeptides that do not normally occur together.

[0104] In addition to exemplary wild-type IgG constant regions that can be used in combination with the VH and VL sequences provided herein, amino acid sequences of monoclonal CLDN4 antibodies are provided herein (see Tables 1-4); the amino acid sequences of the heavy and light chain complementary determining regions (CDRs) of the CLDN4 antibodies are underlined below ( CDR1 ), Underline and bold or Underline, italic, and bold :

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

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

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

[0108] Table 7A. Heavy chain of CLDN4 antibodies (V H ) Complementarity Determining Region (CDR)

[0109] Table 7B. Heavy chain of CLDN4 antibodies (V H ) Complementarity Determining Region (CDR)

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

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

[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 having a specified percentage of identity or similarity to the amino acid or nucleotide sequence of the anti-CLDN4 antibodies described herein. For example, "homology" or "identity" or "similarity" refers to the sequence similarity between two peptides or between two nucleic acid molecules. Homology can be determined by comparing the positions in each sequence that can be aligned for comparison purposes. When 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, when compared to a specified region or the full length of any of the anti-CLDN4 antibodies described herein, the antibody may have 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more amino acid sequence identity. For example, when compared to a specified region or the full length of any of the anti-CLDN4 antibodies described herein, the antibody may have 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more nucleic acid identity. Sequence identity or similarity to the nucleic acids and proteins of the invention can be determined by sequence comparison and / or alignment by methods known in the art, such as using software programs known in the art, such as those described in: Ausubel et al., eds. (2007) Current Protocols in Molecular Biology. [Modern Methods 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 sequence identity or similarity percentages of the nucleic acids and proteins of the invention.

[0113] As used herein, "polypeptide" can encompass singular "polypeptide" as well as plural "polypeptides", and can refer to a molecule consisting of monomers (amino acids) linearly linked by amide bonds (also referred to as peptide bonds). The term "polypeptide" can refer to any one or more chains of two or more amino acids, and does not refer to the specific length of the product. Therefore, peptides, dipeptides, tripeptides, oligopeptides, "proteins", "amino acid chains" or any other terms can be used to refer to one or more chains of two or more amino acids, and can refer to "polypeptides" herein, and the term "polypeptide" can be used to replace any of these terms or to be used interchangeably with any of these terms. "Polypeptide" can also refer to a product modified after the expression of a polypeptide, including but not limited to glycosylation, acetylation, phosphorylation, amidation, derivatization by known protection / blocking groups, proteolytic cleavage or modification by non-naturally occurring amino acids. A polypeptide can be derived from a natural biological source or produced by recombinant technology, but is not necessarily translated from a nucleic acid sequence. It can be produced in any manner, including by chemical synthesis. With respect to amino acid sequences, those skilled in the art will readily recognize that single substitutions, deletions or additions made to nucleic acid, peptide, polypeptide or protein sequences (which change, 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 change results in the substitution 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 may exhibit increased cross-reactivity with CLDN4 compared to unmodified CLDN4 antibodies.

[0114] For example, a "conservative amino acid substitution" is a substitution in which an amino acid residue is replaced by an amino acid residue with a similar side chain. Families of amino acid residues with similar side chains have been defined in the art and include the following side chains: 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), non-polar 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 by another amino acid residue from the same side chain family. In another embodiment, an amino acid chain may be replaced by 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" may refer to a polypeptide or polypeptide complex that specifically recognizes and binds to an antigen.Antibodies may be complete antibodies and any antigen-binding fragments or single chains thereof.For example, "antibodies" may include any protein- or peptide-containing molecule that contains at least a portion of an immunoglobulin molecule with biological activity that binds to an antigen.Non-limiting examples are a complementary determining region (CDR) of a heavy chain or light chain or its ligand-binding portion, a heavy chain or light chain variable region, a heavy chain or light chain constant region, a framework (FR) region, or any portion thereof, or at least a portion of a binding protein.As used herein, the term "antibody" may refer to an immunoglobulin molecule and an immunologically active portion of an immunoglobulin (Ig) molecule, i.e., a molecule containing an antigen binding site that specifically binds to an antigen (immunoreacts with an antigen)."Specific binding" or "immunoreacts with..." means that an antibody reacts with one or more antigenic determinants of an antigen and does not react with other polypeptides.

[0117] As used herein, the term "antibody fragment" or "antigen-binding fragment" is a portion of an antibody, such as an F (ab′)2 、F (ab)2 、F ab ′、F ab , Fv, scFv, etc. Regardless of the structure, antibody fragments will bind to the same antigen recognized by the intact antibody. The term "antibody fragment" may include aptamers (e.g., mirror aptamers), mini antibodies, and diabodies. The term "antibody fragment" may also include any synthetic or genetically engineered protein that acts like an antibody by binding to a specific antigen to form a complex. The antibodies, antigen-binding polypeptides, variants, or derivatives described herein include, but are not limited to, polyclonal antibodies, monoclonal antibodies, multispecific antibodies, human antibodies, humanized antibodies, or chimeric antibodies, single-chain antibodies, epitope-binding fragments, such as Fab, Fab', and F(ab')2, Fd, Fv, single-chain Fv (scFv), single-chain antibodies, dAb (domain antibodies), mini antibodies, disulfide-linked Fv (sdFv), fragments comprising VL or VH domains, fragments produced by Fab expression libraries, and anti-idiotype (anti-Id) antibodies.

[0118] "Single-chain variable fragment" or "scFv" refers to the heavy chain (V H ) and light chain (V L) fusion protein of the variable region of the single-chain Fv ("scFv") polypeptide molecule is a covalently linked VH:VL heterodimer that can be expressed by a gene fusion comprising a VH-encoding gene and a VL-encoding gene connected by a linker encoding a peptide. (See Huston et al. (1988) Proc Nat Acad Sci USA [Proceedings of the National Academy of Sciences of the United States of America] 85(16):5879-5883). In some aspects, these regions are connected by a short peptide of 10 to about 25 amino acids. The linker can be rich in glycine for flexibility, rich in serine or threonine for solubility, and V can be H The N-terminus of L The C-terminus of the constant region is connected, and vice versa. Despite the removal of the constant region and the introduction of the linker, the protein retains the specificity of the original immunoglobulin. Many methods have been described to resolve the chemical structure for converting the naturally aggregated but chemically separated light and heavy polypeptide chains from the antibody V region into scFv molecules, which will fold into a three-dimensional structure that is substantially similar to the structure of the antigen binding site. See, for example, U.S. Patent Nos. 5,091,513; 5,892,019; 5,132,405; and U.S. Patent No. 4,946,778, each of which is incorporated by reference in its entirety.

[0119] Very large naive human scFv libraries have been created and can be created to provide a large resource of rearranged antibody genes for 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 [Proceedings of the National Academy of Sciences of the United States] 89: 9339-43 (1992); Zebedee et al., Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences of the United States] 89: 3175-79 (1992)).

[0120] Antibody molecules obtained from humans are divided into five classes of immunoglobulins: IgG, IgM, IgA, IgE and IgD, which differ from each other by the nature of the heavy chains present in the molecule. It will be appreciated by those skilled in the art that heavy chains are classified as gamma, mu, alpha, delta or epsilon (γ, μ, α, δ, ε) and some subclasses therein (e.g., γ1-γ4). Certain classes also have subclasses, such as IgG1, IgG2, IgG3 and IgG4, etc. Immunoglobulin subclasses (isotypes) such as IgG1, IgG2, IgG3, IgG4, IgG5, etc. have been well characterized and are known to confer functional specialization. For IgG, a standard immunoglobulin molecule comprises 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 Daltons. These four chains are connected by disulfide bonds in a "Y" configuration, with the light chains sandwiched on both sides of the heavy chains like brackets, starting from the mouth of the "Y" and continuing straight to the end of the variable region. The immunoglobulin or antibody molecules described herein can belong to 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 molecules.

[0121] Light chains are classified as kappa or lambda (κ, λ). Each heavy chain category can be combined with a κ or λ light chain. Typically, when immunoglobulins are produced by hybridomas, B cells, or genetically engineered host cells, light chains and heavy chains are covalently bonded to each other and the "tail" portions of the two heavy chains are bonded to each other by covalent disulfide connections or non-covalent connections. In the heavy chain, the amino acid sequence extends from the N-terminal end of the forked end of the Y configuration to the C-terminal end at the bottom of each chain.

[0122] Both light and heavy chains are divided into regions with structural homology and functional homology. The terms "constant" and "variable" are used functionally. The variable domains of the light chain (VL) and heavy chain (VH) parts determine antigen recognition and specificity. In contrast, the constant domains of the light chain (CL) and heavy chain (CH1, CH2 or CH3) confer important biological properties such as secretion, transplacental mobility, Fc receptor binding, complement binding, etc. The term "antigen binding site" or "binding portion" may refer to the part of the immunoglobulin molecule that participates in antigen binding. The antigen binding site is formed by the amino acid residues of the N-terminal variable ("V") region of the heavy chain ("H") and the light chain ("L"). The three highly different segments (called hypervariable regions) in the V region of the heavy chain and the light chain are interspersed between the more conservative flanking segments called "framework regions" or "FR". Therefore, the term "FR" may refer to the amino acid sequence that naturally exists between the hypervariable regions of immunoglobulins 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 the 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 antibodies are shown in Tables 1-4.

[0123] The six CDRs present in each antigen binding domain are short discontinuous amino acid sequences that are specifically positioned to form antigen binding domains when the antibody presents its three-dimensional conformation in an aqueous environment. The remaining amino acids (FR regions) in the antigen binding domain show less intermolecular variability. The framework region mainly adopts a β-folded conformation, and the CDR forms a loop connecting the β-folded structure, and in some cases forms a part of the β-folded structure. The framework region plays a role in forming a scaffold that positions the CDR in the correct orientation through interchain non-covalent interactions. The antigen binding domain formed by the positioned CDR provides a surface complementary to the epitope on the immunoreactive antigen, which promotes the non-covalent binding of the antibody to its cognate epitope. One of ordinary skill in the art can readily identify the amino acids comprising the CDRs and framework regions, respectively, for a heavy or light chain variable region, as they have been previously defined (see, "Sequences of Proteins of Immunological Interest," Kabat, E., et al., U.S. Department of Health and Human Services, (1983); and Chothia and Lesk, J. Mol. Biol., 196:901-917 (1987)).

[0124] When there are two or more definitions of terms used and / or accepted in the art, the definition of the term as used herein is intended to include these meanings, unless clearly stated to the contrary. A particular example is the use of the term "complementarity determining region" ("CDR") to describe the non-continuous antigen combining sites found in the variable region of heavy and light chain polypeptides. This region has been described by Kabat et al., U.S. Department of Health and Human Services, "Sequences of Proteins of Immunological Interest" (1983) and by Chothia et al., J.Mol.Biol. [Journal of Molecular Biology] 196: 901-917 (1987) (which is incorporated herein by reference in its entirety). The CDR definition according to Kabat and Chothia includes overlapping or subsets of amino acid residues when compared to each other. However, the CDR used to refer to an antibody or its variants is intended to fall within the scope of the terms defined and used herein. The appropriate amino acid residues of the CDRs defined by the references cited above are listed in the table below as a comparison. The exact number of residues comprising a CDR will vary depending on the sequence and size of the CDR. Given the variable region amino acid sequence of an antibody, one skilled in the art can routinely determine which residues comprise a CDR.

[0125] Kabat et al. define a numbering system for variable domain sequences that is applicable to any antibody. Technicians can assign this "Kabat numbering" system to any variable domain sequence explicitly without relying on any experimental data other than the sequence itself. As used herein, "Kabat numbering" refers to the numbering system proposed by 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 approximately amino acid 31 (i.e., approximately 9 residues after the first cysteine ​​residue), includes approximately 5-7 amino acids, and ends at the next tryptophan residue. CDR-H2 begins at the fifteenth residue after the end of CDR-H1, includes approximately 16-19 amino acids, and ends at the next arginine or lysine residue. CDR-H3 begins at approximately the thirty-third amino acid residue after the end of CDR-H2; includes 3-25 amino acids; and ends at the sequence WGXG, where X is any amino acid. CDR-L1 begins at approximately residue 24 (i.e., after the cysteine ​​residue); includes approximately 10-17 residues; and ends at the next tryptophan residue. CDR-L2 begins at approximately the sixteenth residue after the end of CDR-L1 and includes approximately 7 residues. CDR-L3 begins at approximately the thirty-third residue after the end of CDR-L2 (ie, after the cysteine ​​residue); includes approximately 7-11 residues and ends at the sequence F or WGXG, where X is any amino acid.

