Claudin 6 antibody and method for treating cancer

Abstract

To provide antigen-binding proteins which bind to Claudin-6 (CLDN6).SOLUTION: Antigen-binding proteins which bind to Claudin-6 (CLDN6) bind to Extracellular Loop 2 (EL2) of the extracellular domain of CLDN6. Related polypeptides, nucleic acids, vectors, host cells, and conjugates are also provided. Kits and pharmaceutical compositions comprising such entities are further provided. Also provided are methods of making antigen-binding proteins and methods of treating subjects having cancer.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] Incorporation by reference of electronically submitted materials A computer-readable nucleotide / amino acid sequence listing, which is submitted simultaneously with this specification and identified hereinafter by a 315,776-byte ASCII (text) file named "51836_SeqListing.txt" (created on September 18, 2018), is hereby incorporated by reference in its entirety. The present disclosure provides an antigen-binding protein that binds to Claudin 6 (CLDN6).

Background Art

[0002] Antibodies constitute powerful therapeutic agents characterized by limited side effects due to the function of the antibody itself that specifically targets different antigens on cells, bacteria, viruses, or toxins. In 1986, Orthoclone OKT3, the first therapeutic monoclonal antibody, was introduced to the market. Since then, the number of biopharmaceuticals in this category has increased significantly. By the end of 2014, 47 monoclonal antibody products had been approved in the United States or Europe for the treatment of various diseases including cancer, inflammatory diseases, cardiovascular diseases, respiratory diseases, and infectious diseases.

[0003] Currently, more than 12 monoclonal antibodies for treating cancer are approved by the US Food and Drug Administration. These include alemtuzumab (Campus®), designated for chronic lymphocytic leukemia (CLL), and trastuzumab (Herceptin®), used to treat breast cancer. Some antibodies are classified as chemotherapeutic agents, such as brentuximab vedotin (Adcetris®) and adtrastuzumab 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, current cancer incidence and mortality rates remain high. The annual incidence of cancer is reported to be over 450 per 100,000 men and women, and the annual cancer mortality rate is a staggering 170 per 100,000 men and women. [Overview of the project]

[0004] This specification provides antigen-binding proteins that bind to claudin 6 (CLDN6). In various embodiments, the antigen-binding proteins of this disclosure bind to human CLDN6, and optionally to mouse CLDN6. In various embodiments, the antigen-binding proteins bind to the extracellular domain (ECD) of CLDN6. In various examples, the antigen-binding proteins bind to the extracellular loop 2 (EL2) of the ECD of CLDN6. In various embodiments, the antigen-binding proteins bind to EL2 and not to the 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 not to any of the other members of the claudin family. In various embodiments, antigen-binding proteins bind to CLDN6 endogenously expressed in human ovarian cancer cells (e.g., OVCA429 cells) and exhibit an IC50 of less than approximately 1200 nM in FACS affinity assays using OVCA429 cells. In various examples, the antigen-binding proteins of this disclosure inhibit tumor growth in a subject (e.g., human) without any other moieties being conjugated to the antigen-binding protein. In various examples, antigen-binding proteins that are not conjugated to non-homologous moieties (e.g., not conjugated to any chemotherapeutic agent, drug, or toxic moiety) inhibit tumor growth in a subject (e.g., human).

[0005] In various embodiments, antigen-binding proteins bind to CLDN6 expressed by human cancer cells. In various embodiments, antigen-binding proteins inhibit the binding interaction between human CLDN6 and a reference anti-CLDN6 antibody. While not bound by any particular theory, the inhibitory effect of antigen-binding proteins provided herein is useful in methods for suppressing tumor growth and for treating subjects with tumors or cancer. As further discussed herein, in various embodiments, the antigen-binding protein is an antibody, its antigen-binding antibody fragment, or an antibody protein product.

[0006] This disclosure also provides antigen-binding proteins comprising at least 3, 4, 5, or all of the amino acid sequences of a particular group of amino acid sequences. In various embodiments, the antigen-binding protein comprises at least 3, 4, 5, or 6 complementarity-determining region (CDR) amino acid sequences of the CLDN6 antibody disclosed herein.

[0007] This disclosure further provides antigen-binding proteins comprising amino acid sequences detailed herein. In various embodiments, the antigen-binding proteins comprise amino acid sequences of the sequence numbers listed in Tables A, A1, B, B1, C, or D, or combinations thereof, as further described herein.

[0008] This specification further provides related polypeptides, nucleic acids, vectors, host cells, and conjugates. Kits and pharmaceutical compositions containing such components are further explored.

[0009] Methods for producing antigen-binding proteins are also provided. In various embodiments, the method involves culturing host cells containing nucleic acids encoding an antigen-binding protein or polypeptide to express the antigen-binding protein or polypeptide described herein.

[0010] This specification further provides methods for treating subjects having cancer. In various embodiments, the methods include administering the pharmaceutical composition of this disclosure to a subject in an amount effective to treat cancer within the subject.

[0011] Methods for treating subjects with CLDN6-expressing cancer, including administering the pharmaceutical compositions described herein, are also provided. Methods for inhibiting tumor growth within subjects, including administering the pharmaceutical compositions described herein, are also further investigated.

[0012] A method for reducing the size of a tumor within a subject or preventing cancer recurrence within a subject includes administering the pharmaceutical composition described herein to the subject.

[0013] This specification also provides a method for treating cancer in subjects diagnosed with low overexpression of CLDN6, comprising administering the pharmaceutical composition described herein to the subject.

[0014] In various embodiments, administration induces apoptosis in tumor cells, for example, in cells expressing CLDN6. In various embodiments, administration 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 explanation of the drawing]

[0015] [Figure 1] This figure shows a graph of CLDN6 expression in normal (non-cancerous) tissue. [Figure 2] This figure shows a graph of CLDN6 expression in cancer cell lines as measured by the Agilent44K method. [Figure 3] This figure shows a graph of CLDN6 expression within cancer cell lines as measured by RNASeq. [Figure 4]This figure shows a set of fluorescence images illustrating the localization of CLDN6-GFP in different cell models. [Figure 5] This figure shows the 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) in mice with endometrial tumors, corresponding to the time (days) after treatment with control IgG2 antibody, AB3, reference Ab1, reference Ab2, reference Ab3, AB2, and AB3. Figure 6B shows a graph of the mean change in tumor volume (mm3) on day 14 in mice with 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) in mice with bladder tumors, corresponding to time (days) after treatment with control IgG2 antibody, AB3, reference Ab1, reference Ab2, and AB3. Figure 7B shows a graph of the mean change in tumor volume (mm3) at day 35 in mice with 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) in mice with ovarian tumors, corresponding to the time (days) after treatment with control IgG2 antibody, AB3, reference Ab1, AB2, and AB3. Figure 8B shows a graph of the mean change in tumor volume (mm3) at day 20 in mice with ovarian tumors treated with control IgG2 antibody, AB3, reference Ab1, AB2, or AB3. [Figure 9]Figure 9A shows a graph of tumor volume (mm3) in mice with melanoma tumors, corresponding to time (days) after treatment with control IgG2 antibody, AB3, reference Ab1, reference Ab2, reference Ab3, and AB3. Figure 9B shows a graph of the mean change in tumor volume (mm3) at day 21 in mice with melanoma tumors treated with control IgG2 antibody, AB3, reference Ab1, reference Ab2, reference Ab3, or AB3. [Figure 10] Figure 10A is a graph showing the tumor growth inhibition rate achieved in tumor-bearing mice treated with AB3 compared to mice treated with a control antibody. Figure 10B is a Western blot image showing different levels of CLDN6 in endometrial cancer cell lines (ARK2), bladder cancer cell line (UMUC4), ovarian cancer cell line (OV90), and melanoma cell line (M202), as well as in control cells. The levels of α-tubulin were nearly identical, indicating equivalent protein levels. [Figure 11] This figure shows a graph of the percentage change in body weight over time (days) in mice with tumors treated with a solvent control, a control antibody, reference Ab1, reference Ab2, reference Ab3, and AB3. [Figure 12A] This figure shows a graph of tumor volume (mm3) in mice with ovarian tumors, corresponding to time (days) after treatment with a solvent control, control IgG2 antibody, AB3, reference Ab1, or one of the anti-CLDN6 antibodies shown. [Figure 12B] This figure shows a graph of the mean change in tumor volume (mm3) at day 28 in mice with ovarian tumors treated with a solvent control, a control IgG2 antibody, AB3, reference Ab1, or one of the anti-CLDN6 antibodies shown. [Figure 13] This figure shows a graph of the percentage change in body weight over time (days) in mice with tumors treated with a solvent control, a control antibody, reference Ab1, and the anti-CLDN6 antibody shown. [Figure 14]This figure shows a series of dose-response curves for several anti-CLDN6 antibodies of the present invention, as well as for reference Ab1 and reference 2. Mouse IgG was used as a control. [Figure 15] Figure 15A is a graph showing the mean change in tumor volume (mm3) at day 35 in mice with bladder tumors treated with a solvent control, a control IgG antibody, a mouse type of AB3, the first humanized form of AB3, and the second humanized form of AB3. Figure 15B is a graph showing the change in tumor volume (mm3) in each of the groups in Figure 15A. [Figure 16] Figure 16A shows graphs of the mean change in tumor volume (mm3) at day 35 in mice with bladder tumors treated with a solvent control, a control IgG antibody, a mouse-type AB3, a first humanized form of AB3, and a second humanized form of AB3. Two control antibodies (one mouse and one chimeric) were also tested in this experiment. Figure 16B shows graphs of the change in tumor volume (mm3) in each group from Figure 16A. [Figure 17] Figure 17A shows a graph of the mean change in tumor volume (mm3) at day 35 in mice with bladder tumors treated with a solvent control, a control IgG antibody, a mouse variant of AB1, and a humanized variant 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) at day 35 in mice with bladder tumors treated with a solvent control, a control IgG antibody, a mouse-type AB4 antibody, and a humanized AB4 antibody. Figure 18B shows a graph of the change in tumor volume (mm3) in each of the groups in Figure 18A. [Figure 19]Figure 19A is a graph showing the mean change in tumor volume (mm3) at day 35 in mice with bladder tumors treated with a solvent control, a control IgG antibody, mouse-type AB3, chimeric AB3, first humanized AB3, second humanized AB3, mouse-type Ab1, humanized AB1, mouse-type AB4, and humanized AB4. Four control antibodies (one antibody having either mouse-type or chimeric form, and one antibody having either mouse-type or human-type form) were also tested in this experiment. Figure 19B is a graph showing the change in tumor volume (mm3) in each group in Figure 19A. [Figure 20] This figure shows a graph of the average change in tumor volume (mm3) at day 55 in mice with bladder tumors treated as described in Figure 19A. [Figure 21] This figure shows a graph of the percentage change in body weight on day 32 of mice with tumors that were treated as described in Figure 19A. [Modes for carrying out the invention]

