Claudin 6 Antibodies and Methods for Treating Cancer - Patent application

Abstract

The present disclosure provides antigen binding proteins that bind to claudin 6 (CLDN6). In various aspects, the antigen binding proteins bind to extracellular loop 2 (EL2) of the extracellular domain of CLDN6. Also provided herein are related polypeptides, nucleic acids, vectors, host cells, and conjugates. Kits and pharmaceutical compositions comprising such components are also provided. Methods for producing the antigen binding proteins and methods for treating a subject with cancer are also provided. [Selected Figure] Figure 1

Description

[Technical field]

[0001] Incorporation by Reference of Electronically Submitted Material The computer readable nucleotide / amino acid sequence listing, identified hereinafter in the 315,776 byte ASCII (text) file entitled "51836_SeqListing.txt" (created on September 18, 2018), submitted contemporaneously herewith, is hereby incorporated by reference in its entirety. The present disclosure provides antigen binding proteins that bind to claudin 6 (CLDN6). [Background technology]

[0002] Antibodies constitute powerful therapeutic agents characterized by limited side effects due to the antibody's own ability to specifically target different antigens on cells, bacteria, viruses or toxins. In 1986, the first therapeutic monoclonal antibody, Orthoclone OKT3, was introduced to the market. Since then, this class of biopharmaceuticals has seen a significant increase. At the end of 2014, 47 monoclonal antibody products were approved in the United States or Europe for the treatment of various diseases, including cancer and inflammatory diseases, cardiovascular diseases, respiratory diseases, and infectious diseases.

[0003] At present, more than 12 monoclonal antibodies have been approved by the US Food and Drug Administration to treat cancer. Among these drugs are alemtuzumab (Campus®), designated for chronic lymphocytic leukemia (CLL), and trastuzumab (Herceptin®), used to treat breast cancer. Some antibodies are classified as chemotherapy drugs, such as brentuximab vedotin (Adcetris®) and ado-trastuzumab emtansine (Kadcyla®). Other antibody products, such as blinatumomab (Blincyto), are designed to recognize and bind to two different antigens. Despite the commercial availability of such antibody products, the current incidence of cancer and cancer deaths remain high. It has been reported that the annual incidence of cancer is more than 450 per 100,000 men and women, and the annual cancer death rate is as high as more than 170 per 100,000 men and women. Summary of the Invention

[0004] Provided herein are antigen binding proteins that bind to claudin 6 (CLDN6). In various aspects, the antigen binding proteins of the present disclosure bind to human CLDN6, and optionally to mouse CLDN6. In various aspects, the antigen binding proteins bind to the extracellular domain (ECD) of CLDN6. In various examples, the antigen binding proteins bind to extracellular loop 2 (EL2) of the ECD of CLDN6. In various aspects, the antigen binding proteins bind to EL2 and do not bind to extracellular loop 1 (EL1) of the ECD of CLDN6. In various examples, the antigen binding proteins bind to another member of the human claudin family, including, for example, claudin 3 (CLDN3), claudin 4 (CLDN4), and claudin 9 (CLDN9). In various examples, the antigen binding proteins bind to CLDN6 and at least one of CLDN4 and CLDN9. In various examples, the antigen binding proteins bind to CLDN6 and do not bind to any other members of the claudin family. In various embodiments, the antigen binding protein binds to CLDN6 endogenously expressed by human ovarian cancer cells (e.g., OVCA429 cells) and exhibits an IC50 of less than about 1200 nM in a FACS affinity assay using OVCA429 cells. In various examples, the antigen binding protein of the present disclosure inhibits tumor growth in a subject (e.g., a human) without any other moiety attached to the antigen binding protein. In various examples, the antigen binding protein that is not conjugated to a heterologous moiety (e.g., not conjugated to any chemotherapeutic agent, drug, or toxic moiety) inhibits tumor growth in a subject (e.g., a human).

[0005] In various embodiments, the antigen binding protein binds to CLDN6 expressed by human cancer cells. In various embodiments, the antigen binding protein inhibits the binding interaction between human CLDN6 and a reference anti-CLDN6 antibody. Without being bound to a particular theory, the inhibitory action of the antigen binding proteins provided herein makes such moieties useful in methods for inhibiting tumor growth and for treating subjects with tumors or cancer. As further discussed herein, in various embodiments, the antigen binding protein is an antibody, an antigen-binding antibody fragment thereof, or an antibody protein product.

[0006] The present disclosure also provides antigen binding proteins that comprise at least three, four, five, or all of the amino acid sequences of a particular group of amino acid sequences. In various embodiments, the antigen binding proteins comprise at least three, four, five, or six complementarity determining region (CDR) amino acid sequences of a CLDN6 antibody disclosed herein.

[0007] The present disclosure further provides antigen binding proteins comprising an amino acid sequence as detailed herein. In various embodiments, the antigen binding protein comprises an amino acid sequence as set forth in Table A, A1, B, B1, C, or D, or a combination thereof, as further described herein.

[0008] Further provided herein are related polypeptides, nucleic acids, vectors, host cells and conjugates. Kits and pharmaceutical compositions containing such components are further contemplated.

[0009] Also provided are methods for producing the antigen binding proteins, in various embodiments, the methods comprise culturing a host cell comprising a nucleic acid encoding the antigen binding protein or polypeptide described herein, so as to express the antigen binding protein or polypeptide.

[0010] Further provided herein are methods for treating a subject having cancer, in various embodiments, the methods comprising administering to the subject a pharmaceutical composition of the present disclosure in an amount effective to treat the cancer in the subject.

[0011] Also provided is a method for treating a subject having a CLDN6-expressing cancer, comprising administering to the subject a pharmaceutical composition as described herein.Further contemplated is a method for inhibiting tumor growth in a subject, comprising administering to the subject a pharmaceutical composition as described herein.

[0012] The method for reducing the size of a tumor in a subject or preventing the recurrence of cancer in a subject comprises administering to the subject a pharmaceutical composition described herein.

[0013] Also provided herein is a method for treating cancer in a subject diagnosed with low overexpression of CLDN6, comprising administering to the subject a pharmaceutical composition described herein.

[0014] In various embodiments, the administering induces apoptosis in tumor cells, e.g., in cells expressing CLDN6, hi various embodiments, the administering induces antibody-dependent cell-mediated cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC), tumor necrosis and cell death or depletion, and / or inhibition of tumor cell adhesion, each of which results in tumor regression or slowing of tumor growth. [Brief description of the drawings]

[0015] [Figure 1] FIG. 1 shows a graph of CLDN6 expression in normal (non-cancerous) tissues. [Diagram 2] FIG. 1 shows a graph of CLDN6 expression in cancer cell lines measured by the Agilent44K method. [Diagram 3] FIG. 1 shows a graph of CLDN6 expression in cancer cell lines measured by RNASeq. [Figure 4]FIG. 1 shows a set of fluorescent images showing the localization of CLDN6-GFP in different cell models. [Diagram 5] Figure 1 shows a sequence alignment of human CLDN6, human CLDN3, human CLDN4, human CLDN9, and mouse CLDN6. The sequences of EL1 and EL2 are shown. [Figure 6] Figure 6A shows a graph of tumor volume (mm3) of tumors as a function of time (days) after treatment with control IgG2 antibody, AB3, Reference Ab1, Reference Ab2, Reference Ab3, AB2, and AB3 in mice bearing endometrial tumors. Figure 6B shows a graph of the mean change in tumor volume (mm3) of tumors at day 14 in mice bearing endometrial tumors treated with control IgG2 antibody, AB3, Reference Ab1, Reference Ab2, Reference Ab3, AB2, or AB3. [Figure 7] Figure 7A shows a graph of tumor volume (mm3) of tumors as a function of time (days) after treatment with control IgG2 antibody, AB3, Reference Ab1, Reference Ab2, and AB3 in mice bearing bladder tumors, and Figure 7B shows a graph of the mean change in tumor volume (mm3) of tumors at day 35 in mice bearing bladder tumors treated with control IgG2 antibody, AB3, Reference Ab1, Reference Ab2, or AB3. [Figure 8] Figure 8A shows a graph of tumor volume (mm3) of tumors as a function of time (days) after treatment with control IgG2 antibody, AB3, reference Ab1, AB2, and AB3 in ovarian tumor-bearing mice, and Figure 8B shows a graph of the mean change in tumor volume (mm3) of tumors at day 20 in ovarian tumor-bearing mice treated with control IgG2 antibody, AB3, reference Ab1, AB2, or AB3. [Figure 9]Figure 9A shows a graph of tumor volume (mm3) of tumors as a function of time (days) after treatment with control IgG2 antibody, AB3, Reference Ab1, Reference Ab2, Reference Ab3, and AB3 in mice bearing melanoma tumors. Figure 9B shows a graph of the mean change in tumor volume (mm3) of tumors at day 21 in mice bearing melanoma tumors treated with control IgG2 antibody, AB3, Reference Ab1, Reference Ab2, Reference Ab3, or AB3. [Figure 10] Figure 10A shows a graph of the percentage of tumor growth inhibition achieved in tumor-bearing mice treated with AB3 compared to mice treated with a control antibody. Figure 10B shows images of Western blots showing different levels of CLDN6 in endometrial cancer cell lines (ARK2), bladder cancer cell lines (UMUC4), ovarian cancer cell lines (OV90), and melanoma cell lines (M202), as well as control cells. The levels of α-tubulin were nearly identical, indicating comparable protein amounts. [Figure 11] FIG. 1 shows a graph of percent weight change over time (days) for tumor-bearing mice treated with vehicle control, control antibody, Reference Ab1, Reference Ab2, Reference Ab3, and AB3. [Figure 12A] FIG. 1 shows a graph of tumor volume (mm3) of tumors in ovarian tumor-bearing mice as a function of time (days) after treatment with vehicle control, control IgG2 antibody, AB3, reference Ab1, or one of the indicated anti-CLDN6 antibodies. [Figure 12B] FIG. 1 shows a graph of the mean change in tumor volume (mm3) of tumors at day 28 in mice bearing ovarian tumors treated with vehicle control, control IgG2 antibody, AB3, reference Ab1, or one of the indicated anti-CLDN6 antibodies. [Figure 13] FIG. 1 shows a graph of the percentage weight change over time (days) for tumor-bearing mice treated with vehicle control, control antibody, reference Ab1, and the indicated anti-CLDN6 antibodies. [Figure 14]Figure 1 shows a series of dose-response curves for several anti-CLDN6 antibodies of the invention, as well as reference Ab1 and reference 2. Mouse IgG was used as a control. [Figure 15] Figure 15A shows a graph of the mean change in tumor volume (mm3) of tumors at day 35 in mice bearing bladder tumors treated with a solvent control, a control IgG antibody, the murine form of AB3, the first humanized form of AB3, and the second humanized form of AB3. Figure 15B shows a graph of the change in tumor volume (mm3) for each of the groups in Figure 15A. [Figure 16] Figure 16A shows a graph of the mean change in tumor volume (mm3) of tumors at day 35 in mice bearing bladder tumors treated with a solvent control, a control IgG antibody, a murine form of AB3, a first humanized form of AB3, and a second humanized form of AB3. Two control antibodies, one murine and one chimeric, were also tested in this experiment. Figure 16B shows a graph of the change in tumor volume (mm3) in each of the groups in Figure 16A. [Figure 17] Figure 17A shows a graph of the mean change in tumor volume (mm3) of tumors at day 35 in mice bearing bladder tumors treated with a solvent control, a control IgG antibody, the murine form of AB1, and the humanized form of AB1. Figure 17B shows a graph of the change in tumor volume (mm3) in each of the groups in Figure 17A. [Figure 18] Figure 18A shows a graph of the mean change in tumor volume (mm3) of tumors at day 35 in mice bearing bladder tumors treated with a solvent control, a control IgG antibody, the murine form of AB4, and the humanized form of AB4, and Figure 18B shows a graph of the change in tumor volume (mm3) for each of the groups in Figure 18A. [Figure 19]Figure 19A shows a graph of the average change in tumor volume (mm3) of tumors at day 35 in mice bearing bladder tumors treated with a solvent control, a control IgG antibody, a murine version of AB3, a chimeric version of AB3, a first humanized version of AB3, a second humanized version of AB3, a murine version of Ab1, a humanized version of AB1, a murine version of AB4, and a humanized version of AB4. Four control antibodies (one antibody having either a murine or chimeric version, and one antibody having either a murine or human version) were also tested in this experiment. Figure 19B shows a graph of the change in tumor volume (mm3) in each of the groups in Figure 19A. [Figure 20] FIG. 19B shows a graph of the mean change in tumor volume (mm3) at day 55 in bladder tumor-bearing mice treated as described in FIG. [Figure 21] FIG. 19B shows a graph of percent body weight change on day 32 for tumor-bearing mice treated as described in FIG. 19A. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0016] Claudine Family Tight junctions, also known as occluding junctions or zonula occludens, are well-organized structures located between two adjacent cells that regulate paracellular permeability and maintain cell polarity in epithelial and endothelial cell sheets. Claudin (CLDN) family genes code for membrane proteins that are key components of tight junctions. CLDN proteins contain four transmembrane (TM) helices (TM1, TM2, TM3, and TM4) and two extracellular loops (EL1 and EL2). The extracellular loops of CLDN proteins from adjacent cells interact with each other to bring the cell sheets into close contact and regulate paracellular transport between the luminal and basolateral spaces.

[0017] CLDN proteins are involved in various human diseases and pathologies. For example, mutations in the CLDN1 gene have been found to result in progressive skin scaling along with bile duct obstruction. Mutations in the CLDN16 gene cause magnesium wasting disease. CLDN19 mutations cause ocular symptoms such as macular coloboma and myopia, while CLDN14 mutations can cause nonsyndromic recessive hearing loss. CLDN3 and CLDN4 are known to be surface receptors for Clostridium perfringens enterotoxin in the gastrointestinal tract, and CLDN1, CLDN6, and CLDN9 are co-receptors for Hepatitis C virus (HCV) entry. Several CLDN proteins have been shown to be aberrantly expressed in cancer. For example, CLDN1 is downregulated in breast and colon cancer, while CLDN3 and CLDN4 are strongly upregulated in multiple cancers.

[0018] Claudin 6 (CLDN6) is a member of the CLDN family. The gene encoding the human CLDN6 protein is located at 16p13.3 on the p arm of human chromosome 16 and is conserved in chimpanzees, rhesus monkeys, dogs, cows, mice, rats, zebrafish, and frogs. CLDN6 is normally expressed in humans as a 220 amino acid precursor protein, the first 21 amino acids of which constitute a signal peptide. The amino acid sequence of the CLDN6 precursor protein is publicly available at the National Center for Biotechnology Information (NCBI) website as NCBI Reference Sequence NP_067018.2 and is provided herein as SEQ ID NO:1. The amino acid at position 143 of SEQ ID NO:1 is Ile. In some instances, due to a single nucleotide polymorphism (SNP) in the DNA sequence encoding CLDN6, the amino acid at position 143 is Val. The amino acid sequence of human CLDN6 with Val at position 143 is provided herein as SEQ ID NO:178.

[0019] Antigen-binding proteins Provided herein is an antigen binding protein that binds to Claudin 6 (CLDN6). The antigen binding protein of the present disclosure can take any one of many forms of antigen binding proteins known in the art. In various embodiments, the antigen binding protein of the present disclosure takes the form of an antibody or an antigen-binding antibody fragment, or an antibody protein product.

[0020] In various embodiments of the present disclosure, the antigen-binding protein comprises, consists essentially of, or consists of an antibody. As used herein, the term "antibody" refers to a protein having a conventional immunoglobulin format, including heavy and light chains, and including a variable region and a constant region. For example, an antibody may be an IgG, which is a pair of two identical polypeptide chains in a "Y-shaped" configuration, each pair having one "light" chain (usually having a molecular weight of about 25 kDa) and one "heavy" chain (usually having a molecular weight of about 50-70 kDa). An antibody has a variable region and a constant region. In the IgG format, the variable region is usually about 100-110 or more amino acids and contains three complementarity determining regions (CDRs) that are primarily responsible for antigen recognition, and are substantially different in other antibodies that bind different antigens. The constant region enables the antibody to recruit cells and molecules of the immune system. The variable region is composed of the N-terminal regions of the light and heavy chains, respectively, while the constant region is composed of the C-terminal portions of the heavy and light chains, respectively. (Janeway et al., “Structure of the Antibody Molecule and the Immunoglobulin Genes”, Immunobiology: The Immune System in Health and Disease, 4th ed. Elsevier Science Ltd. / Garland Publishing, (1999)).

[0021] The general structure and properties of antibody CDRs have been described in the art. Briefly, in an antibody scaffold, the CDRs are embedded within the framework of heavy and light chain variable regions, which constitute the regions primarily responsible for antigen binding and recognition. A variable region typically comprises at least three heavy chain or light chain CDRs (Kabat et al., 1991, Sequences of Proteins of Immunological Interest, Public Health Service NIH, Bethesda, Md.; see also Chothia and Lesk, 1987, J. Mol. Biol. 196:901-917; Chothia et al., 1989, Nature 342:877-883), within framework regions (termed framework regions 1-4, FR1, FR2, FR3 and FR4 by Kabat et al., 1991; see also Chothia and Lesk, 1987, supra).

[0022] The antibody may include any constant region known in the art. Human light chains are classified as kappa and lambda light chains. Heavy chains are classified as μ, δ, γ, α, or ε, defining the antibody isotype as IgM, IgD, IgG, IgA, and IgE, respectively. IgG has several subclasses, including, but not limited to, IgG1, IgG2, IgG3, and IgG4. IgM has subclasses, including, but not limited to, IgM1 and IgM2. Embodiments of the present disclosure include antibodies of all such classes or isotypes. The light chain constant region may be, for example, a kappa or lambda type light chain constant region, for example, a human kappa or lambda type light chain constant region. The heavy chain constant region may be, for example, an α, δ, ε, γ, or μ type heavy chain constant region, for example, a human α, δ, ε, γ, or μ type heavy chain constant region. Thus, in various embodiments, the antibody is of isotype IgA, IgD, IgE, IgG, or IgM, including any one of IgG1, IgG2, IgG3, or IgG4. In various aspects, the antibody comprises a constant region that contains one or more amino acid modifications compared to the natural counterpart to improve half-life / stability or to make the antibody more suitable for expression / manufacturing. In various examples, the antibody comprises a constant region in which the C-terminal Lys residue present in the natural counterpart has been removed or truncated.

[0023] The antibody may be a monoclonal antibody. In some embodiments, the antibody comprises a sequence that is substantially equivalent to a natural antibody produced by a mammal, such as a mouse, rabbit, goat, horse, chicken, hamster, human, etc. In this context, the antibody may be considered to be a mammalian antibody, such as a mouse antibody, a rabbit antibody, a goat antibody, a horse antibody, a chicken antibody, a hamster antibody, a human antibody, etc. In certain aspects, the antigen binding protein is an antibody, such as a human antibody. In certain aspects, the antigen binding protein is a chimeric antibody or a humanized antibody. The term "chimeric antibody" refers to an antibody that contains domains from two or more different antibodies. A chimeric antibody may, for example, contain a constant domain from one species and a variable domain from a second species, or more commonly, may contain stretches of amino acid sequences from at least two species. A chimeric antibody may also contain domains from two or more different antibodies within the same species. The term "humanized" when used in relation to an antibody refers to an antibody that has at least a CDR region derived from a non-human source that has been genetically engineered to have a structure and immunological function that is more similar to a pure human antibody compared to the original source antibody. For example, humanization can include grafting CDRs derived from a non-human antibody, such as a mouse antibody, onto a human antibody. Humanization can also include selecting amino acid substitutions to make the non-human sequence more similar to a human sequence. Information including sequence information of the heavy and light chain constant regions of human antibodies is generally available through the Uniprot database as well as other databases well known to those skilled in the art of antibody engineering and antibody manufacturing. For example, the IgG2 constant region is available from the Uniprot database as Uniprot number P01859, which is incorporated herein by reference.

[0024] Enzymes such as papain and pepsin may be used to cleave antibodies into fragments. Papain cleaves antibodies to generate two Fab fragments and a single Fc fragment. Pepsin cleaves antibodies to generate a F(ab')2 fragment and a pFc' fragment. In various aspects of the disclosure, the antigen-binding proteins of the disclosure are antigen-binding fragments of antibodies (also known as antigen-binding antibody fragments, antigen-binding fragments, antigen-binding sites). In various examples, the antigen-binding antibody fragments are Fab fragments or F(ab')2 fragments.

[0025] The structure of antibodies has been exploited to generate an increasingly wide range of alternative antibody formats, referred to herein as "antibody protein products", with valencies (n) ranging from monomers (n=1), to dimers (n=2), trimers (n=3), tetramers (n=4), and possibly higher, over a molecular weight range of at least about 12-150 kDa. Antibody protein products include antibody protein products based on the complete antibody structure, as well as antibody protein products that mimic antibody fragments that retain complete antigen-binding capacity, such as scFv, Fab, and VHH / VH (discussed below). The smallest antigen-binding fragment that retains its complete antigen-binding site is the Fv fragment, consisting exclusively of the variable (V) region. Either the V region is linked to the scFv (single chain variable fragment) fragment using a soluble, flexible amino acid peptide linker to stabilize the molecule, or a constant (C) domain is added to the V region to generate a Fab fragment [fragment, antigen-binding]. Both scFv and Fab fragments can be easily produced in host cells, e.g., prokaryotic host cells. Other antibody protein products include dimeric and multimeric antibody formats such as disulfide bond stabilized scFv (ds-scFv), single chain Fab (scFab), as well as diabodies, triabodies, tetrabodies, or minibodies (miniAbs), which contain different formats consisting of scFv linked to oligomerization domains. The smallest fragments are the VHH / VH of camelid heavy chain Abs as well as single domain Abs (sdAbs). The building blocks most frequently used to generate new antibody formats are single chain variable (V) domain antibody fragments (scFv), which contain a V domain consisting of heavy and light chains (VH and VL domains) linked by a peptide linker of about 15 amino acid residues. Peptibodies, i.e. peptide-Fc fusions, are yet another antibody protein product. The peptibody structure consists of a biologically active peptide grafted onto an Fc domain and has been well described in the art.See, for example, Shimamoto et al., mAbs 4(5):586-591 (2012).

[0026] Other antibody protein products include single chain antibodies (SCAs), diabodies, triabodies, tetrabodies, bispecific antibodies or triabodies. Bispecific antibodies can be divided into five major classes: BsIgG, adducted IgG, bispecific antibody (BsAb) fragments, bispecific fusion proteins, and BsAb conjugates. See, e.g., Spiess et al., Molecular Immunology 67(2)Part A:97-106(2015).

[0027] In various aspects, the antigen binding proteins of the disclosure comprise, consist essentially of, or consist of any one of these antibody protein products. In various aspects, the antigen binding proteins of the disclosure comprise, consist essentially of, or consist of any one of the following: scFv, Fab VHH / VH, Fv fragment, ds-scFv, scFab, dimeric antibodies, multimeric antibodies (e.g., diabodies, triabodies, tetrabodies), miniAb, camelid heavy chain antibodies, sdAb, peptibody VHH / VH of diabodies, triabodies, tetrabodies, bispecific or triabodies, BsIgG, adduct IgG, BsAb fragments, bispecific fusion proteins, and BsAb conjugates.

[0028] In various examples, the antigen binding proteins of the present disclosure are antibody protein products in monomeric or polymeric, oligomeric or multimeric form. In certain embodiments where an antibody comprises two or more different antigen binding region fragments, the antibody is considered to be bispecific, trispecific or multispecific, or bivalent, trivalent or multivalent, depending on the number of different epitopes that the antibody recognizes and binds.

[0029] In various embodiments, the anti-CLDN6 antibody or antibody variant thereof is selected from the group consisting of a human antibody, a humanized antibody, a chimeric antibody, a monoclonal antibody, a recombinant antibody, an antigen-binding antibody fragment, a single chain antibody, a monomeric antibody, a diabody, a triabody, a tetrabody, a Fab fragment, an IgG1 antibody, an IgG2 antibody, an IgG3 antibody, and an IgG4 antibody.

[0030] In various aspects, the antigen binding proteins of the present disclosure are linked to a therapeutic agent. As described below, the therapeutic agent may be any known in the art, including but not limited to chemotherapeutic agents, cytokines and growth factors, cytotoxic agents, etc. See "Conjugates" below.

[0031] CLDN6 and epitopes The antigen binding proteins of the present disclosure bind to CLDN6. In various embodiments, CLDN6 is Human CLDN6 having the amino acid sequence of TIFF0007599332000001.tif37154 (wherein X is Ile or Val) (SEQ ID NO: 202).

[0032] In various aspects, human CLDN6 comprises any one of the amino acid sequences of SEQ ID NOs: 1, 178, and 200-202.

[0033] In various aspects, the antigen binding protein of the present disclosure binds to an epitope within the amino acid sequence of CLDN6. In various aspects, CLDN6 is human CLDN6, and the antigen binding protein of the present disclosure binds to an epitope within the amino acid sequence of human CLDN6, for example, SEQ ID NOs: 1, 178, and 200-202. "Epitope" refers to a region of CLDN6 or a region within CLDN6 to which an antigen binding protein binds. In some embodiments, the epitope is a linear epitope. "Linear epitope" refers to a region of CLDN6 or a region within CLDN6 to which an antigen binding protein binds, the region being composed of consecutive amino acids in the amino acid sequence of CLDN6. The amino acids of a linear epitope are adjacent to each other in the primary structure of CLDN6. Thus, a linear epitope is an antigen, i.e., a fragment or a portion of the amino acid sequence of CLDN6. In various other embodiments, the epitope is a conformational epitope or a structural epitope. "Conformational epitope" or "structural epitope" refers to an epitope composed of amino acids that are located adjacent to each other only when CLDN6 is in its properly folded state. Unlike a linear epitope, the amino acids of a conformational epitope or structural epitope are not adjacent to each other in the primary structure (i.e., amino acid sequence) of CLDN6. A conformational epitope or structural epitope is not composed of consecutive amino acids in the amino acid sequence of the antigen (CLDN6).

[0034] In various embodiments, the epitope is located within the extracellular domain (ECD) of CLDN6, e.g., human CLDN6. In various embodiments, the antigen binding protein binds to the extracellular loop 2 (EL2) of the ECD of CLDN6, which has the amino acid sequence of WTAHAIIRDFYNPLVAEAQKREL (SEQ ID NO:2). In various embodiments, the epitope to which the antigen binding protein binds is within SEQ ID NO:2. In various embodiments, the antigen binding protein of the present disclosure binds to the N-terminal portion of SEQ ID NO:2, e.g., TAHAIIRDFYNPL (SEQ ID NO:3). In various embodiments, the antigen binding protein of the present disclosure binds to the C-terminal portion of SEQ ID NO:2, e.g., LVAEAQKREL (SEQ ID NO:4). In various examples, the antigen binding protein of the present disclosure binds to EL2 but not to the extracellular loop 1 (EL1) of CLDN6. In various aspects, the epitope(s) bound by an antigen binding protein of the present disclosure is different from the epitope bound by an anti-CLDN6 antibody comprising a light chain variable region comprising the sequence of SEQ ID NO: 185 and a heavy chain variable region comprising the sequence of SEQ ID NO: 186. In various aspects, the epitope(s) bound by an antigen binding protein of the present disclosure is different from the epitope bound by an anti-CLDN6 antibody comprising a light chain variable region comprising the sequence of SEQ ID NO: 181 and a heavy chain variable region comprising the sequence of SEQ ID NO: 182.

[0035] In various embodiments, the antigen binding protein binds to human CLDN6 and non-human CLDN6. In various examples, the non-human CLDN6 is chimpanzee, rhesus monkey, dog, cow, mouse, rat, zebrafish, or frog CLDN6. In various examples, the antigen binding protein binds to human CLDN6 and mouse CLDN6.

[0036] Affinity and avidity The antigen binding proteins provided herein bind to CLDN6 in a non-covalent and reversible manner. In various embodiments, the binding strength of an antigen binding protein to CLDN6 can be described in terms of its affinity, which is an indication of the strength of interaction between the binding site of the antigen binding protein and the epitope. In various aspects, the antigen binding proteins provided herein have high affinity for CLDN6, and therefore bind to a greater amount of CLDN6 in a shorter period of time compared to antigen binding proteins with lower affinity. In various aspects, the antigen binding proteins bind to CLDN6 in a greater amount of CLDN6 in a shorter period of time compared to antigen binding proteins with lower affinity. 5 Mol -1 , at least 10 6 Mol -1 , at least 10 7 Mol -1 , at least 10 8 Mol -1 , at least 10 9 Mol -1 , or at least 10 10 Mol -1 is the equilibrium binding constant K A As will be appreciated by those skilled in the art, K A can be affected by factors including pH, temperature and buffer composition.