[0127] In certain aspects, the CDRs of an antibody can be identified according to the IMGT numbering system. IMGT unique numbers have been defined 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., Pommié, C., Ruiz, M., Giudicelli, V., Foulquier, E., Truong, L., Thouvenin-Contet, V. and Lefranc, Dev. Comp. Immunol., 27, 55-77 (2003)]. In the IMGT unique numbering, conserved amino acids always have the same position, such as 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 unique numbering provides standardized definitions of the framework regions (FR1-IMGT: positions 1 to 26, FR2-IMGT: 39 to 55, FR3-IMGT: 66 to 104 and FR4-IMGT: 118 to 128) and the complementarity determining regions: CDR1-IMGT: 27 to 38, CDR2-IMGT: 56 to 65 and CDR3-IMGT: 105 to 117. Since gaps represent unoccupied positions, the CDR-IMGT length (shown between brackets and separated by dots, e.g. [8.8.13]) becomes key information. IMGT unique numbers are used in 2D graphical representations, designated 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 3D structures in IMGT / 3Dstructure-DB [Kaas, Q., Ruiz, M. and Lefranc, M.-P., T cell receptor and MHC structural data. Nucl. Acids. Res., 32, D208-D210 (2004)].

[0128] As used herein, the term "epitope" may include any protein determinant that can specifically bind to an immunoglobulin, scFv or T cell receptor. The variable region allows an antibody to selectively recognize and specifically bind to an epitope on an antigen. For example, a subset of the VL domain and the VH domain or the complementary determining region (CDR) of an antibody is combined to form a variable region that defines a three-dimensional antigen binding site. This four-level antibody structure forms an antigen binding site present at each arm end of the Y. The epitope determinant can be composed of a chemically active surface molecule group (e.g., amino acids or sugar side chains) and has specific three-dimensional structural features and specific charge characteristics. For example, antibodies against the N-terminal peptide or C-terminal peptide of a polypeptide can be produced. More particularly, 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 chain and the VL chain. In one embodiment, the antibody may be directed against the following: claudin 4 (CLDN4) (209 amino acid residues in length) with UniProtKB ID: O14493CLD4_human, which comprises the following amino acid sequence: MASMGLQVMGIALAVLGWLAVMLCCALPMWRVTAFIGSNIVTSQTIWEG LWMNCVVQSTGQMQCKVYDSLLALPQDLQAARALVIISIIVAALGVLLS VVGGKCTNCLEDESAKAKTMIVAGVVFLLAGLMVIVPVSWTAHNIIQDF YNPLVASGQKREMGASLYVGWAASGLLLLGGGLLCCNCPPRTDKPYSAK YSAARSAAASNYV (SEQ ID NO:94)

[0129] As used herein, the terms "immunobinding" and "immunobinding properties" may refer to the type of non-covalent interaction that occurs between an immunoglobulin molecule and an antigen for which the immunoglobulin is specific. The strength or affinity of the immunobinding interaction can be measured by the dissociation constant (K) of the interaction. d ) is used to represent the smaller K d =K indicates greater affinity. The immunological binding properties of a selected polypeptide can be quantified using methods well known in the art. One such method requires measuring the rates of formation and dissociation of antigen binding sites / antigen complexes, where these rates depend on the concentration of the complex partners, the affinity of the interaction, and geometric parameters that affect the rates equally in both directions. Thus, the "association rate constant" (K on ) and the “dissociation rate constant” (Koff ) can be determined by calculating the concentration and the actual rates of association and dissociation. (See Nature 361: 186-87 (1993)). off / K on The ratio of allows elimination of parameters not related to affinity and is equal to the equilibrium binding constant K D (See, Davies et al. (1990) Annual Rev Biochem 59:439-473). When the equilibrium binding constant (K) is measured by a kinetic assay such as a radioligand binding assay or similar assays known to those skilled in the art such as BIAcore or Octet (BLI), the equilibrium binding constant (K) is the binding constant of interest. D )≤1 μM, ≤10 μM, ≤10 nM, ≤10 pM, or ≤100 pM to about 1 pM, the antibodies of the present invention may specifically bind to the CLDN4 epitope. For example, in some embodiments, K D Between about 1E-12M and about 1E-11M K D In some embodiments, K D Between about 1E-11M and about 1E-10MK D In some embodiments, K D Between about 1E-10M and about 1E-9M K D In some embodiments, K D Between about 1E-9M and about 1E-8M K D In some embodiments, K D Between about 1E-8M and about 1E-7M K D In some embodiments, K D Between about 1E-7M and about 1E-6M K D For example, in some embodiments, K D is about 1E-12M, while in other embodiments, K D is about 1E-11M. In some embodiments, K D is about 1E-10M, while in other embodiments, K D is about 1E-9M. In some embodiments, K D is about 1E-8M, while in other embodiments, K D is about 1E-7M. In some embodiments, K D is about 1E-6M, while in other embodiments, K D is about 1E-5M. For example, in some embodiments, K D is about 3E-11M, while in other embodiments, K D is about 3E-12M. In some embodiments, KD is about 6E-11 M. "Specific binding" or "specific for..." can refer to an antibody that binds to an epitope via its antigen binding domain, and that the binding requires some complementarity between the antigen binding domain and the epitope. For example, an antibody is said to "specifically bind" to an epitope when it binds to the epitope via its antigen binding domain more readily than it binds to a random, unrelated epitope.

[0130] For example, the CLDN4 antibody may be monovalent or divalent, and / or may comprise a single chain or a double chain. Functionally, the binding affinity of the CLDN4 antibody is in the range of 10 -5 M to 10 -12 For example, the binding affinity of CLDN4 antibody is 10 -6 M to 10 -12 M, 10 -7 M to 10 -12 M, 10 -8 M to 10 -12 M, 10 -9 M to 10 -12 M, 10 -5 M to 10 -11 M, 10 -6 M to 10 -11 M, 10 -7 M to 10 -11 M, 10 -8 M to 10 -11 M, 10 -9 M to 10 -11 M, 10 -10 M to 10 -11 M, 10 -5 M to 10 -10 M, 10 -6 M to 10 -10 M, 10 -7 M to 10 - 10 M, 10 -8 M to 10 -10 M, 10 -9 M to 10 -10 M, 10 -5 M to 10 -9 M, 10 -6 M to 10 -9 M, 10 -7 M to 10 -9 M, 10 -8 M to 10 -9 M, 10 -5 M to 10 -8 M, 10 -6 M to 10 -8M, 10 -7 M to 10 -8 M, 10 -5 M to 10 -7 M, 10 -6 M to 10 -7 M or 10 -5 M to 10 -6 M.

[0131] CLDN4 protein or its derivatives, fragments, analogs, homologs or orthologs can be used as immunogens to generate antibodies that immunospecifically bind to these protein components. CLDN4 protein or its derivatives, fragments, analogs, homologs or orthologs coupled to proteoliposomes can be used as immunogens to generate antibodies that immunospecifically bind to these protein components.

[0132] Those skilled in the art will recognize that, without undue experimentation, by determining whether a human monoclonal antibody prevents the binding of a human monoclonal antibody of the invention to CLDN4, it can be determined whether the human monoclonal antibody has the same specificity as the human monoclonal antibody of the invention. For example, if the human monoclonal antibody to be tested competes with the human monoclonal antibody of the invention, as shown by a decrease in binding of the human monoclonal antibody of the invention, then the two monoclonal antibodies bind to the same or closely related epitope.

[0133] Another way to determine whether a human monoclonal antibody has the specificity of the human monoclonal antibody of the present invention is to pre-incubate the human monoclonal antibody of the present invention with a CLDN4 protein that normally reacts therewith, and then add the human monoclonal antibody to be tested to determine whether the ability of the human monoclonal antibody to be tested to bind to CLDN4 is inhibited. 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 performed by utilizing CLDN4 and determining whether the test monoclonal antibody can neutralize CLDN4.

[0134] Various procedures known in the art can be used to generate polyclonal or monoclonal antibodies against the proteins of the invention or 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, New York, 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 provides the IgG fraction of immune serum. Subsequently or alternatively, a specific antigen or an epitope thereof that is the target of the immunoglobulin sought can be fixed on a column to purify the immunospecific antibody by immunoaffinity chromatography. The purification of immunoglobulins is discussed, for example, by D. Wilkinson (The Scientist, Inc., Philadelphia, Pennsylvania, 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" may refer to a population of antibody molecules that contains only one molecular species of antibody molecules 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 in the molecules of the population. MAbs contain an antigen binding site that can immunoreact with an epitope of an antigen that is characterized by having a unique binding affinity for the antigen binding site.

[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, mice, hamsters or other suitable host animals are immunized with an immunizing agent to elicit lymphocytes that produce or can produce antibodies that will specifically bind to the immunizing agent. Alternatively, lymphocytes can be immunized in vitro.

[0138] Immunizing agents can include protein antigens, fragments thereof, or fusion proteins thereof. For example, if cells of human origin are preferred, peripheral blood lymphocytes can be used, or if non-human mammalian origin is preferred, spleen cells or lymph node cells can be used. Lymphocytes are then fused with immortalized cell lines using a suitable fusion agent (e.g., polyethylene glycol) to form hybridoma cells (see Goding, Monoclonal Antibodies: Principles and Practice [monoclonal antibodies: principles and practice], Academic Press, (1986) pages 59-103). Immortalized cell lines can be transformed mammalian cells, such as myeloma cells of rodent, cattle, and human origin. For example, rat or mouse myeloma cell lines are used. Hybridoma cells can be cultured in a suitable culture medium containing one or more substances that inhibit the growth or survival of unfused immortalized cells. For example, if the parental cells lack the enzyme hypoxanthine guanine phosphoribosyltransferase (HGPRT or HPRT), the culture medium for the hybridomas will include hypoxanthine, aminopterin, and thymidine ("HAT medium"), which substances prevent the growth of HGPRT-deficient cells.

[0139] Useful immortalized cell lines are those that effectively fuse, support stable high-level expression of antibodies by selected cells producing antibodies, and are sensitive to culture medium (such as HAT culture medium). For example, immortalized cell lines can be mouse myeloma lines, which can be obtained, for example, from the Salk Institute Cell Distribution Center (Salk Institute Cell Distribution Center) (San Diego, California) and the American Type Culture Collection (American Type Culture Collection) (Manassas, Virginia). Human myeloma and mouse-human heteromyeloma cell lines are also described for the production of human monoclonal antibodies. (See Kozbor, J.Immunol [Journal of Immunology], 133: 3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications [monoclonal antibody production technology and application], Marcel Dekker Company (Marcel Dekker, Inc.), New York, (1987) 51-63 pages)).

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

[0141] After hybridoma cells are identified, clones can be subcloned by limiting dilution procedures and cultured 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, hybridoma cells can be grown in vivo in mammals as ascites.

[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 (eg, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography).

[0143] Monoclonal antibodies can also be prepared by recombinant DNA methods, such as those described in U.S. Patent No. 4,816,567 (incorporated herein by reference in its entirety). Using conventional procedures (e.g., by using oligonucleotide probes that can specifically bind to the genes encoding the heavy and light chains of mouse antibodies), it is easy to separate and sequence the DNA encoding the monoclonal antibodies of the present invention. The hybridoma cells of the present invention are used as the source of such DNA. Once isolated, the DNA can be placed in expression vectors, and then these vectors are transfected into host cells (such as ape COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells) that do not otherwise produce immunoglobulins to obtain the synthesis of monoclonal antibodies in recombinant host cells. DNA can also be modified, such as replacing homologous mouse sequences with coding sequences of human heavy and light chain constant domains (see U.S. Patent No. 4,816,567; Morrison, Nature 368,812-13 (1994)) or by covalently linking all or part of the coding sequences of non-immunoglobulin polypeptides to immunoglobulin coding sequences. Such non-immunoglobulin polypeptides can be substituted for the constant domains of an antibody of the invention, and / or can be substituted for the variable domains of one antigen combining site of an antibody of the invention to generate a chimeric bivalent antibody.

[0144] For example, a fully human antibody is an antibody molecule in which the entire sequence of the light chain and the heavy chain (including CDR) is produced from human genes. Such antibodies are referred to as "human antibodies" or "fully human antibodies". Human monoclonal antibodies, such as fully human antibodies and humanized antibodies, can be prepared using the following techniques: trioma technology; human B cell hybridoma technology (see Kozbor, et al., 1983 Immunol Today [Today's Immunology] 4: 72); and EBV hybridoma technology for producing human monoclonal antibodies (see Cole, et al., 1985 in: MONOCLONAL ANTIBODIES AND CANCER THERAPY [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 in vitro with Epstein Barr virus (see Cole, et al., 1985 In: MONOCLONAL ANTIBODIES AND CANCER THERAPY, Allen R. Liss, Inc., pp. 77-96).