[0016] Claudine Family Tight junctions, also known as closed junctions or closed zones, are orderly structures located between two adjacent cells that maintain cell polarity by regulating paracellular permeability in epithelial and endothelial cell sheets. The claudin (CLDN) family of genes encodes membrane proteins that are essential components of tight junctions. CLDN proteins consist of four transmembrane (TM) helices (TM1, TM2, TM3, and TM4) and two extracellular loops (EL1 and EL2). The extracellular loops of CLDN proteins in adjacent cells interact with each other to tightly bind the cell sheets together and regulate paracellular transport between the luminal and basolateral spaces.

[0017] CLDN proteins are involved in various human diseases and pathological conditions. For example, mutations in the CLDN1 gene have been shown to cause 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 agenesis and myopia, while CLDN14 mutations may cause non-syndromic inferior 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. Abnormal expression of several CLDN proteins has been shown in cancer. For example, CLDN1 is downregulated in breast and colon cancer, while CLDN3 and CLDN4 are strongly upregulated in several 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, cattle, mice, rats, zebrafish, and frogs. CLDN6 is typically 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 generally available on the National Center for Biotechnology Information (NCBI) website as NCBI Reference Sequence NP_067018.2, and is provided herein as Sequence ID: 1. The amino acid at position 143 of Sequence ID: 1 is Ile. In some cases, due to a single nucleotide polymorphism (SNP) within the DNA sequence encoding CLDN6, the amino acid at position 143 is Val. Hereinafter, the amino acid sequence of human CLDN6 with Val at position 143 is provided as Sequence ID: 178.

[0019] Antigen-binding protein This specification provides antigen-binding proteins that bind to claudin 6 (CLDN6). The antigen-binding proteins of this disclosure may take any one of many forms of antigen-binding proteins known in the art. In various embodiments, the antigen-binding proteins of this disclosure may take the form of an antibody, an antigen-binding antibody fragment, or an antibody protein product.

[0020] In various embodiments of this disclosure, the antigen-binding protein comprises, is essentially derived from, or consists of an antibody. As used herein, the term “antibody” means a protein having a typical immunoglobulin format, comprising a heavy chain and a light chain, and comprising a variable region and a constant region. For example, an antibody may be IgG, which is two pairs of identical polypeptide chains in a “Y-shape” structure, each having one “light” chain (typically having a molecular weight of about 25 kDa) and one “heavy” chain (typically having a molecular weight of about 50–70 kDa). Antibodies have a variable region and a constant region. In the IgG format, the variable region is typically about 100–110 or more amino acids and contains three complementarity-determining regions (CDRs) primarily responsible for antigen recognition, which are substantially different in other antibodies that bind to different antigens. The constant region enables the antibody to recruit cells and molecules of the immune system. The variable region consists of the N-terminal regions of the light and heavy chains, respectively, while the constant region consists of the C-terminal regions 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 relevant art. Briefly, in an antibody scaffold, CDRs are incorporated into a framework of heavy chain and light chain variable regions, which primarily constitute the regions responsible for antigen binding and antigen recognition. The variable region typically contains at least three heavy-chain CDRs or light-chain CDRs within the framework region (referred to as framework regions 1-4, FR1, FR2, FR3, and FR4, by Kabat et al., 1991; see also Chothia and Lesk, 1987, (as above)).

[0022] Antibodies may include any constant region known in the art. Human light chains are classified into κ and λ light chains. Heavy chains are classified into μ, δ, γ, α, or ε, defining antibody isotypes 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 this disclosure include antibodies of all such classes or isotypes. The light chain constant region may be, for example, a κ or λ type light chain constant region, e.g., a human κ or λ 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, e.g., a human α, δ, ε, γ, or μ type heavy chain constant region. Therefore, in various embodiments, the antibody is an isotype IgA, IgD, IgE, IgG, or IgM antibody comprising any one of IgG1, IgG2, IgG3, or IgG4. In various embodiments, the antibody includes a constant region having one or more amino acid modifications compared to the natural equivalent in order to improve half-life / stability or to make the antibody more suitable for expression / manufacturing. In various examples, the antibody includes a constant region in which a C-terminal Lys residue present in the natural equivalent has been removed or cleaved.

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

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

[0025] By utilizing the structure of antibodies, an increasingly wide range of alternative antibody formats have been created, with valencies (n) ranging from monomers (n=1) to dimers (n=2), trimers (n=3), tetramers (n=4), and sometimes even more multimers, in a molecular weight range of at least approximately 12 to 150 kDa. In this specification, such alternative antibody formats are referred to as "antibody protein products." Examples of antibody protein products include antibody protein products based on the complete antibody structure, and antibody protein products that mimic antibody fragments that retain complete antigen-binding ability, such as scFv, Fab, and VHH / VH (discussed below). The smallest antigen-binding fragment that retains a complete antigen-binding site is the Fv fragment, which consists exclusively of a variable (V) region. To stabilize the molecule, a soluble and flexible amino acid peptide linker is used to link the V region to an scFv (single-stranded variable fragment) fragment, or a constant (C) domain is added to the V region to create a Fab fragment [fragment, antigen-binding]. Both scFv and Fab fragments can be readily produced in host cells, such as prokaryotic host cells. Other antibody protein products include dimeric and multimeric antibody formats such as diabodies, triabodies, tetrabodies, or minibodies (miniAb), which include different formats consisting of disulfide-stabilized scFv (ds-scFv), single-chain Fab (scFab), and scFv linked to an oligomerized domain. The smallest fragment is VHH / VH, consisting of a camelid heavy chain Ab and a single-domain Ab (sdAb). The most frequently used building block for creating novel antibody formats is the single-chain variable (V)-domain antibody fragment (scFv), which contains a V domain consisting of a heavy chain and a light chain (VH domain and VL domain) linked by a peptide linker of approximately 15 amino acid residues. Peptibodies, or peptide-Fc fusions, are yet another antibody protein product. The structure of a peptide body consists of a biologically active peptide transplanted onto an Fc domain. Peptibodies have been adequately described in the relevant field.For example, see 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 trispecific antibodies. Bispecific antibodies can be classified into five main classes: BsIgG, IgG adducts, bispecific antibody (BsAb) fragments, bispecific fusion proteins, and BsAb conjugates. See, for example, Spiess et al., Molecular Immunology 67(2) Part A:97-106 (2015).

[0027] In various embodiments, the antigen-binding proteins of the Disclosure include, are essentially derived from, or consist of any one of these antibody protein products. In various embodiments, the antigen-binding proteins of the Disclosure include, are essentially derived from, or consist of any one of scFv, Fab VHH / VH, Fv fragment, ds-scFv, scFab, dimeric antibodies, multimeric antibodies (e.g., diabody, triabody, tetrabody), miniAb, camelid heavy chain antibody, sdAb, diabody peptibody VHH / VH, triabody, tetrabody, bispecific or triplicate antibodies, BsIgG, IgG adduct, BsAb fragment, bispecific fusion protein, and BsAb conjugate.

[0028] In various examples, the antigen-binding proteins of this disclosure are antibody-protein products in monomeric, polymeric, oligomeric, or polymeric forms. In certain embodiments in which the antibody comprises two or more different antigen-binding domain fragments, the antibody is considered to be bispecific, triplicate, or multispecific, or bivalent, trivalent, or polyvalent, depending on the number of different epitopes that the antibody recognizes and binds to.

[0029] In various embodiments, the anti-CLDN6 antibody or its antibody variant is selected from the group consisting of human antibodies, humanized antibodies, chimeric antibodies, monoclonal antibodies, recombinant antibodies, antigen-binding antibody fragments, single-chain antibodies, monomeric antibodies, diabodies, triabodies, tetrabodies, Fab fragments, IgG1 antibodies, IgG2 antibodies, IgG3 antibodies, and IgG4 antibodies.

[0030] In various embodiments, the antigen-binding proteins of this disclosure are conjugated to therapeutic agents. These therapeutic agents may include, but are not limited to, any known in the art, such as chemotherapeutic agents, cytokines and growth factors, and cytotoxic agents, as described below. See "Conjugate" below.

[0031] CLDN6 and epitopes The antigen-binding protein of this disclosure binds to CLDN6. In various embodiments, CLDN6 is This is human CLDN6 having the amino acid sequence TIFF0007869831000001.tif37154 (wherein X is Ile or Val) (Sequence ID: 202).

[0032] In various embodiments, human CLDN6 contains one of the amino acid sequences of sequence numbers 1, 178, and 200-202.