[0037] In various embodiments, the binding strength of an antigen binding protein to CLDN6 can be described in terms of its sensitivity. D is the equilibrium dissociation constant between the antigen-binding protein and CLDN6, k off / k on It is the ratio of K D and K. A are inversely correlated. D Since the K value is related to the concentration of antigen-binding protein (the amount of antigen-binding protein desired for a particular experiment), D The lower the value (the lower the concentration), the higher the affinity of the antigen binding protein. In various embodiments, the binding strength of the antigen binding protein to CLDN6 is expressed by the K D In various embodiments, the K of the antigen binding proteins provided herein can be described as D is about 10 -1 , about 10-2 , about 10 -3 , about 10 -4 , about 10 -5 , about 10 -6 In various embodiments, the K of the antigen binding proteins provided herein is equal to or less than 0. D is micromolar, nanomolar, picomolar or femtomolar. In various embodiments, the K of the antigen binding proteins provided herein D is about 10 -4 ~10 -6 , 10 -7 ~10 -9 , 10 -10 ~10 -12 , or 10 -13 ~10 -15 In various embodiments, the K of the antigen binding proteins provided herein is within the range of D is approximately 1.0 x 10 -12 M ~ approx. 1.0×10 -8 In various embodiments, the K of the antigen binding protein is within the range of D is approximately 1.0 x 10 -11 M ~ approx. 1.0×10 -9 It is within the range of M.

[0038] In various embodiments, the affinity of the antigen-binding protein is measured or ranked using flow cytometry or fluorescence-activated cell sorting (FACS)-based assays. Flow cytometry-based binding assays are well known in the art. See, for example, Cedeno-Arias et al., Sci Pharm 79(3):569-581(2011); Rathanaswami et al., Analytical Biochem 373:52-60(2008); and Geuijen et al., J Immunol Methods 302(1-2):68-77(2005). In various embodiments, the affinity of the antigen-binding protein is measured or ranked using a competitive assay as described below in addition to Trikha et al., Int J Cancer 110:326-335(2004) and Tam et al., Circulation 98(11):1085-1091(1998). See the section below entitled "Competition Assays." In Trikh et al., cells expressing the antigen were used in a radioassay. Cells in suspension were used 125 The binding of labeled antigen binding proteins (e.g., antibodies) to cell surface antigens is measured. In various embodiments, the relative affinity of CLDN6 antibodies is measured by a FACS-based assay in which different concentrations of fluorophore-conjugated CLDN6 antibodies are incubated with cells expressing CLDN6 and the emitted fluorescence (which is a direct indicator of antibody-antigen binding) is measured. A curve is generated that plots fluorescence against each dose or concentration. The maximum is the lowest concentration at which the fluorescence reaches a plateau or maximum, which is when binding saturation occurs. Half of the maximum is considered to be the EC50 or IC50, and the antibody with the lowest EC50 / IC50 is considered to have the highest affinity compared to other antibodies tested in the same manner. Such an assay is described in Example 5 herein.

[0039] In various embodiments, the IC measured in a competitive binding inhibition assay 50 The value is the K DIn various examples described below, the competitive assay is a FACS-based assay performed with a reference antibody, a fluorophore-conjugated secondary antibody, and cells expressing CLDN6. In various embodiments, the cells are genetically engineered to overexpress CLDN6. In some embodiments, the cells are HEK293T cells transduced to express CLDN6 using a viral vector. In alternative embodiments, the cells endogenously express CLDN6. In some embodiments, the cells endogenously expressing CLDN6 are predefined as CLDN6 low expressing cells or CLDN6 high expressing cells prior to performing the FACS-based assay. In some embodiments, the cells are cancer cells or tumor cells. In various embodiments, the cells are cells derived from a cell line, such as an ovarian cell line, an endometrial cell line, a bladder cell line, a lung cell line, a gastrointestinal (GI) cell line, a liver cell line, a lung cell line, etc. In various aspects, the cells that endogenously express CLDN6 are selected from the group consisting of OVCA429 ovarian cells, ARK2 endometrial cells, OAW28 ovarian cells, UMUC-4 bladder cells, PEO14 ovarian cells, OV177 ovarian cells, H1693 lung cells, MKN7 upper GI cells, OV-90 ovarian cells, HUH-7 liver cells, JHOS-4 ovarian cells, H1435 lung cells, and NUGC3 upper GI cells. In various aspects, the antigen binding protein inhibits the binding interaction between human CLDN6 expressed by the cells and a reference antibody, which is known to bind to CLDN6 but not to the antigen binding protein of the present disclosure. In various examples, the antigen binding protein of the present disclosure competes with the reference antibody for binding to human CLDN6, thereby reducing the amount of human CLDN6 that binds to the reference antibody as measured in an in vitro competitive binding assay. In various aspects, the antigen binding protein of the present disclosure inhibits the binding interaction between human CLDN6 and the reference antibody, which inhibition is not greater than IC 50 In various embodiments, the antigen binding protein has an IC of less than about 2500 nM in inhibiting the binding interaction between human CLDN6 and the reference antibody. 50 In various embodiments, the antigen binding protein has a molecular weight of less than about 2000 nM, less than about 1500 nM, less than about 1000 nM, less than about 900 nM Less than 800 nM Less than 700 nM Less than 600 nM Less than 500 nM Less than 400 nM Less than 300 nM Less than 200 nM Less than or about 100 nM Less than IC 50 In various embodiments, the antigen binding protein exhibits an IC of less than about 90 nM, less than about 80 nM, less than about 70 nM, less than about 60 nM, less than about 50 nM, less than about 40 nM, less than about 30 nM, less than about 20 nM, or less than about 10 nM. 50 In various examples, the antigen binding proteins of the present disclosure compete for binding to CLDN6 with a reference antibody (wherein the reference antibody is different from any of the antigen binding proteins of the present disclosure) known to bind to CLDN6. See further description of competition assays below.

[0040] Avidity provides an indication of the overall strength of the antibody-antigen complex. Avidity depends on three main parameters: the affinity of the antigen-binding protein to the epitope, the valency of both the antigen-binding protein and CLDN6, and the structural arrangement of the interacting moieties. The higher the valency (number of antigen-binding sites) of the antigen-binding protein, the higher the amount of antigen (CLDN6) that the antigen-binding protein can bind. In various embodiments, the antigen-binding protein has a strong avidity for CLDN6. In various embodiments, the antigen-binding protein is multivalent. In various embodiments, the antigen-binding protein is bivalent. In various examples, the antigen-binding protein is monovalent.

[0041] Cross-reactivity In various embodiments, the antigen binding proteins of the present disclosure bind to CLDN6 and do not bind to any other members of the CLDN family, e.g., do not cross-react with any other members of the CLDN family. In various examples, the antigen binding proteins of the present disclosure are specific for CLDN-6. In various embodiments, the antigen binding proteins of the present disclosure have at least 10-fold, 5-fold, 4-fold, 3-fold, 2-fold higher selectivity for CLDN6 compared to the selectivity of the antigen binding proteins for CLDN3, CLDN4, CLDN9, or a combination thereof. In various embodiments, the antigen binding proteins of the present disclosure have at least 10-fold, 5-fold, 4-fold, 3-fold, 2-fold higher selectivity for CLDN6 compared to the selectivity of the antigen binding proteins for each of CLDN3, CLDN4, and CLDN9. Selectivity is measured by the K of the antigen binding protein for CLDN6 or a CLDN family member. D can be used as a criterion, and K D can be measured by techniques well known in the art, for example, surface plasmon resonance assays, FACS-based affinity assays.

[0042] In various aspects, the antigen binding protein of the disclosure binds to CLDN6 and does not bind to claudin 3 (CLDN3), claudin 4 (CLDN4), and claudin 9 (CLDN9). In various aspects, the antigen binding protein does not bind to CLDN3, CLDN4, and CLDN9, and has an IC of less than about 1200 nM (e.g., less than about 1000 nM, less than about 750 nM, less than about 500 nM, less than about 250 nM) in a FACS-based assay using OVCA429 cells that endogenously express CLDN6. 50In various embodiments, the antigen binding protein does not bind to any of CLDN3, CLDN4, and CLDN9, and the concentration at which 50% binding saturation is achieved in OVCA429 cells that endogenously express CLDN6 is less than about 1200 nM (e.g., less than about 1000 nM, less than about 750 nM, less than about 500 nM, less than about 250 nM). In various embodiments, the antigen binding protein exhibits at least 5-fold higher selectivity for CLDN6 compared to selectivity for CLDN3, CLDN4, and CLDN9, and the concentration at which 50% binding saturation is achieved in OVCA429 cells that endogenously express CLDN6 is less than about 1200 nM (e.g., less than about 1000 nM, less than about 750 nM, less than about 500 nM, less than about 250 nM). In various aspects, the antigen binding protein exhibits an IC50 of less than about 1200 nM (e.g., less than about 1000 nM, less than about 750 nM, less than about 500 nM, less than about 250 nM) to CLDN6 artificial and endogenous models, and exhibits a ratio of CLDN6 IC50 that is more than about 5-fold off from CLDN3, CLDN4 and / or CLDN9. In various examples, the antigen binding protein exhibits an IC50 of less than about 1200 nM (e.g., less than about 1000 nM, less than about 750 nM, less than about 500 nM, less than about 250 nM) to CLDN6, and exhibits an IC50 to any one of CLDN3, CLDN4 and CLDN9 that is at least 5-fold higher compared to its IC50.

[0043] In various embodiments, the antigen binding proteins of the present disclosure bind to CLDN6 and cross-react (e.g., bind) with at least one other member of the CLDN family. In various aspects, the antigen binding proteins of the present disclosure bind to CLDN6 and one or more of CLDN3, CLDN4, and CLDN9. In various aspects, the antigen binding proteins of the present disclosure bind to CLDN6 and CLDN4 or CLDN9, but not to CLDN3. In various examples, the antigen binding proteins of the present disclosure bind to CLDN6 and CLDN4, but not to CLDN3 or CLDN9. In various examples, the antigen binding proteins of the present disclosure bind to CLDN6 and CLDN9, but not to either CLDN3 or CLDN4.

[0044] Competition assay In various embodiments, the antigen binding protein inhibits the binding interaction between human CLDN6 and a reference antibody, which is known to bind CLDN6 but not to an antigen binding protein of the present disclosure. In various examples, the antigen binding protein of the present disclosure competes with the reference antibody for binding to human CLDN6, thereby reducing the amount of human CLDN6 that binds to the reference antibody as measured in an in vitro competitive binding assay. In various embodiments, the reference antibody binds to an epitope within an amino acid sequence of the extracellular domain of human CLDN6, optionally an epitope within EL2 or EL1. In various aspects, the reference antibody comprises a light chain variable sequence encoded by SEQ ID NO: 179 and a heavy chain variable sequence encoded by SEQ ID NO: 180. In various aspects, the reference antibody comprises a light chain variable sequence of SEQ ID NO: 181 and a heavy chain variable sequence of SEQ ID NO: 182. In various aspects, the antigen binding protein of the present disclosure inhibits the binding interaction between human CLDN6 and a reference antibody, which inhibition is not associated with IC 50 In various embodiments, the antigen binding protein has an IC of less than about 2500 nM in inhibiting the binding interaction between human CLDN6 and the reference antibody. 50 In various embodiments, the antigen binding protein has a molecular weight of less than about 2000 nM, less than about 1500 nM, less than about 1000 nM, less than about 900 nM Less than 800 nM Less than 700 nM Less than 600 nM Less than 500 nM Less than 400 nM Less than 300 nM Less than 200 nM Less than or about 100 nM Less than IC 50 In various embodiments, the antigen binding protein exhibits an IC of less than about 90 nM, less than about 80 nM, less than about 70 nM, less than about 60 nM, less than about 50 nM, less than about 40 nM, less than about 30 nM, less than about 20 nM, or less than about 10 nM. 50 Shows.

[0045] In various examples, the antigen binding protein of the present disclosure competes with the reference antibody for binding to human CLDN6, thereby reducing the amount of human CLDN6 bound to the reference antibody as measured in an in vitro competitive binding assay. In various aspects, the in vitro competitive binding assay is a FACS-based assay that measures the fluorescence of a fluorophore-conjugated secondary antibody bound to the Fc of the reference antibody in the presence or absence of a particular amount of an antigen binding protein of the present disclosure. Such a FACS-based assay is described in the Examples herein. In various aspects, the FACS-based assay is performed using a reference antibody, a fluorophore-conjugated secondary antibody, and a cell expressing CLDN6. In various aspects, the cell is genetically engineered to overexpress CLDN6. In some aspects, the cell is a HEK293T cell transduced to express CLDN6 using a viral vector. In alternative aspects, the cell endogenously expresses CLDN6. In some aspects, prior to performing the FACS-based assay, the cell that endogenously expresses CLDN6 is predetermined as a CLDN6 low expressing cell or a CLDN6 high expressing cell. In some aspects, the cell is a cancer cell or tumor cell. In various aspects, the cell is a cell derived from a cell line, such as an ovarian cell line, an endometrial cell line, a bladder cell line, a lung cell line, a gastrointestinal (GI) cell line, a liver cell line, a lung cell line, etc. In various aspects, the cell that endogenously expresses CLDN6 is selected from the group consisting of OVCA429 ovarian cells, ARK2 endometrial cells, OAW28 ovarian cells, UMUC-4 bladder cells, PEO14 ovarian cells, OV177 ovarian cells, H1693 lung cells, MKN7 upper GI cells, OV-90 ovarian cells, HUH-7 liver cells, JHOS-4 ovarian cells, H1435 lung cells, and NUGC3 upper GI cells. In various examples, the antigen binding protein of the present disclosure binds with high affinity to CLDN6 endogenously expressed by one or more of ARK2 cells, OVCA429 cells, LS513 cells, or MCF7 cells. In various embodiments, the antigen binding protein has an IC of less than about 3000 nM as measured in a FACS-based competitive binding inhibition assay using one or more of ARK2 cells, OVCA429 cells, LS513 cells, or MCF7 cells. 50In various embodiments, the antigen binding protein exhibits an IC of less than about 2500 nM, less than about 2000 nM, less than about 1750 nM, less than about 1500 nM, less than about 1250 nM, less than about 1000 nM, less than about 750 nM, or less than about 500 nM as measured in a FACS-based competitive binding inhibition assay using one or more of ARK2 cells, OVCA429 cells, LS513 cells, or MCF7 cells. 50 In various embodiments, the antigen binding protein exhibits an IC of less than about 400 nM, less than about 300 nM, less than about 200 nM, less than about 100 nM, less than about 75 nM, less than about 50 nM, less than about 25 nM, or less than about 10 nM as measured in a FACS-based competitive binding inhibition assay using one or more of ARK2 cells, OVCA429 cells, LS513 cells, or MCF7 cells. 50 Shows.

[0046] Other binding assays, such as competitive binding assays, i.e., competitive assays, in which an antibody is tested for its ability to compete with a second antibody in binding to an antigen or its epitope, are well known in the art.See, for example, Trikha et al., Int J Cancer 110:326-335(2004); Tam et al., Circulation 98(11):1085-1091(1998).US Patent Application Publication No. US20140178905, Chand et al., Biologicals 46:168-171(2017); Liu et al., Anal Biochem 525:89-91(2017); and Goolia et al., J Vet Diagn Invest 29(2):250-253(2017).Other methods for comparing two antibodies are also well known in the art, including, for example, surface plasmon resonance (SPR). SPR can be used to measure the binding constant of an antibody and a second antibody, and the two binding constants can be compared.

[0047] Methods for Producing Antibodies and Related Methods Suitable methods for producing antigen-binding proteins (e.g., antibodies, antigen-binding antibody fragments, and antibody protein products) are well known in the art. For example, standard hybridoma methods for producing antibodies are described, for example, in Harlow and Lane (eds.), Antibodies: A Laboratory Manual, CSH Press (1988), and CA. Janeway et al. (eds.), Immunobiology, 5th Ed., Garland Publishing, New York, NY (2001). The present disclosure Various methods for preparing CLDN6 monoclonal antibodies The law and are described in the Examples herein.

[0048] Depending on the host species, various adjuvants can be used to enhance the immune response, resulting in greater antibody production by the host. Such adjuvants include, but are not limited to, Freund's adjuvant, mineral gels, such as aluminum hydroxide, and surfactants, such as lysolecithin, Pluronic® polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, and dinitrophenol. BCG (bacilli Calmette-Guerin) and Corynebacterium parvum are potentially useful human adjuvants.

[0049] Other methods for producing antibodies are summarized in Table 1.

[0050] [Table 1]

[0051] Methods for testing the ability of an antibody to bind to an epitope of CLDN6, regardless of how the antibody is produced, are well known in the art and include any antibody-antigen binding assay, such as radioimmunoassay (RIA), ELISA, Western blot, immunoprecipitation, SPR, and competitive inhibition assays (see, e.g., Janeway et al., infra, and U.S. Patent Application Publication No. 2002 / 0197266, as well as the section above regarding competitive assays).

[0052] Arrays / Structures As used herein, the term "HC" refers to a heavy chain (HC) CDR1 amino acid sequence as set forth in Table A, i.e., a sequence selected from the group consisting of SEQ ID NOs: 11, 17, 23, 29, 35, 41, 47, 53, 59, 65, 71, 77, 83, 89, 95, 101, 107, 113, 119, 125, and 131, or a variant thereof that differs by only one or two amino acids or has at least or about 70% (e.g., at least about 80%, at least about 85%, at least about 90%, at least about 95%) sequence identity; (c) a CDR2 amino acid sequence, i.e., a sequence selected from the group consisting of SEQ ID NOs: 12, 18, 24, 30, 36, 42, 48, 54, 60, 66, 72, 78, 84, 90, 86, 102, 108, 114, 120, 126, and 132, or a variant thereof differing by only one or two amino acids or having at least or about 70% (e.g., at least about 80%, at least about 85%, at least about 90%, at least about 95%) sequence identity, as set forth in Table A; (d) a CDR3 amino acid sequence, i.e., a sequence selected from the group consisting of SEQ ID NOs: 13, 19, 25, 31, 37, 43, 49, 55, 61, 67, 73, 79, 85, 91, 97, 103, 109, 115, 121, 127, and 133, or a variant thereof differing by only one or two amino acids or having at least or about 70% (e.g., at least about 80%, at least about 85%, at least about 90%, at least about 95%) sequence identity, as set forth in Table A; (e) a LC CDR1 amino acid sequence, i.e., a sequence selected from the group consisting of SEQ ID NOs: 8, 14, 20, 32, 38, 44, 50, 56, 62, 68, 74, 80, 86, 92, 98, 104, 110, 116, 122, and 128, or a variant thereof differing by only one or two amino acids or having at least or about 70% (e.g., at least about 80%, at least about 85%, at least about 90%, at least about 95%) sequence identity, as set forth in Table A;(f) a CDR2 amino acid sequence, i.e., a sequence selected from the group consisting of SEQ ID NOs: 9, 15, 21, 27, 33, 39, 45, 51, 57, 63, 69, 75, 81, 87, 93, 99, 105, 111, 117, 123, and 129, or a variant thereof differing by only one or two amino acids or having at least or about 70% (e.g., at least about 80%, at least about 85%, at least about 90%, at least about 95%) sequence identity, as set forth in Table A; (g) an antigen binding protein comprising a CDR3 amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 16, 22, 28, 34, 40, 46, 52, 58, 64, 70, 76, 82, 88, 94, 100, 106, 112, 118, 124, and 130, or a variant thereof which differs by only one or two amino acids or has at least or about 70% (e.g., at least about 80%, at least about 85%, at least about 90%, at least about 95%) sequence identity; or (g) a combination of any two or more of (a)-(f).

[0053] [Table 2]

[0054] In various aspects, the antigen binding protein comprises an LC CDR1 amino acid sequence, an LC CDR2 amino acid sequence, and an LC CDR3 amino acid sequence set forth in Table A, and at least one or two of the HC CDR amino acid sequences set forth in Table A. In various aspects, the antigen binding protein comprises an HC CDR1 amino acid sequence, an HC CDR2 amino acid sequence, and an HC CDR3 amino acid sequence set forth in Table A, and at least one or two of the LC CDR amino acid sequences set forth in Table A.

[0055] In various embodiments, the antigen binding protein comprises at least three, four, or five of the amino acid sequences set forth in a single line of SEQ ID NO: in Table A. In various embodiments, the antigen binding protein comprises each of the LC CDR amino acid sequences set forth in a single line of SEQ ID NO: in Table A, and at least one or two of the HC CDR amino acid sequences set forth in a single line of SEQ ID NO: in Table A. In various embodiments, the antigen binding protein comprises each of the HC CDR amino acid sequences set forth in a single line of SEQ ID NO: in Table A, and at least one or two of the LC CDR amino acid sequences set forth in a single line of SEQ ID NO: in Table A. In various embodiments, the antigen binding protein comprises all six CDR amino acid sequences set forth in a single line of SEQ ID NO: in Table A. In various embodiments, the antigen binding protein is selected from the group consisting of (a) SEQ ID NOs: 74-79, (b) SEQ ID NOs: 50-55, (c) SEQ ID NOs: 122-127, (d) SEQ ID NOs: 26-31, (e) SEQ ID NOs: 128-133, (f) SEQ ID NOs: 38-43, (g) SEQ ID NOs: 62-67, (h) SEQ ID NOs: 80-85, (i) SEQ ID NOs: 44-49, (j) SEQ ID NOs: 86-91, (k) SEQ ID NOs: 104-106, (l) SEQ ID NOs: 106-108, (l) SEQ ID NOs: 108-109, (l) SEQ ID NOs: 110-122, (l) SEQ ID NOs: 112-124, (l) SEQ ID NOs: 114-126, (l) SEQ ID NOs: 116-122, (l) SEQ ID NOs: 118-124, (l) SEQ ID NOs: 118-126, (l) SEQ ID NOs: 118-128, (l) SEQ ID NOs: 118-132, (l) SEQ ID NOs: 118-134, (l) SEQ ID NOs: 118-136, (l) SEQ ID NOs: 118-138 ...2, (l) SEQ ID NOs: 118-136, (l) SEQ ID NOs 09, (l) SEQ ID NOs: 56 to 61, (m) SEQ ID NOs: 32 to 37, (n) SEQ ID NOs: 110 to 115, (o) SEQ ID NOs: 98 to 103, (p) SEQ ID NOs: 92 to 97, (q) SEQ ID NOs: 116 to 121, (r) SEQ ID NOs: 8 to 13, (s) SEQ ID NOs: 68 to 73, (t) SEQ ID NOs: 14 to 19, and (u) SEQ ID NOs: 20 to 25.

[0056] In various examples, the amino acid sequences of Table A are separated by at least one or more (e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) intervening amino acid(s). In various examples, there are about 10 to about 20 amino acids between the LC CDR1 and LC CDR2 sequences, and about 25 to about 40 amino acids between the LC CDR2 and LC CDR3 sequences. In various examples, there are about 14 to about 16 amino acids between the LC CDR1 and LC CDR2 sequences, and about 30 to about 35 amino acids between the LC CDR2 and LC CDR3 sequences. In various examples, there are about 10 to about 20 amino acids between the HC CDR1 and HC CDR2 sequences, and about 25 to about 40 amino acids between the HC CDR2 and HC CDR3 sequences. In various examples, there are about 14 to about 16 amino acids between the HC CDR1 and HC CDR2 sequences, and about 30 to about 35 amino acids between the HC CDR2 and HC CDR3 sequences.

[0057] In various embodiments, the antigen binding protein comprises: (a) a heavy chain variable region amino acid sequence set forth in Table B, i.e., a sequence selected from the group consisting of SEQ ID NOs: 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, and 175, or a variant thereof that differs by only one or two amino acids or has at least or about 70% (e.g., at least about 80%, at least about 85%, at least about 90%, at least about 95%) sequence identity; or (c) both (a) and (b).

[0058] [Table 3]

[0059] In various embodiments, the antigen binding protein is selected from the group consisting of (a) SEQ ID NO:156 and 157, (b) SEQ ID NO:148 and 149, (c) SEQ ID NO:172 and 173, (d) SEQ ID NO:140 and 141, (e) SEQ ID NO:174 and 175, (f) SEQ ID NO:144 and 145, (g) SEQ ID NO:152 and 153, (h) SEQ ID NO:158 and 159, (i) SEQ ID NO:146 and 147, (j) SEQ ID NO:160 and 161, (k) SEQ ID NO:166 and (i) SEQ ID NO:167, (l) SEQ ID NO:150 and 151, (m) SEQ ID NO:142 and 143, (n) SEQ ID NO:168 and 169, (o) SEQ ID NO:164 and 165, (p) SEQ ID NO:162 and 163, (q) SEQ ID NO:170 and 171, (r) SEQ ID NO:134 and 135, (s) SEQ ID NO:154 and 155, (t) SEQ ID NO:136 and 137, and (u) SEQ ID NO:138 and 139.

[0060] In various aspects, the antigen binding protein does not comprise the pair of amino acid sequences encoded by the sequences of SEQ ID NOs: 179 and 180. In various aspects, the antigen binding protein does not comprise the pair of amino acid sequences encoded by the sequences of SEQ ID NOs: 181 and 182. In various aspects, the antigen binding protein does not comprise the pair of amino acid sequences encoded by the sequences of SEQ ID NOs: 183 and 184. In various aspects, the antigen binding protein does not comprise the pair of amino acid sequences encoded by the sequences of SEQ ID NOs: 185 and 186.

[0061] In various embodiments, the antigen binding protein comprises an amino acid sequence similar to the amino acid sequence referred to above, and further, the antigen binding protein substantially retains its biological function, e.g., its ability to bind to human CLDN6 and inhibit tumor growth to treat cancer.

[0062] In various embodiments, the antigen binding protein comprises an amino acid sequence that differs by only 1, 2, 3, 4, 5, 6, or more amino acids compared to the amino acid sequence(s) mentioned above. In various embodiments, the antigen binding protein comprises a variant of the sequence mentioned, where the variant sequence differs by only 1 or 2 amino acids compared to the sequence mentioned. In various embodiments, the antigen binding protein comprises one or more amino acid substitutions that occur outside the CDRs, for example, one or more amino acid substitutions that occur within the framework region(s) of the heavy or light chain. In various embodiments, the antigen binding protein comprises one or more amino acid substitutions, and furthermore, the antigen binding protein retains the amino acid sequence of the six CDRs. In various embodiments, the antigen binding protein comprises an amino acid sequence with only 1, 2, 3, 4, 5, 6, or more conservative amino acid substitutions compared to the amino acid sequence(s) mentioned above. As used herein, the term "conservative amino acid substitution" refers to the replacement of one amino acid with another amino acid that has similar properties, such as size, charge, hydrophobicity, hydrophilicity and / or aromaticity, and includes the following five groups: I. Small aliphatic non-polar or slightly polar residues: Ala, Ser, Thr, Pro, Gly; II. Polar, negatively charged residues and their amides and esters: Asp, Asn, Glu, Gln, cysteic acid, and homocysteic acid; III. Polar, positively charged residues: His, Arg, Lys, ornithine (Orn); IV. Large aliphatic non-polar residues: Met, Leu, Ile, Val, Cys, norleucine (Nle), homocysteine; V. Large aromatic residues: Phe, Tyr, Trp, acetylphenylalanine; An example of this is an internal exchange between the two.

[0063] In various embodiments, conservative amino acid substitutions are exchanges within one of the following amino acid groups: TIFF0007599332000005.tif46122

[0064] In various embodiments, the antigen binding protein comprises an amino acid sequence having greater than or about 30%, greater than or about 50%, or greater than or about 70% sequence identity to the above-mentioned amino acid sequence. In various embodiments, the antigen binding protein comprises an amino acid sequence having at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90% sequence identity, or greater than 90% sequence identity, to the above-mentioned amino acid sequence. In various embodiments, the antigen binding protein comprises an amino acid sequence having at least 70%, at least 80%, at least 85%, at least 90% sequence identity, or greater than 90% sequence identity, to the full length of the above-mentioned amino acid sequence. In various embodiments, the antigen binding protein comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity to the full length of the above-mentioned amino acid sequence.

[0065] In various embodiments, the antigen binding protein comprises a variant of the referenced sequence, the variant having at least 70% or about 70% sequence identity to the referenced sequence. In various embodiments, the antigen binding protein comprises a variant of the referenced sequence, the variant having at least 80% or about 80% sequence identity to the referenced sequence. In various embodiments, the antigen binding protein comprises a variant of the referenced sequence, the variant having at least 90% or about 90% sequence identity to the referenced sequence. In various embodiments, the antigen binding protein comprises a variant of the referenced sequence, the variant having at least 95% or about 95% sequence identity to the referenced sequence.