[0145] A "humanized antibody" can be an antibody from a non-human species (e.g., a mouse) whose amino acid sequence (e.g., in the CDR region) has been modified to increase its similarity to an antibody variant produced by a human. Antibodies can be humanized by methods known in the art (e.g., CDR transplantation). See also, Safdari et al., (2013) Biotechnol Genet Eng Rev. [Biotechnol Genet Eng Rev.]; 29: 175-86. In addition, humanized antibodies can be produced in transgenic plants as a cheap production 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 by using Agrobacterium tumefaciens or particle bombardment. For example, via transformation, a nucleic acid expression vector containing at least heavy and light chain sequences is expressed in a bacterial culture (i.e., Agrobacterium tumefaciens strain BLA4404). Infiltration of plants can be accomplished by injection. Soluble leaf extracts can be prepared by grinding leaf tissue in a mortar and by centrifugation. Isolation and purification of antibodies can be performed by many methods known to those skilled in the art. Other methods for producing antibodies 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 comprising a vector encoding an antibody of the present invention or producing an antibody of the present invention.

[0146] Antibodies can be humanized using a variety of techniques known in the art, including, for example, CDR grafting (EP 239,400; PCT Publication 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 [Protein Engineering] 7(6):805-814 (1994); Roguska et al., Proc. Natl. Sci. USA [Proceedings of the National Academy of Sciences of the United States of America] 91:969-973 (1994)), and chain shuffling (U.S. Patent No. 5,565,332, which is incorporated by reference in its entirety). “Humanization” (also known as reshaping or CDR grafting) is a well-established technique understood by those skilled in the art for reducing the immunogenicity of monoclonal antibodies (mAbs) from xenogeneic sources (e.g., rodents) and for improving their 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 (e.g., human antibodies) can also be produced using other techniques, including phage display libraries. (See Hoogenboom and Winter, J. Mol. Biol [Journal of Molecular Biology], 227: 381 (1991); Marks et al., J. Mol. Biol [Journal of Molecular Biology], 222: 581 (1991)). Similarly, human antibodies can be prepared by introducing human immunoglobulin loci into transgenic animals (e.g., mice in which endogenous immunoglobulin genes have been partially or completely inactivated). After challenge, the production of human antibodies is observed, which is closely similar to that seen in humans in all aspects, 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; 5,661,016, and in 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 [Nature Biotechnology] 14, 826 (1996); and Lonberg and Huszar, Intern. Rev. Immunol. [International Review of Immunology] 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 antigenic stimulation rather than endogenous antibodies of the animal. (See, PCT Publication No. WO 94 / 02602 and U.S. Patent No. 6,673,986). The endogenous genes encoding heavy and light immunoglobulin chains in the non-human host have been disabled, and the active loci encoding human heavy and light chain immunoglobulins are inserted into the genome of the host. For example, yeast artificial chromosomes containing necessary human DNA are used to incorporate human genes. Preferred modified animals are then obtained as progeny by: crossbreeding intermediate transgenic animals containing less than a complete set of these modifications. A non-limiting example of such a non-human animal is a mouse, and is referred to as a Xenomouse TM, as disclosed in PCT Publication Nos. WO 96 / 33735 and WO 96 / 34096. Such animals produce B cells that secrete complete human immunoglobulins. Antibodies can be obtained directly from animals after immunization with the immunogen of interest, for example as a preparation of polyclonal antibodies, or alternatively from immortalized B cells derived from the animal (e.g., hybridomas that produce monoclonal antibodies). In addition, genes encoding immunoglobulins with human variable regions can be extracted and expressed to directly obtain antibodies, or can be further modified to obtain antibody analogs, such as, for example, single-chain Fv (scFv) molecules.

[0149] Thus, using such techniques, therapeutically useful IgG, IgA, IgM, and IgE antibodies can be produced. For an overview of techniques for producing human antibodies, see Lonberg and Huszar Int. Rev. Immunol. 73:65-93 (1995). For a detailed discussion of this technique for producing human antibodies and human monoclonal antibodies and procedures for producing such antibodies, see, e.g., PCT Publication Nos. WO 98 / 24893; WO 96 / 34096; WO 96 / 33735; U.S. Pat. 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 herein by reference in their entirety. In addition, companies such as Creative BioLabs (Shirley, NY) can be engaged to provide human antibodies against an antigen of choice using techniques similar to those described herein.

[0150] An example of a method for producing a non-human host (using mice as an example) lacking endogenous immunoglobulin heavy chain expression is disclosed in U.S. Pat. No. 5,939,598. It 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 achieved by a targeting vector containing a gene encoding a selectable marker; and generating a transgenic mouse from embryonic stem cells, whose somatic cells and germ cells contain a gene encoding the selectable marker.

[0151] U.S. Patent No. 5,916,771 discloses a method for producing an antibody of interest (e.g., a human antibody). The method comprises introducing an expression vector containing a nucleotide sequence encoding a heavy chain into a mammalian host cell in culture, introducing an expression vector containing a nucleotide sequence encoding a light chain into another mammalian host cell, and fusing the two cells to form a hybrid cell. The hybrid cell expresses an antibody containing a heavy chain and a light chain.

[0152] In a further improvement of this procedure, PCT Publication No. WO 99 / 53049 discloses methods for identifying clinically relevant epitopes on immunogens and related methods for selecting antibodies that immunospecifically bind with high affinity to the relevant epitopes.

[0153] The antibody of interest can also be expressed by a vector containing a DNA segment encoding a single-chain antibody as described herein. Vectors include, but are not limited to, chemical conjugates (e.g., described in WO 93 / 64701, which have a targeting portion (e.g., a ligand for a cell surface receptor) and a nucleic acid binding portion (e.g., polylysine)), viral vectors (e.g., DNA or RNA viral vectors), fusion proteins (e.g., described in PCT / US 95 / 02140 (WO 95 / 22618), which are fusion proteins containing a target portion (e.g., an antibody specific to a target cell) and a nucleic acid binding portion (e.g., protamine)), plasmids, phages, viral vectors, etc. The vector 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, and include poxvirus vectors such as orthopoxvirus vectors or avian poxvirus vectors, herpesvirus vectors such as herpes simplex virus type I (HSV) vectors (see Geller, AI et al., J. Neurochem, 64:487 (1995); Lim, F., et al., in DNA Cloning: Mammalian Systems, D. Glover, ed. (Oxford University 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), adenovirus vectors (see LeGal et al., J. Neurochem, 64:487 (1995); Lim, F., et al., in DNA Cloning: Mammalian Systems, D. Glover, ed. (Oxford University 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), 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 (see Kaplitt, MG et al., Nat. Genet. 8:148 (1994).

[0154] Poxvirus vectors can introduce genes into the cytoplasm. Fowlpox virus vectors only produce short-term expression of nucleic acids. Adenovirus vectors, adeno-associated virus vectors and herpes simplex virus (HSV) vectors can be used to introduce nucleic acids into neural cells. The expression time of adenovirus vectors (about 2 months) is shorter than that of adeno-associated virus (about 4 months), and the expression time of adeno-associated virus is shorter than that of HSV vectors. The selected vector will depend on the target cell and the disease being treated. Introduction can be by standard techniques, such as infection, transfection, transduction or transformation. Examples of gene transfer modes include, for example, naked DNA, CaPO4 precipitation, DEAE dextran, electroporation, protoplast fusion, lipofection, cell microinjection and viral vectors.

[0155] The vector can be used to target substantially any target cell. For example, stereotaxic injection can be used to guide the vector (e.g., adenovirus, HSV) to a preferred position. In addition, a micropump infusion system (e.g., SynchroMed infusion system) can be used to deliver particles by intraventricular (icv) infusion. A method based on bulk flow (referred to as convection) has also been shown to effectively deliver macromolecules to the extended area of ​​the brain, and can be used to deliver the vector to the target cell. (See Bobo et al., Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences of the United States] 91: 2076-2080 (1994); Morrison et al., Am. J. Physiol. [American Journal of Physiology] 266: 292-305 (1994)). Other methods that can be used include catheter, intravenous, parenteral, intraperitoneal and subcutaneous injections and oral or other known routes of administration.

[0156] These vectors can be used to express large quantities of antibodies that can be used in a variety of ways. For example, to detect the presence of CLDN4 in a sample. Antibodies can also be used to attempt to bind to and disrupt CLDN4 activity.

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

[0158] The technique can be adapted to produce single-chain antibodies specific for the antigenic proteins of the present invention (see, e.g., U.S. Pat. No. 4,946,778). In addition, the method can be adapted to construct F ab Expression libraries (see, e.g., Huse, et al., 1989 Science 246:1275-1281) are used to allow rapid and efficient identification of monoclonal F3 proteins with preferred specificity for a protein or its derivatives, fragments, analogs or homologs. ab Fragments. Antibody fragments containing idiotypes directed against protein antigens can be produced by techniques known in the art, including but not limited to: (i) F fragments produced by digestion of antibody molecules with pepsin;(ab')2 fragment; (ii) by reducing F (ab')2 The disulfide bridges of the fragments generate F ab (iii) F fragments produced by treating antibody molecules with papain and a reducing agent ab Fragment and (iv)F v Fragment.

[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. Patent No. 4,676,980), and are used to treat HIV infection (see PCT Publication No. WO 91 / 00360; WO 92 / 20373). Known methods in synthetic protein chemistry, including methods involving cross-linking agents, can be used to prepare antibodies in vitro. For example, immunotoxins can be constructed using disulfide exchange reactions or by forming thioether bonds. Examples of suitable reagents for this purpose include iminothiolate and methyl-4-mercaptobutyrimidate and those disclosed, for example, in U.S. Patent No. 4,676,980.

[0160] The antibodies of the present invention can be modified with respect to effector functions to enhance, for example, the effectiveness of antibodies in treating cancer. For example, one or more cysteine ​​residues can be introduced into the Fc region, thereby allowing the formation of interchain disulfide bonds in the region. The resulting homodimeric antibodies can have improved internalization capacity and / or enhanced complement-mediated cell killing and antibody-dependent cellular cytotoxicity (ADCC). (See Caron et al., J. Exp Med. [Journal of Experimental Medicine], 176: 1 191-1195 (1992) and Shopes, J. Immunol. [Journal of Immunology], 148: 2918-2922 (1992)). Alternatively, antibodies can be engineered to have dual Fc regions and thus can have enhanced complement lysis and ADCC capabilities. (See Stevenson et al., Anti-Cancer Drug Design [Anti-Cancer Drug Design], 3: 219-230 (1989)). In one embodiment, the antibodies of the present invention have modifications in the Fc region so that the Fc region does not bind to Fc receptors. For example, the Fc receptor is an Fcγ receptor. As described herein, antibodies in which the Fc region is modified so that the Fc region does not bind to Fcγ but still binds to the neonatal Fc receptor are useful.

[0161] In an embodiment, the antibody of the present invention may include an Fc variant. See, for example, WO 2018 / 145075 and WO2019 / 183362, which provide Fc variant compositions for enhancing antibody-mediated receptor signaling. In an embodiment, the Fc variant may include amino acid substitutions that change the antigen-dependent effector functions of the antibody (e.g., the circulating half-life of the antibody). When compared to antibodies lacking these substitutions, such antibodies exhibit increased or decreased binding to FcRn, and therefore have increased or decreased serum half-lives, respectively. It is expected that Fc variants with improved affinity for FcRn may have longer serum half-lives, and such molecules have useful applications in methods for treating mammals, wherein the long half-life of the administered antibody is preferred, for example, to treat chronic diseases or disorders. On the contrary, Fc variants with reduced FcRn binding affinity have shorter half-life, and such molecules can also be used, for example, for administration to mammals, where shortened circulation time may be advantageous, for example, for in vivo diagnostic imaging, or for situations where the initial antibody has toxic side effects when it exists in the circulation for a long time. The possibility of Fc variants with reduced FcRn binding affinity passing through the placenta is also less, and therefore can also be used to treat diseases or disorders in pregnant women. In addition, other applications in which FcRn binding affinity reduction can be preferred include those applications in which positioning to the brain, kidney and / or liver is preferred. In one embodiment, antibodies containing Fc variants can show a reduction in transport from the vascular system through the glomerular epithelium. In another embodiment, antibodies containing Fc variants can show a reduction in transport from the brain through the blood-brain barrier (BBB) ​​into the vascular space. In one embodiment, antibodies with altered FcRn binding are included in an Fc domain with one or more amino acid substitutions in the "FcRn binding loop" of the Fc domain. The FcRn binding loop is composed of amino acid residues 280-299 (according to EU numbering). Exemplary amino acid substitutions with altered FcRn binding activity are disclosed in PCT Publication No. WO 05 / 047327, which is incorporated herein by reference. In certain exemplary embodiments, an antibody or fragment thereof of the invention comprises an Fc domain having one or more of the following substitutions: V284E, H285E, N286D, K290E, and S304D (EU numbering).