[0033] In various embodiments, the antigen-binding proteins of this disclosure bind to epitopes within the amino acid sequence of CLDN6. In various embodiments, CLDN6 is human CLDN6, and the antigen-binding proteins of this disclosure bind to epitopes within the amino acid sequences of human CLDN6, e.g., SEQ ID NOs: 1, 178, and 200-202. "Epitope" means a region of CLDN6 or a region within CLDN6 to which the antigen-binding protein binds. In some embodiments, the epitope is a linear epitope. "Linear epitope" means a region of CLDN6 or a region within CLDN6 to which the antigen-binding protein binds, and which consists of a sequence of amino acids in the amino acid sequence of CLDN6. The amino acids of the linear epitope are adjacent to each other in the primary structure of CLDN6. Therefore, the linear epitope is an antigen, i.e., a fragment or part of the amino acid sequence of CLDN6. In various other embodiments, the epitope is a structural epitope or a structural epitope. A "structural epitope" or "structural epitope" refers to an epitope composed of amino acids that are located in close proximity to each other only when CLDN6 is properly folded. Unlike linear epitopes, the amino acids of a structural epitope or structural epitope are not adjacent to each other in the primary structure (i.e., amino acid sequence) of CLDN6. A structural 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, for example, human CLDN6. In various embodiments, the antigen-binding protein binds to extracellular loop 2 (EL2) of the ECD of CLDN6 having the amino acid sequence WTAHAIIRDFYNPLVAEAQKREL (SEQ ID NO: 2). In various embodiments, the epitope to which the antigen-binding protein binds is located inside SEQ ID NO: 2. In various embodiments, the antigen-binding protein of this disclosure binds to the N-terminal portion of SEQ ID NO: 2, for example, THAIIRDFYNPL (SEQ ID NO: 3). In various embodiments, the antigen-binding protein of this disclosure binds to the C-terminal portion of SEQ ID NO: 2, for example, LVAEAQKREL (SEQ ID NO: 4). In various examples, the antigen-binding protein of this disclosure binds to EL2 but not to extracellular loop 1 (EL1) of CLDN6. In various embodiments, the epitope(s) to which the antigen-binding protein of this disclosure binds is different from the epitope to which the anti-CLDN6 antibody, which includes a light chain variable region containing the sequence of SEQ ID NO: 185 and a heavy chain variable region containing the sequence of SEQ ID NO: 186, binds. In various embodiments, the epitope(s) to which the antigen-binding protein of this disclosure binds is different from the epitope to which the anti-CLDN6 antibody, which includes a light chain variable region containing the sequence of SEQ ID NO: 181 and a heavy chain variable region containing the sequence of SEQ ID NO: 182, binds.

[0035] In various embodiments, antigen-binding proteins bind to both human and non-human CLDN6. In various examples, non-human CLDN6 may be CLDN6 from chimpanzees, rhesus monkeys, dogs, cattle, mice, rats, zebrafish, or frogs. In various examples, antigen-binding proteins bind to both human and mouse CLDN6.

[0036] Affinity and binding force The antigen-binding proteins provided herein bind to CLDN6 in a non-covalent and reversible manner. In various embodiments, the binding affinity of the antigen-binding protein to CLDN6 can be described based on its affinity, which is an indicator of the interaction force between the binding site of the antigen-binding protein and the epitope. In various aspects, the antigen-binding proteins provided herein have a high affinity for CLDN6, and thus bind to a larger amount of CLDN6 in a shorter period compared to low-affinity antigen-binding proteins. In various aspects, the antigen-binding protein is at least 10 -1 , D , D , D , D 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 and has an equilibrium binding constant K A . As those skilled in the art will understand, K A may be affected by factors including pH, temperature, and buffer composition.

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

[0038] In various embodiments, the affinity of antigen-binding proteins is measured or ranked using flow cytometry or fluorescence-activated cell isolation (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 antigen-binding proteins is measured or ranked using the competitive assays described below, in addition to those described in Trikha et al., Int J Cancer 110:326-335 (2004) and Tam et al., Circulation 98(11):1085-1091 (1998). Please refer to the following section titled "Competitive Assays." Trikh et al. used antigen-expressing cells in the radioassay. Using cells in suspension, 125 The binding affinity of a 1-labeled antigen-binding protein (e.g., an antibody) to a cell surface antigen is measured. In various embodiments, the relative affinity of a CLDN6 antibody is measured by a FACS-based assay, in which different concentrations of CLDN6 antibody conjugated to a phosphor are incubated with cells expressing CLDN6, and the emitted fluorescence (a direct indicator of antibody-antigen binding) is measured. A curve is created plotting the fluorescence against each dose or concentration. The maximum value is the lowest concentration at which the fluorescence plateaus or reaches its highest point, where binding saturation occurs. Half of the maximum value is considered the EC50 or IC50, and the antibody with the lowest EC50 / IC50 is considered to have the highest affinity compared to other antibodies tested by the same method. Such an assay is described in Example 5 of this specification.

[0039] In various aspects, IC50 is measured by competitive binding inhibition assays. 50 The value is the K of the antigen-binding protein. DThis is similar to the following. In the various examples described below, the competitive assay is a FACS-based assay performed using a reference antibody, a fluoroconjugated secondary antibody, and cells expressing CLDN6. In various embodiments, the cells are genetically modified 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, cells endogenously expressing CLDN6 are pre-defined as CLDN6 low-expression cells or CLDN6 high-expression cells before performing the FACS-based assay. In some embodiments, the cells are cancer cells or tumor cells. In various embodiments, the cells are derived from cell lines, such as ovarian cell lines, endometrial cell lines, bladder cell lines, lung cell lines, gastrointestinal (GI) cell lines, hepatocyte cell lines, etc. In various embodiments, cells endogenously expressing 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 hepatocytes, JHOS-4 ovarian cells, H1435 lung cells, and NUGC3 upper GI cells. In various embodiments, the antigen-binding protein inhibits the binding interaction between human CLDN6 expressed by the cells and a reference antibody, although the reference antibody is known to bind to CLDN6 but not to the antigen-binding protein of this disclosure. In various examples, the antigen-binding protein of this disclosure competes with the reference antibody for binding to human CLDN6, thereby reducing the amount of human CLDN6 bound to the reference antibody when measured in an in vitro competitive binding assay. In various embodiments, the antigen-binding protein of this disclosure inhibits the binding interaction between human CLDN6 and a reference antibody, but this inhibition is not reflected in IC 50 It is represented as follows. In various embodiments, antigen-binding proteins inhibit the binding interaction between human CLDN6 and the reference antibody with an IC of less than approximately 2500 nM. 50 This indicates that, in various embodiments, the antigen-binding protein has a mass of less than approximately 2000 nM, less than approximately 1500 nM, less than approximately 1000 nM, and about 900 nM. nM Less than approximately 800 nM Less than approximately 700 nM Less than approximately 600 nM Less than approximately 500 nM Less than approximately 400 nM Less than approximately 300 nM Less than approximately 200 nM Less than, or about 100 nM ICs less than 50 This indicates that, in various embodiments, the antigen-binding protein has an IC of less than approximately 90 nM, less than approximately 80 nM, less than approximately 70 nM, less than approximately 60 nM, less than approximately 50 nM, less than approximately 40 nM, less than approximately 30 nM, less than approximately 20 nM, or less than approximately 10 nM. 50 This demonstrates that, in various cases, the antigen-binding proteins of this disclosure compete for binding to CLDN6 with a reference antibody known to bind to CLDN6 (the reference antibody being different from any of the antigen-binding proteins of this disclosure). See the following description of the competitive assay.

[0040] Binding strength provides an indicator of the overall strength of the antibody-antigen complex. Binding strength depends on three main parameters: the affinity of the antigen-binding protein to the epitope, the valence of both the antigen-binding protein and CLDN6, and the structural arrangement of the interacting regions. A higher valence (number of antigen-binding sites) of the antigen-binding protein increases the amount of antigen (CLDN6) that the antigen-binding protein can bind to. In various embodiments, antigen-binding proteins have a strong binding affinity to CLDN6. In various embodiments, antigen-binding proteins are polyvalent. In various embodiments, antigen-binding proteins are bivalent. In various examples, antigen-binding proteins are monovalent.

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

[0042] In various embodiments, the antigen-binding protein of this disclosure binds to CLDN6 but not to claudin 3 (CLDN3), claudin 4 (CLDN4), and claudin 9 (CLDN9). In various embodiments, the antigen-binding protein does not bind to CLDN3, CLDN4, and CLDN9 and exhibits IC50 levels of less than approximately 1200 nM (e.g., less than approximately 1000 nM, less than approximately 750 nM, less than approximately 500 nM, less than approximately 250 nM) in FACS-based assays using OVCA429 cells endogenously expressing CLDN6. 50This shows that, in 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 endogenously expressing CLDN6 is less than approximately 1200 nM (e.g., less than approximately 1000 nM, less than approximately 750 nM, less than approximately 500 nM, less than approximately 250 nM). In various embodiments, the antigen-binding protein exhibits at least 5 times 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 endogenously expressing CLDN6 is less than approximately 1200 nM (e.g., less than approximately 1000 nM, less than approximately 750 nM, less than approximately 500 nM, less than approximately 250 nM). In various embodiments, antigen-binding proteins exhibit an IC50 of less than approximately 1200 nM (e.g., less than approximately 1000 nM, less than approximately 750 nM, less than approximately 500 nM, less than approximately 250 nM) to artificial and endogenous models of CLDN6, and a ratio of CLDN6 IC50 that is more than approximately 5 times higher than that of CLDN3, CLDN4, and / or CLDN9. In various examples, antigen-binding proteins exhibit an IC50 of less than approximately 1200 nM (e.g., less than approximately 1000 nM, less than approximately 750 nM, less than approximately 500 nM, less than approximately 250 nM) to CLDN6, and an IC50 to any one of CLDN3, CLDN4, and CLDN9 that is at least 5 times higher compared to that IC50.