[0066] In various embodiments, the antigen binding protein comprises one, two, three, four, or five of the SEQ ID NOs: in a single row of Table A, and at least one variant sequence having at least 70% or about 70% (e.g., at least about 80%, at least about 85%, at least about 90%, at least about 95%) sequence identity to any of SEQ ID NOs:8-133. In various embodiments, the antigen binding protein is selected from the group consisting of (a) SEQ ID NOs: 74-79, (b) SEQ ID NOs: 50-55, (c) SEQ ID NOs: 122-127, (d) SEQ ID NOs: 26-31, (e) SEQ ID NOs: 128-133, (f) SEQ ID NOs: 38-43, (g) SEQ ID NOs: 62-67, (h) SEQ ID NOs: 80-85, (i) SEQ ID NOs: 44-49, (j) SEQ ID NOs: 86-91, (k) SEQ ID NOs: 104-109, (l) SEQ ID NOs: 56-61, (m) SEQ ID NOs: 32-37, (n) SEQ ID NOs: 110-115, (o) SEQ ID NOs: 98-103, (p) SEQ ID NOs: (q) SEQ ID NOs: 92 to 97, (q) SEQ ID NOs: 116 to 121, (r) SEQ ID NOs: 8 to 13, (s) SEQ ID NOs: 68 to 73, (t) SEQ ID NOs: 14 to 19, and (u) SEQ ID NOs: 20 to 25, and the antigen-binding protein further comprises at least one mutant sequence having at least 70% or about 70% (e.g., at least about 80%, at least about 85%, at least about 90%, at least about 95%) sequence identity to at least one of the sequences of the set. For example, in various embodiments, an antigen binding protein comprises four of SEQ ID NOs:74-79, i.e., SEQ ID NOs:74-77, and the antigen binding protein comprises two mutant sequences, one mutant sequence having at least or about 70% (e.g., at least about 80%, at least about 85%, at least about 90%, at least about 95%) sequence identity to SEQ ID NO:78, and another mutant sequence having at least or about 70% (e.g., at least about 80%, at least about 85%, at least about 90%, at least about 95%) sequence identity to SEQ ID NO:79.

[0067] In various embodiments, the antigen binding protein comprises a pair of mutant sequences having at least 70% or about 70% (e.g., at least about 80%, at least about 85%, at least about 90%, at least about 95%) sequence identity to any of SEQ ID NOs: 134-175. In various examples, the antigen binding protein comprises a pair of mutant sequences having at least 70% or about 70% (e.g., at least about 80%, at least about 85%, at least about 90%, at least about 95%) sequence identity to any of SEQ ID NOs: 134-175. In various examples, the antigen binding protein comprises a pair of mutant sequences having at least 70% or about 70% (e.g., at least about 80%, at least about 85%, at least about 90%, at least about 95%) sequence identity to any of SEQ ID NOs: 134-175. In various embodiments, the antigen binding protein comprises a pair of variant sequences having at least or about 70% (e.g., at least about 80%, at least about 85%, at least about 90%, at least about 95%) sequence identity to any of SEQ ID NOs: 134-175, (n) SEQ ID NOs: 168 and 169, (o) SEQ ID NOs: 164 and 165, (p) SEQ ID NOs: 162 and 163, (q) SEQ ID NOs: 170 and 171, (r) SEQ ID NOs: 134 and 135, (s) SEQ ID NOs: 154 and 155, (t) SEQ ID NOs: 136 and 137, and (u) SEQ ID NOs: 138 and 139. In various embodiments, the antigen binding protein comprises a pair of sequences, one sequence of Table B, and another sequence that is a variant sequence having at least or about 70% (e.g., at least about 80%, at least about 85%, at least about 90%, at least about 95%) sequence identity to any of SEQ ID NOs: 134-175.In various embodiments, the antigen binding protein comprises a pair of sequences: (a) SEQ ID NOs: 156 and 157, (b) SEQ ID NOs: 148 and 149, (c) SEQ ID NOs: 172 and 173, (d) SEQ ID NOs: 140 and 141, (e) SEQ ID NOs: 174 and 175, (f) SEQ ID NOs: 144 and 145, (g) SEQ ID NOs: 152 and 153, (h) SEQ ID NOs: 158 and 159, (i) SEQ ID NOs: 146 and 147, (j) SEQ ID NOs: 160 and 161, (k) SEQ ID NOs: 166 and 167, (l) SEQ ID NOs: 150 and 151, (m) SEQ ID NOs: 142 and 143, (n) SEQ ID NOs: (i) SEQ ID NOs: 168 and 169, (o) SEQ ID NOs: 164 and 165, (p) SEQ ID NOs: 162 and 163, (q) SEQ ID NOs: 170 and 171, (r) SEQ ID NOs: 134 and 135, (s) SEQ ID NOs: 154 and 155, (t) SEQ ID NOs: 136 and 137, and (u) SEQ ID NOs: 138 and 139, as well as another sequence which is a mutant sequence having at least 70% or about 70% (e.g., at least about 80%, at least about 85%, at least about 90%, at least about 95%) sequence identity to the sequences (a) to (u). For example, in various embodiments, the antigen binding protein comprises the sequence of SEQ ID NO:134, and the antigen binding protein further comprises a variant sequence having at least 70% or about 70% (e.g., at least about 80%, at least about 85%, at least about 90%, at least about 95%) sequence identity to SEQ ID NO:135.

[0068] In various examples, the antigen binding protein comprises an amino acid sequence of the amino acid sequence mentioned above with one or more amino acid substitutions to reduce or remove reactive amino acids to reduce or prevent undesired side chain reactions. For example, the antigen binding protein comprises an amino acid sequence of the amino acid sequence mentioned above with one or more of: (i) a Trp residue substituted with His, Tyr, or Phe; (ii) an Asn residue substituted with Gln, Ser, Ala, or Asp; (iii) an Asp residue immediately preceding a Pro residue substituted with Ala, Ser, or Glu; (iv) an Asn residue substituted with Gln, Ser, or Ala; and / or (v) a Cys residue substituted with Tyr, Ser, or Ala. In various aspects, the antigen binding protein comprises an amino acid sequence of the amino acid sequence mentioned above with an amino acid substitution that is predicted to have higher binding affinity, higher stability, or other favorable properties based on SHM events or based on statistical analysis of a large number of other similar antibody sequences. In some embodiments, the antigen binding protein comprises: (a) an HC CDR1 amino acid sequence set forth in Table A1, i.e., a sequence selected from the group consisting of SEQ ID NOs: 452, 455, 461, 465, 71, and 472, or a variant thereof that differs by only one or two amino acids or has at least or about 70% (e.g., at least about 80%, at least about 85%, at least about 90%, at least about 95%) sequence identity; (b) an HC CDR2 amino acid sequence set forth in Table A1, i.e., a sequence selected from the group consisting of SEQ ID NOs: 475, 456, 462, 466, 468, and 473, or a variant thereof that differs by only one or two amino acids or has at least or about 70% (e.g., at least about 80%, at least about 85%, at least about 90%, at least about 95%) sequence identity;(d) the LC CDR1 amino acid sequences listed in Table A1, i.e., sequences selected from the group consisting of SEQ ID NOs: 449, 476, 458, 464, 68, and 470, or variants thereof differing by only one or two amino acids or having at least or about 70% (e.g., at least about 80%, at least about 85%, at least about 90%, at least about 95%) sequence identity; (e) the LC CDR2 amino acid sequences listed in Table A1, i.e., sequences selected from the group consisting of SEQ ID NOs: 449, 476, 458, 464, 68, and 470, or variants thereof differing by only one or two amino acids or having at least or about 70% (e.g., at least about 80%, at least about 85%, at least about 90%, at least about 95%) sequence identity; (f) the LC CDR3 amino acid sequences set forth in Table A1, i.e., sequences selected from the group consisting of SEQ ID NOs: 451, 454, 460, 58, 70, and 112, or variants thereof which differ by only one or two amino acids or which have at least or about 70% (e.g., at least about 80%, at least about 85%, at least about 90%, at least about 95%) sequence identity; or (g) a combination of any two or more of (a)-(f).

[0069] [Table 4]

[0070] In some aspects, HC CDR1 comprises a Gly adjacent to the N-terminus of SEQ ID NO:452, and optionally in some aspects, HC CDR1 comprises an MX adjacent to the C-terminus of SEQ ID NO:452, where X is H, N, or S. In various aspects, HC CDR3 comprises an Ala adjacent to the N-terminus of SEQ ID NO:453. In various aspects, LC CDR1 further comprises a TAS adjacent to the N-terminus of SEQ ID NO:449, and optionally an XH adjacent to the C-terminus of SEQ ID NO:449, where X is H, S, Y, or Q. In some aspects, the first amino acid of SEQ ID NO:449 is S or Q, as described below. In some aspects, the first amino acid of SEQ ID NO:451 is S or Q, as described below.

[0071] In various aspects, HC CDR1 comprises a Gly adjacent to the N-terminus of SEQ ID NO:455, and optionally, in various aspects, HC CDR1 comprises an MX adjacent to the C-terminus of SEQ ID NO:455, where X is N, S, or H. In some aspects, HC CDR2 comprises a Gln adjacent to the N-terminus of SEQ ID NO:456, and optionally an H adjacent to the C-terminus of SEQ ID NO:456. In various aspects, LC CDR1 comprises a RIS adjacent to the N-terminus of SEQ ID NO:476, and optionally an LA adjacent to the C-terminus of SEQ ID NO:476. In various aspects, LC CDR2 comprises an XLVE adjacent to the C-terminus of SEQ ID NO:477, where X is I or S.

[0072] In various embodiments, HC CDR1 comprises an MH adjacent to the C-terminus of SEQ ID NO:461. In various embodiments, HC CDR2 comprises a Tyr adjacent to the N-terminus of SEQ ID NO:462, and optionally a TH adjacent to the C-terminus of SEQ ID NO:462. In exemplary embodiments, HC CDR3 does not include the first two amino acids of SEQ ID NO:463. In various embodiments, LC CDR1 comprises an RSS adjacent to the N-terminus of SEQ ID NO:458, and optionally an LN adjacent to the C-terminus of SEQ ID NO:458. In various embodiments, LC CDR2 comprises an XRFS adjacent to the C-terminus of SEQ ID NO:459, where X is Q, S, A, or D.

[0073] In various embodiments, HC CDR1 comprises an MH adjacent to the C-terminus of SEQ ID NO:465. In various embodiments, HC CDR2 comprises a YI adjacent to the N-terminus of SEQ ID NO:466, and optionally, a Xaa adjacent to the C-terminus of SEQ ID NO:466, where Xaa is N, S, Q, or A. In various embodiments, LC CDR1 comprises an LAS adjacent to the N-terminus of SEQ ID NO:464, and optionally, a LA adjacent to the C-terminus of SEQ ID NO:464. In various embodiments, LC CDR2 comprises a SLAD adjacent to the C-terminus of SEQ ID NO:57.

[0074] In various embodiments, HC CDR1 comprises MH adjacent to the C-terminus of SEQ ID NO:71. In various embodiments, HC CDR2 comprises Tyr adjacent to the N-terminus of SEQ ID NO:468, and optionally IY adjacent to the C-terminus of SEQ ID NO:468. In various embodiments, LC CDR1 comprises RAS adjacent to the N-terminus of SEQ ID NO:68, and optionally SYIH adjacent to the C-terminus of SEQ ID NO:68. In various embodiments, LC CDR2 comprises XLES adjacent to the C-terminus of SEQ ID NO:69, where X is N, Q, S, A, or D.

[0075] In various aspects, the LC CDR1 comprises a KSS adjacent to the N-terminus of SEQ ID NO:470, and optionally a YLA adjacent to the C-terminus of SEQ ID NO:470. In various aspects, the LC CDR2 comprises a TRES adjacent to the C-terminus of SEQ ID NO:471. In various aspects, the HC CDR1 comprises a MN adjacent to the C-terminus of SEQ ID NO:472. In various aspects, the HC CDR2 comprises a Xaa adjacent to the N-terminus of SEQ ID NO:473, where Xaa is N, Q, S, or A, and optionally a Thr adjacent to the C-terminus of SEQ ID NO:473.

[0076] In various aspects, the antigen binding protein comprises an LC CDR1, LC CDR2, and LC CDR3 amino acid sequence set forth in Table A1, and at least one or two of the HC CDR amino acid sequences set forth in Table A1. In various aspects, the antigen binding protein comprises an HC CDR1, HC CDR2, and HC CDR3 amino acid sequence set forth in Table A1, and at least one or two of the LC CDR amino acid sequences set forth in Table A1.

[0077] In various embodiments, the antigen binding protein comprises at least three, four, or five of the amino acid sequences set forth in a single line of SEQ ID NO: in Table A1. In various embodiments, the antigen binding protein comprises each of the LC CDR amino acid sequences set forth in a single line of SEQ ID NO: in Table A1, and at least one or two of the HC CDR amino acid sequences set forth in a single line of SEQ ID NO: in Table A1. In various embodiments, the antigen binding protein comprises each of the HC CDR amino acid sequences set forth in a single line of SEQ ID NO: in Table A1, and at least one or two of the LC CDR amino acid sequences set forth in a single line of SEQ ID NO: in Table A1. In various embodiments, the antigen binding protein comprises all six CDR amino acid sequences set forth in a single line of SEQ ID NO: in Table A1. In various embodiments, the antigen binding protein comprises six CDR amino acid sequences selected from the group consisting of: (a) SEQ ID NOs: 449-453 and 475; (b) SEQ ID NOs: 476-477, 454-457; (c) SEQ ID NOs: 458-463; (d) SEQ ID NOs: 57, 58, 464-467; (e) SEQ ID NOs: 68-71 and 468-469; and (f) SEQ ID NOs: 112 and 470-474.

[0078] In various examples, the amino acid sequences of Table A1 are separated by at least one or more (e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) intervening amino acid(s). In various examples, there are about 10 to about 20 amino acids between the LC CDR1 and LC CDR2 sequences, and about 25 to about 40 amino acids between the LC CDR2 and LC CDR3 sequences. In various examples, there are about 14 to about 16 amino acids between the LC CDR1 and LC CDR2 sequences, and about 30 to about 35 amino acids between the LC CDR2 and LC CDR3 sequences. In various examples, there are about 10 to about 20 amino acids between the HC CDR1 and HC CDR2 sequences, and about 25 to about 40 amino acids between the HC CDR2 and HC CDR3 sequences. In various examples, there are about 14 to about 16 amino acids between the HC CDR1 and HC CDR2 sequences, and about 30 to about 35 amino acids between the HC CDR2 and HC CDR3 sequences.

[0079] In various embodiments, the antigen binding protein comprises: (a) a heavy chain variable region amino acid sequence set forth in Table B1, i.e., a sequence selected from the group consisting of SEQ ID NOs: 478, 480, 482, 484, 486, and 488, or a variant thereof which differs by only one or two amino acids or has at least or about 70% (e.g., at least about 80%, at least about 85%, at least about 90%, at least about 95%) sequence identity; (b) a light chain variable region amino acid sequence set forth in Table B1, i.e., a sequence selected from the group consisting of SEQ ID NOs: 479, 481, 483, 485, 487, and 489, or a variant thereof which differs by only one or two amino acids or has at least or about 70% (e.g., at least about 80%, at least about 85%, at least about 90%, at least about 95%) sequence identity; or (c) both (a) and (b).

[0080] [Table 5]

[0081] In various embodiments, the antigen binding protein comprises a pair of amino acid sequences selected from the group consisting of: (a) SEQ ID NOs: 478 and 479, (b) SEQ ID NOs: 480 and 481, (c) SEQ ID NOs: 482 and 483, (d) SEQ ID NOs: 484 and 485, (e) SEQ ID NOs: 486 and 487, and (f) SEQ ID NOs: 488 and 489. In various aspects, the antigen binding protein comprises a variant of a sequence having a SEQ ID NO: as set forth in Table B1, differing by only one or two amino acids or having at least 70% or about 70% (e.g., at least about 80%, at least about 85%, at least about 90%, at least about 95%) sequence identity, where the different amino acid(s) are present at the positions set forth below in the "humanized antibody."

[0082] Humanized antibodies In various embodiments, the antigen binding protein is a humanized version of an antigen binding protein described in Table A, Table A1, Table B or Table B1.

[0083] Humanized AB1 In various aspects, the antigen binding protein comprises one or more of the following positions in the heavy chain variable region: 5, 8, 11, 12, 13, 20, 31, 33, 35, 38, 40, 48, 50, 55, 57, 59, 61, 65, 66, 67, 68, 70, 72, 74, 76, 79, 80, 82, 87, 90, 91, 98, 101, and 116. In various examples, the antigen binding protein is a humanized form of AB1 as described in Table B or B1 having one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35) amino acid substitutions in number. In various embodiments, the antigen binding protein is a humanized version of AB1 as set forth in Table B or B1, having one or more amino acid substitutions at one or more of the following positions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12) in the heavy chain variable region: 20, 31, 35, 48, 50, 59, 67, 70, 74, 79, 98, 101. In various examples, the antigen binding protein comprises the amino acid sequence of SEQ ID NO: 429. In various embodiments, the amino acids at the above listed positions are selected from those set forth in the table below.

[0084] [Table 6]

[0085] In various embodiments, the antigen binding protein is located at one or more of the following positions in the light chain variable region: 1, 3, 4, 9, 10, 11, 15, 17, 21, 24, 27, 29, 32, 34, 35, 43, 44, 48, 51, 52, 53, 54, 55, 56, 61, 67, 71, 72, 73, 79, 80, 81, 84, 90, 92, 93, 94, 95, 96, 101, 107. (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, or 41). In various examples, the antigen binding protein comprises the amino acid sequence of SEQ ID NO:430. In various embodiments, the antigen binding protein is a humanized version of AB1 as set forth in Table B or B1, having one or more amino acid substitutions at one or more of the following positions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13) in the light chain variable region: 4, 21, 32, 34, 48, 51, 53, 61, 67, 79, 84, 91, and 93. In various examples, the antigen binding protein comprises the amino acid sequence of SEQ ID NO: 431. In various embodiments, the amino acids at the above listed positions are selected from the amino acids set forth in the table below.

[0086] [Table 7]

[0087] Humanized AB3 In various embodiments, the antigen binding protein is a humanized form of AB3 as described in Table B or B1 having one or more amino acid substitutions at one or more of the following positions in the heavy chain variable region: 3, 5, 18, 19, 23, 31, 33, 35, 40, 42, 49, 50, 52, 53, 54, 55, 56, 57, 58, 59, 61, 64, 76, 79, 80, 81, 87, 94, 95, 99, 106, 112, 114 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33). In various examples, the antigen binding protein comprises the amino acid sequence of SEQ ID NO: 432. In various embodiments, the antigen binding protein is a humanized version of AB3 as set forth in Table B or B1, having one or more amino acid substitutions at one or more of the following positions (e.g., 1, 2, 3, 4, 5, 6, or 7) in the heavy chain variable region: 31, 35, 50, 55, 79, 99, 106. In various examples, the antigen binding protein comprises the amino acid sequence of SEQ ID NO: 433. In various embodiments, the amino acids at the above listed positions are selected from the amino acids set forth in the table below.

[0088] [Table 8]

[0089] In various embodiments, the antigen binding protein is a humanized form of AB3 as described in Table B or B1 having one or more amino acid substitutions at one or more of the following positions in the light chain variable region: 9, 17, 18, 25, 27, 28, 30, 34, 40, 43, 45, 48, 50, 52, 53, 55, 56, 70, 72, 74, 76, 84, 85, 90, 91, 93, 94, 97, and 100 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29). In various examples, the antigen binding protein comprises the amino acid sequence of SEQ ID NO:434. In various embodiments, the antigen binding protein is a humanized version of AB3 as set forth in Table B or B1, having one or more amino acid substitutions at one or more of the following positions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, or 9) in the light chain variable region: 25, 34, 48, 53, 55, 84, 85, 90, and 93. In various examples, the antigen binding protein comprises the amino acid sequence of SEQ ID NO: 435. In various embodiments, the amino acids at the above listed positions are selected from the amino acids set forth in the table below.

[0090] [Table 9]

[0091] Humanized AB4 In various embodiments, the antigen binding protein is located at one of the following positions in the heavy chain variable region: 5, 11, 12, 13, 20, 29, 31, 33, 37, 38, 40, 45, 48, 50, 55, 56, 57, 59, 61, 62, 65, 66, 67, 68, 70, 72, 74, 76, 79, 82, 84, 87, 91, 97, 101, 117 or A humanized form of AB4 as described in Table B or B1 having one or more amino acid substitutions at multiple (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36) positions. In various examples, the antigen binding protein comprises the amino acid sequence of SEQ ID NO:436. In various aspects, the antigen binding protein is a humanized version of AB4 as set forth in Table B or B1, having one or more amino acid substitutions at one or more of the following positions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19) in the heavy chain variable region: 20, 29, 31, 37, 45, 48, 56, 59, 61, 62, 65, 66, 68, 70, 74, 79, 84, 97, and 101. In various examples, the antigen binding protein comprises the amino acid sequence of SEQ ID NO: 437. In various aspects, the amino acids at the above listed positions are selected from the amino acids set forth in the table below.

[0092] [Table 10]

[0093] In various embodiments, the antigen binding protein is a humanized version of AB4 as described in Table B or B1, having one or more amino acid substitutions at one or more of the following positions in the light chain variable region (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24). In various examples, the antigen binding protein comprises the amino acid sequence of SEQ ID NO:438. In various embodiments, the antigen binding protein is a humanized version of AB4 as set forth in Table B or B1, having one or more amino acid substitutions at one or more of the following positions (e.g., 1, 2, 3, 4, 5, 6, or 7) in the light chain variable region: 33, 39, 55, 57, 81, 95, and 96. In various examples, the antigen binding protein comprises the amino acid sequence of SEQ ID NO: 439. In various embodiments, the amino acids at the above listed positions are selected from those set forth in the table below.

[0094] [Table 11]

[0095] Humanized AB18 In various aspects, the antigen binding protein is a humanized form of AB18 as described in Table B or B1, having one or more amino acid substitutions at one or more of the following positions in the heavy chain variable region: 5, 9, 11, 12, 20, 38, 40, 41, 43, 44, 48, 61, 65, 67, 68, 70, 72, 74, 76, 79, 82, 84, 87, 91, and 116 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25), and optionally at one or more of the following positions: 20, 48, 68, 70, 79 (e.g., 1, 2, 3, 4, or 5). In various examples, the antigen binding protein comprises the amino acid sequence of SEQ ID NO: 440 or 441. In various embodiments, the amino acids at the positions listed above are selected from the amino acids set forth in the table below.

[0096] [Table 12]

[0097] In various embodiments, the antigen binding protein is a humanized version of AB18 as described in Table B or B1, having one or more amino acid substitutions at one or more of the following positions in the light chain variable region: 1, 3, 9, 15, 18, 19, 21, 22, 49, 51, 69, 93, 84, 78, 105, and 111 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16), and optionally at one or more of the following positions: 19, 21, or 84 (e.g., 1, 2, or 3). In various examples, the antigen binding protein comprises the amino acid sequence of SEQ ID NO: 442 or 443. In various embodiments, the amino acids at the above listed positions are selected from the amino acids set forth in the table below.

[0098] [Table 13]

[0099] Humanized AB9 In various embodiments, the antigen binding protein is a humanized version of AB9 as described in Table B or B1 having one or more amino acid substitutions at one or more of the following positions in the heavy chain variable region: 1, 5, 9, 11, 12, 20, 38, 40, 41, 43, 44, 48, 61, 63, 65, 67, 69, 70, 72, 73, 74, 76, 79, 84, 87, 91, 93, 112, and 113 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29). In various examples, the antigen binding protein comprises the amino acid sequence of SEQ ID NO:444. In various aspects, the amino acids at the positions listed above are selected from the amino acids set forth in the table below.

[0100] [Table 14]

[0101] In various embodiments, the antigen binding protein is a humanized version of AB9 as described in Table B or B1 having one or more amino acid substitutions at one or more of the following positions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15) in the light chain variable region: 9, 11, 15, 17, 18, 43, 45, 70, 72, 73, 74, 80, 84, 85, and 100. In various examples, the antigen binding protein comprises the amino acid sequence of SEQ ID NO: 445. In various embodiments, the amino acids at the above listed positions are selected from the amino acids set forth in the table below.

[0102] [Table 15]

[0103] Humanized AB11 In various embodiments, the antigen binding protein is a humanized version of AB11 as described in Table B or B1, having one or more amino acid substitutions at one or more of the following positions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15) in the heavy chain variable region: 1, 15, 18, 19, 42, 49, 63, 75, 76, 78, 80, 84, 88, and 93. In various examples, the antigen binding protein comprises the amino acid sequence of SEQ ID NO: 446. In various embodiments, the amino acids at the above listed positions are selected from the amino acids set forth in the table below.

[0104] [Table 16]

[0105] In various aspects, the antigen binding protein is a humanized version of AB11 as described in Table B or B1, having one or more amino acid substitutions at one or more of the following positions in the light chain variable region: 4, 9, 17, 22, 64, 78, 80, 81, 82, 83, 84, 87, 89, 104, and 110 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15), optionally at one or more of the following positions: 4, 82, 110. In various examples, the antigen binding protein comprises the amino acid sequence of SEQ ID NO: 447 or 448. In various aspects, the amino acids at the above listed positions are selected from the amino acids set forth in the table below.

[0106] [Table 17]

[0107] In various embodiments, the antigen binding protein comprises: (a) a heavy chain variable region amino acid sequence as set forth in Table C, i.e., a sequence selected from the group consisting of SEQ ID NOs: 376-379, 384-387, 391-396, 403-408, 412, 413, 416-419, and 422-427, or a variant thereof which differs by only one or two amino acids or has at least or about 70%, about 80%, about 85%, about 90%, or about 95% sequence identity. (b) a light chain variable region amino acid sequence set forth in Table C, i.e., a sequence selected from the group consisting of SEQ ID NOs: 380-383, 388-390, 397-402, 409-411, 414, 415, 420, and 421, or a variant thereof which differs by only one or two amino acids or has at least or about 70%, about 80%, about 85%, about 90%, or about 95% sequence identity; or (c) both (a) and (b).

[0108] [Table 18]

[0109] In various embodiments, the humanized antigen binding protein comprises a pair of amino acid sequences shown in Table D.

[0110] [Table 19] TIFF0007599332000022.tif4443

[0111] In various embodiments, the antigen binding protein comprises a pair of mutant sequences, each having at least 70% or about 70% (e.g., at least about 80%, at least about 85%, at least about 90%, at least about 95%) sequence identity to a SEQ ID NO: in Table C. In various embodiments, the antigen binding protein comprises a pair of sequences, one sequence selected from the SEQ ID NOs in Table C and a sequence having a SEQ ID NO: in Table D. orand another sequence which is a variant sequence having at least 70% or about 70% (e.g., at least about 80%, at least about 85%, at least about 90%, at least about 95%) sequence identity to a sequence having a sequence number set forth in Table C:

[0112] In various embodiments, the antigen binding protein comprises a pair of sequences, one sequence selected from the SEQ ID NOs: set forth in Table D, and another sequence which is a variant sequence having at least or about 70% (e.g., at least about 80%, at least about 85%, at least about 90%, at least about 95%) sequence identity to the sequence having the SEQ ID NO: set forth in Table D. For example, in various aspects, the antigen binding protein comprises the sequence of SEQ ID NO:419, and the antigen binding protein further comprises a variant sequence having at least or about 70% (e.g., at least about 80%, at least about 85%, at least about 90%, at least about 95%) sequence identity to SEQ ID NO:421.