[0162] In certain embodiments, mutation is introduced into the constant region of mAb so that the antibody-dependent cell-mediated cytotoxicity (ADCC) activity of the mAb is changed. For example, the mutation is the LALA mutation in the CH2 domain. In one embodiment, an antibody (for example, human mAb or bispecific Ab) contains mutation on a scFv unit of heterodimeric mAb, which reduces ADCC activity. In another embodiment, mAb contains mutation on both chains of heterodimeric mAb, which completely eliminates ADCC activity. For example, the mutation introduced into one or two scFv units of mAb is the LALA mutation in the CH2 domain. These mAbs with variable ADCC activity can be optimized so that mAb shows maximum selective killing to cells expressing an antigen recognized by mAb, but shows minimum killing to the second antigen recognized by mAb.

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

[0164] Exemplary amino acid substitutions that confer reduced or altered glycosylation are described in PCT Publication No. WO 05 / 018572, which is incorporated herein by reference in its entirety. In some embodiments, the antibodies or fragments thereof of the present invention are modified to eliminate glycosylation. Such antibodies or fragments thereof may be referred to as "agly" antibodies or fragments thereof (e.g., "aglycosylated" antibodies). Although not wishing to be bound by theory, "aglycosylated" antibodies or fragments thereof may have improved in vivo safety and stability profiles. Exemplary aglycosylated antibodies or fragments thereof include an aglycosylated Fc region of an IgG4 antibody, which lacks Fc effector functions, thereby eliminating the possibility of Fc-mediated toxicity to normal important tissues and cells expressing CLDN4. In yet another embodiment, the antibodies or fragments thereof of the present invention include altered glycans. For example, the antibody may have a reduced number of fucose residues on the N-glycan at Asn297 of the Fc region, i.e., it is afucosylated. In another embodiment, the antibody may have an altered number of sialic acid residues on the N-glycan at Asn297 of the Fc region.

[0165] The invention also relates to immunoconjugates comprising an antibody conjugated to a cytotoxic agent, such as a toxin (eg, an enzymatically active toxin of bacterial, fungal, plant or animal origin, or fragments thereof) or a radioactive isotope (ie, a radioconjugate).

[0166] Enzymatically active toxins and fragments thereof that can be used include diphtheria A chain, non-binding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, Dianthus caryophyllus proteins, Phytolacca americana proteins (PAPI, PAPII and PAP-S), Momordica charantia inhibitor, curculin, crotonin, saponin, gelonin, mitomycin, restrictocin, phenomycin, enomycin and trichothecenes. A variety of radionuclides can be used to produce radioconjugated antibodies. Non-limiting examples include 212 Bi, 131 I. 131 In, 90 Y and 186 Re.

[0167] Conjugates of antibodies and cytotoxic agents are prepared using a variety of bifunctional protein coupling agents, such as N-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (e.g., dimethyl diimidoadipate HCL), active esters (e.g., disuccinimidyl suberate), aldehydes (e.g., glutaraldehyde), bis-azido compounds (e.g., bis(p-azidobenzoyl)hexanediamine), bis-diazo derivatives (e.g., bis-(p-diazobenzoyl)-ethylenediamine), diisocyanates (e.g., toluene 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 chelator for conjugating radionucleotides to antibodies (see PCT Publication No. WO 94 / 11026 and U.S. Pat. No. 5,736,137).

[0168] Those of ordinary skill in the art will appreciate that a wide variety of possible moieties may be coupled to the resulting antibodies or other molecules of the invention (see, e.g., "Conjugate Vaccines," in Contributions to Microbiology and Immunology, JM Cruse and RE Lewis, Jr (eds.), Carger Press, New York, (1989), the entire contents of which are incorporated herein by reference).

[0169] As long as the antibody and the other part retain their respective activities, coupling can be completed by any chemical reaction that can bind two molecules. The connection can include many chemical mechanisms, such as covalent bonding, affinity bonding, insertion, coordination bonding and complexing. In one embodiment, the binding is covalent bonding. Covalent bonding can be achieved by direct condensation of existing side chains or by incorporation of external bridging molecules. Many divalent or multivalent linking agents can be used to couple protein molecules (such as antibodies of the present invention) with other molecules. For example, representative coupling agents can include organic compounds, such as thioesters, carbodiimides, succinimide esters, diisocyanates, glutaraldehyde, diazobenzene and hexamethylenediamine. This list is not intended to exhaust various coupling agents known in the art, but is an example of more common coupling 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, e.g., 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 acetyl hydrazide derivatives coupled 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-dimethylamino-propyl) carbodiimide hydrochloride; (ii) SMPT (4-succinimidyloxycarbonyl-α-methyl-α-(2-pyridyl-dithio)-toluene (Pierce Chemical Company, Inc., MD)) Chem. Co.), catalog number (21558G)); (iii) SPDP (succinimidyl-6 [3-(2-pyridyldithio) propionamido] hexanoate (Pierce Chemical Company, catalog number #21651G)); (iv) Sulfo-LC-SPDP (sulfosuccinimidyl 6 [3-(2-pyridyldithio) propionamido] hexanoate (Pierce Chemical Company, catalog number #2165-G)); and (v) Sulfo-NHS (-hydroxysulfo-succinimide: Pierce Chemical Company, catalog number #24510), conjugated with EDC.

[0170] The joints described herein include components with different properties, 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. The solubility of joints containing NHS esters is not as good as sulfo-NHS esters. Further, the joint SMPT contains a sterically hindered disulfide bond and can form a conjugate with enhanced stability. Disulfide connections are generally less stable than other connections because disulfide connections are cut in vitro, resulting in fewer available conjugates. Sulfo-NHS can enhance the stability of carbodiimide coupling. When used in conjunction with sulfo-NHS, carbodiimide coupling (e.g., EDC) forms an ester that is more resistant to hydrolysis than a separate carbodiimide coupling reaction.

[0171] The antibodies disclosed herein can also be formulated as immunoliposomes. Liposomes containing antibodies are 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. Pat. Nos. 4,485,045 and 4,544,545. Liposomes with extended circulation time are disclosed in U.S. Pat. No. 5,013,556.

[0172] Non-limiting examples of useful liposomes can be generated by reverse phase evaporation using a lipid composition comprising phosphatidylcholine, cholesterol and PEG-derived phosphatidylethanolamine (PEG-PE). The liposomes are extruded through a filter of a determined pore size to produce liposomes with a preferred diameter. The Fab' fragments of the antibodies of the present invention can be conjugated to liposomes via a disulfide exchange reaction as described in Martin et al., J. Biol. Chem. [Journal of Biological Chemistry], 257: 286-288 (1982).

[0173] Multispecific antibodies (bispecific and trispecific)

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

[0175] A monospecific antibody is an antibody that has one or more binding sites that specifically bind a single antigen.

[0176] Multispecific antibodies are antibodies that can recognize two or more different antigens. For example, a bispecific antibody (bsAb) is an antibody comprising two variable domains or scFv units, so that the resulting antibody recognizes two different antigens. For example, a trispecific antibody (tsAb) is an antibody comprising two variable domains or scFv units, so 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 antigen and / or a third antigen. In one embodiment, the multispecific antibody (e.g., bispecific and trispecific antibodies) may comprise a CLDN4-specific fusion protein that encompasses the antibodies 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 with NCBI reference number NP_000873.2; IL-12B (p40 subunit) protein sequence with NCBI reference number NP_002178.2); IL-18 (protein sequence with NCBI reference number NP_001553.1); IL-15 (protein sequence with NCBI reference number NP_000576.1); 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)), cytokine cognate receptors (e.g., IL-12R), and cell surface receptors. Non-limiting examples of the 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 CLDN4 fusion proteins. For example, the fusion protein comprises an antibody comprising a variable domain or scFv unit and a ligand or antigen and / or a third ligand or antigen as described herein, 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) that can be used to design CLDN4 fusion proteins as described herein include, but are not limited to, anti-CAIX antibodies described in PCT / US2006 / 046350 and PCT / US2015 / 067178; anti-CXCR4 antibodies described in PCT / US20006 / 005691 and PCT / US2019 / 022272; anti-CCR4 antibodies described in PCT / US2008 / 088435, PCT / US2013 / 039744, and PCT / US2015 / 054202 ; Anti-PD-L1 antibodies described in PCT / US2008 / 088435 and PCT / US2020 / 062815; Anti-PD-1 antibodies described in PCT / US2020 / 037791 and PCT / US2020 / 037781; Anti-GITR antibodies described in PCT / US2017 / 043504; Anti-claudin-4 antibodies described in PCT / US2019 / 022272; and Anti-MUC1 antibodies described in PCT / US2020 / 037783 (each application 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 forms of multispecific antibodies (e.g., bispecific antibodies and trispecific antibodies, such as fusion proteins comprising antibodies that recognize CLDN4 and a ligand) are described herein.The ligand can be a tumor-associated antigen (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-CAIX, 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 also PCT / US2015 / 067225 and PCT / US2019 / 022272 for additional tumor-associated surface antigens, which are incorporated by reference in their entireties)); cytokines (e.g., IL-12 (IL-12A (p35 subunit) protein sequence with NCBI reference number NP_000873.2; IL-12B (p40 subunit) protein sequence with NCBI reference number NP_002178.2); IL-18 (protein sequence with NCBI reference number NP_001553.1); IL-15 (protein sequence with NCBI reference number NP_001553.1); 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, ICOSL, GITR, GITRL, TIGIT, CD27, VISTA, B7H3, B7H4, HEVM (or BTLA), CD4 7 and CD73. Different forms of bispecific antibodies or trispecific antibodies are also provided herein. In some embodiments, each 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 antibody or trispecific antibody further includes 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 antibody or trispecific antibody of the present invention may comprise a heavy chain and light chain combination or scFv of a CLDN4 antibody described herein.

[0177] The multispecific antibodies (e.g., bispecific antibodies 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, the bispecific antibody is a single polypeptide in which two scFv fragments are connected by a long linker polypeptide, the length of which is sufficient to allow intramolecular association between two scFv units to form an antibody. In other embodiments, the bispecific antibody is more than one polypeptide connected by covalent or non-covalent bonds. In some embodiments, the amino acid linker described herein (GGGGSGGGGS; "(G4S)2") can be generated using a longer G4S linker to improve flexibility. For example, a connector could also be: “(G4S)3” (for example, GGGGSGGGGSGGGGS); "(G4S)4" (e.g., GGGGSGGGGSGGGGSGGGGS); "(G4S)5" (e.g., GGGGSGGGGSGGGGSGGGGSGGGGS); "(G4S)6" (e.g., GGGGSGGGGSGGGGSGGGGSGGGGSGGGGS); "(G4S)7" (e.g., GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS); etc. For example, use of a (G4S)5 linker can provide greater flexibility for the ligands described herein and can improve expression. In some embodiments, the linker can also be (GS) n 、(GGS) n 、(GGGS) n 、(GGSG) n 、(GGSGG) n or (GGGGS) n , wherein 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. Patent No. 9,708,412; U.S. Patent Application Publication Nos. US20180134789 and US20200148771; and PCT Publication No. WO 2019051122 (each of which is incorporated by reference in its entirety).

[0178] In another embodiment, multispecific antibodies (e.g., bispecific antibodies and trispecific antibodies, such as anti-CLDN4-scFv fusions) can be constructed using the "knobs-in-holes" approach (Ridgway et al., Protein Eng 7:617-621 (1996)). In this approach, the Ig heavy chains of two different variable domains are reduced to selectively disrupt heavy chain pairing while retaining heavy chain-light chain pairing. Two heavy chain-light chain heterodimers recognizing two different antigens / ligands or three different antigens / ligands are mixed to promote heterologous junction pairing, which is mediated by the engineered "knobs-in-holes" of the CH3 domain.

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

[0180] The multispecific antibodies (e.g., bispecific antibodies and trispecific antibodies, such as anti-CLDN4-scFv fusions) of the present invention can be produced by introducing the R409 residue in the CH3 domain of an antibody that recognizes CLDN4 or a second and / or third antigen and introducing a Cys-Pro-Ser-Cys sequence in the hinge region of the antibody, so that the heavy chain-light chain dimer is exchanged to produce an antibody molecule having one heavy chain-light chain dimer that recognizes CLDN4 and a second heavy chain-light chain dimer that recognizes a second and / or third antigen, wherein the second and / or third antigen (or ligand) is any antigen (or ligand) disclosed herein. Known IgG4 molecules can also be altered so that the heavy chain and light chain recognize CLDN4 or a second and / or third antigen as disclosed herein. Due to the intrinsic characteristics of IgG4 molecules, it can be beneficial to use this method to construct the multispecific antibodies (e.g., bispecific antibodies and trispecific antibodies, such as anti-CLDN4-scFv fusions) of the present invention, in which the Fc region is different from other IgG subtypes in that it interacts poorly with the effector systems of the immune response (e.g., complement and Fc receptors expressed by certain leukocytes). This particular property makes these IgG4-based multispecific antibodies (e.g., bispecific antibodies and trispecific antibodies, such as anti-CLDN4-scFv fusions) attractive for therapeutic applications, in which the antibody needs to bind to one or more targets and functionally alter a signaling pathway associated with the one or more targets, but does not trigger effector activity.