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

[0044] Competitive assay In various embodiments, the antigen-binding protein inhibits the binding interaction between human CLDN6 and a reference antibody, while the reference antibody is known to bind to CLDN6 but not to the antigen-binding protein of this disclosure. In various examples, the antigen-binding protein of this disclosure reduces the amount of human CLDN6 bound to the reference antibody when measured in an in vitro competitive binding assay by competing with the reference antibody for binding to human CLDN6. In various embodiments, the reference antibody binds to an epitope in the amino acid sequence of the extracellular domain of human CLDN6, optionally to an epitope in EL2 or EL1. In various embodiments, the reference antibody includes 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 embodiments, the reference antibody includes a light chain variable sequence of SEQ ID NO: 181 and a heavy chain variable sequence of SEQ ID NO: 182. In various embodiments, the antigen-binding protein of this disclosure inhibits the binding interaction between human CLDN6 and the reference antibody, but this inhibition is not measured in IC 50 It is represented as follows. In various embodiments, antigen-binding proteins inhibit the binding interaction between human CLDN6 and the reference antibody with an IC of less than approximately 2500 nM. 50 This indicates that, in various embodiments, the antigen-binding protein has a mass of less than approximately 2000 nM, less than approximately 1500 nM, less than approximately 1000 nM, and about 900 nM. nM Less than approximately 800 nM Less than approximately 700 nM Less than approximately 600 nM Less than approximately 500 nM Less than approximately 400 nM Less than approximately 300 nM Less than approximately 200 nM Less than, or about 100 nM ICs less than 50 This indicates that, in various embodiments, the antigen-binding protein has an IC of less than approximately 90 nM, less than approximately 80 nM, less than approximately 70 nM, less than approximately 60 nM, less than approximately 50 nM, less than approximately 40 nM, less than approximately 30 nM, less than approximately 20 nM, or less than approximately 10 nM. 50 This indicates.

[0045] In various embodiments, the antigen-binding protein of this disclosure competes with the reference antibody for binding to human CLDN6, thereby reducing the amount of human CLDN6 bound to the reference antibody when measured in an in vitro competitive binding assay. In various embodiments, the in vitro competitive binding assay is a FACS-based assay that measures the fluorescence of a fluorescent-conjugated secondary antibody bound to the Fc of a reference antibody in the presence or absence of a specific amount of the antigen-binding protein of this disclosure. Such FACS-based assays are described in the examples herein. In various embodiments, the FACS-based assay is performed using a reference antibody, a fluorescent-conjugated secondary antibody, and cells expressing CLDN6. In various embodiments, the cells are genetically modified 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, before performing the FACS-based assay, the cells endogenously expressing CLDN6 are pre-determined as either CLDN6-low-expressing or CLDN6-high-expressing cells. In some embodiments, the cells are cancer cells or tumor cells. In various embodiments, the cells are cells derived from cell lines, such as ovarian cell lines, endometrial cell lines, bladder cell lines, lung cell lines, gastrointestinal (GI) cell lines, hepatocyte cell lines, and lung cell lines. In various embodiments, cells endogenously expressing 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 hepatocytes, JHOS-4 ovarian cells, H1435 lung cells, and NUGC3 upper GI cells. In various examples, the antigen-binding protein of this disclosure binds with high affinity to CLDN6 endogenously expressed in one or more of the following cells: ARK2 cells, OVCA429 cells, LS513 cells, or MCF7 cells. In various embodiments, antigen-binding proteins exhibit IC50s of less than approximately 3000 nM when measured by a FACS-based competitive binding inhibition assay using one or more ARK2, OVCA429, LS513, or MCF7 cells. 50In various embodiments, antigen-binding proteins show ICs of less than approximately 2500 nM, less than approximately 2000 nM, less than approximately 1750 nM, less than approximately 1500 nM, less than approximately 1250 nM, less than approximately 1000 nM, less than approximately 750 nM, or less than approximately 500 nM when measured by 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, antigen-binding proteins exhibit IC values ​​of less than approximately 400 nM, less than approximately 300 nM, less than approximately 200 nM, less than approximately 100 nM, less than approximately 75 nM, less than approximately 50 nM, less than approximately 25 nM, or less than approximately 10 nM when measured by a FACS-based competitive binding inhibition assay using one or more of ARK2, OVCA429, LS513, or MCF7 cells. 50 This indicates.

[0046] Other binding assays, such as competitive binding assays, which test the ability of an antibody to compete with a second antibody for 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). U.S. Patent Application Publication 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, such as surface plasmon resonance (SPR). Using SPR, the binding constants of one antibody and a second antibody can be measured, 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 antibody production are described, for instance, 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). This disclosure Various methods for preparing CLDN6 monoclonal antibodies The law Examples are provided herein.

[0048] Depending on the host species, various adjuvants can be used to enhance the immune response and lead to greater antibody production by the host. Examples of such adjuvants include, but are not limited to, Freund's adjuvants, mineral gels such as aluminum hydroxide, and surfactants such as lysolecithin, Pluronic® polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanins, 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 the CLDN6 epitope, regardless of how the antibody is manufactured, are well known in the art and include any antibody-antigen binding assay, such as radioimmunoassay (RIA), ELISA, Western blotting, immunoprecipitation, SPR, and competitive inhibition assays (see, for example, Janeway et al., infra, and U.S. Patent Application Publication No. 2002 / 0197266, as well as the above section on competitive assays).

[0052] Array / Structure This specification refers to (a) sequences selected from the group consisting of heavy chain (HC) complementarity-determining region (CDR) 1 amino acid sequences listed in Table A, i.e., sequence numbers: 11, 17, 23, 29, 35, 41, 47, 53, 59, 65, 71, 77, 83, 89, 95, 101, 107, 113, 119, 125, and 131, or variant sequences having at least one or two amino acids different or 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, and (b) HC sequences listed in Table A. (c) HC as listed in Table A, 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, and 132, or variant sequences having at least one or two different amino acids or 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, (c) HC as listed in Table A (d) A sequence selected from the group consisting of CDR3 amino acid sequences, i.e., sequence numbers: 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 that differs by only one or two amino acids or has 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, (d) a variant as described in Table A (e) LC (LC) CDR1 amino acid sequences, i.e., sequences 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 variant 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, (e) LC listed in Table A(f) LCs listed in Table A, which are CDR2 amino acid sequences, i.e., sequences 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 variant sequences thereof that differ by only one or two amino acids or have 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, (f) LCs listed in Table A The present invention provides an antigen-binding protein comprising a CDR3 amino acid sequence, 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; a variant thereof that differs by only one or two amino acids or has 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; or a combination of any two or more of (g)(a) to (f).

[0053] [Table 2]

[0054] In various embodiments, the antigen-binding protein includes at least one or two of the LC CDR1 amino acid sequence, LC CDR2 amino acid sequence, and LC CDR3 amino acid sequence described in Table A, as well as the HC CDR amino acid sequence described in Table A.

[0055] In various embodiments, the antigen-binding protein comprises at least three, four, or five of the amino acid sequences shown by the single-row sequence numbers in Table A. In various embodiments, the antigen-binding protein comprises each of the LC CDR amino acid sequences shown by the single-row sequence numbers in Table A, and at least one or two of the HC CDR amino acid sequences shown by the single-row sequence numbers in Table A. In various embodiments, the antigen-binding protein comprises each of the HC CDR amino acid sequences shown by the single-row sequence numbers in Table A, and at least one or two of the LC CDR amino acid sequences shown by the single-row sequence numbers in Table A. In various embodiments, the antigen-binding protein comprises all six CDR amino acid sequences shown by the single-row sequence numbers in Table A. In various embodiments, the antigen-binding protein is (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: 104-1 It contains six CDR amino acid sequences selected from the group consisting of 09, (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: 92-97, (q) SEQ ID NOs: 116-121, (r) SEQ ID NOs: 8-13, (s) SEQ ID NOs: 68-73, (t) SEQ ID NOs: 14-19, and (u) SEQ ID NOs: 20-25.

[0056] In various examples, the amino acid sequences in 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 acids. In various examples, there are approximately 10 to 20 amino acids between the LC CDR1 sequence and the LC CDR2 sequence, and approximately 25 to 40 amino acids between the LC CDR2 sequence and the LC CDR3 sequence. In various examples, there are approximately 14 to 16 amino acids between the LC CDR1 sequence and the LC CDR2 sequence, and approximately 30 to 35 amino acids between the LC CDR2 sequence and the LC CDR3 sequence. In various examples, there are approximately 10 to 20 amino acids between the HC CDR1 sequence and the HC CDR2 sequence, and approximately 25 to 40 amino acids between the HC CDR2 sequence and the HC CDR3 sequence. In various examples, there are approximately 14 to 16 amino acids between the HC CDR1 sequence and the HC CDR2 sequence, and approximately 30 to 35 amino acids between the HC CDR2 sequence and the HC CDR3 sequence.

[0057] In various embodiments, the antigen-binding protein is (a) a sequence selected from the group consisting of the heavy chain variable region amino acid sequences listed in Table B, namely, 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 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, (b) The light chain variable region amino acid sequences listed in Table B, i.e., sequences 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 variant sequences thereof that differ by only one or two amino acids or have 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, or (c) including both (a) and (b).

[0058] [Table 3]

[0059] In various embodiments, the antigen-binding protein is (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 It contains a pair of amino acid sequences selected from the group consisting of 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 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.

[0060] In various embodiments, the antigen-binding protein does not contain the pair of amino acid sequences encoded by sequence numbers 179 and 180. In various embodiments, the antigen-binding protein does not contain the pair of amino acid sequences encoded by sequence numbers 181 and 182. In various embodiments, the antigen-binding protein does not contain the pair of amino acid sequences encoded by sequence numbers 183 and 184. In various embodiments, the antigen-binding protein does not contain the pair of amino acid sequences encoded by sequence numbers 185 and 186.

[0061] In various embodiments, antigen-binding proteins contain amino acid sequences similar to those mentioned above, and furthermore, antigen-binding proteins substantially retain their biological function, for example, their ability to bind to human CLDN6, inhibit tumor growth, and treat cancer.