[0113] nucleic acid The present disclosure further provides a nucleic acid comprising a nucleotide sequence encoding an antigen binding protein of the present disclosure. As used herein, "nucleic acid" includes "polynucleotides," "oligonucleotides," and "nucleic acid molecules," and generally refers to a polymer of DNA or RNA, or modified forms thereof, that may be single-stranded or double-stranded, that may be synthesized or obtained (e.g., isolated and / or purified) from a natural source, that may contain natural, non-natural, or modified nucleotides, and that may contain natural, non-natural, or modified internucleotide linkages, such as phosphoramidate or phosphorothioate linkages, in place of the phosphodiesters present between the nucleotides of unmodified oligonucleotides. A nucleic acid may comprise any nucleotide sequence that encodes any of the antigen binding proteins of the present disclosure. In various embodiments, the nucleic acid comprises: (a) a heavy chain (HC) complementarity determining region (CDR) 1 amino acid sequence set forth in Table A or A1, i.e., a sequence selected from the group consisting of SEQ ID NOs: 11, 17, 23, 29, 35, 41, 47, 53, 59, 65, 71, 77, 83, 89, 95, 101, 107, 113, 119, 125, 131, 452, 455, 461, 465, and 472, or a variant thereof that differs by only one or two amino acids or has at least or about 70% (e.g., at least or about 80%, at least or about 85%, at least or about 90%, at least or about 95%) sequence identity; (b) a heavy chain (HC) CDR 1 amino acid sequence set forth in Table A or A1, i.e., a sequence selected from the group consisting of SEQ ID NOs: 11, 17, 23, 29, 35, 41, 47, 53, 59, 65, 71, 77, 83, 89, 95, 101, 107, 113, 119, 125, 131, 452, 455, 461, 465, and 472, or a variant thereof that differs by only one or two amino acids or has at least or about 70% (e.g., at least or about 80%, at least or about 85%, at least or about 90%, at least or about 95%) sequence identity; CDR2 amino acid sequences, i.e., sequences selected from the group consisting of SEQ ID NOs: 12, 18, 24, 30, 36, 42, 48, 54, 60, 66, 72, 78, 84, 90, 86, 102, 108, 114, 120, 126, 132, 475, 456, 462, 466, 468, and 473, or variants thereof which differ by only one or two amino acids or have at least or about 70% (e.g., at least or about 80%, at least or about 85%, at least or about 90%, at least or about 95%) sequence identity;(c) a HC CDR3 amino acid sequence set forth in Table A or A1, i.e., a sequence selected from the group consisting of SEQ ID NOs: 13, 19, 25, 31, 37, 43, 49, 55, 61, 67, 73, 79, 85, 91, 97, 103, 109, 115, 121, 127, 133, 453, 457, 463, 467, 469, and 474, or a variant thereof differing by only one or two amino acids or having at least or about 70% (e.g., at least or about 80%, at least or about 85%, at least or about 90%, at least or about 95%) sequence identity; (d) a HC CDR3 amino acid sequence set forth in Table A or A1, i.e., a sequence selected from the group consisting of SEQ ID NOs: 13, 19, 25, 31, 37, 43, 49, 55, 61, 67, 73, 79, 85, 91, 97, 103, 109, 115, 121, 127, 133, 453, 457, 463, 467, 469, and 474, or a variant thereof differing by only one or two amino acids or having at least or about 70% (e.g., at least or about 80%, at least or about 85%, at least or about 90%, at least or about 95%) sequence identity; (e) a light chain (LC) CDR1 amino acid sequence selected from the group consisting of SEQ ID NOs: 8, 14, 20, 32, 38, 44, 50, 56, 62, 68, 74, 80, 86, 92, 98, 104, 110, 116, 122, 128, 449, 476, 458, 464, and 470, or a variant thereof differing by only one or two amino acids or having at least or about 70% (e.g., at least or about 80%, at least or about 85%, at least or about 90%, at least or about 95%) sequence identity to a LC CDR1 amino acid sequence as set forth in Table A or A1; (f) a CDR2 amino acid sequence, i.e., a sequence selected from the group consisting of SEQ ID NOs: 9, 15, 21, 27, 33, 39, 45, 51, 57, 63, 69, 75, 81, 87, 93, 99, 105, 111, 117, 123, 129, 450, 477, 459, and 471, or a variant thereof differing by only one or two amino acids or having at least or about 70% (e.g., at least or about 80%, at least or about 85%, at least or about 90%, at least or about 95%) sequence identity, as set forth in Table A or A1; CDR3 amino acid sequences, i.e., sequences selected from the group consisting of SEQ ID NOs: 10, 16, 22, 28, 34, 40, 46, 52, 58, 64, 70, 76, 82, 88, 94, 100, 106, 112, 118, 124, 130, 451, 454, and 460; oror (g) a combination of any two or more of (a)-(f). In various embodiments, the nucleic acid comprises a nucleotide sequence encoding an antigen binding protein comprising an LC CDR1 amino acid sequence, an LC CDR2 amino acid sequence, and an LC CDR3 amino acid sequence set forth in Table A or A1, and at least one or two of the HC CDR amino acid sequences set forth in Table A or A1. In various embodiments, the nucleic acid comprises a nucleotide sequence encoding an antigen binding protein comprising an HC CDR1 amino acid sequence, an HC CDR2 amino acid sequence, and an HC CDR3 amino acid sequence set forth in Table A or A1, and at least one or two of the LC CDR amino acid sequences set forth in Table A or A1. In various embodiments, the nucleic acid comprises at least three, four, or five of the amino acid sequences set forth in a single line of SEQ ID NO: in Table A or A1, (b) at least one or two of the LC CDR amino acid sequences set forth in a single line of SEQ ID NO: in Table A or A1, and at least one or two of the HC CDR amino acid sequences set forth in a single line of SEQ ID NO: in Table A or A1, (c) at least one of the HC CDR amino acid sequences set forth in a single line of SEQ ID NO: in Table A or A1, and at least one or two of the LC CDR amino acid sequences set forth in a single line of SEQ ID NO: in Table A or A1, at least one or two of the CDR amino acid sequences, (d) all six CDR amino acid sequences shown in the SEQ ID NOs: in a single row of Table A, and / or (e) (a) SEQ ID NOs: 74 to 79, (b) SEQ ID NOs: 50 to 55, (c) SEQ ID NOs: 122 to 127, (d) SEQ ID NOs: 26 to 31, (e) SEQ ID NOs: 128 to 133, (f) SEQ ID NOs: 38 to 43, (g) SEQ ID NOs: 62 to 67, (h) SEQ ID NOs: 80 to 85, (i) SEQ ID NOs: 44 to 49, (j) SEQ ID NOs: 86 to 91, (k) SEQ ID NOs: 104 to 109, (l) SEQ ID NOs: 56 to 61, (m) SEQ ID NOs: 32 to 37, (n) SEQ ID NOs: 110 to 115, (o) SEQ ID NOs: 98 to 103, (p) SEQ ID NOs: 92 to 97,(q) a nucleotide sequence encoding an antigen-binding protein comprising six CDR amino acid sequences selected from the group consisting of SEQ ID NOs: 116 to 121, (r) SEQ ID NOs: 8 to 13, (s) SEQ ID NOs: 68 to 73, (t) SEQ ID NOs: 14 to 19, (u) SEQ ID NOs: 20 to 25, (v) SEQ ID NOs: 449 to 453 and 475, (w) SEQ ID NOs: 476 to 477, 454 to 457, (x) SEQ ID NOs: 458 to 463, (y) SEQ ID NOs: 57, 58, 464 to 467, (z) SEQ ID NOs: 68 to 71 and 468 to 469, and (aa) SEQ ID NOs: 112, and 470 to 474. In various embodiments, the nucleic acid comprises: (a) a heavy chain variable region amino acid sequence set forth in Table B or B1, i.e., a sequence selected from the group consisting of SEQ ID NOs: 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 478, 480, 482, 484, 486, and 488, or a variant thereof that differs by only one or two amino acids or has at least or about 70% (e.g., at least or about 80%, at least or about 85%, at least or about 90%, at least or about 95%) sequence identity; (b) a light chain variable region amino acid sequence set forth in Table B or B1, i.e., a sequence selected from the group consisting of SEQ ID NOs: 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 478, 480, 482, 484, 486, and 488; or a variant thereof which differs by only one or two amino acids or has at least or about 70% (e.g., at least or about 80%, at least or about 85%, at least or about 90%, at least or about 95%) sequence identity; or (c) both (a) and (b). In various embodiments, the nucleic acid is selected from the group consisting of: (a) SEQ ID NOs: 156 and 157; (b) SEQ ID NOs: 148 and 149; (c) SEQ ID NOs: 172 and 173; (d) SEQ ID NOs: 140 and 141;(e) SEQ ID NO:174 and 175; (f) SEQ ID NO:144 and 145; (g) SEQ ID NO:152 and 153; (h) SEQ ID NO:158 and 159; (i) SEQ ID NO:146 and 147; (j) SEQ ID NO:160 and 161; (k) SEQ ID NO:166 and 167; (l) SEQ ID NO:150 and 151; (m) SEQ ID NO:142 and 143; (n) SEQ ID NO:168 and 169; (o) SEQ ID NO:164 and 165; (p) SEQ ID NO:162 and 163; (q) SEQ ID NO:170 and 171; (r) SEQ ID NO:134 and 135; (s) SEQ ID NO:154 and 155; (t) SEQ ID NO:136 and 137; and (u) SEQ ID NO:138 and 139. In various embodiments, the nucleic acid comprises a nucleotide sequence encoding an antigen binding protein comprising a pair of amino acid sequences selected from the group consisting of the pairs set forth in Table D. In various aspects, the nucleic acid comprises a nucleotide sequence comprising any one or more of SEQ ID NOs: 208-375. In some embodiments, the nucleic acid does not contain any insertions, deletions, inversions, and / or substitutions. In other embodiments, the nucleic acid comprises one or more insertions, deletions, inversions, and / or substitutions.

[0114] In some aspects, the nucleic acid of the present disclosure is recombinant. As used herein, the term "recombinant" refers to (i) a molecule constructed outside a living cell by combining a natural or synthetic nucleic acid segment into a nucleic acid molecule capable of replicating in the living cell, or (ii) a molecule obtained by replicating the molecule described in (i) above. For the purposes of this specification, the replicating can be in vitro or in vivo.

[0115] In some aspects, nucleic acids are constructed based on chemical synthesis and / or enzymatic ligation reactions using methods well known in the art. See, for example, Sambrook et al., supra; and Ausubel et al., supra. For example, nucleic acids may be chemically synthesized using natural nucleotides or variously modified nucleotides designed to increase the biological stability of the molecule or to increase the physical stability of the hybridization duplex (e.g., phosphorothioate derivatives and acridine-substituted nucleotides). Examples of modified nucleotides that can be used to generate nucleic acids include 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxymethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, β-D-galactosyl eosin, inosine, N 6 -Isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N-substituted adenines, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, β-D-mannosyl eosin, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N 6 -isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxocine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methyl ester, 3-(3-amino-3-N-2-carboxypropyl)uracil, and 2,6-diaminopurine. Alternatively, one or more of the nucleic acids of the present disclosure may be purchased from companies such as Macromolecular Resources (Fort Collins, CO) and Synthegen (Houston, TX).

[0116] vector In some aspects, the nucleic acid of the disclosure is incorporated into a vector. In this regard, the disclosure provides a vector comprising any of the nucleic acids disclosed herein. In various aspects, the vector is a recombinant expression vector. For purposes of this specification, the term "recombinant expression vector" refers to a recombinant oligonucleotide or polynucleotide construct that allows expression of an mRNA, protein, polypeptide, or peptide by a host cell when the recombinant oligonucleotide or polynucleotide construct comprises a nucleotide sequence encoding an mRNA, protein, polypeptide, or peptide and contacts the cell under conditions sufficient to express the mRNA, protein, polypeptide, or peptide in the cell. The vector of the disclosure is not entirely natural. However, portions of the vector may be natural. The vectors disclosed herein may contain any type of nucleotide, including, but not limited to, DNA and RNA, which may be single-stranded or double-stranded and may be partially synthetic or obtained from natural sources, and DNA and RNA, which may contain natural, non-natural, or modified nucleotides. The vector may contain natural or non-natural internucleotide linkages, or both types of linkages. In some aspects, the modified nucleotides or non-natural internucleotide linkages do not interfere with the transcription or replication of the vector.

[0117] The vector of the present disclosure may be any suitable vector and may be used to transduce, transform or transfect any suitable host. Suitable vectors include vectors for propagation and propagation, vectors for expression, or both, such as plasmids and viruses. The vector may be a plasmid-based expression vector. In various aspects, the vector is selected from the group consisting of pUC series (Fermentas Life Sciences), pBluescript series (Stratagene, LaJolla, CA), pET series (Novagen, Madison, WI), pGEX series (Pharmacia Biotech, Uppsala, Sweden), and pEX series (Clontech, Palo Alto, CA). Bacteriophage vectors such as λGT10, λGT11, λZapII (Stratagene), λEMBL4, and λNMI149 may also be used. Examples of plant expression vectors include pBI101, pBI101.2, pBI101.3, pBI121, and pBIN19 (Clontech). Examples of animal expression vectors include pEUK-C1, pMAM, and pMAMneo (Clontech). In some aspects, the vector is a viral vector, e.g., a retroviral vector. In various aspects, the vector is an adenoviral vector, an adeno-associated viral (AAV) vector, a herpes simplex viral (HSV) vector, a vesicular stomatitis viral (VSV) vector, a vaccinia viral vector, or a lentiviral vector. See, e.g., Howarth et al., Cell Biol. Toxicol. 26(1):1-20 (2010). In various aspects, the vector is a baculoviral vector that infects arthropods, e.g., insects. In various embodiments, the baculovirus vector is Autographa californica polyhedrosis virus (AcMNPV) or Bombyx mori nuclear polyhedrosis virus (BmNPV).See, e.g., Khan, Adv Pharm Bull 3(2):257-263 (2013); Miller, Bioessays 11(4):91-96 (1989); Atkinson et al., Pestic Sci 28:215-224 (1990).

[0118] The vectors of the present disclosure can be prepared using standard recombinant DNA techniques, for example, as described in Sambrook et al., (supra) and Ausubel et al., (supra). Circular or linear expression vector constructs can be prepared to contain a replication system that functions in prokaryotic or eukaryotic host cells. Replication systems can be derived, for example, from CoIE1, 2μ plasmid, lambda, SV40, bovine papilloma virus, and the like.

[0119] In some aspects, vectors include regulatory sequences, such as transcription and translation initiation and termination codons, that are specific to the type of host (e.g., bacterial, fungal, plant, or animal) into which the vector will be introduced, as appropriate, considering whether the vector is DNA- or RNA-based.

[0120] A vector may contain one or more marker genes to allow for the selection of transformed or transfected hosts. Marker genes include biocide resistance, resistance to, e.g., antibiotics, heavy metals, etc., complementation in auxotrophic hosts resulting in prototrophy, etc. Suitable marker genes for use in the expression vectors disclosed herein include, for example, the neomycin / G418 resistance gene, the hygromycin resistance gene, the histidinol resistance gene, the tetracycline resistance gene, and the ampicillin resistance gene.

[0121] The vector may contain a native or non-native promoter operably linked to a nucleotide sequence encoding a polypeptide (including a functional portion or a functional variant thereof), or a nucleotide sequence complementary to or hybridizing to a nucleotide sequence encoding a polypeptide. The selection of a promoter (e.g., strong, weak, inducible, tissue-specific, and development-specific) is within the knowledge of one skilled in the art. Similarly, the combination of a nucleotide sequence with a promoter is within the knowledge of one skilled in the art. The promoter may be a non-viral promoter or a viral promoter, such as a cytomegalovirus (CMV) promoter, an SV40 promoter, an RSV promoter, and a promoter present in the long terminal repeat of the murine stem cell virus.

[0122] host cell Provided herein is a host cell comprising the nucleic acid or vector of the present disclosure. As used herein, the term "host cell" refers to any type of cell that can contain the vector disclosed herein and produce the expression product (e.g., mRNA, protein) encoded by the nucleic acid. In some aspects, the host cell is an adherent cell or a suspension cell, i.e., a cell that grows in suspension. In various aspects, the host cell is a cultured cell or a primary cell, i.e., directly isolated from an organism, e.g., a human. The host cell can be any cell type, originate from any type of tissue, and can be at any developmental stage.

[0123] In various embodiments, the antigen binding protein is a glycosylated protein and the host cell is a glycosylation competent cell. In various embodiments, the glycosylation competent cell is a eukaryotic cell, including but not limited to a yeast cell, a filamentous fungal cell, a protozoan cell, an algae cell, an insect cell, or a mammalian cell. Such host cells are described in the art. See, for example, Frenzel, et al., Front Immunol 4:217 (2013). In various embodiments, the eukaryotic cell is a mammalian cell. In various embodiments, the mammalian cell is a non-human mammalian cell. In some embodiments, the cells are selected from the group consisting of Chinese hamster ovary (CHO) cells and derivatives thereof (e.g., CHO-K1, CHO pro-3), mouse myeloma cells (e.g., NS0, GS-NS0, Sp2 / 0), cells genetically engineered to lack dihydrofolate reductase (DHFR) activity (e.g., DUKX-X11, DG44), human embryonic kidney 293 (HEK293) cells or derivatives thereof (e.g., HEK293T, HEK293-EBNA), African green monkey kidney cells (e.g., COS cells, VERO cells), human cervical carcinoma cells (e.g., HeLa), human bone osteosarcoma epithelial cells U2-OS, adenocarcinoma human alveolar basal epithelial cells A549, human fibrosarcoma cells HT1080, mouse brain tumor cells CAD, embryonal carcinoma cells P19, mouse embryonic fibroblast cells NIH 3T3, mouse fibroblast L929, mouse neuroblastoma N2a, human breast cancer MCF-7, retinoblastoma Y79, human retinoblastoma SO-Rb50, human hepatocellular carcinoma Hep G2, mouse B myeloma J558L, or baby hamster kidney (BHK) cells (Gaillet et al. 2007; Khan, Adv Pharm Bull 3(2):257-263(2013)).

[0124] In some aspects, a host cell for propagating or replicating a vector is a prokaryotic cell, such as a bacterial cell.

[0125] Further provided in the present disclosure is a population of cells comprising at least one host cell as described herein. In some aspects, the population of cells is a heterogeneous population, comprising host cells comprising the vectors described, plus at least one other cell that does not comprise any of the vectors. Alternatively, in some aspects, the population of cells is a substantially homogeneous population, where the population comprises primarily (e.g., consists essentially of) host cells comprising the vector. In some aspects, the population is a clonal population of cells, where all cells of the population are clones of a single host cell comprising the vector, such that all cells of the population comprise the vector. In various embodiments of the present disclosure, the population of cells is a clonal population comprising host cells comprising the vectors described herein.

[0126] Manufacturing method Also provided herein is a method for producing an antigen binding protein that binds to CLDN6. In various embodiments, the method comprises culturing a host cell comprising a nucleic acid comprising a nucleotide sequence encoding an antigen binding protein described herein in a cell culture medium, and recovering the antigen binding protein from the cell culture medium. The host cell may be any of the host cells described herein. In various aspects, the host cell is selected from the group consisting of CHO cells, NS0 cells, COS cells, VERO cells, and BHK cells. In various aspects, culturing the host cell comprises culturing the host cell in a growth medium to sustain the growth and proliferation of the host cell. In various aspects, the growth medium increases cell density, viability, and productivity of the culture in a timely manner. In various aspects, the growth medium comprises amino acids, vitamins, inorganic salts, glucose, and serum as a source of growth factors, hormones, and attachment factors. In various aspects, the growth medium is a synthetic medium consisting of amino acids, vitamins, trace elements, inorganic salts, lipids, and insulin or insulin-like growth factors. In addition to nutrients, the growth medium also helps maintain pH and osmolality. Several types of growth media are commercially available and described in the art. See, for example, Arora, "Cell Culture Media: A Review" MATER METHODS 3:175 (2013).

[0127] In various embodiments, the method comprises culturing the host cells in a feed medium. In various embodiments, the method comprises culturing in a fed-batch culture in a feed medium. Methods for producing recombinant proteins are well known in the art. See, e.g., Li et al., "Cell culture processes for monoclonal antibody production" MAbs 2(5):466-477 (2010).

[0128] The method for producing an antigen-binding protein may include one or more steps for purifying the protein from the cell culture or its supernatant, and preferably recovering the purified protein. In various embodiments, the method includes one or more chromatography steps, such as affinity chromatography (e.g., Protein A affinity chromatography), ion exchange chromatography, hydrophobic interaction chromatography. In various embodiments, the method includes purifying the protein using a Protein A affinity chromatography resin.

[0129] In various embodiments, the method further comprises a step of formulating the purified protein, etc., to obtain a formulation comprising the purified protein, such as described in Formulation and Process Development Strategies for Manufacturing, eds. Jameel and Hershenson, John Wiley & Sons, Inc. (Hoboken, NJ), 2010.

[0130] In various aspects, the antigen binding protein is linked to a polypeptide, and the antigen binding protein is part of a fusion protein. Thus, the present disclosure further provides a method for producing a fusion protein comprising an antigen binding protein that binds to CLDN6. In various embodiments, the method comprises culturing in a cell culture medium a host cell comprising a nucleic acid comprising a nucleotide sequence encoding a fusion protein as described herein, and recovering the fusion protein from the cell culture medium.

[0131] Conjugates The present disclosure also provides antigen binding proteins bound, linked or conjugated to a second moiety (e.g., a heterologous moiety, a conjugate moiety). Thus, the present disclosure provides a conjugate comprising an antigen binding protein and a heterologous moiety. As used herein, the term "heterologous moiety" is synonymous with "conjugate moiety" and means any molecule (chemical or biochemical, natural or non-encoded) that is distinct from the antigen binding protein of the present disclosure. Various heterologous moieties include, but are not limited to, polymers, carbohydrates, lipids, nucleic acids, oligonucleotides, DNA or RNA, amino acids, peptides, polypeptides, proteins, therapeutic agents (e.g., cytotoxic agents, cytokines), or diagnostic agents.

[0132] In some embodiments, the heterologous moiety is a polymer. The polymer may be branched or unbranched. The polymer may be of any molecular weight. In some embodiments, the polymer has an average molecular weight of about 2 kDa to about 100 kDa (the term "about" indicates that in a formulation of water-soluble polymers, some molecules will be heavier and some will be lighter compared to the stated molecular weight). In some aspects, the average molecular weight of the polymer is about 5 kDa to about 50 kDa, about 12 kDa to about 40 kDa, or about 20 kDa to about 35 kDa.

[0133] In some embodiments, the polymer is modified to have a single reactive group (e.g., an active ester for acylation or an aldehyde for alkylation, etc.), so that the degree of polymerization can be controlled. In some embodiments, the polymer is water-soluble, so that the protein to which the polymer is attached does not precipitate in an aqueous environment, such as a physiological environment. In some embodiments, the polymer is pharma- ceutically acceptable, for example, when the composition is used for therapeutic applications. Additionally, in some aspects, the polymer is a mixture of polymers, e.g., a copolymer, a block copolymer.

[0134] In some embodiments, the polymer is selected from the group consisting of polyamides, polycarbonates, polyalkylenes and their derivatives, including polyalkylene glycols, polyalkylene oxides, and polyalkylene terephthalates, polymers of acrylic and methacrylic esters, including poly(methyl methacrylate), poly(ethyl methacrylate), poly(butyl methacrylate), poly(isobutyl methacrylate), poly(hexymethacrylate), poly(isodecyl methacrylate), poly(lauryl methacrylate), poly(phenyl methacrylate), poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutyl acrylate), and poly(octadecyl acrylate), polyvinyl polymers, including polyvinyl alcohols, polyvinyl ethers, polyvinyl esters, and polyvinyl halides. , poly(vinyl acetate) and polyvinylpyrrolidone), polyglycolides, polysiloxanes, polyurethanes and their copolymers, celluloses (including alkyl celluloses, hydroxyalkyl celluloses, cellulose ethers, cellulose esters, nitrocellulose, methyl cellulose, ethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, hydroxybutyl methyl cellulose, cellulose acetate, cellulose propionate, cellulose acetate butyrate, cellulose acetate phthalate, carboxyethyl cellulose, cellulose triacetate and cellulose sulfate sodium salt), polypropylene, polyethylenes (including poly(ethylene) glycol, poly(ethylene oxide) and poly(ethylene terephthalate)), and polystyrene.

[0135] A particularly preferred water-soluble polymer for use herein is polyethylene glycol (PEG). As used herein, polyethylene glycol is meant to encompass any of the forms of PEG that can be used to derivatize other proteins, such as mono-(C1-C10) alkoxy- or aryloxy-polyethylene glycol. PEG is a linear or branched neutral polyether, is available in a wide range of molecular weights, and is soluble in water and most organic solvents.

[0136] In some embodiments, the heterologous moiety is a carbohydrate. In some embodiments, the carbohydrate is a monosaccharide (e.g., glucose, galactose, fructose), a disaccharide (e.g., sucrose, lactose, maltose), an oligosaccharide (e.g., raffinose, stachyose), a polysaccharide (starch, amylase, amylopectin, cellulose, chitin, callose, laminarin, xylan, mannan, fucoidan, galactomannan).

[0137] In some embodiments, the heterologous moiety is a lipid. In some embodiments, the lipid is a fatty acid (eicosanoid, prostaglandin, leukotriene, thromboxane, N-acylethanolamine), a glycerolipid (e.g., mono-, di-, tri-substituted glycerol), a glycerophospholipid (e.g., phosphatidylcholine, phosphatidylinositol, phosphatidylethanolamine, phosphatidylserine), a sphingolipid (e.g., sphingosine, ceramide), a sterol lipid (e.g., steroid, cholesterol), a prenol lipid, a glycolipid, or a polyketide, an oil, a wax, a cholesterol, a sterol, a fat-soluble vitamin, a monoglyceride, a diglyceride, a triglyceride, a phospholipid.

[0138] In some embodiments, the heterologous moiety is a therapeutic agent. The therapeutic agent may be any of the therapeutic agents known in the art. Examples of therapeutic agents contemplated herein include natural enzymes, proteins derived from natural sources, recombinant proteins, natural peptides, synthetic peptides, cyclic peptides, antibodies, receptor agonists, cytotoxic agents, immunoglobulins, beta-adrenergic blockers, calcium channel blockers, coronary vasodilators, cardiac glycosides, antiarrhythmics, cardiac sympathomimetics, angiotensin-converting enzyme (ACE) inhibitors, diuretics, inotropes, cholesterol- and triglyceride-lowering agents, bile acid sequestrants, fibrates, 3-hydroxy-3-methylglutaryl (H) Mg-CoA reductase inhibitors, niacin derivatives, antiadrenergics, alpha adrenergic blockers, centrally acting antiadrenergics, vasodilators, potassium-sparing agents, thiazides and related drugs, angiotensin II receptor antagonists, peripheral vasodilators, antiandrogens, estrogens, antibiotics, retinoids, insulin and analogs, alpha-glucosidase inhibitors, biguanides, meglitinides, sulfonylureas, thiazolidinediones, androgens, progestogens, bone metabolism regulators, anterior pituitary hormones, hypothalamic peripheral hormones, posterior pituitary hormones, gonadotropins, gonadotropin releasing hormone antagonists, ovulation inducers, selective estrogen receptor modulators, antithyroid drugs, thyroid hormones, bulk forming agents, laxatives, antiperistaltic agents, microbiota regulators, intestinal adsorbents, intestinal anti-infectives, anti-anorexic drugs, anti-cachexia drugs, anti-bulimia drugs, appetite suppressants, anti-obesity drugs, antacids, upper gastrointestinal drugs, anticholinergic drugs, aminosalicylic acid derivatives, biological response modifiers, corticosteroids, antispasmodics, 5-HT4 partial agonists, antihistamines, cannabinoids, dopamine antagonists, Serotonin antagonists, cell protective agents, histamine H2 receptor antagonists, mucosal protective agents, proton pump inhibitors, H. pylori eradication drugs, erythropoiesis stimulators, hematopoietic agents, anemia drugs, heparin, antifibrinolytic agents, hemostatic agents, blood coagulation factors, adenosine diphosphate inhibitors, glycoprotein receptor inhibitors, fibrinogen-platelet binding inhibitors, thromboxane A2 inhibitors, plasminogen activators, antithrombotic agents, glucocorticoids, mineralocorticoids, corticosteroids, selective immunosuppressants, antifungal agents, preventive treatment, AIDS-related infections,Agents for cytomegalovirus, non-nucleoside reverse transcriptase inhibitors, nucleoside analogue reverse transcriptase inhibitors, protease inhibitors, anemia, Kaposi's sarcoma, aminoglycosides, carbapenems, cephalosporins, glycopeptides, lincosamides, macrolides, oxazolidinones, penicillins, streptogramins, sulfonamides, trimethoprim and derivatives, tetracyclines, anthelmintics, amoebics, biguanides, cinchona alkaloids, folate antagonists, quinoline derivatives, Pneumocystis carinii drugs, hydrazides, imidazoles, triazoles, nitroimidazoles, cyclic amines, neuraminidase inhibitors, nucleosides, phosphate binders, cholinesterase inhibitors, adjunctive therapy drugs, barbiturates and derivatives, benzodiazepines, gamma aminobutyric acid derivatives, hydantoin derivatives, iminostilbene derivatives, succinimide derivatives, anticonvulsants, ergot alkaloids, antimigraine preparations, biological response modifiers, carbamates, tricyclic derivatives, depolarizing agents, nondepolarizing agents, neuromuscular paralytic agents, CNS stimulants, dopaminergic agents, monoamine oxidase inhibitors, COMT inhibitors, alkylsulfonates, ethylenimines, imidazotetrazines, nitrogen mustard analogues, nitrosoureas, platinum-containing compounds, antimetabolites, purine analogues, pyrimidine analogues, urea derivatives, anthracyclines, actinomycin, camptothecin derivatives, epipods Phyllotoxins, taxanes, vinca alkaloids and analogs, antiandrogens, antiestrogens, nonsteroidal aromatase inhibitors, protein kinase inhibitor antitumor agents, azaspirodecanedione derivatives, anxiolytics, stimulants, monoamine reuptake inhibitors, selective serotonin reuptake inhibitors, antidepressants, benzisoxazole derivatives, butyrophenone derivatives, dibenzodiazepine derivatives, dibenzothiazepine derivatives, diphenylbutylpiperidine derivatives, phenothiazines, thienobenzodiazepine derivatives, thioxanthene derivatives, allergen extracts, nonsteroids, leukotriene receptor antagonists, xanthines, endothelin receptor antagonists, prostaglandins, pulmonary surfactants, mucolytic agents, antimitotics, uricosuric agents, xanthine oxidase inhibitors, phosphodiesterase inhibitors, methenamine salts, nitrofuran derivatives, quinolones, smooth muscle relaxants,These include, but are not limited to, parasympathomimetics, halogenated hydrocarbons, esters of aminobenzoic acid, amides (e.g., lidocaine, articaine hydrochloride, bupivacaine hydrochloride), antipyretics, hypnotics and sedatives, cyclopyrrolones, pyrazolopyrimidines, nonsteroidal anti-inflammatory drugs, opioids, paraaminophenol derivatives, alcohol dehydrogenase inhibitors, heparin antagonists, adsorbents, emetics, opioid antagonists, cholinesterase reactivators, nicotine replacement therapy, vitamin A analogs and antagonists, vitamin B analogs and antagonists, vitamin C analogs and antagonists, vitamin D analogs and antagonists, vitamin E analogs and antagonists, and vitamin K analogs and antagonists.