[0181] The multispecific antibodies described herein (e.g., bispecific antibodies and trispecific antibodies, such as anti-CLDN4-scFv fusions) can be engineered with a non-depleting heavy chain isotype (e.g., 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. On the one hand, the multispecific antibodies (e.g., bispecific antibodies and trispecific antibodies, such as anti-CLDN4-scFv fusions) contain mutations on one scFv unit of the heterodimeric multispecific antibody, which reduces ADCC activity. On the other hand, the multispecific antibodies (e.g., bispecific antibodies and trispecific antibodies, such as anti-CLDN4-scFv fusions) contain mutations on both chains of the heterodimeric multispecific antibody, which completely eliminates ADCC activity. For example, the mutation introduced in one or two scFv units of a multispecific antibody (e.g., bispecific and trispecific antibodies, such as anti-CLDN4-scFv fusions) is a LALA mutation in the CH2 domain. These multispecific antibodies (e.g., bispecific and trispecific antibodies, such as anti-CLDN4-scFv fusions) with variable ADCC activity can be optimized so that the multispecific antibody exhibits maximum selective killing of cells expressing one antigen recognized by the multispecific antibody; however, minimal killing is exhibited for a second antigen recognized by the multispecific antibody.

[0182] The multispecific antibodies (e.g., bispecific antibodies) described herein can be engineered into modular tetrameric bispecific antibodies (tBsAbs). See, e.g., WO 2018 / 071913, which is incorporated herein by reference in its entirety. For example, a tetravalent antibody can be a dimer of a bispecific scFv fragment comprising a first binding site for a first antigen and a second binding site for a second antigen. In an embodiment, an anti-CLDN4 antibody can be a first binding site for a first antigen. In an embodiment, an anti-CLDN4 antibody can be a second binding site for a second antigen. The two binding sites can be connected together by a linker domain. In an embodiment, 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 an embodiment, the flank of the immunoglobulin hinge region amino acid sequence can be a flexible linker amino acid sequence, for example, having a linker amino acid sequence (GGGS) x1-6 、(GGGGS) x1-6Or GSAGSAAGSGEF. In an embodiment, the linker domain includes at least a portion of an immunoglobulin Fc domain (e.g., an IgG1, IgG2, IgG3, or IgG4 Fc domain). In an embodiment, at least a portion of the immunoglobulin Fc domain does not include a CH2 domain. In an embodiment, at least a portion of the immunoglobulin Fc domain may be a CH2 domain. Exemplary CH2 domain amino acid sequences include APELLGGPDVFLF (SEQ ID NO: 95). The Fc domain can be connected 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 may include a flexible linker amino acid sequence (e.g., (GGGS)) at one or both ends. x1-6 、(GGGGS) x1-6 or GSAGSAAGSGEF).

[0183] In an embodiment, the tBsAb may be 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 an embodiment, the multispecific antibody can 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 can include a CLDN4 antibody or a functional fragment thereof disclosed herein, and an antibody or fragment thereof that binds to a T cell antigen. For example, an antibody or fragment thereof that binds to a T cell antigen can be specific for CD3.

[0185] In an embodiment, the multispecific antibody can be a trispecific T cell engager (TriTE). The term "TriTE" (trispecific T cell engager) can 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 can include a CLDN4 antibody or a functional fragment thereof disclosed herein, and an antibody or fragment thereof that binds to a T cell antigen. For example, an antibody or fragment thereof that binds to a T cell antigen can be specific to CD3, CD28, or both.

[0186] The multispecific antibodies disclosed herein (eg, bispecific antibodies and trispecific antibodies, such as anti-CLDN4-scFv fusions) can be used to treat chronic infections, diseases or medical conditions, such as cancer.

[0187] Fusion Protein

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

[0189] In some embodiments, the CLDN4 antibody or its functional fragment disclosed herein is directly linked to a second protein. In other embodiments, the CLDN4 antibody or its functional fragment is linked to a second protein via a linker, such as a flexible polypeptide chain. The linker can be any suitable linker of any length, but the length can be at least 1, 2, 3, 4, 5, 10, 15, 20, 25 or 30 amino acids. In one embodiment, the linker is an amino acid sequence naturally present in the host's immunoglobulin molecule, such that the presence of the linker cannot cause a mammal to produce an immune response to the linker sequence. A fusion protein of the present invention that includes more than one additional protein in addition to the CLDN4 antibody may have multiple linker sequences connecting each additional protein or peptide sequence.

[0190] The fusion protein 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 can be introduced into an expression system to translate and produce a fusion protein. Alternatively, those skilled in the art can easily use de novo protein synthesis technology to produce the fusion protein described herein.

[0191] Use of antibodies against CLDN4

[0192] Antibodies of the invention that specifically bind to CLDN4 proteins or fragments thereof can be administered for the treatment of CLDN4-related diseases or disorders. "CLDN4-related diseases or disorders" include disease states and / or symptoms associated with disease states in which increased levels of CLDN4 and / or activation of cell signaling pathways involving CLDN4 are found. Exemplary CLDN4-related diseases or disorders include, but are not limited to, diseases in which T cells are suppressed, such as cancer and infectious diseases. In some embodiments, 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 gastric cancer. In embodiments, the cancer can be a CLDN4-related cancer. In embodiments, the CLDN-4-related cancer can be triple-negative breast cancer.

[0193] The antibodies of the present invention, including bispecific antibodies, polyclonal antibodies, monoclonal antibodies, humanized antibodies and fully human antibodies, can be used as therapeutic agents. Such agents can be used to treat cancer in a subject, improve vaccine efficiency or enhance natural immune responses. Antibody preparations, such as antibody preparations with high specificity and high affinity for their target antigens, are administered to a subject and will have an effect due to their binding to the target. Administration of antibodies can eliminate or inhibit or interfere with the activity of CLDN4 protein.

[0194] Pharmaceutical composition

[0195] Antibodies of the invention that specifically bind to CLDN4 protein or fragments thereof can be administered in the form of pharmaceutical compositions for the treatment of cancer. Principles and considerations involved in preparing therapeutic pharmaceutical compositions comprising antibodies and guidance for the selection of components are provided, for example, in Remington: The Science And Practice Of Pharmacy 20th Edition (Alfonso R. Gennaro, et al., eds.) 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 any patient will depend on a variety of factors, including the specific antibody used, its variant or derivative, the patient's age, weight, overall health, sex and diet, as well as the time of administration, excretion rate, drug combination and the severity of the disease being treated. The judgment of these factors by the medical caregiver is within the scope of those of ordinary skill in the art. The amount will also depend on the individual patient to be treated, the route of administration, the type of formulation, the characteristics of the compound used, the severity of the disease and the preferred effect. The amount used can be determined by pharmacological and pharmacokinetic principles well known in the art.

[0197] The therapeutically effective amount of the antibody of the present invention can be the amount required to achieve therapeutic purposes. As described herein, this can be the binding interaction between the antibody and its target antigen, in some cases, the binding interaction interferes with the function of the target. In addition, the amount to be applied will depend on the binding affinity of the antibody to its specific antigen, and will also depend on the rate at which the antibody applied is exhausted from the free volume of other subjects to which it is applied. The dosage of the antigen-binding polypeptides described herein applied to a subject (e.g., a patient) is about 0.1 mg / kg to 100 mg / kg of patient weight, 0.1 mg / kg to 20 mg / kg of patient weight, or 1 mg / kg to 10 mg / kg of patient weight. Due to the immune response to foreign polypeptides, human antibodies have a longer half-life in the human body than antibodies from other species. Therefore, it is generally possible to use lower doses of human antibodies and lower frequencies. Further, the dosage and frequency of the antibodies of the present disclosure can be reduced by enhancing the uptake of these antibodies and tissue permeability (e.g., entering the brain) (by modification such as, for example, lipidation). By way of non-limiting example, a typical range of therapeutically effective doses of the antibodies or antibody fragments of the invention may be from about 0.1 mg / kg body weight to about 50 mg / kg body weight. Typical dosing frequencies may be, for example, from twice a day to once a week.

[0198] In the case of using antibody fragments, it is preferred to specifically bind to the minimum inhibitory fragment of the target protein binding domain. For example, based on the variable region sequence of the antibody, a peptide molecule can be designed to retain the ability to bind to the target protein sequence. Such peptides can be produced by chemical synthesis and / or produced by recombinant DNA technology. (See, for example, Marasco et al., Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences of the United States], 90: 7889-7893 (1993)). The formulation can also contain more than one active compound according to the needs of the indication to be treated, such as those with complementary activities that do not adversely affect each other. Alternatively or in addition, the composition may include an agent that enhances its function, such as, for example, a cytotoxic agent, a cytokine (e.g., IL-15), a chemotherapeutic agent or a growth inhibitor. Such molecules are suitably present in an amount effective for the intended purpose.

[0199] The active ingredients can also be embedded in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, such as 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 for in vivo administration must be sterile. This can be readily accomplished by filtration through sterile filtration membranes.

[0201] Sustained release formulations can be prepared. Suitable examples of sustained release formulations include semipermeable matrices of solid hydrophobic polymers containing antibodies, which are in the form of shaped articles, such as films or microcapsules. Examples of sustained release matrices include polyesters, hydrogels (e.g., poly(2-hydroxyethyl-methacrylate) or poly(vinyl alcohol)), polylactic acid (U.S. Patent No. 3,773,919), copolymers of L-glutamic acid and γ-ethyl-L-glutamic acid, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers (e.g., LUPRON DEPOT TM (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 allow release of molecules for more than 100 days, some hydrogels release proteins for shorter durations.

[0202] The antibodies or agents of the present invention (also referred to herein as "active compounds") and their derivatives, fragments, analogs and homologues can be incorporated into pharmaceutical compositions suitable for administration. Such pharmaceutical compositions can comprise an antibody or agent and a pharmaceutically acceptable carrier. As used herein, the term "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents and absorption delaying agents, etc. that are compatible with drug administration. Suitable carriers are described in the latest edition of Remington's Pharmaceutical Sciences, which is a standard reference text in the field and is incorporated herein by reference. Non-limiting examples of such carriers or diluents include water, saline, Ringer's solution, dextrose solution and 5% human serum albumin. Liposomes and non-aqueous vehicles such as fixed oils can also be used. The use of such media and agents for pharmaceutically active substances is well known in the art. Unless any conventional media or agents are incompatible with the active compound, it is expected that they will be used in the composition. Supplementary active compounds can also be incorporated into these compositions.

[0203] The pharmaceutical composition of the present invention is formulated to be compatible with the intended route of administration. Examples of routes of administration include parenteral administration, such as intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (i.e., topical), transmucosal, and rectal administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous applications may include the following components: sterile diluents, such as water for injection, saline solutions, fixed oils, polyethylene glycols, glycerol, propylene glycol, or other synthetic solvents; antibacterial agents, such as benzyl alcohol or methyl parabens; antioxidants, such as ascorbic acid or sodium bisulfite; chelating agents, such as ethylenediaminetetraacetic acid (EDTA); buffers, such as acetates, citrates, or phosphates; and agents for adjusting tension, such as sodium chloride or dextrose. pH can be adjusted with an acid or base (such as hydrochloric acid or sodium hydroxide). Parenteral preparations may be packaged in ampoules, disposable syringes, or multidose 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 TM(BASF, Parsippany, NJ) or phosphate buffered saline (PBS). In an embodiment, the composition is sterile and fluid to the extent that it is easy to inject. It can be stable under the conditions of manufacture and storage and can prevent the contaminating effects of microorganisms (such as bacteria and fungi). The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, a polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, etc.) and a suitable mixture thereof. Appropriate fluidity can be maintained, for example, by using a coating such as lecithin, by maintaining the desired particle size in the case of a dispersion, and by using a surfactant. Prevention of the effects of microorganisms can be achieved by various antibacterial and antifungal agents (e.g., parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc.). In many cases, isotonic agents such as sugars, polyols (such as mannitol, sorbitol), and sodium chloride may be included in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.

[0205] Sterile injectable solutions can be prepared by incorporating the required amount of active compound into a suitable solvent as needed with one or a combination of the ingredients listed above, followed by sterilization by filtration. For example, dispersions are prepared by incorporating the active compound into a sterile vehicle containing a basic dispersion medium and other required ingredients from those listed above. In the case of sterile powders for the preparation of sterile injectable solutions, the preparation methods are vacuum drying and freeze drying, which produce a powder of the active ingredient plus any additional ingredients from its previously sterile filtered solution.