[0062] In various embodiments, the antigen-binding protein contains an amino acid sequence that differs by only one, two, three, four, five, six, or more amino acids compared to the amino acid sequence(s) mentioned above. In various embodiments, the antigen-binding protein contains a variant sequence of the mentioned sequence, the variant sequence differing by only one or two amino acids compared to the mentioned sequence. In various embodiments, the antigen-binding protein contains one or more amino acid substitutions occurring outside the CDR, for example, one or more amino acid substitutions occurring inside the framework region(s) of the heavy or light chain. In various embodiments, the antigen-binding protein contains 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 contains an amino acid sequence having only one, two, three, four, five, six, or more conserved amino acid substitutions compared to the amino acid sequence(s) mentioned above. As used herein, the term “conservative amino acid substitution” means substituting one amino acid with another amino acid having similar properties, such as size, charge, hydrophobicity, hydrophilicity, and / or aromaticity, and includes the following five groups: I. Small aliphatic nonpolar or slightly polar residues: Ala, Ser, Thr, Pro, Gly; II. Polar and negatively charged residues, and their amides and esters: Asp, Asn, Glu, Gln, cysteic acid, and homocysteic acid; III. Polar and positively charged residues: His, Arg, Lys, Ornithine (Orn); IV. Large aliphatic nonpolar residues: Met, Leu, Ile, Val, Cys, norleucine (Nle), homocysteine; V. Large aromatic residues: Phe, Tyr, Trp, acetylphenylalanine; One example is an internal exchange.

[0063] In various forms, a conservative amino acid substitution is an exchange within one of the following amino acid groups: TIFF0007869831000005.tif46122

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

[0065] In various embodiments, the antigen-binding protein contains a variant sequence of the sequence mentioned, and the variant sequence has at least 70% or about 70% sequence identity with respect to the sequence mentioned above. In various embodiments, the antigen-binding protein contains a variant sequence of the sequence mentioned, and the variant sequence has at least 80% or about 80% sequence identity with respect to the sequence mentioned above. In various embodiments, the antigen-binding protein contains a variant sequence of the sequence mentioned, and the variant sequence has at least 90% or about 90% sequence identity with respect to the sequence mentioned above. In various embodiments, the antigen-binding protein contains a variant sequence of the sequence mentioned, and the variant sequence has at least 95% or about 95% sequence identity with respect to the sequence mentioned above.

[0066] In various embodiments, the antigen-binding protein includes one, two, three, four, or five sequences from the single row sequence numbers in Table A, and 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 any of sequence numbers 8-133. In various embodiments, the antigen-binding protein is (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) sequence The antigen-binding protein comprises one, two, three, four, or five sequences from a set of sequences selected from (q)Sequence IDs: 92-97, (r)Sequence IDs: 8-13, (s)Sequence IDs: 68-73, (t)Sequence IDs: 14-19, and (u)Sequence IDs: 20-25, and 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 with respect to at least one of the sequences in the set. For example, in various embodiments, the antigen-binding protein comprises four sequences from 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 70% or about 70% (e.g., at least about 80%, at least about 85%, at least about 90%, at least about 95%) sequence identity with SEQ ID NO: 78, and another 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 with SEQ ID NO: 79.

[0067] In various embodiments, the antigen-binding protein contains 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 includes (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: 14 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 (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 from Table B, and 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 any of SEQ ID NOs: 134-175.In various embodiments, the antigen-binding protein is 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) sequence The sequence comprises one sequence selected from (a) to (u) sequence numbers: 168 and 169, (o) sequence numbers: 164 and 165, (p) sequence numbers: 162 and 163, (q) sequence numbers: 170 and 171, (r) sequence numbers: 134 and 135, (s) sequence numbers: 154 and 155, (t) sequence numbers: 136 and 137, and (u) sequence numbers: 138 and 139, and 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 with sequences (a) to (u). For example, in various embodiments, the antigen-binding protein comprises the sequence of SEQ ID NO: 134, and 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 with respect to SEQ ID NO: 135.

[0068] In various embodiments, antigen-binding proteins include amino acid sequences of the amino acid sequences mentioned above, having one or more amino acid substitutions to reduce or remove reactive amino acids and thereby reduce or prevent undesirable side-chain reactions. For example, antigen-binding proteins include amino acid sequences of the amino acid sequences mentioned above, having one or more of the following: (i) Trp residues substituted with His, Tyr, or Phe; (ii) Asn residues substituted with Gln, Ser, Ala, or Asp; (iii) Asp residues immediately preceding Pro residues substituted with Ala, Ser, or Glu; (iv) Asn residues substituted with Gln, Ser, or Ala; and / or (v) Cys residues substituted with Tyr, Ser, or Ala. In various embodiments, antigen-binding proteins include amino acid sequences of the amino acid sequences mentioned above, having amino acid substitutions that are predicted to have higher binding affinity, higher stability, or other desirable properties based on SHM events or statistical analysis of a large number of other similar antibody sequences. In some embodiments, the antigen-binding protein is (a) a sequence selected from the group consisting of HC CDR1 amino acid sequences listed in Table A1, i.e., sequence numbers 452, 455, 461, 465, 71, and 472, or a variant thereof that differs by only one or two amino acids or has 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; (b) a sequence selected from the group consisting of HC CDR2 amino acid sequences listed in Table A1, i.e., sequence numbers 475, 456, 462, 466, 468, and 473, or a variant thereof that differs by only one or two amino acids or has 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; (c) an HC(d) LC CDR3 amino acid sequences, i.e., sequences selected from the group consisting of SEQ ID NOs: 453, 457, 463, 467, 469, and 474, or variant sequences thereof that differ by only one or two amino acids or have 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, (e) 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 variant sequences thereof that differ by only one or two amino acids or have 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, (e) LC CDR1 amino acid sequences listed in Table A1 (f) A CDR2 amino acid sequence, i.e., a sequence selected from the group consisting of SEQ ID NOs: 450, 477, 459, 57, 69, and 471, or a variant thereof that differs by only one or two amino acids or has 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, or (g) a combination of any two or more of (a) to (f).

[0069] [Table 4]

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

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

[0072] In various embodiments, HC CDR1 contains MH adjacent to the C-terminus of SEQ ID NO: 461. In various embodiments, HC CDR2 contains Tyr adjacent to the N-terminus of SEQ ID NO: 462, and optionally TH adjacent to the C-terminus of SEQ ID NO: 462. In exemplary embodiments, HC CDR3 does not contain the first two amino acids of SEQ ID NO: 463. In various embodiments, LC CDR1 contains RSS adjacent to the N-terminus of SEQ ID NO: 458, and optionally LONDON adjacent to the C-terminus of SEQ ID NO: 458. In various embodiments, LC CDR2 contains 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 includes MH adjacent to the C-terminus of SEQ ID NO: 465. In various embodiments, HC CDR2 includes YI adjacent to the N-terminus of SEQ ID NO: 466, and optionally Xaa adjacent to the C-terminus of SEQ ID NO: 466, where Xaa is N, S, Q, or A. In various embodiments, LC CDR1 includes LAS adjacent to the N-terminus of SEQ ID NO: 464, and optionally LA adjacent to the C-terminus of SEQ ID NO: 464. In various embodiments, LC CDR2 includes SLAD adjacent to the C-terminus of SEQ ID NO: 57.

[0074] In various embodiments, HC CDR1 contains MH adjacent to the C-terminus of SEQ ID NO: 71. In various embodiments, HC CDR2 contains 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 contains 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 contains XLES adjacent to the C-terminus of SEQ ID NO: 69, where X is N, Q, S, A, or D.

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

[0076] In various embodiments, the antigen-binding protein includes at least one or two of the LC CDR1 amino acid sequence, LC CDR2 amino acid sequence, and LC CDR3 amino acid sequence described in Table A1, as well as the HC CDR amino acid sequence described in Table A1.

[0077] In various embodiments, the antigen-binding protein includes at least three, four, or five of the amino acid sequences shown by the single-row sequence numbers in Table A1. In various embodiments, the antigen-binding protein includes each of the LC CDR amino acid sequences shown by the single-row sequence numbers in Table A1, and at least one or two of the HC CDR amino acid sequences shown by the single-row sequence numbers in Table A1. In various embodiments, the antigen-binding protein includes each of the HC CDR amino acid sequences shown by the single-row sequence numbers in Table A1, and at least one or two of the LC CDR amino acid sequences shown by the single-row sequence numbers in Table A1. In various embodiments, the antigen-binding protein includes all six CDR amino acid sequences shown by the single-row sequence numbers in Table A1. In various embodiments, the antigen-binding protein contains 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 and 454-457, (c) SEQ ID NOs: 458-463, (d) SEQ ID NOs: 57, 58 and 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 in 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 acids. In various examples, there are approximately 10 to 20 amino acids between the LC CDR1 sequence and the LC CDR2 sequence, and approximately 25 to 40 amino acids between the LC CDR2 sequence and the LC CDR3 sequence. In various examples, there are approximately 14 to 16 amino acids between the LC CDR1 sequence and the LC CDR2 sequence, and approximately 30 to 35 amino acids between the LC CDR2 sequence and the LC CDR3 sequence. In various examples, there are approximately 10 to 20 amino acids between the HC CDR1 sequence and the HC CDR2 sequence, and approximately 25 to 40 amino acids between the HC CDR2 sequence and the HC CDR3 sequence. In various examples, there are approximately 14 to 16 amino acids between the HC CDR1 sequence and the HC CDR2 sequence, and approximately 30 to 35 amino acids between the HC CDR2 sequence and the HC CDR3 sequence.

[0079] In various embodiments, the antigen-binding protein includes (a) a heavy chain variable region amino acid sequence listed 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 that differs by only one or two amino acids or has 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, (b) a light chain variable region amino acid sequence listed 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 that differs by only one or two amino acids or has 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, or (c) both of (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 embodiments, the antigen-binding protein comprises a variant sequence of the sequence having SEQ ID NOs listed in Table B1, which differs by only one or two amino acids or has 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, and the differing amino acids(s) are located at the positions described below within the "humanized antibody".