[0139] The antigen binding proteins of the present disclosure may be conjugated to one or more cytokines and growth factors that are effective in inhibiting tumor metastasis and that have been shown to have a growth-inhibitory effect on at least one cell population, including, but not limited to, M-CSF, GM-CSF, TNF, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IFN, TNFα, TNF1, TNF2, G-CSF, Meg-CSF, GM-CSF, thrombopoietin, stem cell factor, and erythropoietin.Additional growth factors for use herein include angiogenin, bone morphogenetic protein-1, bone morphogenetic protein-2, bone morphogenetic protein-3, bone morphogenetic protein-4, bone morphogenetic protein-5, bone morphogenetic protein-6, bone morphogenetic protein-7, bone morphogenetic protein-8, bone morphogenetic protein-9, bone morphogenetic protein-10, bone morphogenetic protein-11, bone morphogenetic protein-12, bone morphogenetic protein-13, bone morphogenetic protein-14, bone morphogenetic protein-15, bone morphogenetic protein receptor IA, bone morphogenetic protein receptor IB, brain derived neurotrophic factor, ciliary neurotrophic factor, ciliary neurotrophic factor receptor α, cytokine-induced neutrophil chemotactic factor 1, cytokine-induced neutrophil chemotactic factor 2α, cytokine-induced neutrophil chemotactic factor 2β, beta endothelial cell growth factor, endothelin 1, epithelium-derived neutrophil attractant, glial cell line-derived neurotrophic factor receptor α1, glial cell line-derived neurotrophic factor receptor α2, growth-associated protein, growth-associated protein α, growth-associated protein β, growth-associated protein γ, heparin-binding epidermal growth factor, hepatocyte growth factor, hepatocyte growth factor receptor , insulin-like growth factor I, insulin-like growth factor receptor, insulin-like growth factor II, insulin-like growth factor binding protein, keratinocyte growth factor, leukemia inhibitory factor, leukemia inhibitory factor receptor alpha, nerve growth factor, nerve growth factor receptor, neurotrophin-3, neurotrophin-4, pre-B cell growth stimulatory factor, stem cell factor, stem cell factor receptor, transforming growth factor alpha, transforming growth factor beta, transforming growth factor beta 1, transforming growth factor beta 1.2, transforming growth factor beta 2, transforming growth factor beta 3, transforming growth factor beta 5, latent transforming growth factor beta 1, transforming growth factor beta binding protein I, transforming growth factor beta binding protein II, transforming growth factor beta binding protein III, tumor necrosis factor receptor type I, tumor necrosis factor receptor type II, urokinase-type plasminogen activator receptor, and chimeric proteins and biologically or immunologically active fragments thereof.

[0140] In some embodiments, the conjugate comprises an antigen binding protein as described herein and a cytotoxic agent. A cytotoxic agent is any molecule (chemical or biochemical) that is toxic to cells. In some aspects, when a cytotoxic agent is conjugated to an antigen binding protein of the present disclosure, the results obtained are synergistic. In other words, the effect of the combined treatment of the antigen binding protein and the cytotoxic agent is synergistic, that is, the effect is higher than would be expected from the additive effect of each individually. Therefore, the dose of the cytotoxic agent can be reduced, which simultaneously reduces the risk of toxicity issues and other side effects. In some embodiments, the cytotoxic agent is a chemotherapeutic agent. Chemotherapeutic agents are well known in the art and include, but are not limited to, platinum coordination compounds as described in U.S. Pat. No. 6,630,124, topoisomerase inhibitors, antibiotics, antimitotic alkaloids, and difluoronucleosides.

[0141] In some embodiments, the chemotherapeutic agent is a platinum coordination compound. The term "platinum coordination compound" refers to any tumor cell growth inhibiting platinum coordination compound that provides platinum in ionic form. In some embodiments, cisplatin is a platinum coordination compound employed in the compositions and methods of the present disclosure. In some embodiments, the chemotherapeutic agent is a topoisomerase inhibitor. In some aspects, the topoisomerase inhibitor is a camptothecin or a camptothecin analog. In still further other embodiments of the present disclosure, the chemotherapeutic agent is an antibiotic compound. Suitable antibiotics include, but are not limited to, doxorubicin, mitomycin, bleomycin, daunorubicin, and streptozocin. In some embodiments, the chemotherapeutic agent is an antimitotic alkaloid. Antimitotic alkaloids are generally extractable from Catharanthus roseus and have been shown to be effective as anticancer chemotherapeutic agents. In other embodiments of the present disclosure, the chemotherapeutic agent is a difluoronucleoside. It is well known in the art that 2'-deoxy-2',2'-difluoronucleosides have antiviral activity. Such compounds are disclosed and taught in U.S. Patent Nos. 4,526,988 and 4,808,614. European Patent Application Publication No. 184,365 discloses that these same difluoronucleosides have oncolytic activity.

[0142] The present disclosure also provides a conjugate comprising an antigen-binding protein of the present disclosure linked to a polypeptide, such that the conjugate is a fusion protein. Thus, the present disclosure provides a fusion protein comprising an antigen-binding protein of the present disclosure linked to a polypeptide. In various embodiments, the polypeptide is a diagnostic label, for example, a fluorescent protein such as green fluorescent protein, or other tag, for example, a Myc tag. In various aspects, the polypeptide is one of the above cytokines, lymphokines, growth factors, or other hematopoietic factors.

[0143] Linker In some embodiments, the conjugate is directly linked to the heterologous moiety. In alternative embodiments, the conjugate comprises a linker that connects the compound of the present disclosure to the heterologous moiety. In some aspects, the linker comprises a molecular chain that is 1 to about 60 atoms, 1 to 30 atoms, or more, 2 to 5 atoms, 2 to 10 atoms, 5 to 10 atoms, or 10 to 20 atoms long. In some embodiments, the atoms of the molecular chain are all carbon atoms. In some embodiments, the atoms of the molecular chain in the backbone of the linker are selected from the group consisting of C, O, N, and S. The atoms of the molecular chain and the linker may be selected according to their predicted solubility (hydrophilicity) to provide a more soluble conjugate. In some embodiments, the linker provides a functional group that is susceptible to cleavage by an enzyme or other catalyst or hydrolytic conditions present in the target tissue or organ or within the target cell. In some embodiments, the length of the linker is long enough to reduce the possibility of steric hindrance. In some embodiments, the linker is an amino acid linker or a peptidyl linker. Such peptidyl linkers may be of any length. Various linkers are from about 1-50 amino acids in length, 5-50, 3-5, 5-10, 5-15, or 10-30 amino acids in length.

[0144] Compositions, pharmaceutical compositions and formulations Provided herein are compositions comprising an antigen binding protein, nucleic acid, vector, host cell, or conjugate disclosed herein. In some aspects, the compositions comprise the antigen binding protein in isolated and / or purified form. In some aspects, the compositions comprise a single type (e.g., structure) of the antigen binding protein of the disclosure, or a combination of two or more antigen binding proteins of the disclosure, where the combination comprises two or more antigen binding proteins of different types (e.g., structures).

[0145] In some aspects, the composition comprises an agent that improves the chemical-physical properties of the antigen binding protein, e.g., by stabilizing the antigen binding protein at a particular temperature, e.g., room temperature, extending the shelf life, inhibiting degradation, e.g., oxidative protease mediated degradation, extending the half-life of the antigen binding protein, etc. In some aspects, the composition optionally comprises any of the agents disclosed herein as a heterologous or conjugated moiety in admixture with or conjugated to an antigen binding protein of the disclosure.

[0146] In various aspects of the present disclosure, the composition further comprises a pharma- ceutically acceptable carrier, diluent or excipient. In some embodiments, the antigen-binding protein, nucleic acid, vector, host cell, or conjugate disclosed herein (hereinafter referred to as "active agent") is formulated into a pharmaceutical composition comprising the active agent together with a pharma- ceutically acceptable carrier, diluent or excipient. In this regard, the present disclosure further provides a pharmaceutical composition comprising the active agent intended for administration to a subject, e.g., a mammal.

[0147] In some embodiments, the active agent is included in the pharmaceutical composition at a purity level suitable for administration to a patient. In some embodiments, the active agent has a purity level of at least about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% and a pharma- ceutically acceptable diluent, carrier, or excipient. In some embodiments, the composition contains the active agent at a concentration of about 0.001 to about 30.0 mg / ml.

[0148] In various embodiments, the pharmaceutical composition comprises a pharma- ceutically acceptable carrier. As used herein, the term "pharmaceutically acceptable carrier" includes any of the standard pharmaceutical carriers, such as phosphate buffered saline, water, emulsions such as oil / water emulsions or water / oil emulsions, and various types of wetting agents. The term also includes any of the agents approved by a regulatory agency of the U.S. Federal Government or listed in the United States Pharmacopeia for use in animals, including humans.

[0149] Pharmaceutical compositions may contain, for example, an acidifier, an additive, an adsorbent, an aerosol propellant, an exhaust agent, an alkalizing agent, an anti-caking agent, an anticoagulant, an antimicrobial preservative, an antioxidant, a preservative, a base, a binder, a buffer, a chelating agent, a coating agent, a colorant, a drying agent, a detergent, a diluent, a bactericide, a disintegrant, a dispersant, a dissolution enhancer, a dye, an emollient, an emulsifier, an emulsion stabilizer, a filler, a film former, a flavor enhancer, a flavoring agent, a glidant, a gelling agent, a granulating agent, a humectant, a lubricant, a mucoadhesive agent, an ointment base, an ointment, an oily vehicle, an organic base, a pastel base, a pigment, a plasticizer, a polishing agent, a preservative, a sequestering agent, a skin penetration agent, a solubilizing agent, a solvent, a stabilizer, a suppository base, a surface active agent, a surfactant, a suspending agent, a sweetener, a therapeutic agent, a thickening agent ... suspending agent, a sweetener, a therapeutic agent, a thickening agent, a suppository base, a surface active agent, a thickening agent, a suppository base, a surface active agent, a suppository base, a surface active agent, a suspending agent, a sweetener, a therapeutic agent, a thickening agent, a suppository base, a surface active agent, a thickening The composition may contain any pharma- ceutically acceptable ingredients, including, for example, glycerol, tonicity agents, toxicity agents, viscosity-increasing agents, water-absorbing agents, water-miscible cosolvents, water-softening agents, or wetting agents. See, e.g., the Handbook of Pharmaceutical Excipients, Third Edition, A.H. Kibbe (Pharmaceutical Press, London, UK, 2000), which is incorporated by reference in its entirety. Remington's Pharmaceutical Sciences, Sixteenth Edition, E.W. Martin (Mack Publishing Co., Easton, Pa., 1980), which is incorporated by reference in its entirety.

[0150] In various aspects, pharmaceutical compositions include compounding substances that are non-toxic to the recipient at the dosages and concentrations employed. In certain embodiments, pharmaceutical compositions include an active agent and one or more pharma- ceutically acceptable salts, polyols, surfactants, osmotic balancing agents, tonicity agents, antioxidants, antibiotics, antifungal agents, bulking agents, lyoprotectants, antifoaming agents, chelating agents, preservatives, coloring agents, analgesics, or additional pharmaceutical agents. In various aspects, pharmaceutical compositions optionally include one or more polyols and / or one or more surfactants in addition to one or more additives, including, but not limited to, pharma- ceutically acceptable salts, osmotic balancing agents (tonicity agents), antioxidants, antibiotics, antifungal agents, bulking agents, lyoprotectants, antifoaming agents, chelating agents, preservatives, coloring agents, and analgesics.

[0151] In certain embodiments, pharmaceutical compositions may contain formulation substances to adjust, maintain or preserve, for example, the pH, osmolality, viscosity, clarity, color, isotonicity, aroma, sterility, stability, dissolution or release rate, adsorption, or permeability of the composition. Suitable compounding substances in such embodiments include amino acids (such as, for example, glycine, glutamine, asparagine, arginine, or lysine), antimicrobial agents, antioxidants (such as, for example, ascorbic acid, sodium sulfite, or sodium bisulfite), buffers (such as, for example, borate, bicarbonate, Tris-HCl, citrate, phosphate, or other organic acids), bulking agents (such as, for example, mannitol or glycine), chelating agents (such as, for example, ethylenediaminetetraacetic acid (EDTA)), complexing agents (such as, for example, caffeine, polyvinylpyrrolidone, β-cyclodextrin, or hydroxypropyl-β-cyclodextrin), fillers, monosaccharides, disaccharides, and other carbohydrates (such as, for example, glucose, mannose, or dextrin), proteins (such as, for example, serum albumin, gelatin, or immunoglobulins), colorants, flavoring agents, and diluents, emulsifiers, hydrophilic polymers (such as, for example, polyvinylpyrrolidone), low molecular weight polypeptides, salt forming agents, and the like. These include, but are not limited to, counterions (such as, for example, sodium), preservatives (such as, for example, benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid or hydrogen peroxide), solvents (such as, for example, glycerin, propylene glycol or polyethylene glycol), sugar alcohols (such as, for example, mannitol or sorbitol), suspending agents, surfactants or wetting agents (such as, for example, Pluronic®, PEG, sorbitan esters, polysorbates such as polysorbate 20, polysorbatc, triton, tromethamine, lecithin, cholesterol, tyloxapol, etc.), stability enhancers (such as, for example, sucrose or sorbitol), tonicity enhancers (such as, for example, alkali metal halides, preferably sodium or potassium chloride, mannitol, sorbitol, etc.), delivery vehicles, diluents, additives and / or pharmaceutical adjuvants.See REMINGTON'S PHARMACEUTICAL SCIENCES, 18th Edition, (AR Genrmo, ed.), 1990, Mack Publishing Company.

[0152] The pharmaceutical composition may be formulated to achieve a physiologically compatible pH. In some embodiments, the pH of the pharmaceutical composition may be, for example, about 4 or about 5 to about 8.0, about 4.5 to about 7.5, or about 5.0 to about 7.5. In various embodiments, the pH of the pharmaceutical composition is 5.5 to 7.5.

[0153] The present disclosure provides methods for producing pharmaceutical compositions. In various aspects, the methods include mixing an antigen binding protein, a conjugate, a fusion protein, a nucleic acid, a vector, a host cell, or a combination thereof with a pharma- ceutically acceptable carrier, diluent, or excipient.

[0154] Route of administration In the present disclosure, the active agent or pharmaceutical composition comprising the active agent may be administered to a subject via any suitable administration route. For example, the active agent may be administered to a subject via parenteral, nasal, oral, pulmonary, topical, or rectal administration. The following description of the administration route is provided merely to illustrate various embodiments, but should not be construed as limiting the scope in any manner.

[0155] Formulations suitable for parenteral administration include aqueous and non-aqueous isotonic sterile injection solutions that may contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, as well as aqueous and non-aqueous sterile suspensions that may contain suspending agents, solubilizing agents, thickening agents, stabilizers, and preservatives. The term "parenteral" means not through the digestive tract, but through some other route such as subcutaneous, intramuscular, intraspinal, or intravenous. The active agents of the present disclosure may be administered with a pharmaceutical carrier, for example, a physiologically acceptable diluent including a sterile liquid or mixture of liquids including water, saline, aqueous dextrose and related sugar solutions, alcohols, such as ethanol or hexadecyl alcohol, glycols, such as propylene glycol or polyethylene glycol, dimethylsulfoxide, glycerol, ketals, such as 2,2-dimethyl-1,3-dioxolane-4-methanol, ethers, poly(ethylene glycol) 400, oils, fatty acids, fatty acid esters or glycerides, or acetylated fatty acid glycerides, suspending agents, such as pectin, carbomer, methylcellulose, hydroxypropylmethylcellulose or carboxymethylcellulose, or emulsifying agents and other pharmaceutical auxiliary agents, with or without the addition of a pharma- ceutically acceptable surfactant, such as soap or detergent.

[0156] The oils that can be used in parenteral formulations include petroleum, animal, vegetable, or synthetic oils.Specific examples of oils include peanut, soybean, sesame, cottonseed, corn, olive, petrolatum, and mineral.Suitable fatty acids for use in parenteral formulations include oleic acid, stearic acid, and isostearic acid.Ethyl oleate and isopropyl myristate are examples of suitable fatty acid esters.

[0157] Suitable soaps for use in parenteral formulations include alkali metal, ammonium, and triethanolamine salts of fatty acids, and suitable surfactants include (a) cationic surfactants, such as dimethyldialkylammonium halides and alkylpyridinium halides; (b) anionic surfactants, such as alkyl, aryl, and olefin sulfonates, alkyl sulfates, olefin sulfates, ether sulfates, and monoglyceride sulfates, as well as alkyl, olefin sulfosuccinates, ether sulfosuccinates, and monoglyceride sulfosuccinates; (c) nonionic surfactants, such as fatty amine oxides, fatty acid alkanolamides, and polyoxyethylene polypropylene copolymers; (d) amphoteric surfactants, such as alkyl-β-aminopropionates and 2-alkyl-imidazoline quaternary ammonium salts; and (e) mixtures thereof.

[0158] In some embodiments, the parenteral formulation contains about 0.5% to about 25% by weight of the active agent of the present disclosure in a liquid formulation. Preservatives and buffers may be used. To minimize or eliminate inflammation at the injection site, such compositions may contain one or more non-ionic surfactants having a hydrophilic-lipophilic balance (HLB) of about 12 to about 17. The amount of surfactant in such formulations typically ranges from about 5% to about 15% by weight. Suitable surfactants include polyethylene glycol sorbitan fatty acid esters, such as sorbitan monooleate, and high molecular weight addition compounds of ethylene oxide with a hydrophobic base, formed by condensing propylene oxide with propylene glycol. In some aspects, the parenteral formulations are provided in single or multi-dose sealed containers, such as ampoules and vials, and may be stored in a freeze-dried (lyophilized) state requiring only the addition of a sterile liquid excipient for injection (e.g., water) immediately prior to use. In some aspects, extemporaneous injection solutions and suspensions are prepared from sterile powders, granules and tablets of the type described above.

[0159] Injectable formulations are subject to the present disclosure. The requirements for effective pharmaceutical carriers for injectable compositions are well known to those skilled in the art (see, for example, Pharmaceutics and Pharmacy Practice, JBLippincott Company, Philadelphia, PA, Banker and Chalmers, eds., pages 238-250 (1982), and ASHP Handbook on Injectable Drugs, Toissel, 4th ed., pages 622-630 (1986)).

[0160] dose The active agents of the present disclosure are believed to be useful in methods for inhibiting tumor growth as well as other methods further described herein, including methods for treating or preventing cancer. For purposes of this disclosure, the amount or dose of the active agent administered should be sufficient, for example, to achieve a therapeutic or prophylactic effect in the subject or animal for an appropriate period of time. For example, a dose of the active agent of the present disclosure should be sufficient to treat the cancers described herein for a period of about 1-4 minutes, 1-4 hours, or 1-4 weeks, or longer, such as 5-20 or more weeks, from the time of administration. In certain embodiments, the period may be even longer. The dose is determined by the effect of the particular active agent as well as the condition of the animal (e.g., human) to be treated and the body weight of the animal (e.g., human).

[0161] Many assays for determining dosage are well known in the art. For the purposes of this specification, the starting dosage for administration to a mammal can be determined using an assay that includes comparing the degree of cancer treatment by administration of a given dose of the active agent of the present disclosure to a mammal between a set of mammals, each set of mammals being administered a different dose of the active agent. The degree of cancer treatment by administration of a given dose can be represented, for example, by the degree of tumor regression achieved by the active agent in a mouse xenograft model. Methods for evaluating tumor regression are well known in the art and are described in the examples of this specification.

[0162] The dosage of the active agent of the present disclosure will also be determined by the presence, nature and extent of any side effects that may accompany the administration of a particular active agent of the present disclosure. Typically, the dosage of the active agent of the present disclosure for treating each individual patient will be determined by the attending physician, taking into consideration various factors such as age, body weight, general health, nutrition, sex, the active agent of the present disclosure to be administered, the route of administration, and the severity of the condition being treated. By way of example and not intended to limit the present disclosure, the dosage of the active agent of the present disclosure may be about 0.0001 to about 1 g / kg (body weight of the subject to be treated) / day, about 0.0001 to about 0.001 g / kg (body weight) / day, or about 0.01 mg to about 1 g / kg (body weight) / day.

[0163] Controlled-release formulations In some embodiments, the active agents described herein may be modified into a depot form (see, for example, U.S. Pat. No. 4,450,150) so that the release of the active agent of the present disclosure into the body to which the active agent of the present disclosure is administered is controlled according to time and location within the body. The depot form of the active agent of the present disclosure may be, for example, an implantable composition comprising the active agent and a porous or non-porous material, such as a polymer, in which the active agent is encapsulated or diffused within the porous material and / or diffuses due to the degradation of the non-porous material. Thus, when the depot is implanted into a desired location within the subject's body, the active agent is released from the implant at a predetermined rate.

[0164] In certain embodiments, the pharmaceutical composition comprising the active agent is modified to have any type of in vivo release characteristics.In some embodiments, the pharmaceutical composition is an immediate release formulation, a controlled release formulation, a sustained release formulation, a slow release formulation, a delayed release formulation, or a biphasic release formulation.Methods for formulating peptides for controlled release are well known in the art.See, for example, Qian et al., J Pharm 374:46-52(2009), and International Patent Publication Nos. WO2008 / 130158, WO2004 / 033036, WO2000 / 032218, and WO1999 / 040942.

[0165] The composition may further comprise, for example, a micelle or liposome or some other encapsulated form, or may be administered in a sustained release form providing a long term storage and / or delivery effect.

[0166] use The antigen binding proteins of the present disclosure are useful for inhibiting tumor growth. Without being bound to a particular theory, the inhibitory effects of the antigen binding proteins provided herein make such moieties useful in methods for treating cancer.

[0167] Thus, provided herein are methods for inhibiting tumor growth in a subject and for reducing the size of a tumor in a subject. In various embodiments, the methods include administering to the subject a pharmaceutical composition of the present disclosure in an amount effective to inhibit tumor growth or reduce the size of a tumor in the subject. In various aspects, the growth of an ovarian, melanoma, bladder, or endometrial tumor is inhibited. In various aspects, the size of an ovarian, melanoma, bladder, or endometrial tumor is reduced.

[0168] As used herein, the terms "inhibit" or "reduce" and words derived therefrom may not be 100% or complete inhibition or reduction. Rather, there are various degrees of inhibition or reduction that one of skill in the art would recognize as having potential benefits or therapeutic effects. In this regard, the antigen binding proteins of the present disclosure may inhibit tumor growth or reduce tumor size to any amount or level. In various embodiments, the inhibition provided by the methods of the present disclosure is at least 10% or about 10% inhibition (e.g., at least 20% or about 20% inhibition, at least 30% or about 30% inhibition, at least 40% or about 40% inhibition, at least 50% or about 50% inhibition, at least 60% or about 60% inhibition, at least 70% or about 70% inhibition, at least 80% or about 80% inhibition, at least 90% or about 90% inhibition, at least 95% or about 95% inhibition, at least 98% or about 98% inhibition). In various embodiments, the reduction provided by the methods of the present disclosure is at least or about a 10% reduction (e.g., at least or about a 20% reduction, at least or about a 30% reduction, at least or about a 30% reduction, at least or about a 40% reduction, at least or about a 50% reduction, at least or about a 50% reduction, at least or about a 60% reduction, at least or about a 70% reduction, at least or about a 70% reduction, at least or about a 80% reduction, at least or about a 90% reduction, at least or about a 95% reduction, at least or about a 95% reduction, at least or about a 98% reduction).

[0169] Further provided herein is a method for treating a subject having cancer, e.g., a CLDN6-expressing cancer, in various embodiments, the method comprises administering to the subject a pharmaceutical composition of the present disclosure in an amount effective to treat the cancer in the subject.

[0170] For purposes herein, the cancer of the methods disclosed herein may be any cancer, for example, any malignant growth or tumor resulting from abnormal and unregulated cell division that can metastasize to another part of the body via the lymphatic system or bloodstream. In some aspects, the cancer is selected from the group consisting of acute lymphocytic cancer, acute myeloid leukemia, alveolar rhabdomyosarcoma, bone cancer, brain cancer, breast cancer, cancer of the anus, anal canal, or anorectal, cancer of the eye, cancer of the intrahepatic bile duct, cancer of the joints, cancer of the neck, gallbladder, or pleura, cancer of the nose, nasal cavity, or middle ear, cancer of the oral cavity, cancer of the vulva, chronic lymphocytic leukemia, chronic myeloid cancer, colon cancer, esophageal cancer, cervical cancer, gastrointestinal carcinoid tumor, Hodgkin's lymphoma, hypopharyngeal cancer, kidney cancer, laryngeal cancer, liver cancer, lung cancer, malignant mesothelioma, melanoma, multiple myeloma, nasopharyngeal cancer, non-Hodgkin's lymphoma, ovarian cancer, pancreatic cancer, cancer of the peritoneum, omentum, and mesenteric cancer, pharyngeal cancer, prostate cancer, rectal cancer, renal cancer, and / or rectal cancer. In certain embodiments, the cancer is selected from the group consisting of head and neck cancer, ovarian cancer, cervical cancer, bladder and esophageal cancer, pancreatic cancer, gastrointestinal cancer, gastric cancer, breast cancer, endometrial and colorectal cancer, hepatocellular carcinoma, glioblastoma, bladder cancer, lung cancer, e.g., non-small cell lung cancer (NSCLC), bronchioloalveolar carcinoma. In various embodiments, the cancer is ovarian cancer, melanoma, bladder cancer, lung cancer, liver cancer, endometrial cancer. In various embodiments, the cancer is any cancer characterized by moderate to high expression of CLDN6. See, e.g., Figures 1-3. In various embodiments, the cancer is acute myeloid leukemia, large B-cell lymphoma, gastric cancer, prostate cancer, melanoma, colon cancer, rectal cancer, bladder cancer, cervical cancer, liver cancer, breast cancer, renal clear cell carcinoma, head and neck cancer, sarcoma, renal non-chromatinized carcinoma, low grade glioma, adrenocortical carcinoma, glioblastoma, renal papillary cell carcinoma, lung squamous cell carcinoma, thyroid cancer, lung adenocarcinoma, pancreatic cancer, endometrial cancer, uterine sarcoma, or ovarian cancer. In various embodiments, the cancer is selected from ovarian cancer, endometrial cancer, uterine cancer, lung cancer, gastric cancer, breast cancer, head and neck squamous cell carcinoma (HNSCC), cervical cancer, and bladder cancer.