[0206] Oral compositions may include inert diluents or edible carriers. They may be encapsulated in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound may be incorporated with an excipient and used in the form of tablets, lozenges, or capsules. Oral compositions may also be prepared using a fluid carrier used as a mouthwash, wherein the compound in the fluid carrier is orally applied and swished and spit out or swallowed. Pharmaceutically compatible adhesives and / or adjuvant materials may be included as part of the composition. Tablets, pills, capsules, lozenges, etc. may contain any of the following ingredients or compounds with similar properties: adhesives, such as microcrystalline cellulose, tragacanth or gelatin; excipients, such as starch or lactose; disintegrants, such as alginic acid, Primogel or corn starch; lubricants, such as magnesium stearate or Sterotes; glidants, such as colloidal silicon dioxide; sweeteners, such as sucrose or saccharin; or flavorings, such as mint, methyl salicylate or orange flavoring.

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

[0208] Systemic administration can also be carried out by transmucosal or transdermal means. For transmucosal or transdermal administration, a penetrant suitable for the barrier to be penetrated is used in the formulation. Such penetrants are well known in the art and include, for example, detergents, bile salts, and fusidic acid derivatives for transmucosal administration. Transmucosal administration can be accomplished by using nasal sprays or suppositories. For transdermal administration, these active compounds are formulated into ointments, salves, gels, or creams (as known in the art).

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

[0210] In one embodiment, these active compounds are prepared together with a carrier that protects the compound from being quickly eliminated from the body, such as a controlled release formulation, including an implant and a microencapsulated delivery system. Biodegradable biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. The method for preparing such a formulation is obvious to those skilled in the art. These materials can also be commercially available from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes with monoclonal antibodies to viral antigens targeting infected cells) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those of ordinary skill in the art, for example, as described in U.S. Patent No. 4,522,811.

[0211] Oral or parenteral compositions may be formulated in dosage unit form for ease of administration and uniformity of dosage. As used herein, dosage unit form refers to physically discrete units suitable as single dosages for the subject to be treated; each unit contains a predetermined quantity of active compound calculated to produce the preferred therapeutic effect associated with the required pharmaceutical carrier. The specifications for the dosage unit forms of the present invention may be determined by and are directly dependent on the unique characteristics of the active compound and the therapeutic effect to be achieved, as well as the inherent limitations of the art in compounding such active compounds for treating individuals.

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

[0213] diagnosis

[0214] The antibodies according to the present invention can be used as agents 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 gastric cancer. For example, the antibody can contain a detectable label. The antibody can be a polyclonal antibody or a monoclonal antibody. The whole antibody or its fragment (e.g., F ab , scFv or F (ab)2 ). With respect to probes or antibodies, the term "labeled" may encompass direct labeling of the probe or antibody by coupling (i.e., physically connecting) a detectable substance to the probe or antibody, and indirect labeling of the probe or antibody by reacting with another directly labeled reagent. Examples of indirect labeling include detection of a primary antibody using a fluorescently labeled secondary antibody, and end-labeling of a DNA probe using biotin, so that detection can be performed using fluorescently labeled streptavidin. The term "biological sample" may include tissues, cells, and biological fluids separated from a subject, as well as tissues, cells, and fluids present in a subject. Therefore, the use of the term "biological sample" includes blood and fractions or components of blood, including serum, plasma, or lymph. That is, the detection method of the present invention may be used for in vitro and in vivo detection of analyte mRNA, protein, or genomic DNA in a biological sample. For example, in vitro techniques for detecting analyte mRNA include RNA hybridization and in situ hybridization. In vitro techniques for detecting analyte proteins include enzyme-linked immunosorbent assay (ELISA), protein blotting, immunoprecipitation, and immunofluorescence. In vitro techniques for detecting analyte genomic DNA include DNA hybridization.

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

[0216] Antibodies against CLDN4 protein (or fragments thereof) can be used in methods known in the art involving localization and / or quantification of CLDN4 protein (e.g., for measuring the level of CLDN4 protein in an appropriate physiological sample, for diagnostic methods, for imaging the protein, etc.). In a given embodiment, antibodies specific for CLDN4 protein or derivatives, fragments, analogs or homologs thereof containing an antibody-derived antigen binding domain are used as pharmacologically active compounds (referred to herein as "therapeutic agents").

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

[0218] Detection can be facilitated by coupling (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, dichlorotriazineamine 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.

[0219] Screening methods

[0220] The present invention provides methods for identifying modulators, i.e., candidate or test compounds or agents (e.g., peptides, peptidomimetics, small molecules or other drugs) that modulate or otherwise interfere with CLDN4 activity (also referred to herein as "screening assays"). Methods for identifying compounds that can be used to treat 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 CLDN4 expression and / or activity. The test compounds of the present invention can be obtained using any of a variety of approaches in combinatorial library methods known in the art, including but not limited to: biological libraries; spatially addressable parallel solid phase or solution phase libraries; synthetic library methods requiring deconvolution; "one bead one compound" library methods; and synthetic library methods using affinity chromatography selection. The biological library approach is limited to peptide libraries, while 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 [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 and most preferably less than about 4 kD. A small molecule may be, for example, a nucleic acid, a peptide, a polypeptide, a peptide mimetic, a carbohydrate, a lipid, or other organic or inorganic molecule. Libraries of chemical and / or biological mixtures (e.g., fungal, bacterial, or algal extracts) are known in the art and may be screened using any assay of the invention.

[0223] Examples of methods for synthesizing molecular libraries can be found in the art, for example in: DeWitt, et al., 1993. Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences of the United States of America] 90:6909; Erb, et al., 1994. Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences of the United States of America] 91:11422; Zuckermann, et al., 1994. J. Med. Chem. [Journal of Medicinal Chemistry] 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 presented in solution (see, e.g., Houghten, 1992. Biotechniques 13:412-421), or on beads (see Lam, 1991. Nature 354:82-84), on chips (see Fodor, 1993. Nature 364:555-556), in bacteria (see U.S. Pat. No. 5,223,409), in spores (see U.S. Pat. No. 5,233,409), in plasmids (see Cull, et al., 1992. Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences of the United States of America] 89:1865-1869) or on bacteriophage (see Scott and Smith, 1990. Science [Science] 249:386-390; Devlin, 1990. Science [Science] 249:404-406; Cwirla, et al., 1990. Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences of the United States of America] 87:6378-6382; Felici, 1991. J. Mol. Biol. [Journal of Molecular Biology] 222:301-310; and U.S. Patent No. 5,233,409).

[0225] In one embodiment, a candidate compound is introduced into an antibody-antigen complex and it is determined whether the candidate compound disrupts the antibody-antigen complex, wherein disruption of the complex indicates that the candidate compound modulates CLDN4 activity.

[0226] In another embodiment, at least one CLDN4 protein is provided, which 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 disrupted after the introduction of the one or more candidate compounds, the candidate compound can be used to treat cancer or a proliferative disease or disorder.

[0227] Determine that the ability of test compound to interfere or destroy antibody-antigen complex can be realized by, for example, coupling the test compound with radioisotope or enzyme labeling, so that the combination of the test compound and antigen or its biologically active part can be determined by the labeled compound in the detection complex. For example, 125r, 35S, 14C or 3H can be used to directly or indirectly label the test compound, and the radioisotope is detected by direct counting of radio radiation or by scintillation counting. Alternatively, the test compound can be enzymatically labeled with, for example, horseradish peroxidase, alkaline phosphatase or luciferase, and the enzyme labeling is detected by determining the conversion of suitable 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 otherwise disrupt an 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 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. Additionally, the antigen may be a CLDN4 protein or a portion thereof.

[0231] The screening methods disclosed herein can be performed as cell-based assays or cell-free assays. In the case of a cell-free assay comprising a membrane-bound form of the CLDN4 protein, it is desirable to use a solubilizing agent such that the membrane-bound form of the protein is maintained in solution. Examples of such solubilizing agents include non-ionic detergents such as n-octyl glucoside, n-dodecyl glucoside, n-dodecyl maltoside, octanoyl 1-N-methyl glucoside, decanoyl-N-methyl glucoside, X-100, X-114, Isotridecyl poly(ethylene glycol ether) n, N-dodecyl 1--N,N-dimethyl 1-3-ammonio-1-propane sulfonate, 3-(3-cholamidopropyl) dimethylamino alcohol (dimethylamminiol)-1-propane sulfonate (CHAPS) or 3-(3-cholamidopropyl) dimethylamino alcohol-2-hydroxy-1-propane sulfonate (CHAPSO).

[0232] In more than one embodiment, it is desirable to fix the antibody or antigen to facilitate the separation of the composite form of one or both of the antibody or antigen from the non-complex form after the candidate compound is introduced, and to adapt to the automation of the assay. The observation of the antibody-antigen complex in the presence and absence of the candidate compound can be completed in any container suitable for containing the reactant. The example of such a container includes a microtiter plate, a test tube, and a microcentrifuge tube. In one embodiment, a fusion protein can be provided, which adds one or both of the domains that allow the protein to be bound to the matrix. For example, GST-antibody fusion protein or GST-antigen fusion protein can be adsorbed onto glutathione agarose beads (Sigma Chemical, St. Louis, Missouri) or glutathione derivatization microtiter plates, then combined with the test compound, and the mixture is incubated under conditions conducive to the formation of the complex (for example, under the physiological conditions of salt and pH). After incubation, wash the beads or microtiter plate wells to remove any unbound components, fix the matrix in the case of beads, and directly or indirectly determine the complex. 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 fixing proteins on a matrix can also be used in the screening assay of the present invention. For example, antibodies or antigens (e.g., CLDN4 proteins) can be fixed using the conjugation of biotin and streptavidin. Biotinylated antibodies or antigen molecules can be prepared from biotin-NHS (N-hydroxy-succinimide) using techniques well known in the art (e.g., biotinylation kit, Pierce Chemicals, Rockford, Illinois) and fixed in the wells of a 96-well plate (Pierce Chemicals) coated with streptavidin. Alternatively, other antibodies that react with the target antibody or antigen but do not interfere with the formation of the target antibody-antigen complex can be derivatized to the wells of the plate, and unbound antibodies or antigens can be trapped in the wells by antibody conjugation. In addition to the method for fixing the complex for GST described herein, the method for detecting such complexes also includes immunodetection of the complex using such other antibodies that react with the antibody or antigen.

[0234] The present invention further relates to novel agents identified by any of the screening assays described herein and their use in the treatments described herein.

[0235] Chimeric Antigen Receptor (CAR) Cell Therapy

[0236] Cell therapy, such as chimeric antigen receptor (CAR) cell therapy, is also provided herein. For example, the cell can be a CART cell or a CAR NK cell.

[0237] For example, CAR cell therapy redirects the patient's T cells and / or NK cells to kill tumor cells through exogenous expression of CAR on T cells or NK cells. CAR can be a transmembrane fusion protein that connects the antigen recognition domain of the antibody to the intracellular signaling domain of the T cell receptor and the auxiliary receptor or NK cell receptor.

[0238] In one embodiment, a monospecific CAR cell is provided. For example, the anti-CLDN4 antibodies described herein can be used as the targeting portion of the CAR cell. For example, the CLDN4 antibody has low affinity but high avidity for its antigen. In another example, the CLDN4 antibody may have high affinity but low avidity for its antigen. Antibodies with fewer binding sites may have high affinity and low avidity, while antibodies with more binding sites may have low affinity and high avidity.

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

[0240] In an embodiment, the anti-CLDN4 antibody or CLDN4 fusion protein described herein can be used as a payload for armored CAR cell therapy. Suitable cells can be used, for example, which can secrete the anti-CLDN4 antibody of the present invention (or alternatively engineered to express the anti-CLDN4 antibody to be secreted as described herein). The anti-CLDN4 "payload" to be secreted can be, for example, a mini antibody, scFv, IgG molecule, bispecific fusion molecule, and other antibody fragments described herein. After contact or engineering, the cells described herein can then be introduced into patients who need to be treated by infusion therapy known to those skilled in the art.

[0241] In an embodiment, the patient may suffer from a CLDN4-related disease or disorder as described herein, such as cancer. Cells (e.g., T cells) may be, for example, T lymphocytes, CD4+T cells, CD8+T cells, or a combination thereof, but are not limited thereto. Exemplary CARs and CAR production platforms that can be used in various 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, the CLDN4 antibodies discussed herein can be used in constructing multispecific antibodies or as a payload for CAR-T cells or CAR NK cells. For example, in one embodiment, the anti-CLDN4 antibodies discussed herein can be used for targeting of CAR (i.e., as a targeting moiety). In another embodiment, the anti-CLDN4 antibodies discussed herein can be used as a targeting moiety, and different CLDN4 antibodies targeting different epitopes can be used as payloads. In another embodiment, the payload can be an immunomodulatory antibody payload.