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

[0083] Humanized AB1 In various embodiments, the antigen-binding protein is located at one or more of the following positions within 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. The number 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 various examples, the antigen-binding protein contains the amino acid sequence of SEQ ID NO: 428. In various embodiments, the antigen-binding protein is a humanized form of AB1 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: 20, 31, 35, 48, 50, 59, 67, 70, 74, 79, 98, 101 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12). In various examples, the antigen-binding protein contains the amino acid sequence of SEQ ID NO: 429. In various embodiments, the amino acids at the positions listed above are selected from the amino acids listed in the following table.

[0084] [Table 6]

[0085] In various embodiments, the antigen-binding protein is located at one or more of the following positions within 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. Humanized forms of AB1 as described in Table B or B1, having one or more amino acid substitutions in (for example, 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 contains the amino acid sequence of SEQ ID NO: 430. In various embodiments, the antigen-binding protein is a humanized form of AB1 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, 21, 32, 34, 48, 51, 53, 61, 67, 79, 84, 91, and 93 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13). In various examples, the antigen-binding protein contains the amino acid sequence of SEQ ID NO: 431. In various embodiments, the amino acids at the positions listed above are selected from the amino acids listed in the following table.

[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 contains the amino acid sequence of SEQ ID NO: 432. 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: 31, 35, 50, 55, 79, 99, 106 (e.g., 1, 2, 3, 4, 5, 6, or 7). In various examples, the antigen-binding protein contains the amino acid sequence of SEQ ID NO: 433. In various embodiments, the amino acids at the positions listed above are selected from the amino acids listed in the following table.

[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 contains the amino acid sequence of SEQ ID NO: 434. 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: 25, 34, 48, 53, 55, 84, 85, 90, and 93 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, or 9). In various examples, the antigen-binding protein contains the amino acid sequence of SEQ ID NO: 435. In various embodiments, the amino acids at the positions listed above are selected from the amino acids listed in the following table.

[0090] [Table 9]

[0091] Humanized AB4 In various embodiments, the antigen-binding protein is located at one of the following positions within 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 It is a humanized form of AB4 as described in Table B or B1, having one or more amino acid substitutions in 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). In various examples, the antigen-binding protein contains the amino acid sequence of SEQ ID NO: 436. In various embodiments, the antigen-binding protein is a humanized form 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 heavy chain variable region: 20, 29, 31, 37, 45, 48, 56, 59, 61, 62, 65, 66, 68, 70, 74, 79, 84, 97, and 101 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19). In various examples, the antigen-binding protein contains the amino acid sequence of SEQ ID NO: 437. In various embodiments, the amino acids at the positions listed above are selected from the amino acids listed in the following table.

[0092] [Table 10]

[0093] In various embodiments, the antigen-binding protein is a humanized form 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: 7, 14, 17, 18, 31, 33, 39, 41, 42, 44, 50, 51, 55, 57, 60, 81, 88, 92, 94, 95, 96, 99, 100, 105 (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 contains the amino acid sequence of SEQ ID NO: 438. In various embodiments, the antigen-binding protein is a humanized form 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: 33, 39, 55, 57, 81, 95, and 96 (e.g., 1, 2, 3, 4, 5, 6, or 7). In various examples, the antigen-binding protein contains the amino acid sequence of SEQ ID NO: 439. In various embodiments, the amino acids at the positions listed above are selected from the amino acids listed in the following table.

[0094] [Table 11]

[0095] Humanized AB18 In various embodiments, 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 contains 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 listed in the table below.

[0096] [Table 12]

[0097] In various embodiments, 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 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 contains the amino acid sequence of SEQ ID NO: 442 or 443. In various embodiments, the amino acids at the positions listed above are selected from the amino acids listed in the following table.

[0098] [Table 13]

[0099] Humanized AB9 In various embodiments, the antigen-binding protein is a humanized form 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 contains the amino acid sequence of SEQ ID NO: 444. In various embodiments, the amino acids at the positions listed above are selected from the amino acids listed in the table below.

[0100] [Table 14]

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

[0102] [Table 15]

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

[0104] [Table 16]

[0105] In various embodiments, the antigen-binding protein is a humanized form 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), and optionally at one or more of the following positions: 4, 82, and 110. In various examples, the antigen-binding protein contains the amino acid sequence of SEQ ID NO: 447 or 448. In various embodiments, the amino acids at the positions listed above are selected from the amino acids listed in the following table.

[0106] [Table 17]

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

[0108] [Table 18]

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

[0110] [Table 19] TIFF0007869831000022.tif48170

[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 with respect to the sequence numbers listed in Table C. In various embodiments, the antigen-binding protein comprises a pair of sequences, one sequence selected from the sequence numbers listed in Table C, and a sequence having the sequence numbers listed in Table D. orThe sequence comprises 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 with the sequence having the sequence number listed in Table C.

[0112] In various embodiments, the antigen-binding protein comprises a pair of sequences, one sequence selected from the sequence numbers listed in Table D, and 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 sequence having the sequence numbers listed in Table D. For example, in various embodiments, the antigen-binding protein comprises the sequence of sequence number 419, and the antigen-binding protein further comprises 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 sequence number 421.

[0113] nucleic acid This disclosure further provides nucleic acids comprising nucleotide sequences encoding antigen-binding proteins of this disclosure. As used herein, “nucleic acid” includes “polynucleotide,” “oligonucleotide,” and “nucleic acid molecule,” and generally means a polymer or modified form of DNA or RNA which may be single-stranded or double-stranded and may be synthesized or obtained (e.g., isolated and / or purified) from natural resources, a polymer or modified form of DNA or RNA which may contain natural, unnatural or modified nucleotides, and a polymer or modified form of DNA or RNA which may contain natural, unnatural or modified internucleotide junctions, such as phosphoramidate junctions or phosphorothioate junctions, instead of phosphodiesters present between nucleotides in unmodified oligonucleotides. A nucleic acid may comprise any nucleotide sequence encoding any of the antigen-binding proteins of this disclosure. In various embodiments, the nucleic acid is (a) a sequence selected from the group consisting of heavy chain (HC) complementarity-determining region (CDR) 1 amino acid sequences listed in Table A or A1, i.e., 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 sequence having sequence identity of at least 70% or about 70% (e.g., at least 80% or about 80%, at least 85% or about 85%, at least 90% or about 90%, at least 95% or about 95%), or (b) an HC sequence listed in Table A or A1 A sequence selected from the group consisting of CDR2 amino acid sequences: 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 sequence having at least 70% or approximately 70% (e.g., at least 80% or approximately 80%, at least 85% or approximately 85%, at least 90% or approximately 90%, at least 95% or approximately 95%) sequence identity,(c) A sequence selected from the group consisting of HC CDR3 amino acid sequences listed in Table A or A1, i.e., sequence numbers: 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 sequence having at least 70% or about 70% (e.g., at least 80% or about 80%, at least 85% or about 85%, at least 90% or about 90%, at least 95% or about 95%) sequence identity, (d) As listed in Table A or A1 (e) LCs listed in Table A or A1, which are LCs selected from the group consisting of the light chain (LC) CDR1 amino acid sequences, i.e., sequences 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 variant sequences having sequence identity of at least 70% or about 70% (e.g., at least 80% or about 80%, at least 85% or about 85%, at least 90% or about 90%, at least 95% or about 95%), (f) LCs listed in Table A or A1 A CDR3 amino acid sequence, specifically 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, 130, 451, 454, and 460, orThe nucleic acid comprises a nucleotide sequence encoding an antigen-binding protein, which includes a variant sequence having at least 70% or about 70% (e.g., at least 80% or about 80%, at least 85% or about 85%, at least 90% or about 90%, at least 95% or about 95%) sequence identity, or any combination of two or more of (g)(a) to (f). In various embodiments, the nucleic acid comprises a nucleotide sequence encoding an antigen-binding protein, which includes at least one or two of the LC CDR1 amino acid sequences, LC CDR2 amino acid sequences, and LC CDR3 amino acid sequences described in Table A or A1, as well as the HC CDR amino acid sequences described in Table A or A1. In various embodiments, the nucleic acid comprises a nucleotide sequence encoding an antigen-binding protein, which includes at least one or two of the HC CDR1 amino acid sequences, HC CDR2 amino acid sequences, and HC CDR3 amino acid sequences described in Table A or A1, as well as the LC CDR amino acid sequences described in Table A or A1. In various embodiments, the nucleic acid is (a) at least three, four, or five of the amino acid sequences shown by the single row sequence number in Table A or A1: (b) each of the LC CDR amino acid sequences shown by the single row sequence number in Table A or A1: and at least one or two of the HC CDR amino acid sequences shown by the single row sequence number in Table A or A1: (c) each of the HC CDR amino acid sequences shown by the single row sequence number in Table A or A1: and the LC CDR amino acid sequences shown by the single row sequence number in Table A or A1: (d) All six CDR amino acid sequences indicated by the single row number in Table A, and / or (e) (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: 92-97,The nucleotide sequence encoding an antigen-binding protein includes six CDR amino acid sequences selected from the group consisting of (q) SEQ ID NOs: 116-121, (r) SEQ ID NOs: 8-13, (s) SEQ ID NOs: 68-73, (t) SEQ ID NOs: 14-19, (u) SEQ ID NOs: 20-25, (v) SEQ ID NOs: 449-453 and 475, (w) SEQ ID NOs: 476-477, 454-457, (x) SEQ ID NOs: 458-463, (y) SEQ ID NOs: 57, 58, 464-467, (z) SEQ ID NOs: 68-71 and 468-469, and (aa) SEQ ID NOs: 112 and 470-474. In various embodiments, the nucleic acid is (a) a sequence selected from the group consisting of heavy chain variable region amino acid sequences listed in Table B or B1, i.e., 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 sequence having at least 70% or about 70% (e.g., at least 80% or about 80%, at least 85% or about 85%, at least 90% or about 90%, at least 95% or about 95%) sequence identity, or (b) the light chain variable region listed in Table B or B1. The amino acid sequence, 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, 176, 479, 481, 483, 485, 487, and 489; or a variant thereof that differs by only one or two amino acids or has at least 70% or about 70% (e.g., at least 80% or about 80%, at least 85% or about 85%, at least 90% or about 90%, at least 95% or about 95%) sequence identity; or a nucleotide sequence encoding an antigen-binding protein, including both (a) and (b). In various embodiments, the nucleic acids are (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,The sequence comprises a nucleotide sequence encoding an antigen-binding protein, which includes a pair of amino acid sequences selected from the group consisting of (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 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 nucleic acid comprises a nucleotide sequence encoding an antigen-binding protein, comprising a pair of amino acid sequences selected from the group consisting of pairs listed in Table D. In various embodiments, the nucleic acid comprises a nucleotide sequence comprising one or more sequences from SEQ ID NOs: 208 to 375. In some embodiments, the nucleic acid contains no insertions, deletions, inversions, and / or substitutions. In other embodiments, the nucleic acid contains one or more insertions, deletions, inversions, and / or substitutions.