[0171] As used herein, the term "treat" and related words do not necessarily mean 100% or complete treatment. Rather, there are various degrees of treatment that one skilled in the art will recognize as having potential benefits or therapeutic effects. In this regard, the disclosed method for treating cancer can provide any amount or level of treatment. Furthermore, the treatment provided by the disclosed method can include treating one or more symptoms or signs or symptoms of the cancer being treated. The treatment provided by the disclosed method can also include slowing the progression of the cancer. For example, the disclosed method can treat cancer by improving T cell activity or immune response against the cancer, inhibiting tumor or cancer growth, inhibiting metastasis of tumor cells, promoting cell death of tumor or cancer cells, etc. In various embodiments, the methods are for treating with the intent of delaying the onset or recurrence of cancer for at least 1 day, 2 days, 4 days, 6 days, 8 days, 10 days, 15 days, 30 days, 2 months, 3 months, 4 months, 6 months, 1 year, 2 years, 3 years, 4 years, or more. In various embodiments, the methods are for treating with the intent of extending the survival of the subject.

[0172] The antigen binding proteins of the present disclosure may also be used to detect CLDN6 in a sample or to diagnose CLDN6-positive cancer. Thus, the present disclosure provides a method for detecting claudin 6 (CLDN6) in a sample. In various embodiments, the method comprises contacting the sample with an antigen binding protein, conjugate, or fusion protein described herein, and assaying an immune complex comprising the antigen binding protein, conjugate, or fusion protein bound to CLDN6. The present disclosure also provides a method for diagnosing claudin 6 (CLDN6)-positive cancer in a subject. In various embodiments, the method comprises contacting a biological sample comprising cells or tissue taken from a subject with an antigen binding protein, conjugate, or fusion protein described herein, and assaying an immune complex comprising the antigen binding protein, conjugate, or fusion protein bound to CLDN6.

[0173] subject In some embodiments of the present disclosure, the subject is a mammal, including but not limited to, a mammal of the order Rodentia, such as mice and hamsters, a mammal of the order Lagomorpha, such as rabbits, a mammal of the order Carnivora, including Felidae (cats) and Canidae (dogs), a mammal of the order Artiodactyla, including Bovidae (cows) and Swines (pigs), or a mammal of the order Perissodactyla, including Equidae (horses). In some aspects, the mammal is a mammal of the order Primates, Ceboids, or Simoids (monkeys), or a mammal of the order Apes (humans and apes). In some aspects, the mammal is a human.

[0174] kit In some embodiments, the antigen binding protein of the present disclosure is provided in a kit. In various aspects, the kit includes the antigen binding protein(s) as a unit dose. For purposes herein, a "unit dose" refers to an individual amount dispersed in a suitable carrier. In various aspects, a unit dose is an amount sufficient to produce a desired effect in a subject, e.g., inhibiting tumor growth, reducing tumor size, treating cancer. Thus, provided herein is a kit including an antigen binding protein of the present disclosure, optionally provided in a unit dose. In various aspects, the kit includes several unit doses, e.g., a weekly or monthly supply of unit doses, each of which is optionally packaged individually or otherwise separated from the other unit doses. In some embodiments, the components / unit doses of the kit are packaged with instructions for administration to a patient. In some embodiments, the kit includes one or more devices for administration to a patient, e.g., a needle and a syringe, etc. In some aspects, the antigen binding protein of the present disclosure, a pharma- ceutically acceptable salt thereof, a conjugate comprising the antigen binding protein, or a multimer or dimer comprising the antigen binding protein is prepackaged in a ready-to-use form, e.g., a syringe, an intravenous infusion bag, etc. In some aspects, the kit further comprises other therapeutic or diagnostic agents or a pharma- ceutically acceptable carrier (e.g., a solvent, a buffer, a diluent, etc.), including any of those described herein. In certain aspects, the kit comprises an antigen binding protein of the present disclosure together with an agent, e.g., a therapeutic agent for use in chemotherapy or radiation therapy.

[0175] Various embodiments In various embodiments of the disclosure, the antigen binding protein binds to human claudin 6 (CLDN6) protein (SEQ ID NO:200), and (a) the antigen binding protein binds to extracellular loop 2 (EL2) of the extracellular domain (ECD) of CLDN6 and does not bind to extracellular loop 1 (EL1) of the ECD of CLDN6, (b) does not bind to any of claudin 3 (CLDN3), claudin 4 (CLDN4) and claudin 9 (CLDN9) and inhibits binding of a reference antibody to CLDN6 endogenously expressed by OVCA429 cells at less than about 1200 nM, or (c) a combination thereof. In various examples, the antigen binding protein binds to an epitope within the amino acid sequence WTAHAIIRDFYNPLVAEAQKREL (SEQ ID NO:2) or binds to the amino acid sequence TAHAIIRDFYNPL (SEQ ID NO:3) or LVAEAQKREL (SEQ ID NO:4) of CLDN6. In various aspects, the antigen binding protein does not bind to any one or more of claudin 3 (CLDN3), claudin 4 (CLDN4) and claudin 9 (CLDN9). In various examples, the antigen binding protein does not bind to CLDN3. In various examples, the antigen binding protein binds to CLDN6, CLDN4 and CLDN9 but not to CLDN3. In various examples, the antigen binding protein binds to CLDN6 and CLDN4 but not to CLDN3 or CLDN9. In various aspects, the antigen binding protein binds to CLDN6 and CLDN9 but not to CLDN3 or CLDN4.

[0176] In various examples, the antigen binding proteins of the disclosure inhibit binding of a reference antibody to CLDN6 endogenously expressed by OVCA429 cells at less than about 1200 nM, where the reference antibody comprises a light chain variable sequence of SEQ ID NO: 181 and a heavy chain variable sequence of SEQ ID NO: 182, or a light chain variable sequence of SEQ ID NO: 185 and a heavy chain variable sequence of SEQ ID NO: 186. In various aspects, the antigen binding proteins of the disclosure inhibit binding of a reference antibody to CLDN6 endogenously expressed by OVCA429 cells at less than about 1000 nM or less than 750 nM (e.g., less than about 500 nM, less than about 250 nM, less than about 100 nM), where the reference antibody comprises a light chain variable sequence of SEQ ID NO: 181 and a heavy chain variable sequence of SEQ ID NO: 182, or a light chain variable sequence of SEQ ID NO: 185 and a heavy chain variable sequence of SEQ ID NO: 186.

[0177] In various embodiments, the antigen binding protein comprises: (a) a heavy chain CDR1 amino acid sequence set forth in Table A or A1, i.e., a sequence selected from the group consisting of SEQ ID NOs: 11, 17, 23, 29, 35, 41, 47, 53, 59, 65, 71, 77, 83, 89, 95, 101, 107, 113, 119, 125, 131, 452, 455, 461, 465, and 472, or a sequence that differs by only one or two amino acids or by at least or about 70% (e.g., at least or about 85%, at least or about 90%) of the heavy chain CDR1 amino acid sequence set forth in Table A or A1. (b) a heavy chain CDR2 amino acid sequence set forth in Table A or A1, i.e., a sequence selected from the group consisting of SEQ ID NOs: 12, 18, 24, 30, 36, 42, 48, 54, 60, 66, 72, 78, 84, 90, 86, 102, 108, 114, 120, 126, 132, 475, 456, 462, 466, 468, and 473, or a variant thereof having sequence identity by only one or two amino acids or at least or about 70% (e.g., at least or about 85%, at least or about 90%). (c) a heavy chain CDR3 amino acid sequence set forth in Table A or A1, i.e., a sequence selected from the group consisting of SEQ ID NOs: 13, 19, 25, 31, 37, 43, 49, 55, 61, 67, 73, 79, 85, 91, 97, 103, 109, 115, 121, 127, 133, 453, 457, 463, 467, 469, and 474, or a sequence that differs by only one or two amino acids or has at least or about 70% (e.g., at least or about 85%, at least or about 90%) sequence identity. (d) a light chain CDR1 amino acid sequence set forth in Table A or A1, i.e., a sequence selected from the group consisting of SEQ ID NOs: 8, 14, 20, 32, 38, 44, 50, 56, 62, 68, 74, 80, 86, 92, 98, 104, 110, 116, 122, 128, 449, 476, 458, 464, and 470, or a variant thereof which differs by only one or two amino acids or has at least or about 70% (e.g., at least or about 85%, at least or about 90%) sequence identity;(e) a light chain CDR2 amino acid sequence set forth in Table A or A1, i.e., a sequence selected from the group consisting of SEQ ID NOs: 9, 15, 21, 27, 33, 39, 45, 51, 57, 63, 69, 75, 81, 87, 93, 99, 105, 111, 117, 123, 129, 450, 477, 459, and 471, or a variant thereof differing by only one or two amino acids or having at least or about 70% (e.g., at least 85% or about 85%, at least 90% or about 90%) sequence identity; (f) a light chain CDR2 amino acid sequence set forth in Table A or A1, i.e., a sequence selected from the group consisting of SEQ ID NOs: 9, 15, 21, 27, 33, 39, 45, 51, 57, 63, 69, 75, 81, 87, 93, 99, 105, 111, 117, 123, 129, 450, 477, 459, and 471, or a variant thereof differing by only one or two amino acids or having at least or about 70% (e.g., at least 85% or about 85%, at least 90% or about 90%) sequence identity; light chain CDR3 amino acid sequences, i.e., sequences selected from the group consisting of SEQ ID NOs: 10, 16, 22, 28, 34, 40, 46, 52, 58, 64, 70, 76, 82, 88, 94, 100, 106, 112, 118, 124, 130, 451, 454, and 460, or variants thereof differing by only one or two amino acids or having at least or about 70% (e.g., at least or about 85%, at least or about 90%) sequence identity; (g) a combination of any two or more of (a) to (f);

[0178] In various embodiments, the antigen binding protein comprises a light chain CDR1 amino acid sequence, a light chain CDR2 amino acid sequence, and a light chain CDR3 amino acid sequence set forth in Table A or A1, and one or two of the heavy chain CDR amino acid sequences set forth in Table A or A1. In some examples, the antigen binding protein comprises a heavy chain CDR1 amino acid sequence, a heavy chain CDR2 amino acid sequence, and a heavy chain CDR3 amino acid sequence set forth in Table A or A1, and one or two of the light chain CDR amino acid sequences set forth in Table A or A1. In various embodiments, the antigen binding protein is selected from the group consisting of (a) SEQ ID NOs: 74-79, (b) SEQ ID NOs: 50-55, (c) SEQ ID NOs: 122-127, (d) SEQ ID NOs: 26-31, (e) SEQ ID NOs: 128-133, (f) SEQ ID NOs: 38-43, (g) SEQ ID NOs: 62-67, (h) SEQ ID NOs: 80-85, (i) SEQ ID NOs: 44-49, (j) SEQ ID NOs: 86-91, (k) SEQ ID NOs: 104-109, (l) SEQ ID NOs: 56-61, (m) SEQ ID NOs: 32-37, (n) SEQ ID NOs: 110-115, (o) SEQ ID NOs: 98-103, (p) SEQ ID NOs: (c) SEQ ID NOs: 92 to 97, (q) SEQ ID NOs: 116 to 121, (r) SEQ ID NOs: 8 to 13, (s) SEQ ID NOs: 68 to 73, (t) SEQ ID NOs: 14 to 19, (u) SEQ ID NOs: 20 to 25, (v) SEQ ID NOs: 449 to 453 and 475, (w) SEQ ID NOs: 476 to 477, 454 to 457, (x) SEQ ID NOs: 458 to 463, (y) SEQ ID NOs: 57, 58, 464 to 467, (z) SEQ ID NOs: 68 to 71 and 468 to 469, and (aa) SEQ ID NOs: 112, and 470 to 474.In various embodiments, the antigen binding protein comprises: (a) a heavy chain variable region amino acid sequence set forth in Table B, i.e., a sequence selected from the group consisting of SEQ ID NOs: 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, and 175, or a variant thereof that differs by only one or two amino acids or has at least or about 70% (e.g., at least 85% or about 85%, at least 90% or about 90%) sequence identity; ) a light chain variable region amino acid sequence as set forth in Table B, i.e., a sequence selected from the group consisting of SEQ ID NOs:134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, and 176, or a variant thereof which differs by only one or two amino acids or has at least or about 70% (e.g., at least 85% or about 85%, at least 90% or about 90%) sequence identity, or both (a) and (b). In various aspects, the antigen binding proteins are selected from the group consisting of (a) SEQ ID NOs: 156 and 157, (b) SEQ ID NOs: 148 and 149, (c) SEQ ID NOs: 172 and 173, (d) SEQ ID NOs: 140 and 141, (e) SEQ ID NOs: 174 and 175, (f) SEQ ID NOs: 144 and 145, (g) SEQ ID NOs: 152 and 153, (h) SEQ ID NOs: 158 and 159, (i) SEQ ID NOs: 146 and 147, (j) SEQ ID NOs: 160 and 161, (k) SEQ ID NOs: 166 and 167, and (l) SEQ ID NOs: 168 and 169. 67, (l) SEQ ID NOs: 150 and 151, (m) SEQ ID NOs: 142 and 143, (n) SEQ ID NOs: 168 and 169, (o) SEQ ID NOs: 164 and 165, (p) SEQ ID NOs: 162 and 163, (q) SEQ ID NOs: 170 and 171, (r) SEQ ID NOs: 134 and 135, (s) SEQ ID NOs: 154 and 155, (t) SEQ ID NOs: 136 and 137, and (u) SEQ ID NOs: 138 and 139.

[0179] In various embodiments, the antigen binding protein comprises: (a) a heavy chain variable region amino acid sequence set forth in Table B1 or C, i.e., a sequence selected from the group consisting of SEQ ID NOs: 376-379, 384-387, 391-396, 403-408, 412, 413, 416-419, 422-427, 478, 480, 482, 484, 486, and 488, or a variant thereof which differs by only one or two amino acids or has at least or about 70%, about 80%, about 90%, or about 95% sequence identity. In various embodiments, the antigen binding protein comprises a pair of amino acid sequences set forth in Table D, (b) a light chain variable region amino acid sequence set forth in Table B1 or C, i.e., a sequence selected from the group consisting of SEQ ID NOs: 380-383, 388-390, 397-402, 409-411, 414, 415, 420, 421, 479, 481, 483, 485, 487, and 489, or a variant thereof which differs by only one or two amino acids or has at least or about 70%, about 80%, about 90%, or about 95% sequence identity, or (c) both (a) and (b).

[0180] The present disclosure provides a HC CDR1 comprising, optionally, an amino acid sequence of YTFTXYT (wherein X is T, V, D, or S) (SEQ ID NO: 452), which optionally comprises the amino acid sequence of YTFTTYT (SEQ ID NO: 11); (B) an HC CDR2 comprising, optionally, an amino acid sequence of IXPSSGYT (wherein X is Q, S, A, or N) (SEQ ID NO: 475), which optionally comprises the amino acid sequence of INPSSGYT (SEQ ID NO: 12); (C) an HC CDR3 comprising, optionally, an amino acid sequence of AXGDYYVAY (wherein X is N, Q, H, or D) (SEQ ID NO: 453), which optionally comprises the amino acid sequence of ANGDYYVAY (SEQ ID NO: 13). (D) an LC CDR1 comprising an amino acid sequence of SSVSSXY (wherein X is T, V, F or D) (SEQ ID NO: 449), optionally comprising the amino acid sequence of SSVSSTY (SEQ ID NO: 8); (E) an LC CDR2 comprising an amino acid sequence of XTX (wherein X at position 1 is S, T, Q or A and X at position 3 is S, T, D or Q) (SEQ ID NO: 450), optionally comprising the amino acid sequence of STS (SEQ ID NO: 9); and (F) an LC CDR3 comprising an amino acid sequence of HXYXRSPLT (wherein X at position 2 is Q, H or S and X at position 4 is H, Y, Q or S) (SEQ ID NO: 451), optionally comprising the amino acid sequence of HQYHRSPLT (SEQ ID NO: 10).

[0181] The antigen binding protein may comprise: (A) an HC CDR1 optionally comprising the amino acid sequence of FTFSXYX (wherein X at position 5 is N, S, R, Q, or A, and X at position 7 is W, H, Y, or F) (SEQ ID NO: 455), which comprises the amino acid sequence of FTFSNYW (SEQ ID NO: 23); (B) an HC CDR2 optionally comprising the amino acid sequence of IRLKXDXYAT (wherein X at position 5 is S, N, A, or T, and X at position 7 is Q, S, A, or N) (SEQ ID NO: 456), which comprises the amino acid sequence of IRLKSDNYAT (SEQ ID NO: 24). CDR2, (C) optionally comprising the amino acid sequence of XDGPPSGX (wherein X at position 1 is N, D, or T and X at position 8 is S, T, A, C, or Y) (SEQ ID NO: 457), which comprises the amino acid sequence of NDGPPSGC (SEQ ID NO: 25); CDR3, (D) optionally comprising the amino acid sequence of EXIYSY (wherein X is Q, S, A, D, or N) (SEQ ID NO: 476), which comprises the amino acid sequence of ENIYSY (SEQ ID NO: 20); CDR1, (E) optionally comprising the amino acid sequence of XAK (wherein X at position 1 is Q, S, A, D, or N) (SEQ ID NO: 477), which comprises the amino acid sequence of NAK (SEQ ID NO: 21); and (F) optionally, a LC CDR3 comprising the amino acid sequence of QXHYXVPWT (wherein X at position 2 is H, Q, S, or T and X at position 5 is T, S, N, or G) (SEQ ID NO:454), which comprises the amino acid sequence of QHHYTVPWT (SEQ ID NO:22).

[0182] The antigen binding protein may comprise: (A) an HC CDR1 comprising an amino acid sequence of YTXTXYT (wherein X at position 3 is F, Y, S, or T and X at position 5 is S, T, Y, or D) (SEQ ID NO: 461), optionally comprising the amino acid sequence of YTFTSYT (SEQ ID NO: 29); (B) an HC CDR2 comprising an amino acid sequence of IXPSSXYT (wherein X at position 2 is Q, S, A, or N and X at position 6 is T, S, V, D, or G) (SEQ ID NO: 462), optionally comprising the amino acid sequence of INPSSTYT (SEQ ID NO: 30). CDR2, (C) optionally comprising the amino acid sequence of XRGEXGGFAY (wherein X at position 1 is S, A, T, or V, and X at position 5 is L, V, or F) (SEQ ID NO: 463), which comprises the amino acid sequence of SRGELGGFAY (SEQ ID NO: 31); CDR3, (D) optionally comprising the amino acid sequence of QSLVHSXGXTY (wherein X at position 7 is D, N, E, Q, S, or A, and X at position 9 is Q, S, A, D, or N) (SEQ ID NO: 458), which comprises the amino acid sequence of QSLVHSDGNTY (SEQ ID NO: 26); CDR1, (E) an LC CDR2 optionally comprising an amino acid sequence of XVX (wherein X at position 1 is K, Q, or R and X at position 3 is S, T, or V) (SEQ ID NO: 459), which comprises the amino acid sequence of KVS (SEQ ID NO: 27); and (F) an LC CDR3 optionally comprising an amino acid sequence of SXXTHVPYT (wherein X at position 2 is Q, H, or T and X at position 3 is S, G, T, or D) (SEQ ID NO: 460), which comprises the amino acid sequence of SQSTHVPYT (SEQ ID NO: 28).

[0183] In various embodiments, the antigen binding protein of the present disclosure is an antibody, e.g., a monoclonal antibody. In various examples, the antigen binding protein is an IgG. In various embodiments, the antigen binding protein inhibits colony growth in a soft agar 3D growth assay by at least about 50%, or inhibits tumor growth in xenograft mice injected with human cancer cells. In various embodiments, the antigen binding protein inhibits tumor growth in xenograft mice injected with ovarian cancer cells, melanoma cancer cells, bladder cancer cells, or endometrial cancer cells. In various examples, the antigen binding protein inhibits tumor growth in xenograft mice injected with ovarian cancer cells, bladder cancer cells, or endometrial cancer cells by at least 50%.

[0184] The present disclosure provides conjugates comprising the antigen binding proteins described herein and heterologous portions. The present disclosure also provides fusion proteins comprising the antigen binding proteins described herein. The present disclosure further provides a nucleic acid comprising a nucleotide sequence encoding the antigen binding protein, conjugate, or fusion protein of the present disclosure. The present disclosure provides a vector comprising a nucleic acid comprising a nucleotide sequence encoding the antigen binding protein, conjugate, or fusion protein of the present disclosure. The present disclosure further provides a host cell comprising a nucleic acid or vector of the present disclosure.

[0185] The present disclosure provides a method for producing an antigen binding protein that binds to claudin 6 (CLDN6) protein, comprising (i) culturing a host cell of the present disclosure in a cell culture medium, the host cell comprising a nucleic acid comprising a nucleotide sequence encoding an antigen binding protein of any one of the preceding claims, and (ii) recovering the antigen binding protein from the cell culture medium. Also provided is a method for producing a fusion protein comprising an antigen binding protein that binds to claudin 6 (CLDN6) protein, comprising (i) culturing a host cell of the present disclosure in a cell culture medium, the host cell comprising a nucleic acid comprising a nucleotide sequence encoding a fusion protein of the present disclosure, and (ii) recovering the fusion protein from the cell culture medium.

[0186] The present disclosure further provides a method for producing a pharmaceutical composition comprising mixing an antigen binding protein, conjugate, fusion protein, nucleic acid, vector, host cell, or combination thereof of the present disclosure with a pharma- ceutically acceptable carrier, diluent, or excipient. Also provided is a pharmaceutical composition comprising an antigen binding protein, conjugate, fusion protein, nucleic acid, vector, host cell, or combination thereof of the present disclosure and a pharma- ceutically acceptable carrier, diluent, or excipient.

[0187] Provided herein is a method for treating a subject having a CLDN6-expressing cancer, comprising administering to the subject a pharmaceutical composition described herein in an amount effective to treat the cancer. Also provided is a method for inhibiting tumor growth in a subject, comprising administering to the subject a pharmaceutical composition described herein in an amount effective to inhibit tumor growth. The present disclosure provides a method for reducing the size of a tumor in a subject, comprising administering to the subject a pharmaceutical composition described herein in an amount effective to reduce the size of the tumor. Further provided is a method for preventing the recurrence of cancer in a subject, comprising administering to the subject a pharmaceutical composition described herein in an amount effective to prevent the recurrence of the cancer.

[0188] The present disclosure provides a method for detecting claudin 6 (CLDN6) in a sample, comprising contacting the sample with an antigen binding protein, conjugate, or fusion protein of the present disclosure, and assaying for an immune complex comprising the antigen binding protein, conjugate, or fusion protein bound to CLDN6. Also provided herein is a method for diagnosing claudin 6 (CLDN6)-positive cancer in a subject, comprising contacting a biological sample comprising cells or tissue taken from the subject with an antigen binding protein, conjugate, or fusion protein of the present disclosure, and assaying for an immune complex comprising the antigen binding protein, conjugate, or fusion protein bound to CLDN6.

[0189] The present disclosure also provides a method for treating cancer in a subject diagnosed with low overexpression of CLDN6. In various embodiments, the method comprises administering to the subject a pharmaceutical composition disclosed herein in an amount effective to prevent recurrence of the cancer. In some aspects, the administering induces apoptosis in tumor cells, and optionally, the administering induces apoptosis in cells expressing CLDN6. In various aspects, the subject has a tumor, and the tumor is semi-quantitatively classified into one of four groups: high expression, medium expression, low expression, and non-expression. In various examples, high expression is defined as CLDN6 RNA greater than 12 log Fragments Per Kilobase Million (FPKM), CLDN6 RNA measured by RNASeq, or CLDN6 protein levels greater than 3+ measured by immunohistochemistry (IHC). In various examples, medium expression is defined as CLDN6 RNA greater than 10 log FPKM, CLDN6 RNA measured by RNASeq, or CLDN6 protein level measured by IHC greater than 2+. In various examples, low expression is defined as CLDN6 RNA greater than 6 log FPKM, CLDN6 RNA measured by RNASeq, or CLDN6 protein level measured by IHC greater than 1+. In various examples, non-expression is defined as CLDN6 RNA less than 6 log FPKM, CLDN6 RNA measured by RNASeq, or CLDN6 protein level below the detection limit of IHC. In various embodiments, subjects with the above tumors are also described as having high, medium, low, or no expression of CLDN6.

[0190] The following examples are provided merely to illustrate the present disclosure and are not intended to limit its scope in any way. EXAMPLES

[0191] [Example 1] This example describes the analysis of CLDN6 RNA levels in different sources of cells and tissues.

[0192] To establish a baseline for CLDN6 expression in different source materials, we assessed the expression levels of CLDN6 expression in patient samples, normal tissues, and cell lines generated by the Translational Oncology Research laboratory (TORL).

[0193] Information contained in The Cancer Genome Atlas (TCGA) database maintained by the National Cancer Institute (NCI) was used to measure CLDN6 RNA levels in patient samples. Information in the Genotype-Tissue Expression (GTEX) database maintained by the Common Fund was used to measure CLDN6 levels in normal tissues. Analysis of tissues from the GTEX database revealed that CLDN6 was detectable in a variety of sites, including brain, pituitary, pancreas, kidney, lung, thyroid, and cervix, among other tissues (Figure 1).

[0194] CLDN6 expression levels were measured in TORL cancer cell lines using Agilent 44K microarrays (4 × 44K array chips, Agilent Technologies, Santa Clara, CA) and RNA sequencing (RNA-Seq) assays. RNASeq was performed at BGI Americas (Cambridge, MA) using BGI Americas' "RNASeq for Quantitative" service. As shown in Figures 2 and 3, CLDN6 expression levels were detectable in breast, kidney, colon, sarcoma, and liver cancer cells, while ovarian, head and neck, lung, and bladder cancer cells expressed the highest levels of CLDN6.

[0195] [Example 2] This example describes the generation of cells genetically engineered to overexpress CLDN6.

[0196] Models genetically engineered to overexpress CLDN6 were generated. These models were used to confirm the efficacy of the CLDN6 antibody described in Example 5. Briefly, the nucleotide sequence encoding CLDN6 was introduced into a bicistronic vector with a CMV promoter and an attenuated internal ribosome entry site (IRES) of encephalomyocarditis virus (EMCV). The IRES was placed between the gene of interest (GOI) cDNA (CLDN6) and the puromycin cDNA. A woodchuck posttranscriptional regulator (WPRE) was placed downstream of the puromycin cDNA. The vectors also expressed either a GFP marker sequence or a MycDDK tag. The sequence of the expression vector containing GFP is provided herein as SEQ ID NO:189.

[0197] Expression vectors were virally transduced into HEK293T cells (for screening purposes) and NIH3T3 cells (for immunization). Positively transduced cells were selected based on survival in medium containing puromycin (1 μg / ml). Positive cells were subcloned to obtain a stable, homogenous, clonal population of CLDN6-overexpressing cells.

[0198] BD Biosciences Accuri (商標) Subclone CLDN6 expression was confirmed by flow cytometry using a reference CLDN6 monoclonal antibody (mAb) on a flow cytometer (San Jose, Calif.). Secondary antibodies and conjugates: Alexa Fluor® 647 goat anti-mouse IgG (minimal x-reactivity) antibody (Biolegend, San Diego, Calif.; catalog number 405322) was used to detect binding activity between the reference CLDN6 mAb and the CLDN6 expressed by the subclone.

[0199] The cellular localization of CLDN6 was confirmed by fluorescence microscopy using a Cellavista® Imaging System (Synentec, Mountain View, CA) with cells expressing CLDN6-green fluorescent protein (GFP) fusion protein. As shown in Figure 4, GFP fluorescence was detected in the cell membrane, demonstrating that CLDN6 is localized in the cell membrane.

[0200] [Example 3] This example describes the generation of reference and control antibodies.

[0201] The heavy and light chain variable regions of the antibody are (商標) Benchmark (reference) CLDN6-specific and control antibodies were generated by cloning into the Expression System (ThermoFisher Scientific, Waltham, Mass.) to generate recombinant mouse IgG2A chimeric antibodies. These antibodies were tested alongside the newly generated CLDN6-specific antibodies described in Example 5.