[0242] Solid tumors bring unique challenges to CAR-T therapy. Some obstacles to the effectiveness of CAR-T in solid tumors include heterogeneous antigen expression, insufficient tissue homing, activation, persistence and immunosuppressive tumor microenvironment. Unlike blood cancers, tumor-associated target proteins are overexpressed between tumors and healthy tissues, resulting in T cell killing of healthy tissues outside the target / tumor. In addition, immunosuppression in the tumor microenvironment (TME) limits the activation of CAR-T cells towards killing tumors. After such contact or engineering, cells can then be introduced into cancer patients who need to be treated by infusion therapy known to those skilled in the art. Cancer patients can suffer from any type of cancer disclosed herein. Cells (e.g., T cells) can be, for example, tumor-infiltrating T lymphocytes, CD4+T cells, CD8+T cells, or a combination thereof, but are not limited thereto.

[0243] In an embodiment, CAR cells (ie, CAR T cells or CAR NK cells) can be generated using a lentiviral system (via transduction), a retroviral system (via transfection (electroporation)) and a transposon system (via PiggyBac) according to methods known in the art. Promoters useful for the payload used in generating CAR-T include, for example, a constitutive promoter (wherein the promoter is the same as the promoter for CAR-T, such as EF1a, followed by IRES or 2A); an inducible promoter (the promoter is different from the promoter for CAR-T, such as NFAT, IL-2prom); and a genetically engineered promoter (eg, a promoter "knocked in" of the CLDN4 locus of a cytokine and / or under the control of an endogenous promoter). In one embodiment, the CLDN4 antibody or CLDN4 fusion protein discussed herein can be used in constructing a multispecific antibody or as a payload of a CAR cell. For example, in one embodiment, the anti-CLDN4 antibody or CLDN4 fusion protein discussed herein can be used for targeting of CARS (ie, as a targeting moiety). In one embodiment, the anti-CLDN4 antibody or CLDN4 fusion protein discussed herein can be used as a payload secreted by CAR cells. In another embodiment, the anti-CLDN4 antibody or CLDN4 fusion protein discussed herein can be used as a targeting portion, and different CLDN4 antibodies targeting different epitopes can be used as a payload. In another embodiment, the payload can be an immunomodulatory antibody payload. In some embodiments, the CLDN4 antibody or CLDN4 fusion protein used in the CAR-T composition as described herein is not a high-affinity CLDN4 antibody (e.g., so that the antibody does not strongly bind to its CLDN4 target). For example, the CLDN4 antibody or CLDN4 fusion protein described herein can be used as a payload secreted by CAR cells, wherein there are two targeting portions (e.g., tumor-associated surface antigens) selected for a specific 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, 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 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.

[0244] Technicians should recognize that CAR cells can be generated by cell sources known to technicians. 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), cord blood cells, cell lines (e.g., NK92 cell lines), 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. [From CAR-T cells to CAR-NK cells: a developing method for immunotherapy of blood malignancies]" Frontiers in Oncology [oncology frontiers] (2021): 3151.

[0245] Chimeric B cell receptor

[0246] Modified B cell receptors, such as B cell receptors containing antibodies or antibody fragments previously selected by high affinity for specific disease-related antigens, are powerful new ways to fight disease. Because B cells act as specialized antigen-presenting cells, they can process antigens and present antigens on MHC class II molecules, enhance immune cell recognition of tumors and assist in the spread of new antigens. As a key component of immune memory, chimeric antibody signaling and secretion (CASS) type B cells will simultaneously recruit a wide range of immune cells and reverse tumor-infiltrating lymphocyte exhaustion, thereby providing a robust and lifelong surveillance program to prevent tumor metastasis and recurrence. In an embodiment, B cells may include chimeric, non-natural and at least partially artificially engineered receptors. In some cases, the engineered chimeric B cell receptor has one, two, three, four or more components, and in some embodiments, the one or more components promote B cell targeting or binding to one or more cells containing antigens.

[0247] Aspects of the present invention include genetically engineered B cells that are modified to express and carry a chimeric B cell receptor on their surface. In an embodiment, the genetically modified B cells may include a single chimeric B cell receptor targeting an antigen (e.g., CLDN4), or a single chimeric B cell receptor targeting two or more antigens (e.g., a bispecific chimeric B cell receptor or a multispecific chimeric B cell receptor). In some embodiments, the cell includes a split chimeric B cell receptor, such as two different scFvs with different costimulatory domains expressed on the surface of the B cell. Further, some embodiments include a fine-tuned chimeric B cell receptor.

[0248] In embodiments, the chimeric B cell receptor comprises an extracellular domain, a transmembrane domain, and an intracellular signaling domain; such that 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 a target cell 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 an embodiment, the extracellular ligand binding domain may comprise an antigen binding domain or antigen recognition domain derived from an antibody directed against an antigen of a target (eg, an anti-CLDN4 antibody described herein). In an embodiment, the extracellular ligand binding domain may comprise an antibody or fragment thereof described herein.

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

[0252] The signaling domain or intracellular signaling domain of the chimeric B cell receptor of the present invention is responsible for intracellular signaling after the extracellular ligand binding domain binds to the target, resulting in the activation of immune cells and immune responses. In other words, the signal transduction domain is responsible for the activation of at least one normal function of the B cell expressing the chimeric B cell receptor. Therefore, the term "signaling domain" can refer to the part of the protein that guides the cell to perform a specialized function (such as the early activation of Lyn and Syk and the late activation of NFAT and NFκB as examples).

[0253] Chimeric B cell receptors may comprise a native transmembrane domain and an intracellular domain. In native B cells, engagement of the B cell receptor results in rapid tyrosine phosphorylation and calcium ion polarization of the intracellular domain, leading to downstream activation of NFAT and NF-kB. Using NFAT / NF-kB response elements to drive expression of our secretory proteins, we have designed an inducible expression system that will be activated by antigens associated with disease states (e.g., cancer).

[0254] The salient features of suitable transmembrane polypeptides include the ability to be expressed on the surface of immune cells (e.g., B cells) and interact together to direct the cellular response of immune cells to predetermined target cells. Different transmembrane polypeptides of chimeric B cell receptors comprising extracellular ligand binding domains and / or signal transduction domains interact together to participate in signal transduction after binding to the target ligand and induce an immune response. The transmembrane domain can be derived from a natural source or a synthetic source. The transmembrane domain can be derived from any membrane-bound protein or transmembrane protein.

[0255] Treatment

[0256] As used herein, the term "treat" or "treatment" refers to both therapeutic treatment, and prophylactic or preventive measures, wherein the goal is to prevent or slow down (reduce) unsought physiological changes or disorders, such as the progression of cancer. Beneficial or preferred clinical results include, but are not limited to, alleviation of symptoms, reduction in the extent of the disease, a stable state of the disease (i.e., not worsening), a delay or slowing of disease progression, an improvement or slowing of the disease state, and relief (whether partial or overall), whether these results are detectable or undetectable. "Treatment" may mean prolonged survival compared to the expected survival if not receiving treatment. Those in need of treatment include those already suffering from a condition or disorder, as well as those susceptible to a condition or disorder, or those in which a condition or disorder is to be prevented.

[0257] The present invention provides preventive and therapeutic methods for treating subjects at risk (or susceptible to cancer) for cancer (e.g., if an early detection cancer biomarker is identified in such a subject) or subjects at risk for other cell proliferation-related diseases or disorders. Such diseases or disorders include, but are not limited to, those associated with, for example, abnormal expression of CLDN4 and / or abnormal activation of cell signaling pathways involving CLDN4. Such diseases or disorders are included in CLDN-related diseases or disorders. For example, the method is used to treat, prevent or alleviate the symptoms of cancer. In an 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 by 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. In addition, the methods of the present invention can be used to treat hematological cancers, such as leukemias and lymphomas. 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 also be treated or prevented or whose symptoms can be alleviated include those solid tumors with high mutation load and WBC in the filtrate.

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

[0259] Subjects at risk for cancer or a cell proliferation-related disease or disorder may include patients with a family history of cancer or subjects exposed to known or suspected carcinogens. Administration of a prophylactic agent may occur prior to the manifestation of cancer, thereby preventing the disease or alternatively delaying its progression.

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

[0261] The present invention further provides a preventive and therapeutic method for treating a subject at risk of chronic or acute viral, bacterial or parasitic infection (or susceptible to chronic or acute viral, bacterial or parasitic infection). The present invention also provides a therapeutic method for treating a subject at risk of a disease or disorder or condition associated with T cell exhaustion or at risk of T cell exhaustion. The present invention also provides a therapeutic method for treating a subject at risk of a disease or disorder or condition associated with T cell exhaustion or at risk of T cell exhaustion. 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 those caused by, for example, hepatitis B virus (HBV), hepatitis C virus (HCV), herpes simplex virus 1 (HSV-1), Helicobacter pylori or Toxoplasma. Other acute infections included are those caused by, for example, microorganisms, such as Gram-positive bacteria, Gram-negative bacteria, protozoa or fungi as described herein.

[0262] The present invention also includes methods for increasing or enhancing the immune response to an antigen. Increase or enhance response immunity by administering the monoclonal antibody, scFv antibody or bispecific antibody of the present invention to a subject. For example, enhance response immunity by enhancing antigen-specific T effector function. Antigen is a virus (e.g., HIV), bacteria, parasite or tumor antigen. The immune response is a natural immune response. The natural immune response refers to an immune response caused by infection. Infection is a chronic infection. Increasing or enhancing the immune response to an antigen can be measured by a variety of methods known in the art. For example, an immune response can be measured by measuring any of the following: T cell activity, T cell proliferation, T cell activation, the generation of effector cytokines and T cell transcriptional profiles. Alternatively, the immune response is a response due to vaccination.

[0263] Therefore, in another aspect, the present invention provides a method for increasing vaccine efficiency by administering a monoclonal antibody or scFv antibody of the present invention and a vaccine to a subject. The antibody and the vaccine are administered sequentially or in parallel. The vaccine is a tumor vaccine, a bacterial vaccine or a viral vaccine.

[0264] Combination Methods

[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 disclosed herein include, but are not limited to, antibiotic derivatives (e.g., doxorubicin, bleomycin, daunorubicin, and actinomycin); 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, cytarabine, cyclophosphamide, estramustine, hydroxyurea, procarbaz ... hydrazine, mitomycin, busulfan, cisplatin, and vincristine sulfate); hormones (e.g., medroxyprogesterone, estramustine sodium phosphate, ethinyl estradiol, estradiol, megestrol acetate, methyltestosterone, diethylstilbestrol diphosphate, chlorethoxyquin and testolactone); nitrogen mustard derivatives (e.g., melphalan, chlorambucil, mechlorethamine (nitrogen mustard), and thiotepa); steroids and combinations thereof (e.g., betamethasone sodium phosphate); and others (e.g., dacarbazine, asparaginase, mitotane, vincristine sulfate, vinblastine sulfate, and etoposide).

[0266] In other embodiments, the compositions of the present invention described herein can be administered in combination with cytokines. Cytokines that can be administered with the compositions include, but are 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 further embodiments, the compositions described herein may be administered in combination with other therapeutic or prophylactic regimens, such as radiation therapy.

[0268] In some embodiments, the compositions described herein can be administered in combination with other immunotherapeutic agents. Non-limiting examples of immunotherapeutic agents include cintuzumab, abavotumab, adetumumab, atozumab, alemtuzumab, atumomab, atoximab, maanatumomab, acitumomab, baviximab, betumomab, bevacizumab, bivacizumab, binatoumab, brentuximab, cantuzumab, catumaxomab, cetuximab, situximab, citutumumab, clivatuzumab, conatumumab, daratumumab, drozitumab, duligotumab, dusig itumab), demuximab, daclizumab, dalotuzumab, emeticholic acid, elotuzumab, ensituximab, ertuinomab, daclizumab, farletuzumab, ficlatuzumab, figitumumab, flanvotumab, futuximab, ganitumab, gemtuzumab, gemtuzumab, glembatumumab, ibrimoduzumab monoclonal antibody, igovomab, imgatuzumab, indatuximab, inotuzumab, intumumab, ipilimumab, itumumab, labetuzumab, lexatumumab, lintuzumab, lorvotuzumab, lucatumumab, mapatumumab, matuzumab, milatuzumab, mitrazumab, moxetumomab, narnatumab, necitumumab ), nimotuzumab, nofetumomab, ocaratuzumab, ofatumumab, olaratumab, onartuzumab, oportuzumab, oregovomab, penicillin, parsatuzumab, patritumab, pemtumomab, pertuzumab, pintuzumab, protumumab, racotumomab, radretumab,Rilotumumab, rituximab, rotuximab, satumumab, cilocumab, siltuximab, solitomab, tacatuzumab, taplitumomab, tenatumomab, teprotumumab, gacilizumab, tositumomab, trastuzumab, tucotuzumab, ublituximab, veltuzumab, vorsetuzumab, votumumab, zalutumumab, CC49, 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 of a CLDN4 protein or, alternatively, to two different epitopes of a 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 other embodiments, cancer can be treated by administering a bispecific antibody that binds to CLDN4 and to a protein other than CLDN4. For example, another protein other than CLDN4 may 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, another protein other than CLDN4 is a tumor-associated antigen; another protein other than CLDN4 may also be a cytokine. Non-limiting examples of another protein 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 an anti-PD-1 antibody alone or in combination with: an additional antibody that recognizes another protein other than CLDN4, and cells that can affect or enhance an immune response. For example, these cells can be melanoma peripheral blood mononuclear cells (PBMCs), or any cell type found in PBMCs, such as cytotoxic T cells, macrophages, and natural killer (NK) cells.