[0114] In certain embodiments, the nucleic acids of this disclosure are recombinant. As used herein, the term “recombinant” means (i) a molecule constructed outside of a living cell by attaching a natural or synthetic nucleic acid segment to a nucleic acid molecule that can replicate in a living cell, or (ii) a molecule obtained from replication of the molecule described in (i) above. For the purposes of this specification, replication may be in vitro or in vivo.

[0115] In some embodiments, 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. (as above); and Ausubel et al. (as above). For example, nucleic acids may be chemically synthesized using native nucleotides or variously modified nucleotides designed to improve the biological stability of the molecule or the physical stability of the double-stranded nucleotides formed by hybridization (e.g., phosphorothioate derivatives and acridine-substituted nucleotides). Examples of modified nucleotides that can be used to produce 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 -Isopentenyl adenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N-substituted adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, β-D-mannosyl eosin, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N 6 Examples include, but are not limited to, isopentenyl adenine, uracil-5-oxyacetic acid(v), wibutoxosin, pseudouracil, queosin, 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 this disclosure may be purchased from companies such as Macromolecular Resources (Fort Collins, CO) and Synthegen (Houston, TX).

[0116] vector In some embodiments, the nucleic acids of this disclosure are incorporated into a vector. In this regard, this disclosure provides a vector comprising one of the nucleic acids disclosed herein. In various embodiments, the vector is a recombinant expression vector. For the purposes of this specification, the term “recombinant expression vector” means a recombinant oligonucleotide or polynucleotide construct comprising a nucleotide sequence encoding mRNA, protein, polypeptide, or peptide, which enables the expression of mRNA, protein, polypeptide, or peptide by a host cell when the vector comes into contact with the cell under conditions sufficient to express mRNA, protein, polypeptide, or peptide in the cell. The vectors of this disclosure are not natural as a whole. However, parts of the vectors may be natural. The vectors disclosed herein may be single-stranded or double-stranded and may contain any type of nucleotide, including but not limited to DNA and RNA that may be partially synthesized or obtained from natural resources, and DNA and RNA that may contain natural, non-natural, or modified nucleotides. The vectors may contain natural or non-natural nucleotide junctions, or both types of junctions. In some embodiments, modified nucleotides or unnatural internucleotide junctions do not interfere with the transcription or replication of the vector.

[0117] The vectors of this disclosure may be any suitable vectors that may be used to transduce, transform, or transfect any suitable host. Suitable vectors include vectors intended for propagation and proliferation, vectors intended for expression, or both, such as plasmids and viruses. The vector may be a plasmid-based expression vector. In various embodiments, the vector is selected from the group consisting of the pUC series (Fermentas Life Sciences), the pBluescript series (Stratagene, LaJoIIa, CA), the pET series (Novagen, Madison, WI), the pGEX series (Pharmacia Biotech, Uppsala, Sweden), and the 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 embodiments, the vector is a viral vector, such as a retroviral vector. In various embodiments, the vector is an adenovirus vector, an adeno-associated virus (AAV) vector, a herpes simplex virus (HSV) vector, a vesicular stomatitis virus (VSV) vector, a vaccinia virus vector, or a lentivirus vector. See, for example, Howarth et al., Cell Biol. Toxicol. 26(1):1-20 (2010). In various embodiments, the vector is a baculovirus vector that infects arthropods, such as insects. In various forms, the baculovirus vector is autographa California polynuclear virus (AcMNPV) or silkworm polyhedra disease (BmNPV).For example, see 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 this disclosure can be prepared, for example, using standard recombinant DNA techniques described in Sambrook et al., (as described above) and Ausubel et al., (as described above). Expression vector constructs, which may be circular or linear, can be prepared to contain a replication system that functions within a prokaryotic or eukaryotic host cell. The replication system may be derived, for example, from CoIEl, 2μ plasmid, λ, SV40, bovine papillomavirus, etc.

[0119] In some embodiments, the vector may include regulatory sequences, such as start and stop codons for transcription and translation, which are specific to the type of host (e.g., bacteria, fungi, plants, or animals) into which the vector is introduced, taking into consideration whether the vector is DNA-based or RNA-based, as necessary.

[0120] The vector may contain one or more marker genes that allow selection of a transformed or transfected host. Examples of marker genes include biocide resistance, such as resistance to antibiotics and heavy metals, and complementarity within a nutrient-requiring host that provides protonation. Suitable marker genes for expression vectors disclosed herein include, for example, neomycin / G418 resistance genes, hygromycin resistance genes, histidinol resistance genes, tetracycline resistance genes, and ampicillin resistance genes.

[0121] The vector may contain a native or non-native promoter functionally ligated to a nucleotide sequence encoding a polypeptide (including its functional site or functional variant), a nucleotide sequence complementary to the polypeptide-encoding sequence, or a nucleotide sequence hybridizing to the polypeptide-encoding sequence. The selection of promoters (e.g., strong, weak, inducible, tissue-specific, and development-specific) is known to those skilled in the art. Similarly, the combination of nucleotide sequences with promoters is also known to those 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 within the long-terminal repeat sequence of a mouse stem cell virus.

[0122] host cell This specification provides host cells containing the nucleic acids or vectors disclosed herein. As used herein, the term “host cell” means any type of cell that may contain the vectors disclosed herein and may produce expression products encoded by the nucleic acids (e.g., mRNA, proteins). In some embodiments, the host cell is an adherent cell or a suspension cell, i.e., a cell that grows in a suspension. In various embodiments, the host cell is a cultured cell or a primary cell, i.e., one isolated directly from an organism, e.g., a human. The host cell may be any cell type, may originate from any type of tissue, and may be at any developmental stage.

[0123] In various embodiments, the antigen-binding protein is a glycosylated protein, and the host cell is a glycosylated competent cell. In various embodiments, the glycosylated competent cell is a eukaryotic cell, including but not limited to yeast cells, filamentous fungal cells, protozoan cells, algal cells, insect cells, or mammalian cells. Such host cells have been 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 include Chinese hamster ovary (CHO) cells and their derivatives (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 fetal kidney 293 (HEK293) cells or their derivatives (e.g., HEK293T, HEK293-EBNA), African green monkey kidney cells (e.g., COS cells, VERO cells), human cervical cancer cells (e.g., HeLa), human osteosarcoma epithelial cells U2-OS, adenocarcinoma human alveolar basal epithelial cells A549, human fibrosarcoma cells HT1080, mouse brain tumor cells CAD, embryonic cancer cells P19, and mouse embryonic fibroblasts NIH. These include 3T3, mouse fibroblasts L929, mouse neuroblastoma cells N2a, human breast cancer cells MCF-7, retinoblastoma cells Y79, human retinoblastoma cells SO-Rb50, human liver cancer cells Hep G2, mouse B myeloma cells J558L, or baby hamster kidney (BHK) cells (Gaillet et al. 2007; Khan, Adv Pharm Bull 3(2):257-263(2013)).

[0124] In some embodiments, the host cell for amplifying or replicating the vector is a prokaryotic cell, such as a bacterial cell.

[0125] Further provided in this disclosure are populations of cells comprising at least one type of host cell described herein. In some embodiments, the population of cells is a heterogeneous population comprising at least one other type of cell that does not contain any of the vectors, in addition to the vector-containing host cells described herein. Alternatively, in some embodiments, the population of cells is a substantially homogeneous population in which the population comprises primarily (e.g., essentially consisting of) vector-containing host cells. In some embodiments, the population is a clonal population of cells in which all cells of the population are clones of a single host cell containing a vector, and as a result, all cells of the population contain a vector. In various embodiments of this disclosure, the population of cells is a clonal population comprising vector-containing host cells described herein.

[0126] Manufacturing method This specification also provides a method for producing an antigen-binding protein that binds to CLDN6. In various embodiments, the method comprises culturing a host cell containing a nucleic acid comprising a nucleotide sequence encoding the 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 embodiments, the host cell is selected from the group consisting of CHO cells, NS0 cells, COS cells, VERO cells, and BHK cells. In various embodiments, the step of 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 embodiments, the growth medium improves cell density, culture viability, and productivity in a timely manner. In various embodiments, the growth medium contains amino acids, vitamins, inorganic salts, glucose, and serum as sources of growth factors, hormones, and adhesion factors. In various embodiments, the growth medium is a fully synthetic medium consisting of amino acids, vitamins, trace elements, inorganic salts, lipids, and insulin or insulin-like growth factor. In addition to nutrients, growth media also help maintain pH and osmotic pressure. Several types of growth media are commercially available and have been described in the field. For example, see Arora, “Cell Culture Media: A Review” MATER METHODS 3:175 (2013).