[0202] Briefly, ExpiCHO was performed according to the manufacturer's protocol. (商標) Plasmids containing the control and benchmark antibody sequences were transfected using the Expression System (Cat. No.: A29133, ThermoFisher Scientific, USA). Cells were cultured in the medium provided in the kit at 37°C and 8% CO2 on day 1, then at 32°C and 5% CO2 after transfection. ExpiCHO was eluted by centrifugation at 1,000g for 10 min followed by 5,000g for 30 min. (商標)Antibodies were purified by clarifying the culture medium. The supernatant was then filtered using a 0.45 μm filter followed by a 0.22 μm filter. Affinity purification was then performed on the supernatant using Protein A / G resin (Life Technologies, Carlsbad, CA; Cat. No. 20424) according to the manufacturer's protocol. Prior to ELISA purification, the antibody titer in the culture medium was roughly determined to ensure that the amount of medium injected accounted for less than 80% of the binding capacity of the resin. After incubation, the resin was washed with PBS and eluted with elution buffer (Life Technologies, Cat. No. 21004). The eluted fraction was immediately adjusted to physiological pH by adding Tris buffer (pH 8.0). Buffer exchange and protein concentration were then performed on the purified antibody using an Amicon Ultra-15 Centrifugal Filter Unit (Life Technologies, Cat. No. UFC900324) with PBS buffer. Antibody concentration was measured by BCA protein assay. SDS-PAGE and Coomassie staining were performed to check the purity of the antibodies. The purified protein was aliquoted and stored at -80°C for long-term storage or at 4°C for immediate use.

[0203] Antibody integrity was confirmed by SDS-PAGE under non-reducing versus reducing conditions followed by Coomassie staining, which showed one major band around 150 kDa under non-reducing conditions, whereas two bands at 50 kDa and 25 kDa were observed under reducing conditions.

[0204] Antibodies specific to other CLDN family members having sequence similarity (Figure 5), i.e., CLDN3, CLDN4 and CLDN9, were produced in a manner essentially similar to that described above except that the antibody sequence contained in the plasmid was an antibody sequence specific to CLDN3, CLDN4 or CLDN9.

[0205] [Example 4] In this example, characterization of cell lines that highly express endogenous CLDN6 is described.

[0206] A panel of cancer cell lines was analyzed for endogenous expression of CLDN6 in the cell lines by FACS and Western blot. Briefly, antibody binding to the target was confirmed by FACS using cells overexpressing CLDN6 (e.g., HEK293T cells overexpressing CLDN6 as described in Example 2) and cell lines endogenously expressing high or low levels of CLDN6 as measured in Example 1. CLDN6-expressing cells were incubated with reference or control antibodies (described in Example 3) for 30 minutes on ice, washed, and then incubated with Alexa Fluor® 647-conjugated goat anti-mouse IgG (minimal x-reactivity) antibody (Biolegend Cat. No. 405322) for 30 minutes on ice. BD Biosciences Accuri (商標) Fluorescence was read on a flow cytometer (San Jose, Calif.).

[0207] Western blots were performed on nitrocellulose using reference and control antibodies. Briefly, samples derived from cell lysates were boiled to denature protein components. SDS-PAGE (SDS polyacrylamide gel electrophoresis) was used to separate the denatured proteins by polypeptide length. The separated proteins were then transferred from the acrylamide gel to a nitrocellulose membrane. A 2% bovine serum albumin (BSA) solution was used to block the membrane to minimize nonspecific antibody binding. The membrane was incubated with the reference or control antibody. The membrane was stained with a horseradish peroxidase (HRP)-conjugated secondary antibody that recognizes the reference or control antibody, and the secondary antibody was detected by chemiluminescence.

[0208] The overexpressing lines were used to validate the control and reference antibodies, and once validated, the control and reference antibodies were used to characterize the endogenous cell lines. Cells overexpressing CLDN6 were included in these assays as a positive control.

[0209] In addition to endometrial, bladder, lung, and upper GI cancer cell lines, four ovarian cancer cell lines were found to express high levels of CLDN6 on their surfaces by FACS assay. High levels of CLDN6 expression were also detected by Western blot. Two other ovarian cancer cell lines, another liver cancer cell line, another lung cancer cell line, and another upper GI cancer cell line were found to express intermediate levels of CLDN6 on their surfaces by Western blot detection. Endometrial and bladder tumor cells also expressed high levels of CLDN6 as xenografts in vivo. The endogenous expression levels of CLDN6 by the cancer cell lines tested are summarized in Table 2.

[0210] [Table 20]

[0211] [Example 5] In this example, immunization of mice to generate CLDN6-specific antibodies is described.

[0212] CLDN6-specific antibodies were generated by immunizing Balb / c and CD1 mice with a mixture of three different peptide immunogens according to the techniques of the Fred Hutchinson Cancer Research Center. The three peptides spanned the second loop (i.e., EL2) within the CLDN6 extracellular domain. The peptides included full-length EL2, a peptide spanning the first (N-terminal) half of EL2, and a peptide spanning the second (C-terminal) half of EL2. The sequences of the three peptides are listed in Table 3.

[0213] [Table 21]

[0214] We also immunized mice with 3T3 cells overexpressing full-length CLDN6 using a plasmid containing the human CLDN6-myc-DDK expression vector.

[0215] Splenocytes were harvested from immunized mice and fused with myeloma lines using BTX Electrofusion (BTX, Holliston, MA) to generate hybridomas. 7680 primary hybridoma cultures were generated and grown in 384-well plates. The ability of antibodies to bind peptides was assessed by bead array using beads expressing three different peptide targets. 1920 potential positive antibodies were re-arrayed into 96-well plates and further screened against endogenous and artificial cell line models using flow cytometry.

[0216] The positive hybridoma supernatants were then counter-screened against endogenous and artificial model proteins with sequence similarity to the target region (e.g., other CLDN proteins) using flow cytometry. From the second screening and counter-screening, about 20 CLDN6-specific antibodies were selected for further testing. These antibodies were subcloned to identify the variable heavy and variable light chain sequences. See Table B and the Sequence Listing.

[0217] ExpiCHO (商標) The CLDN6 antibody was formatted as a full-length IgG antibody using Expression. The heavy and light chain variable regions of the antibody were encoded by pcDNA (商標) 3.4-TOPO (登録商標) The antibody was cloned into an antibody expression vector genetically engineered in the laboratory based on a vector (catalog number: A14697, ThermoFisher Scientific, USA) and expressed using a kit (ExpiCHO (商標)CHO cells were transfected according to the protocol provided in the ThermoFisher Scientific Expression System, Catalog No. A29133, ThermoFisher Scientific, USA). After purifying the antibodies, cell surface binding of the antibodies to CLDN6 and antibody IC50 were measured by FACS with Alexa Fluor® 647 NHS ester (succinimidyl ester) (Cat. No. A20106, ThermoFisher Scientific) directly conjugated to the CLDN6 antibody according to the manufacturer's protocol. CLDN6 antibodies were tested from 0.32 nM to 1000 nM (serial dilution 1:5, 6 points) using a 50 μl volume system containing 150,000 cells.

[0218] In a FACS assay, CLDN6-expressing cells were used to measure the ability of CLDN6 antibodies to bind to CLDN6 on the cell surface and cross-react with other CLDN family members. HEK293T cells genetically engineered to express human CLDN6 fused to GFP, mouse CLDN6 fused to GFP, CLDN9-GFP, CLDN4-GFP, or CLDN3-GFP, or GFP alone (without CLDN6) were used as artificial models of CLDN6 expression. ARK2 cells, OVCA429 cells, LS513 cells, and MCF7 cells were used as endogenous models of CLDN6 expression and models for CLDN3 / 4 expression.

[0219] For each type of cell and each mAb tested, cells were detached from the surface of the culture flask using EDTA (instead of trypsin) to protect cell surface proteins. The detached cells were then incubated with Alexa Fluor®-labeled CLDN6 mAb at the indicated concentrations for 30 minutes on ice in the dark. CLDN6 mAb was directly labeled with Alexa Fluor® 647 NHS ester (succinimidyl ester). After washing, cells were read using a BD Accuri™ Flow Cytometer C6 that detects antibody-antigen protein binding in the FL4H channel. Each antibody was tested at various concentrations to generate a dose-fluorescence curve. The EC50 / IC50 (concentration of antibody at half maximum) of the antibody was calculated based on the FL4H (gated on live singlet cells) values ​​using a very simple IC50 toolkit available online that can plot biological dose-response data and fit it to a curve type to obtain the EC50 / IC50. The maximum was the lowest concentration of antibody where the fluorescence was maximal. The antibodies were also screened for their ability to cross-react with other CLDN proteins, such as CLDN9, CLDN3, and CLDN4. These values ​​were used to measure the relative affinity of each antibody in the antibody set tested. Cross-reactivity data was obtained using a similar method, but with cells with different expression profiles of CLDN6, CLDN3, CLDN4, and CLDN9.

[0220] Relative affinity and cross-reactivity data determined by this method are listed in Tables 4 and 5.

[0221] [Table 22]

[0222] [Table 23]

[0223] [Example 6] In this example, the characterization of a chimeric mouse IgG mAb is described.

[0224] Soft agar 3D growth assays and xenograft binding assays were performed to further characterize the mAbs described in Example 5. Briefly, for each well of a 48-well plate, 250 μL of top layer mixture containing 10,000 cells in 0.6% SeaPlaque agarose in 1× RPMI medium was seeded on top of a bottom layer of solidified 250 uL of 0.6% SeaPlaque agarose in 1× RPMI medium. A liquid feeder layer of 250 uL containing 1× RPMI medium was placed on top of the solidified top layer. All three layers of the soft agar assay were prepared with or without trastuzumab, CLDN6 mAb, or mouse IgG2a control, diluted 1:10 starting at 150 ng / mL (1 uM) and ending at 1.5 ng / mL. Each test condition was performed in duplicate. Cells were allowed to form colonies for 3 weeks before staining with 0.05% neutral red and viewing under an EVOS XL inverted light microscope. Cell lines that showed a ≥ 20% reduction in colony numbers in the treated vs. control were considered sensitive.

[0225] As shown in Table 6, many of the cell lines showed a reduction in colony numbers when treated with the indicated antibodies.

[0226] [Table 24]

[0227] In vivo binding studies were performed using xenograft mice injected with human cancer cell lines. Briefly, 6-week-old CD-1 athymic nude mice (Charles River Laboratories) were used to create xenograft models of human cancer cell lines. Each cell line was subcutaneously injected under the following conditions: 0.75 × 10 for ARK2; 7 cells, 1.0 × 10 for UMUC4 cells, 7 cells, 1.0 × 10 for OV90 7 cells, and 0.5 × 10 for M202.7 The cells were then cultured in 100% PBS-C100 medium (all containing 50% Matrigel (BD Biosciences)). A sufficient number of mice were injected to obtain 8 mice per treatment group. Tumors were 150–300 mm 3 Mice were randomized into treatment groups when tumor size reached an average size of 100 mm. For treatment, each therapeutic antibody (AB3, AB2, ref. Ab1, ref. Ab2, ref. Ab3 (trastuzumab), and non-targeting IgG2 control) was diluted in sterile saline to a working concentration of 1 mg / ml for tail vein (IV) injection. For the M202 study, trametinib (DMSO solvate, MedChem Express) was administered PO at 1.0 mg / kg (10% Cremaphor, 10% PEG400) on a 5-day on, 2-day off schedule for the first weekly cycle, then reduced to 0.5 mg / kg for the remaining 2 weeks. Tumor xenografts were measured with calipers 3 times weekly and tumor volume (mm) was calculated by multiplying by height x width x length. 3 ) were determined. Mice were treated for 2 to 7 weeks. At the end of the study, animals were euthanized and tumor tissues were excised, sectioned, and stored as flash frozen or formalin-fixed paraffin-embedded (FFPE) tissues for biomarker analysis. All animal experiments were performed under protocols approved by the IACUC and the University of California, Los Angeles Animal Research Committee. Data were analyzed using StudyLog software from StudyDirector (San Francisco, CA). Results are presented as the mean volume for each group. Error bars indicate standard error of the mean (SE).

[0228] The results of the xenograft assay are shown in Figures 6-10. As shown in Figures 6A and 6B, AB2 and AB3 each caused a significant mean change in tumor volume at day 14 in mice bearing endometrial tumors compared to the control IgG2 antibody. As shown in Figures 7A and 7B, AB3 caused a significant mean change in tumor volume at day 35 in mice bearing bladder tumors compared to the control IgG2 antibody. Figures 8A and 8B show that AB2 and AB3 each caused a significant mean change in tumor volume at day 20 in mice bearing ovarian tumors compared to the control IgG2 antibody. Figures 9A and 9B show that AB3 acted in a CLDN-specific manner, since the model used in Figures 9A and 9B does not express any of CLDN6, CLDN3, CLDN4, or CLDN9, and was therefore used as a negative control. The data in Figures 9A and 9B also suggest that AB3 has less off-target activity compared to Reference Ab1 and Reference Ab2. Figure 10A summarizes the results of Figures 6 to 9. As shown in Figure 10A, AB3 significantly inhibited tumor growth in mice bearing endometrial, bladder, and ovarian tumors, each of which expresses CLDN6, but did not inhibit tumor growth in mice bearing melanoma tumors (Figure 10B), which do not express CLDN6. As shown in Figure 11, the average change in mouse body weight during treatment did not change significantly, suggesting the safety of the treatment.

[0229] A second set of experiments was performed using a xenograft model of OV90, a human ovarian cancer cell line. Mice were injected with one of the 10 mAbs described in Example 5, a control antibody (mouse IgG2a antibody, reference CLDN6 Ab), or a PBS vehicle control. There were eight mice per group, and each animal was injected intravenously with 10 mg / kg of antibody every four days. As shown in Figures 12A and 12B, several antibodies described in Example 5 reduced tumor volume in mice bearing ovarian tumors. All antibodies tested reduced tumor volume compared to the vehicle control, with AB3, AB4, AB7, and AB10 performing the best. As shown in Figure 13, the body weight of animals treated with AB3, AB4, AB7, or AB10 did not change significantly, suggesting their safety.

[0230] [Example 7] In this example, further characterization of a chimeric mouse IgG mAb is described.

[0231] Quantitative internalization assays were performed. Briefly, binding of reference Ab1, AB3 or AB4 induced internalization of CLDN6 protein was tested using a positive control (the ubiquitously expressed transferrin receptor (TfR), a well-known cell surface receptor that is internalized after antibody binding).

[0232] TfR and CLDN6 antibodies were obtained from Texas Red (商標)CLDN6- or TfR-labeled cells were labeled with -X, succinimidyl ester, mixed isomers, catalog no. T6134 (ThermoFisher Scientific). Cells were seeded in μ-Slide 8 Well chambers (catalog no. 80826, ibidi Cells In Focus Inc.) one day prior to antibody treatment to allow cells to attach and grow. Cells were incubated with labeled antibodies for 30 minutes on ice in the dark. Chambers containing CLDN6- or TfR-labeled cells were then read on an Echo Lab Fluorescence Microscope and pre-internalization images were collected. Chambers were then incubated at 37°C for 40 minutes to allow internalization to proceed before images were collected again on an Echo Lab Fluorescence Microscope. AB3 and AB4 produced a greater degree of CLDN6 internalization compared to that produced by reference Ab1 (not shown).

[0233] [Example 8] In this example, further characterization of a chimeric mouse IgG mAb is described.

[0234] Two-dimensional (2D) proliferation assays were performed using select antibodies as described in Example 5 as follows. Cells were seeded in duplicate in 24-well plates at 5,000-20,000 cells per well. The following day, cells were treated with six 1-5 dilutions of mAb (starting at 100 nM of either trastuzumab, CLDN6 mAb, or mouse IgG2a control) and a fixed concentration of 1 ng / μL monomethyl auristatin E (MMAE)-conjugated anti-mouse secondary (Moradec, LLC) to generate dose-response curves. Cells in untreated wells were quantified on day 1, the day of antibody treatment, and later on day 6, the range of cell proliferation was measured. mAb-treated wells were quantified on day 6, and proliferation in each treatment condition was determined as a normalized percentage of proliferation in untreated cells. Quantification was performed on a Z1 particle counter (Beckman Coulter, Inc.).

[0235] The results are shown in Figure 14. AB2, AB3, AB4, and AB5 showed the highest growth inhibitory effect. The IC50 for each of these antibodies was 0.1 nM to 1 nM. AB7, AB10, AB11, and AB15 also showed growth inhibitory ability in this assay, although to a relatively lower extent than AB2, AB3, AB4, and AB5.

[0236] [Example 9] This example illustrates the humanization of an antibody of the present disclosure.

[0237] A subset of antibodies listed in Table A was selected for humanization analysis. The heavy chain variable (VH) and light chain variable (VL) sequences of the AB1, AB3, AB4, AB9, AB11, and AB18 antibodies were compared to a library of known human germline sequences derived from human VH genes and human VL Kappa genes (IMGT® the international ImMunoGeneTics information system® www.imgt.org; founder and curator: Marie-Paule Lefranc, Montpellier, France) using the following databases: IMGT human VH genes (F+ORF, 273 germline sequences) and IMGT human VL Kappa genes (F+ORF, 74 germline sequences). The recipient human germline was selected from the recipient human germline that was closest to the sequence of the parent antibody.

[0238] Table 7 lists information about the human germline sequences selected as receptor sequences and the selected human heavy chain joining regions (J genes) for each VH and VL of each antibody. The joining regions (J genes) were selected from the human joining region sequences deposited in IMGT®, the international ImMunoGeneTics information system®, www.imgt.org (Founder and curator: Marie-Paule Lefranc, Montpellier, France).

[0239] [Table 25]

[0240] CDRs were defined according to the AbM definitions (see Dr Andrew CRMartin's website at www.bioinf.org.uk / abs / for a table comparing CDR definitions).

[0241] Modifications of human germline framework (i.e., non-CDR residues within VH and VL) sites to the corresponding parental murine sequences may be necessary to optimize binding of the humanized antibody. Sequences for types of humanized antibodies are provided as SEQ ID NOs:376-421.

[0242] For AB1, Asn52 (consecutive number) of CDR2 of HC and Asn54 of CDR2 of LC were each determined to have a low probability of deamidation based on sequence and conformation.

[0243] For AB3, Asn31 (consecutive number) of CDR1 of HC, Asn57 of CDR2 of HC, Asn28 of CDR1 of LC, and Asn50 of CDR2 of LC were determined to have low potential for deamidation based on sequence and conformation. Trp33 of CDR1 of HC was determined to be potentially solvent exposed and potentially oxidized, especially under stress conditions. CDR3 of HC was determined to have a free Cys106 in the CDR, which may be problematic during antibody production due to potential solvent exposure. This Cys residue was recommended for modification to Tyr, Ser, or Ala. Maintenance of binding of these modified antibodies was tested. Ile53 of CDR2 of LC was determined to be solvent exposed and may result in non-specific binding. It was suggested that this Ile residue be modified to Ser. Maintenance of binding of these modified antibodies was tested.

[0244] For AB4, Asn52 (sequential number) of CDR2 of HC and Asn58 of CDR2 of LC were each determined to have a low probability of deamidation based on sequence and conformation. The sequence DGNT in CDR1 of LC was determined to be just as problematic as having a high probability of isoaspartate formation (in sequence DG) as well as a probability of deamidation (in sequence NT). This sequence was recommended for modification.

[0245] For AB9, Asn33 (sequential number) in CDR1 of HC and Asn52 and Asn59 in CDR2 of HC were determined to have a low probability of deamidation based on sequence and conformation. Asn54 was determined to have a medium probability of deamidation based on sequence and conformation. The NGG sequence in CDR2 of HC was determined to have a high / medium probability of deamidation followed by isoaspartate formation. Therefore, it was recommended to modify this amino acid sequence. Free Cys106 in CDR3 of HC was determined to be potentially solvent exposed and thus could be problematic during antibody manufacturing. It is suggested that this Cys residue be modified to Tyr, Ser, or Ala. The maintenance of binding of these modified antibodies was tested. Arg28 in CDR1 of HC is not frequently present in human antibodies. This residue was modified to Thr and tested for maintenance of binding. For AB9, Trp32 (sequential numbering) in CDR1 of LC was determined to be potentially solvent exposed and susceptible to oxidation, especially under stress conditions. Leu24 in the same CDR is not frequently present in human antibodies. This residue was altered to Arg and tested for retention of binding.

[0246] For AB11, Asp54-Ser55 (sequential numbering) in CDR2 of the HC was determined to have a low probability of isoaspartate formation. Asn57 in CDR2 of the LC was determined to have a low probability of deamidation based on sequence and conformation.

[0247] For AB18, Asn33 in CDR1 of HC and CDR-H2 Asn50 (consecutive numbers) were determined to have a low probability of deamidation based on sequence and conformation. The Asp-Pro (DP) sequence in CDR2 of HC was determined to have a probability of fragmentation under acidic conditions. Within the VL domain, Asn34 and Asn37 in CDR1 of LC were determined to have a low probability of deamidation based on sequence and conformation. Trp56 in CDR3 of LC was determined to have a probability of being solvent exposed and to be oxidized, especially under stress conditions.

[0248] Table 8 shows the scheme for combining humanized VH and humanized VL. In the absence of humanization, it is equivalent to a chimeric mAb. Preferred pairs are shown in bold underlined text.

[0249] [Table 26] TIFF0007599332000030.tif239157

[0250] The humanized antibodies listed in Table 8 were constructed and expressed essentially as described in Example 5. FACS assays were performed to measure the relative antigen binding avidity of the humanized antibodies essentially as described in Example 5. Two doses (1.5 μg or 0.3 μg) of the humanized antibodies were tested for binding to either human CLDN6 or mouse CLDN6 (protein expressed by engineered 293T clone). The results of the assay are listed in Table 9.

[0251] [Table 27]

[0252] Similarly, FACS assays were performed to measure the relative antigen binding avidity of the humanized antibodies (either 1.5 μg or 0.3 μg) in binding to CLDN6 expressed by the indicated cancer cell lines. The results of the assays are listed in Table 10. 2nd Ab only was used as a negative control. 64A-chim, h64A, and SC27-108-chim were used as reference antibodies. The corresponding parent antibody (antibody before humanization) was used as a control and was named "chim".

[0253] [Table 28]

[0254] Based on the in vitro antigen binding data, three humanized antibodies were selected for further testing and development. These antibodies were derived from AB1, AB3 and AB4.

[0255] In vivo binding studies of humanized versions of AB1, AB3 and AB4 were performed using xenograft mice injected with the bladder cancer cell line UMUC4 essentially as described in Example 6. Briefly, UMUC4 xenograft models were generated using 6-week-old CD-1 athymic nude mice (Charles River Laboratories). 3 Mice were randomized into treatment groups after reaching an average size of 1 mg / ml. Humanized antibodies were diluted in sterile saline to a working concentration of 1 mg / ml for tail vein (IV) injection. Tumor xenografts were measured with calipers three times a week and tumor volume (mm) was calculated by multiplying height x width x length. 3 Mice were treated for 2 to 7 weeks. At the end of the study, animals were euthanized and tumor tissues were excised, sectioned, and stored as flash-frozen or formalin-fixed, paraffin-embedded (FFPE) tissue for biomarker analysis.

[0256] The results of the xenograft assay are shown in Figures 15-21. Figure 15 shows the results of the xenograft assay of two forms of humanized AB3 (AB3-2 and AB3-4), where the treatment included 10 mg / kg administered Q4D. Controls included a solvent control (PBS), human IgG (10 mg / kg Q4D), and the murine form of AB3 (10 mg / kg Q4D). As shown in this figure, humanized AB3-4 showed a reduction in tumor volume over the 35-day treatment period. Figure 16 shows the results of the xenograft assay of the same treatment as the experiment shown in Figure 15, except that two additional controls were performed: 64A MSE and its chimeric form (64A-CHIM). Similar to Figure 15, Figure 16 shows a significant reduction in tumor volume after treatment with humanized AB3-4.

[0257] Figure 17 shows the results of a xenograft assay of humanized AB1-5. Controls included a solvent control (PBS), human IgG (10 mg / kg Q4D), and the murine form of AB1 (10 mg / kg Q4D). As shown in Figure 17, mice treated with humanized AB1-5 showed a significant reduction in tumor volume.

[0258] Figure 18 shows the results of a xenograft assay of humanized AB4-3. Controls included a solvent control (PBS), human IgG (10 mg / kg Q4D), and the murine form of AB4 (10 mg / kg Q4D). Mice treated with humanized AB4-3 did not show a significant reduction in tumor volume.

[0259] Figures 19-21 show the results of a xenograft assay testing all four humanized antibodies from Figures 15-18. The murine and chimeric versions of the reference CLDN6 antibody were used as controls. As shown in Figure 19, mice treated with humanized AB3-4 and AB1-5 showed a reduction in tumor volume. Figure 20 shows the tumor volume over time up to day 55. The body weights of the mice in the assay are shown in Figure 21.

[0260] [Example 10] An in silico analysis was performed using the different sequences of AB1, AB3, and AB4. In particular, for each antibody, the sequences of (a) the original parent clone, (b) the closest mouse germline sequence, (c) the closest human germline sequence, and (d) the humanized sequence were aligned. Amino acids believed to have undergone affinity maturation are marked with an asterisk, while amino acids that differ from the amino acid at that position according to the antibody database information are marked with a hashtag. The CDRs of each sequence are boxed. Based on this analysis, several humanized antibodies with the sequences listed in Table 10 are generated.

[0261] [Table 29]

[0262] Multiple antibodies having sequences defined by these consensus sequences are generated essentially as described in Example 5 and tested in vitro by FACS for antigen binding (essentially as described in Example 5) and in vivo for ability to reduce tumor volume in mice (essentially as described in Example 6).

[0263] All references cited herein, including publications, patent applications, and patents, are hereby incorporated by reference to the same extent as if each reference was individually and specifically indicated to be incorporated by reference and was set forth in its entirety herein.

[0264] In the context of describing this disclosure (particularly in the context of the claims which follow), use of the terms "a," "an," "the," and similar referents should be construed to cover both the singular and the plural, unless otherwise stated herein or otherwise clearly contradicted by context. The terms "comprising," "having," "including," and "containing" should be construed as open-ended terms (i.e., meaning "including but not limited to"), unless otherwise indicated.

[0265] The recitation of ranges of values ​​herein is intended to serve merely as a shorthand method for referring individually to each individual value and each endpoint within the range, unless otherwise stated herein, and each individual value and endpoint is incorporated herein as if it were individually recited herein.

[0266] All methods described herein can be carried out in any suitable order unless otherwise specified herein or otherwise clearly contradicted by the context.All examples provided herein or the use of various terms (e.g., "etc.") are merely intended to better clarify the disclosure and do not impose limitations on the scope of the disclosure unless specifically claimed.No term in this specification should be interpreted as indicating any non-claimed element essential to the implementation of the disclosure.