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

[0272] Diagnostic tests

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

[0274] In some embodiments, an anti-CLDN4 antibody of the invention is linked to a detectable moiety for diagnostic purposes, eg, to provide a method for detecting cancer cells in a subject at risk for or suffering from cancer.

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

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

[0277] In the case of a "targeted" conjugate (i.e., a conjugate containing a targeting moiety (the targeting moiety is a molecule or feature designed to localize the conjugate to a specific site or sites in the body of a subject or animal)), localization can refer to a state when equilibrium has been substantially achieved between bound, "localized" entity and unbound, "free" entity in the subject. The speed with which such equilibrium is achieved depends on the route of administration. For example, a conjugate administered by intravenous injection can achieve localization within a few minutes of injection. On the other hand, oral administration of the conjugate may require several hours to achieve localization. Alternatively, localization can simply refer to the location of the entity in the body of a subject or animal within a selected period of time following administration of the entity. As another example, localization is achieved when the moiety becomes distributed following administration.

[0278] Those skilled in the art can make reasonable estimates of the time to achieve localization. In addition, the change of the localization state over time can be tracked by imaging the detectable part (e.g., luminescent conjugate) according to the method of the present invention, for example, using a photodetector device. The "photodetector device" used can have a high enough sensitivity to allow imaging of weak light from the mammal in a reasonable time, and use the signal from such a device to construct an image.

[0279] In cases where extremely bright light-producing moieties can be used and / or light-producing fusion proteins located near the surface of the subject or animal being imaged can be detected, a pair of "night vision" goggles or a standard high-sensitivity camera, such as a silicon intensified tube (SIT) camera (e.g., from 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 imaged scene no longer appears continuous. Instead, it is represented by individual photons that are distinct from each other in time and space. When viewed on a monitor, such an image appears as flashing 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 has no meaning. The goal is to simply detect the presence of a signal (photons) and count its occurrences based on its position over time.

[0281] At least two types of photodetector devices described herein can detect single photons and generate signals that can be analyzed by an image processor. Noise reduction photodetector devices achieve sensitivity by reducing background noise in the photon detector (rather than amplifying the photon signal). Noise is reduced primarily by cooling the detector array. These devices include CCD cameras known as "back-thinned" cooled charge-coupled device (CCD) cameras. In more sensitive instruments, cooling can be achieved using, for example, liquid nitrogen, so that the temperature of the CCD array reaches approximately -120°C. "Back-thinned" refers to an ultra-thin backplane that reduces the length of the path followed by the photons to be detected, thereby improving quantum efficiency. A sensitive back-thinned cryogenic CCD camera is the "TECH 512," a 200 series camera available from Photometries, Ltd. (Tucson, Arizona).

[0282] "Photon amplification devices" amplify photons before they reach the detection screen. This category includes CCD cameras with intensifiers such as microchannel intensifiers. Microchannel intensifiers consist of an array of metal channels perpendicular to and coextensive with the camera's detection screen. The microchannel array is placed between the sample, subject, or animal to be imaged and the camera. Most photons that enter a channel of the array contact one side of the channel before exiting. A voltage applied to the array causes many electrons to be released per photon collision. These electrons from the collisions leave their originating channel in a "shotgun" pattern and are detected by the camera.

[0283] Even higher sensitivity can be achieved by placing an array of intensifying microchannels in series so that electrons generated in the first stage in turn produce an amplified electron signal in the second stage. However, the increase in sensitivity comes at the expense of spatial resolution, which decreases with each additional stage of amplification. An exemplary microchannel intensifier-based single-photon detection device is the C2400 series available from Hamamatsu.

[0284] The image processor processes the signal generated by the photodetector device, which counts photons to construct an image that can be displayed on a monitor or printed on a video printer, for example. Such an image processor is sold as part of a system including a sensitive photon counting camera as described herein, and can therefore be obtained from the same source. The image processor is connected to a personal computer, such as an IBM compatible PC or an Apple Macintosh computer (Apple Computer, Cupertino, California), which may or may not be included as part of the purchased imaging system. Once the images are in the form of digital files, they can be manipulated and printed by various image processing programs (e.g., "ADOBE PHOTOSHOP", Adobe Systems, Adobe Systems, Mountain View, California).

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

[0286] The present invention also encompasses a kit 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) that can detect cancer or tumor cells in a biological sample; a means for determining the amount of CLDN4 in a sample; and a means for comparing the amount of CLDN4 in a sample with a standard. In some embodiments, the standard is a non-cancerous cell or a cell extract thereof. The compound or agent may 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] Although the invention has been described in conjunction with specific embodiments thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages and modifications are within the scope of the following claims.

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

[0290] Provide example below to help more fully understand the present invention.The following example illustrates the exemplary mode of making and practicing the present invention.However, the scope of the present invention is not limited to the specific embodiment disclosed in these examples, and these examples are only for illustrative purposes, because alternative methods can be utilized to obtain similar results. Example 1 - Claudin 4 as a therapeutic target

[0291] We identified two anti-claudin4 scFvs (Gly1-4-G3 and Gly1-2-F4) by whole-cell panning. We performed three rounds of panning, alternating between 293T and Cf2Th cells transduced to stably express CLDN4. Our scFvs showed minimal cross-reactivity with the structurally related protein claudin3.

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

[0293] These scFvs can be developed as monoclonal antibodies for therapeutic or diagnostic purposes. In addition, they can also be used as targeting moieties for CAR T cells against cancers that overexpress CLND4. Example 2

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

[0295] - Low to no background binding of the antibody to 293T in the absence of CLDN-3 or CLDN-4

[0296] – CLDN-3 antibodies specifically recognize only CLDN-3 cell lines

[0297] – CLDN-4 antibody weakly binds to CLDN-3 cell lines

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

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

[0300] 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. Such equivalents are considered to be within the scope of the invention and are covered 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, the antibody or antigen-binding fragment thereof comprising a heavy chain, a light chain, or both a heavy chain and a light chain, The heavy chain comprises a HCDR1 comprising GFTFNNYA (SEQ ID NO: 9), GFTFGGYA (SEQ ID NO: 12), GGTFSSYA (SEQ ID NO: 15) or GGTFNNYA (SEQ ID NO: 18), A HCDR2 comprising IRDSGGST (SEQ ID NO: 10), LSNSGSNA (SEQ ID NO: 13) or IIPIVDIA (SEQ ID NO: 16), a HCDR3 comprising ARRGYSSSWYGDGYYYGMDV (SEQ ID NO: 11), ARAVMSSSWYMRRYYYYYMDV (SEQ ID NO: 14) or ARGGSQGAYYMDV (SEQ ID NO: 17), or a combination of its CDRs; and The light chain comprises LCDR1 comprising SGSIASSF (SEQ ID NO: 19), RSNIGSNT (SEQ ID NO: 22), SGSIASNY (SEQ ID NO: 25) or QSVSNY (SEQ ID NO: 28), a LCDR2 comprising ENN (SEQ ID NO: 20), SNN (SEQ ID NO: 23), EDN (SEQ ID NO: 26) or GAS (SEQ ID NO: 29), a 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 their CDRs.

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), the therapeutic antibody comprising a variable domain and a constant domain, wherein the constant domain is IgG and the variable domain comprises a framework region and a complementarity determining means for binding to claudin 4. 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, the isolated 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 VLCDR1 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 VHCDR2 comprising the amino acid sequence of SEQ ID NO: 13, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 14, a VLCDR1 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 VLCDR1 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 VHCDR2 comprising the amino acid sequence of SEQ ID NO: 16, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 17, a VLCDR1 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.

7. An isolated scFv antibody that binds to human claudin 4 protein, the isolated scFv antibody 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 VLCDR1 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 VHCDR2 comprising the amino acid sequence of SEQ ID NO: 13, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 14, a VLCDR1 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 VLCDR1 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 VHCDR2 comprising the amino acid sequence of SEQ ID NO: 16, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 17, a VLCDR1 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.

8. An isolated antibody or fragment thereof that binds to human claudin 4 protein, the isolated antibody or fragment thereof comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising an amino acid sequence selected from the group consisting of: SEQ ID NO: 1, 3, 5 and 7, or a sequence at least 90% identical thereto, the light chain variable region comprising an amino acid sequence selected from the group consisting of: SEQ ID NO: 2, 4, 6 and 8, or a sequence at least 90% identical thereto.

9. An isolated scFv that binds to human claudin 4 protein, the isolated scFv comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising an amino acid sequence selected from the group consisting of: SEQ ID NO: 1, 3, 5 and 7, or a sequence at least 90% identical thereto, and the light chain variable region comprising an amino acid sequence selected from the group consisting of: SEQ ID NO: 2, 4, 6 and 8, or a sequence at least 90% identical thereto.

10. An F(ab) that binds to claudin 4, the F(ab) 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 VLCDR1 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 VHCDR2 comprising the amino acid sequence of SEQ ID NO: 13, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 14, a VLCDR1 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 VLCDR1 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 VHCDR2 comprising the amino acid sequence of SEQ ID NO: 16, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 17, a VLCDR1 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.

11. A VhH that binds to claudin 4, the VhH 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, 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) 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, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO:

17.

12. An isolated bispecific antibody comprising a fragment as claimed in any one of claims 1 to 11 and a second antigen binding fragment specific for a molecule on an immune cell.

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. 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. 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, the BiTE 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 VLCDR1 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 VHCDR2 comprising the amino acid sequence of SEQ ID NO: 13, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 14, a VLCDR1 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 VLCDR1 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 VHCDR2 comprising the amino acid sequence of SEQ ID NO: 16, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 17, a VLCDR1 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.

17. A bispecific T cell engager (BiTE) that binds to human claudin 4 protein, the BiTE comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising an amino acid sequence selected from the group consisting of: SEQ ID NO: 1, 3, 5 and 7, or a sequence at least 90% identical thereto, and the light chain variable region comprising an amino acid sequence selected from the group consisting of: SEQ ID NO: 2, 4, 6 and 8, or a sequence at least 90% identical thereto.

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

19. A nucleic acid encoding the antibody of any one of claims 1 to 11, wherein: 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; and 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.

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

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

22. The pharmaceutical composition of claim 21, 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 VLCDR1 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 VHCDR2 comprising the amino acid sequence of SEQ ID NO: 13, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 14, a VLCDR1 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 VLCDR1 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 VHCDR2 comprising the amino acid sequence of SEQ ID NO: 16, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 17, a VLCDR1 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.

23. The pharmaceutical composition of claim 21, comprising 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 1-11.

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

26. A nucleic acid encoding a BiTE according to any one of claims 16-17.

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

28. 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, wherein the antigen comprises claudin 4, wherein the extracellular ligand binding domain comprises an antibody or a 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 VLCDR1 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 VHCDR2 comprising the amino acid sequence of SEQ ID NO: 13, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 14, a VLCDR1 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 VLCDR1 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 VHCDR2 comprising the amino acid sequence of SEQ ID NO: 16, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 17, a VLCDR1 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.

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. The engineered cell of claim 28, wherein the cell comprises a T cell, a NK cell, a NKT cell, an iPS cell, an iPS-derived cell, a cell line, or a B cell.

31. The engineered cell of claim 28, wherein the cell comprises a CD4+, CD8+, CD3+ pan T cell, or any combination thereof.

32. 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 1-11, or (b) the pharmaceutical composition of claim 21.

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

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) the antibody of any one of claims 1-11 or (b) the pharmaceutical composition of claim 21 for treating cancer.

36. The antibody of any one of claims 1-11 or (b) the pharmaceutical composition of claim 21 for use in therapy.

37. The antibody of any one of claims 1-11 or (b) the pharmaceutical composition of claim 21 for use in treating cancer.

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