[0267] Preferred embodiments of the present disclosure are described herein, including the best mode for carrying out the present disclosure known to the inventors. Variations of these preferred embodiments may become apparent to those skilled in the art upon reading and understanding the above description. The inventors anticipate that such variations will be adopted by those skilled in the art as necessary, and the inventors intend for the present disclosure to be practiced otherwise than as expressly described herein. Accordingly, the present disclosure includes all modifications and equivalents of the subject matter recited in the claims appended hereto. Moreover, any combination of the above-described elements, including all possible variations thereof, is encompassed by the present disclosure unless otherwise expressly stated herein or otherwise clearly contradicted by context. The present invention provides the following: 1. An antigen binding protein that binds to human claudin 6 (CLDN6) protein (SEQ ID NO: 200), a. the antigen binding protein binds to extracellular loop 2 (EL2) of the extracellular domain (ECD) of CLDN6 and does not bind to extracellular loop 1 (EL1) of the ECD of CLDN6; b. does not bind to claudin 3 (CLDN3), claudin 4 (CLDN4) and claudin 9 (CLDN9) and inhibits binding of the reference antibody to CLDN6 endogenously expressed by OVCA429 cells at less than about 1200 nM; or c. A combination of these The antigen-binding protein. 2. The antigen-binding protein according to claim 1, which binds to an epitope within the amino acid sequence of WTAHAIIRDFYNPLVAEAQKREL (SEQ ID NO:2). 3. The antigen-binding protein according to 2 above, which binds to the amino acid sequence of TAHAIIRDFYNPL (SEQ ID NO: 3) or LVAEAQKREL (SEQ ID NO: 4) of CLDN6. 4. An antigen-binding protein according to any one of 1 to 3 above, which does not bind to any one or more of claudin 3 (CLDN3), claudin 4 (CLDN4) and claudin 9 (CLDN9). 5. An antigen-binding protein according to 4 above, which does not bind to CLDN3. 6. The antigen-binding protein according to 5 above, which binds to CLDN6, CLDN4 and CLDN9. 7. The antigen-binding protein according to 5 above, which does not bind to CLDN9. 8. The antigen-binding protein according to 7 above, which binds to CLDN6 and CLDN4. 9. The antigen-binding protein according to 5 above, which does not bind to CLDN4. 10. The antigen-binding protein according to 9 above, which binds to CLDN6 and CLDN9. 11. The antigen binding protein of any of the preceding claims, wherein the reference antibody comprises a light chain variable sequence of SEQ ID NO:181 and a heavy chain variable sequence of SEQ ID NO:182, or a light chain variable sequence of SEQ ID NO:185 and a heavy chain variable sequence of SEQ ID NO:186. 12. a. A heavy chain CDR1 amino acid sequence as set forth in Table A, i.e. a sequence selected from the group consisting of SEQ ID NOs: 11, 17, 23, 29, 35, 41, 47, 53, 59, 65, 71, 77, 83, 89, 95, 101, 107, 113, 119, 125, and 131, or a variant thereof differing by only one or two amino acids or having at least or about 70% sequence identity; b. A heavy chain CDR2 amino acid sequence as set forth in Table A, i.e., a sequence selected from the group consisting of SEQ ID NOs: 12, 18, 24, 30, 36, 42, 48, 54, 60, 66, 72, 78, 84, 90, 86, 102, 108, 114, 120, 126, and 132, or a variant thereof differing by only one or two amino acids or having at least or about 70% sequence identity; c. A heavy chain CDR3 amino acid sequence as set forth in Table A, i.e., a sequence selected from the group consisting of SEQ ID NOs: 13, 19, 25, 31, 37, 43, 49, 55, 61, 67, 73, 79, 85, 91, 97, 103, 109, 115, 121, 127, and 133, or a variant thereof differing by only one or two amino acids or having at least or about 70% sequence identity; d. A light chain CDR1 amino acid sequence as set forth in Table A, i.e., a sequence selected from the group consisting of SEQ ID NOs: 8, 14, 20, 32, 38, 44, 50, 56, 62, 68, 74, 80, 86, 92, 98, 104, 110, 116, 122, and 128, or a variant thereof differing by only one or two amino acids or having at least or about 70% sequence identity; e. A light chain CDR2 amino acid sequence set forth in Table A, i.e., a sequence selected from the group consisting of SEQ ID NOs: 9, 15, 21, 27, 33, 39, 45, 51, 57, 63, 69, 75, 81, 87, 93, 99, 105, 111, 117, 123, and 129, or a variant thereof differing by only one or two amino acids or having at least or about 70% sequence identity; f. A light chain CDR3 amino acid sequence as set forth in Table A, i.e., a sequence selected from the group consisting of SEQ ID NOs: 10, 16, 22, 28, 34, 40, 46, 52, 58, 64, 70, 76, 82, 88, 94, 100, 106, 112, 118, 124, and 130, or a variant thereof differing by only one or two amino acids or having at least or about 70% sequence identity; g. A combination of two or more of (a) to (f). The antigen-binding protein according to any of the above, 13. The antigen-binding protein according to claim 12, wherein said variant sequence has at least about 80% or at least 85% or about 85% sequence identity. 14. An antigen-binding protein according to claim 13, wherein said variant sequence has at least about 90% sequence identity or at least or about 95% sequence identity. 15. The antigen-binding protein according to claim 12, comprising a light chain CDR1 amino acid sequence, a light chain CDR2 amino acid sequence, and a light chain CDR3 amino acid sequence set out in Table A, and one or two of the heavy chain CDR amino acid sequences set out in Table A. 16. The antigen-binding protein according to claim 12, comprising a heavy chain CDR1 amino acid sequence, a heavy chain CDR2 amino acid sequence, and a heavy chain CDR3 amino acid sequence set out in Table A, and one or two of the light chain CDR amino acid sequences set out in Table A. 17. a. SEQ ID NO: 74 to 79, b. SEQ ID NO: 50 to 55, c. SEQ ID NO: 122 to 127, d. SEQ ID NO: 26 to 31, e. SEQ ID NO: 128 to 133, f. SEQ ID NO: 38 to 43, g. SEQ ID NO: 62 to 67, h. SEQ ID NO: 80 to 85, i. SEQ ID NO: 44 to 49, j. SEQ ID NO: 86 to 91, k. SEQ ID NO: 104 to 109, l. SEQ ID NO: 56 to 61, m. SEQ ID NO: 32 to 37, n. SEQ ID NO: 110 to 115, SEQ ID NO: 98 to 103, p. SEQ ID NO: 92 to 97, q. SEQ ID NO: 116 to 121, r. SEQ ID NO: 8 to 13, s. SEQ ID NO: 68 to 73, t. SEQ ID NOs: 14 to 19, and u. SEQ ID NO: 20 to 25, 17. An antigen-binding protein according to any one of claims 12 to 16, comprising six CDR amino acid sequences selected from the group consisting of: 18. a. A heavy chain variable region amino acid sequence as set forth in Table B, i.e., a sequence selected from the group consisting of SEQ ID NOs: 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, and 175, or a variant thereof which differs by only one or two amino acids or has at least or about 70% sequence identity; b. a light chain variable region amino acid sequence set forth in Table B, i.e., a sequence selected from the group consisting of SEQ ID NOs: 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, and 176, or a variant thereof which differs by only one or two amino acids or has at least or about 70% sequence identity; or c. Both (a) and (b); 20. The antigen-binding protein according to any one of claims 12 to 19, comprising: 19. The antigen-binding protein according to claim 18, wherein the variant sequence has at least about 80% or at least about 85% sequence identity. 20. The antigen-binding protein according to claim 19, wherein the variant sequence has at least about 90% or at least about 95% sequence identity. 21. a. SEQ ID NO:156 and 157, b. SEQ ID NOs:148 and 149, c. SEQ ID NOs:172 and 173, d. SEQ ID NOs:140 and 141, e. SEQ ID NOs:174 and 175, f. SEQ ID NOs:144 and 145, g. SEQ ID NOs:152 and 153, h. SEQ ID NOs:158 and 159, i. SEQ ID NOs:146 and 147, j. SEQ ID NOs:160 and 161, k. SEQ ID NOs:166 and 167, l. SEQ ID NOs:150 and 151, m. SEQ ID NOs:142 and 143, n. SEQ ID NOs: 168 and 169, o. SEQ ID NO:164 and 165, p. SEQ ID NO: 162 and 163, q. SEQ ID NO:170 and 171, r. SEQ ID NO:134 and 135, s. SEQ ID NO:154 and 155, t. SEQ ID NOs:136 and 137, and SEQ ID NOs: 138 and 139, 19. The antigen-binding protein according to claim 18, comprising a pair of amino acid sequences selected from the group consisting of: 22. An antigen-binding protein according to any of the above, which is an antibody. 23. The antigen-binding protein according to claim 22, which is a monoclonal antibody. 24. The antigen-binding protein according to claim 22 or 23, which is an IgG. 25. An antigen-binding protein according to any of the preceding claims, which inhibits colony growth in a soft agar 3D growth assay by at least about 50%. 26. An antigen-binding protein according to any of the above, which inhibits tumor growth in xenograft mice injected with human cancer cells. 27. An antigen-binding protein according to claim 26, which inhibits tumor growth in xenograft mice injected with ovarian, melanoma, bladder or endometrial cancer cells. 28. The antigen-binding protein according to claim 27, which inhibits tumor growth by at least 50% in xenograft mice injected with ovarian cancer cells, bladder cancer cells, or endometrial cancer cells. 29. (a) a HC CDR1 amino acid sequence as set forth in Table A or A1, i.e., a sequence selected from the group consisting of SEQ ID NOs: 11, 17, 23, 29, 35, 41, 47, 53, 59, 65, 71, 77, 83, 89, 95, 101, 107, 113, 119, 125, 131, 452, 455, 461, 465, and 472, or a variant thereof differing by only one or two amino acids or having at least or about 70% sequence identity; (b) a HC CDR1 amino acid sequence as set forth in Table A or A1, (c) a CDR2 amino acid sequence, i.e., a sequence selected from the group consisting of SEQ ID NOs: 12, 18, 24, 30, 36, 42, 48, 54, 60, 66, 72, 78, 84, 90, 86, 102, 108, 114, 120, 126, 132, 475, 456, 462, 466, 468, and 473, or a variant thereof differing by only one or two amino acids or having at least or about 70% sequence identity; (d) a CDR3 amino acid sequence, i.e., a sequence selected from the group consisting of SEQ ID NOs: 13, 19, 25, 31, 37, 43, 49, 55, 61, 67, 73, 79, 85, 91, 97, 103, 109, 115, 121, 127, 133, 453, 457, 463, 467, 469, and 474, or a variant thereof which differs by only one or two amino acids or has at least or about 70% sequence identity; (e) a CDR1 amino acid sequence, i.e., a sequence selected from the group consisting of SEQ ID NOs: 8, 14, 20, 32, 38, 44, 50, 56, 62, 68, 74, 80, 86, 92, 98, 104, 110, 116, 122, 128, 449, 476, 458, 464, and 470, or a variant thereof which differs by only one or two amino acids or has at least or about 70% sequence identity;(f) a CDR2 amino acid sequence, i.e., a sequence selected from the group consisting of SEQ ID NOs: 9, 15, 21, 27, 33, 39, 45, 51, 57, 63, 69, 75, 81, 87, 93, 99, 105, 111, 117, 123, 129, 450, 477, 459, and 471, or a variant thereof which differs by only one or two amino acids or has at least or about 70% sequence identity; (g) an antigen binding protein comprising a CDR3 amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 16, 22, 28, 34, 40, 46, 52, 58, 64, 70, 76, 82, 88, 94, 100, 106, 112, 118, 124, 130, 451, 454, and 460, or a variant thereof which differs by only one or two amino acids or has at least or about 70% sequence identity; or (g) a combination of any two or more of (a) to (f). 30. a. SEQ ID NO: 74 to 79, b. SEQ ID NO: 50 to 55, c. SEQ ID NO: 122 to 127, d. SEQ ID NO: 26 to 31, e. SEQ ID NO: 128 to 133, f. SEQ ID NO: 38 to 43, g. SEQ ID NO: 62 to 67, h. SEQ ID NO: 80 to 85, i. SEQ ID NO: 44 to 49, j. SEQ ID NO: 86 to 91, k. SEQ ID NO: 104 to 109, l. SEQ ID NO: 56 to 61, m. SEQ ID NO: 32 to 37, n. SEQ ID NO: 110 to 115, SEQ ID NO: 98 to 103, p. SEQ ID NO: 92 to 97, q. SEQ ID NO: 116 to 121, r. SEQ ID NO: 8 to 13, s. SEQ ID NO: 68 to 73, t. SEQ ID NO: 14 to 19, u. SEQ ID NO: 20 to 25, v. SEQ ID NOs: 449 to 453 and 475, w. SEQ ID NO: 476-477, 454-457, x. SEQ ID NO: 458 to 463, y. SEQ ID NO: 57, 58, 464 to 467, z. SEQ ID NOs: 68 to 71 and 468 to 469, and aa. SEQ ID NO: 112 and 470 to 474, An antigen-binding protein comprising six CDR amino acid sequences selected from the group consisting of: 31. a. A heavy chain variable region amino acid sequence as set forth in Table B, i.e., a sequence selected from the group consisting of SEQ ID NOs: 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, and 175, or a variant thereof which differs by only one or two amino acids or has at least or about 70% sequence identity; b. a light chain variable region amino acid sequence set forth in Table B, i.e., a sequence selected from the group consisting of SEQ ID NOs: 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, and 176, or a variant thereof which differs by only one or two amino acids or has at least or about 70% sequence identity; or c. Both (a) and (b); 1. An antigen-binding protein comprising: 32. The antigen-binding protein according to claim 31, wherein the variant sequence has at least about 85% sequence identity, or about 90% or about 95% sequence identity. 33. a. SEQ ID NOs: 156 and 157; b. SEQ ID NOs:148 and 149, c. SEQ ID NOs:172 and 173, d. SEQ ID NOs:140 and 141, e. SEQ ID NOs:174 and 175, f. SEQ ID NOs:144 and 145, g. SEQ ID NOs:152 and 153, h. SEQ ID NOs:158 and 159, i. SEQ ID NOs:146 and 147, j. SEQ ID NOs:160 and 161, k. SEQ ID NOs:166 and 167, l. SEQ ID NOs:150 and 151, m. SEQ ID NOs:142 and 143, n. SEQ ID NOs: 168 and 169, o. SEQ ID NO:164 and 165, p. SEQ ID NO: 162 and 163, q. SEQ ID NO:170 and 171, r. SEQ ID NO:134 and 135, s. SEQ ID NO:154 and 155, t. SEQ ID NOs:136 and 137, and SEQ ID NOs: 138 and 139, An antigen-binding protein comprising a pair of amino acid sequences selected from the group consisting of: 34. a. A heavy chain variable region amino acid sequence as set forth in Table C, i.e., a sequence selected from the group consisting of SEQ ID NOs: 376-379, 384-387, 391-396, 403-408, 412-413, 416-419, and 422-427, or a variant thereof which differs by only one or two amino acids or has at least or about 70% sequence identity; b. A light chain variable region amino acid sequence as set forth in Table C, i.e., a sequence selected from the group consisting of SEQ ID NOs: 380-383, 388-390, 397-402, 409-411, 414-415, and 420-421, or a variant thereof that differs by only one or two amino acids or has at least or about 70% sequence identity; or c. Both (a) and (b); 1. An antigen-binding protein comprising: 35. The antigen-binding protein according to claim 6, wherein the variant sequence has at least about 85% sequence identity. 36. The antigen-binding protein according to claim 7, wherein the variant sequence has at least about 90% or about 95% sequence identity. 37. a. SEQ ID NO:376 and 380, b. SEQ ID NOs:377 and 380; c. SEQ ID NOs:377 and 381, d. SEQ ID NOs:377 and 382, e. SEQ ID NOs:377 and 383, f. SEQ ID NOs:378 and 381, g. SEQ ID NOs:378 and 382, h. SEQ ID NOs:378 and 383, i. SEQ ID NOs:379 and 381, j. SEQ ID NOs:379 and 382, k. SEQ ID NOs:379 and 383, l. SEQ ID NOs: 384 and 388, m. SEQ ID NO:385 and 388, n. SEQ ID NO: 385 and 389, o. SEQ ID NO:386 and 388; p. SEQ ID NO: 386 and 389, q. SEQ ID NO: 387 and 389, r. SEQ ID NO: 422 and 389, s. SEQ ID NO: 391 and 397, t. SEQ ID NOs: 392 and 397; u. SEQ ID NO:393 and 398, v. SEQ ID NO: 394 and 398, w. SEQ ID NO: 395 and 398, x. SEQ ID NOs: 396 and 398, y. SEQ ID NO: 423 and 398, z. SEQ ID NOs: 424 and 398, aa. SEQ ID NO: 425 and 398, bb. SEQ ID NO:426 and 398, cc. SEQ ID NO: 427 and 398, dd. SEQ ID NO: 403 and 409, ee. SEQ ID NO: 404 and 409, ff. SEQ ID NO: 405 and 410, gg. SEQ ID NO: 405 and 411, hh. SEQ ID NO:406 and 410, ii. SEQ ID NOs:406 and 411, jj. SEQ ID NO: 407 and 410, kk. SEQ ID NO: 407 and 411, ll. SEQ ID NO: 408 and 410, mm. SEQ ID NO: 408 and 411, nn. SEQ ID NO: 412 and 414, oo. SEQ ID NO: 413 and 414, pp. SEQ ID NO: 416 and 420, qq. SEQ ID NO: 417 and 420, rr. SEQ ID NO: 417 and 421, ss. SEQ ID NO: 418 and 420, tt. SEQ ID NO: 418 and 421, SEQ ID NO: 419 and 420, and vv. SEQ ID NO: 419 and 421, An antigen-binding protein comprising a pair of amino acid sequences selected from the group consisting of: 38. (A) optionally, an HC CDR1 comprising the amino acid sequence of YTFTXYT (wherein X is T, V, D, or S) (SEQ ID NO: 452), which comprises the amino acid sequence of YTFTTYT (SEQ ID NO: 11); (B) optionally, an HC CDR2 comprising the amino acid sequence of IXPSSGYT (wherein X is Q, S, A, or N) (SEQ ID NO:475), which comprises the amino acid sequence of INPSSGYT (SEQ ID NO:12); (C) optionally, an HC CDR3 comprising the amino acid sequence of AXGDYYVAY (wherein X is N, Q, H, or D) (SEQ ID NO:453), which comprises the amino acid sequence of ANGDYYVAY (SEQ ID NO:13); (D) optionally, a LC CDR1 comprising the amino acid sequence of SSVSSXY (wherein X is T, V, F, or D) (SEQ ID NO: 449), which comprises the amino acid sequence of SSVSSTY (SEQ ID NO: 8); (E) an LC CDR2 comprising the amino acid sequence of XTX (wherein X at position 1 is S, T, Q, or A and X at position 3 is S, T, D, or Q) (SEQ ID NO: 450), optionally comprising the amino acid sequence of STS (SEQ ID NO: 9); and (F) an LC CDR3 comprising an amino acid sequence of HXYXRSPLT (wherein X at position 2 is Q, H, or S and X at position 4 is H, Y, Q, or S) (SEQ ID NO:451), optionally comprising an amino acid sequence of HQYHRSPLT (SEQ ID NO:10); 1. An antigen-binding protein comprising: 39. (A) optionally, an HC CDR1 comprising an amino acid sequence of FTFSXYX (wherein X at position 5 is N, S, R, Q, or A, and X at position 7 is W, H, Y, or F) (SEQ ID NO: 455), which comprises the amino acid sequence of FTFSNYW (SEQ ID NO: 23); (B) optionally, an HC CDR2 comprising the amino acid sequence of IRLKXDXYAT (wherein X at position 5 is S, N, A, or T and X at position 7 is Q, S, A, or N) (SEQ ID NO: 456), which comprises the amino acid sequence of IRLKSDNYAT (SEQ ID NO: 24); (C) optionally, an HC CDR3 comprising an amino acid sequence of XDGPPSGX (wherein X at position 1 is N, D, or T and X at position 8 is S, T, A, C, or Y) (SEQ ID NO: 457), which comprises the amino acid sequence of NDGPPSGC (SEQ ID NO: 25); (D) LC CDR1 comprising the amino acid sequence of EXIYSY (wherein X is Q, S, A, D, or N) (SEQ ID NO:476), optionally comprising the amino acid sequence of ENIYSY (SEQ ID NO:20); (E) an LC CDR2 comprising the amino acid sequence of XAK (wherein X at position 1 is Q, S, A, D, or N) (SEQ ID NO: 477), optionally comprising the amino acid sequence of NAK (SEQ ID NO: 21); and (F) optionally, a LC CDR3 comprising the amino acid sequence of QXHYXVPWT (wherein X at position 2 is H, Q, S, or T and X at position 5 is T, S, N, or G) (SEQ ID NO:454), which comprises the amino acid sequence of QHHYTVPWT (SEQ ID NO:22); 1. An antigen-binding protein comprising: 40. (A) HC CDR1 optionally comprising the amino acid sequence of YTXTXYT (wherein X at position 3 is F, Y, S, or T, and X at position 5 is S, T, Y, or D) (SEQ ID NO: 461), which comprises the amino acid sequence of YTFTSYT (SEQ ID NO: 29); (B) HC CDR2 optionally comprising the amino acid sequence of IXPSSXYT (wherein X at position 2 is Q, S, A, or N, and X at position 6 is T, S, V, D, or G) (SEQ ID NO: 462), which comprises the amino acid sequence of INPSSTYT (SEQ ID NO: 30); (C) optionally, an HC CDR3 comprising the amino acid sequence of XRGEXGGFAY (wherein X at position 1 is S, A, T, or V, and X at position 5 is L, V, or F) (SEQ ID NO:463), which comprises the amino acid sequence of SRGELGGFAY (SEQ ID NO:31); (D) LC CDR1 comprising the amino acid sequence of QSLVHSXGXTY (wherein X at position 7 is D, N, E, Q, S, or A, and X at position 9 is Q, S, A, D, or N) (SEQ ID NO: 458), optionally comprising the amino acid sequence of QSLVHSDGNTY (SEQ ID NO: 26); (E) an LC CDR2 comprising an amino acid sequence of XVX (wherein X at position 1 is K, Q, or R and X at position 3 is S, T, or V) (SEQ ID NO: 459), optionally comprising the amino acid sequence of KVS (SEQ ID NO: 27); and (F) optionally, a LC CDR3 comprising an amino acid sequence of SXXTHVPYT (wherein X at position 2 is Q, H, or T and X at position 3 is S, G, T, or D) (SEQ ID NO:460), which comprises the amino acid sequence of SQSTHVPYT (SEQ ID NO:28); 1. An antigen-binding protein comprising: 41. A conjugate comprising an antigen-binding protein according to any of the above. 42. A fusion protein comprising any of the antigen-binding proteins described above. 43. A nucleic acid comprising a nucleotide sequence encoding any of the above antigen-binding proteins, the conjugates described in 41, or the fusion proteins described in 42. 44. A vector comprising the nucleic acid described in 43 above. 45. A host cell comprising a nucleic acid according to 43 above or a vector according to 44 above. 46. ​​A method for producing an antigen-binding protein that binds to claudin 6 (CLDN6) protein, comprising: (i) culturing in a cell culture medium a host cell as described in 45 above, wherein the host cell contains a nucleic acid comprising a nucleotide sequence encoding any of the antigen-binding proteins described above; and (ii) recovering the antigen-binding protein from the cell culture medium. 47. A method for producing a fusion protein comprising an antigen-binding protein that binds to claudin 6 (CLDN6) protein, the method comprising: (i) culturing in a cell culture medium a host cell as described in claim 45, wherein the host cell comprises a nucleic acid comprising a nucleotide sequence encoding the fusion protein as described in claim 30; and (ii) recovering the fusion protein from the cell culture medium. 48. A method for producing a pharmaceutical composition, comprising mixing an antigen-binding protein according to any one of 1 to 40, a conjugate according to 41, a fusion protein according to 42, a nucleic acid according to 43, a vector according to 44, a host cell according to 45, or a combination thereof, with a pharma- ceutically acceptable carrier, diluent or excipient. 49. A pharmaceutical composition comprising an antigen-binding protein according to any one of 1 to 40, a conjugate according to 41, a fusion protein according to 42, a nucleic acid according to 43, a vector according to 44, a host cell according to 45, or a combination thereof, together with a pharma- ceutically acceptable carrier, diluent or excipient. 50. A method for treating a subject having a CLDN6-expressing cancer, comprising administering to the subject a pharmaceutical composition described in 49 above in an amount effective to treat the cancer. 51. A method for inhibiting tumor growth in a subject, comprising administering to the subject a pharmaceutical composition described in 49 above in an amount effective to inhibit tumor growth. 52. A method for reducing the size of a tumor in a subject, comprising administering to the subject a pharmaceutical composition described in 49 above in an amount effective to reduce the size of the tumor. 53. A method for preventing recurrence of cancer in a subject, comprising administering to the subject a pharmaceutical composition described in 49 above in an amount effective to prevent recurrence of cancer. 54. A method for treating cancer in a subject diagnosed with low overexpression of CLDN6, comprising administering to the subject a pharmaceutical composition described in 49 above in an amount effective to prevent recurrence of the cancer. 55. A method according to any one of claims 50 to 54, wherein the administering induces apoptosis in tumor cells. 56. A method according to any one of claims 50 to 55, wherein the administering induces apoptosis in cells expressing CLDN6. 57. A method for detecting claudin 6 (CLDN6) in a sample, comprising contacting the sample with an antigen-binding protein described in any one of 1 to 40, a conjugate described in 41, or a fusion protein described in 42, and assaying an immune complex comprising the antigen-binding protein, conjugate, or fusion protein bound to CLDN6. 58. A method for diagnosing claudin 6 (CLDN6)-positive cancer in a subject, comprising contacting a biological sample containing cells or tissue taken from the subject with an antigen-binding protein described in any one of 1 to 40, a conjugate described in 41, or a fusion protein described in 42, and assaying an immune complex comprising the antigen-binding protein, conjugate, or fusion protein bound to CLDN6.

Claims

1. An antibody or antigen-binding fragment thereof that binds to human claudin 6 (CLDN6) protein (SEQ ID NO: 200), An antibody or antigen-binding fragment thereof comprising: a HC CDR1 comprising the amino acid sequence of FTFSNYW (SEQ ID NO: 455); a HC CDR2 comprising the amino acid sequence of IRLKSDNYAT (SEQ ID NO: 456); a HC CDR3 comprising the amino acid sequence of NDGPPSGS or NDGPPSGC (SEQ ID NO: 457); a LC CDR1 comprising the amino acid sequence of ENIYSY (SEQ ID NO: 476); a LC CDR2 comprising the amino acid sequence of NAK (SEQ ID NO: 477); and a LC CDR3 comprising the amino acid sequence of QHHYTVPWT (SEQ ID NO: 454).

2. The antibody or antigen-binding fragment thereof of claim 1 , wherein HC CDR1 comprises the amino acid sequence of GFTFSNYW (SEQ ID NO: 23).

3. The antibody or antigen-binding fragment thereof described in claim 1, wherein the antibody is a monoclonal antibody.

4. The antibody or antigen-binding fragment thereof described in claim 1, wherein the antibody is a chimeric antibody or a humanized antibody.

5. The antibody or antigen-binding fragment thereof described in claim 1, wherein the antibody is a humanized antibody comprising a heavy chain variable domain amino acid sequence of SEQ ID NO:387 and a light chain variable domain amino acid sequence of SEQ ID NO:

389.

6. A conjugate comprising the antibody or antigen-binding fragment thereof described in claim 1.

7. A fusion protein comprising the antibody or antigen-binding fragment thereof described in claim 1.

8. A nucleic acid comprising a nucleotide sequence encoding the antibody or antigen-binding fragment thereof of claim 1, or the fusion protein of claim 7.

9. A vector comprising the nucleic acid of claim 8.

10. A host cell comprising the nucleic acid of claim 8.

11. A method for producing an antibody or antigen-binding fragment thereof that binds to human claudin 6 (CLDN6) protein (sequence number 200), comprising: (i) culturing a host cell described in claim 10 in a cell culture medium; and (ii) recovering the antibody or antigen-binding fragment thereof from the cell culture medium.

12. A method for producing a fusion protein comprising an antibody or antigen-binding fragment thereof that binds to human claudin 6 (CLDN6) protein (SEQ ID NO: 200), the method comprising: (i) culturing a host cell described in claim 10 in a cell culture medium; and (ii) recovering the fusion protein from the cell culture medium.

13. A method for producing a pharmaceutical composition comprising mixing an antibody or antigen-binding fragment thereof described in any one of claims 1 to 5, a conjugate described in claim 6, a fusion protein described in claim 7, a nucleic acid described in claim 8, a vector described in claim 9, a host cell described in claim 10, or a combination thereof, with a pharma- ceutically acceptable carrier, diluent or excipient.

14. A pharmaceutical composition comprising an antibody or antigen-binding fragment thereof described in any one of claims 1 to 5, a conjugate described in claim 6, a fusion protein described in claim 7, a nucleic acid described in claim 8, a vector described in claim 9, a host cell described in claim 10, or a combination thereof, together with a pharma- ceutically acceptable carrier, diluent or excipient.

15. 15. The pharmaceutical composition of claim 14 for use in treating a subject with a CLDN6-expressing cancer.

16. Use of the pharmaceutical composition described in claim 14 in the manufacture of a medicament for the treatment of CLDN6-expressing cancer.

17. A method for inhibiting tumor growth in vitro or ex vivo, comprising contacting CLDN6-positive cancer cells with a pharmaceutical composition described in claim 14 in an amount effective for inhibiting tumor growth.

18. The pharmaceutical composition according to claim 14, for use in preventing recurrence of cancer in a subject.

19. The pharmaceutical composition of claim 15, wherein the CLDN6-expressing cancer is a CLDN6 low-overexpressing cancer.

20. The pharmaceutical composition of claim 14, which induces apoptosis in tumor cells.

21. The pharmaceutical composition of claim 14, which induces apoptosis in cells expressing CLDN6.

22. A method for detecting claudin 6 (CLDN6) in a sample, comprising contacting the sample with an antibody or antigen-binding fragment thereof described in any one of claims 1 to 5, a conjugate described in claim 6, or a fusion protein described in claim 7, and assaying an immune complex comprising the antibody or antigen-binding fragment thereof, conjugate, or fusion protein bound to CLDN6.

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