[0127] In various embodiments, the method includes culturing host cells in a feed medium. In various embodiments, the method includes culturing in a feed medium using a fed-batch culture method. Methods for producing recombinant proteins are well known in the art. For example, see Li et al., “Cell culture processes for monoclonal antibody production” MAbs 2(5):466-477 (2010).

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

[0129] In various embodiments, the method further includes a step to obtain a formulation containing purified protein by incorporating purified protein or the like. Such a step is described in Formulation and Process Development Strategies for Manufacturing, eds. Jameel and Hershenson, John Wiley & Sons, Inc. (Hoboken, NJ), 2010.

[0130] In various embodiments, the antigen-binding protein is linked to a polypeptide, and the antigen-binding protein is part of the fusion protein. Therefore, 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 a host cell containing a nucleic acid comprising a nucleotide sequence encoding the fusion protein described herein in a cell culture medium, and recovering the fusion protein from the cell culture medium.

[0131] Conjugate This disclosure also provides antigen-binding proteins bound, linked, or conjugated to a second portion (e.g., a non-homologous portion, a conjugate portion). Therefore, this disclosure provides a conjugate comprising an antigen-binding protein and a non-homologous portion. As used herein, the term “non-homologous portion” is synonymous with “conjugate portion” and means any molecule (chemical or biochemical, natural or non-coding) that is different from the antigen-binding protein of this disclosure. Various non-homologous portions 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 non-homologous portion is a polymer. The polymer may be branched or unbranched. The polymer may have 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 a water-soluble polymer, some molecules are heavier and some are lighter compared to the stated molecular weight). In some embodiments, 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), thereby allowing control over the degree of polymerization. In some embodiments, the polymer is water-soluble, and as a result, the proteins to which the polymer binds do not precipitate in aquatic environments such as physiological environments. In some embodiments, the polymer is pharmaceutically acceptable, for example, when the composition is used for therapeutic purposes. In addition, in some embodiments, the polymer is a mixture of polymers, such as a copolymer or block copolymer.

[0134] In some embodiments, the polymers include polyamides, polycarbonates, polyalkylenes and their derivatives (including polyalkylene glycols, polyalkylene oxides, and polyalkylene terephthalates), acrylic acid ester polymers and methacrylic acid ester polymers (including poly(methyl methacrylate), poly(ethyl methacrylate), poly(butyl methacrylate), poly(isobutyl methacrylate), poly(hexy methacrylate), poly(isodecyl methacrylate), poly(lauryl methacrylate), poly(phenyl methacrylate), poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutyl acrylate, and poly(octadecyl acrylate)), and polyvinyl polymers (polyvinyl alcohol, polyvinyl ether, polyvinyl ester, and polyvinyl halide). The following materials are selected from the group consisting of poly(vinyl acetate, including polyvinylpyrrolidone), polyglycolides, polysiloxanes, polyurethanes and their copolymers, cellulose (including alkylcellulose, hydroxyalkylcellulose, cellulose ethers, cellulose esters, nitrocellulose, methylcellulose, ethylcellulose, hydroxypropylcellulose, hydroxypropyl methylcellulose, hydroxybutyl methylcellulose, cellulose acetate, cellulose propionate, cellulose acetate butyrate, cellulose acetate phthalate, carboxyethylcellulose, cellulose triacetate, and sodium cellulose sulfate), polypropylene, polyethylene (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 means encompassing any form 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, available in a wide range of molecular weights, and soluble in water and most organic solvents.

[0136] In some embodiments, the non-homologous portion 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), or a polysaccharide (starch, amylase, amylopectin, cellulose, chitin, callose, laminarin, xylan, mannan, fucoidan, galactomannan).

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

[0138] In some embodiments, the non-homologous portion is a therapeutic agent. The therapeutic agent may be any therapeutic agent known in the art. Examples of therapeutic agents considered herein include natural enzymes, proteins derived from natural resources, recombinant proteins, natural peptides, synthetic peptides, cyclic peptides, antibodies, receptor agonists, cytotoxic agents, immunoglobulins, β-adrenergic blockers, calcium channel blockers, coronary vasodilators, cardiac glycosides, antiarrhythmics, cardiac sympathomimetic agents, angiotensin-converting enzyme (ACE) inhibitors, diuretics, inotropes, cholesterol-lowering agents and triglyceride-lowering agents, bile acid sequesters, fibrates, and 3-hydroxy-3-methylglutaryl (H MG)-CoA reductase inhibitors, niacin derivatives, anti-adrenergic agonists, α-adrenergic blockers, centrally acting anti-adrenergic agonists, vasodilators, potassium-sparing agents, thiazides and related drugs, angiotensin II receptor antagonists, peripheral vasodilators, anti-androgens, estrogens, antibiotics, retinoids, insulin and analogs, α-glucosidase inhibitors, biguanides, meglitinides, sulfonylureas, thiazolidinediones, androgens, progestogens, bone metabolism regulators, anterior pituitary hormones, hypothalamus Partial hormones, posterior pituitary hormones, gonadotropins, gonadotropin-releasing hormone antagonists, ovulation inducers, selective estrogen receptor modulators, antithyroid drugs, thyroid hormones, bulk-forming agents, laxatives, peristalsis inhibitors, bacterial flora modulators, intestinal adsorbents, intestinal anti-infective agents, anti-anorexia drugs, anti-cachexia drugs, anti-bulimia drugs, appetite suppressants, anti-obesity drugs, antacids, upper gastrointestinal drugs, anticholinergics, aminosalicylic acid derivatives, biological response modulators, corticosteroids, antispasmodics, 5-HT4 partial agonists, antihistamines, cannabinoids, dopamine antagonists, Serotonin antagonists, cytoprotective agents, histamine H2 receptor antagonists, mucosal protective agents, proton pump inhibitors, H. pylori eradication agents, erythrocyte production stimulants, 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, prophylactic treatment, AIDS-related infections.Drugs for cytomegalovirus, non-nucleoside reverse transcriptase inhibitors, nucleoside analog reverse transcriptase inhibitors, protease inhibitors, anemia, Kaposi's sarcoma, aminoglycosides, carbapenems, cephalosporins, glycopeptides, lincosamides, macrolides, oxazolidinones, penicillin, streptogramin, sulfonamides, trimethoprim and derivatives, tetracyclines, anthelmintics, anti-amebic drugs, biguanides, cinchona bark alkaloids, folic acid antagonists, quinoline derivatives, Pneumocystis Carinii therapy drugs, hydrazide, imidazole, triazole, nitroimidazole, cyclic amines, neuraminidase inhibitors, nucleosides, phosphate binders, cholinesterase inhibitors, adjunctive therapies, barbiturates and derivatives, benzodiazepines, gamma-aminobutyric acid derivatives, hydantoin derivatives, iminostilbene derivatives, succinimide derivatives, anticonvulsants, ergot alkaloids, anti-migraine preparations, biological response modifiers, carbamates, tricyclic derivatives, depolarizers, non-depolarizers, neuromuscular paralytics, CNS stimulants, dopaminergic reagents, monoamine oxidase inhibitors, COMT inhibitors, alkyl sulfonates, ethyleneimine, imidazotetrazine, nitrogen mustard analogs, nitrosourea, platinum-containing compounds, antimetabolites, purine analogs, pyrimidine analogs, urea derivatives, anthracyclines, actinomycin, camptothecin derivatives, epipod Phylotoxin, taxanes, vinca alkaloids and analogs, antiandrogens, antiestrogens, nonsteroidal aromatase inhibitors, protein kinase inhibitory antitumor agents, azaspirodecandione 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, nonsteroidal drugs, leukotriene receptor antagonists, xanthines, endothelin receptor antagonists, prostaglandins, pulmonary surfactants, mucolytics, antimitotics, uric acid excretion agents, xanthine oxidase inhibitors, phosphodiesterase inhibitors, methenamine salts, nitrofuran derivatives, quinolones, smooth muscle relaxants,Examples of such substances include, but are not limited to, parasympathetic agonists, halogenated hydrocarbons, aminobenzoic acid esters and amides (e.g., lidocaine, articaine hydrochloride, bupivacaine hydrochloride), antipyretics, hypnotics and sedatives, cyclopyrrolone, pyrazolopyrimidines, nonsteroidal anti-inflammatory drugs, opioids, para-aminophenol derivatives, alcohol dehydrogenase inhibitors, heparin antagonists, adsorbents, emetics, opioid antagonists, cholinesterase reactivators, nicotine replacement therapies, 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 this disclosure may be conjugated to one or more cytokines and growth factors effective in inhibiting tumor metastasis, and the cytokines or growth factors have been shown to have an inhibitory effect on the growth of at least one cell population. Examples of such cytokines, lymphokines, growth factors, or other hematopoietic factors include, but are 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 factors, and erythropoietin.Additional growth factors for use in this specification include angiogenin, osteomorphic protein-1, osteomorphic protein-2, osteomorphic protein-3, osteomorphic protein-4, osteomorphic protein-5, osteomorphic protein-6, osteomorphic protein-7, osteomorphic protein-8, osteomorphic protein-9, osteomorphic protein-10, osteomorphic protein-11, osteomorphic protein-12, osteomorphic protein-13, osteomorphic protein-14, osteomorphic protein-15, osteomorphic protein receptor IA, osteomorphic protein receptor IB, and brain. Derived neurotrophic factors, ciliary neurotrophic factor, ciliary neurotrophic factor receptor α, cytokine-induced neutrophil migration factor 1, cytokine-induced neutrophil migration factor 2α, cytokine-induced neutrophil migration factor 2β, β-endothelial growth factor, endothelin 1, epithelial neutrophil attractant, glial cell line-derived neurotrophic factor receptor α1, glial cell line-derived neurotrophic factor receptor α2, growth-related proteins, growth-related proteins α, growth-related proteins β, growth-related proteins γ, heparin-binding epithelial growth factor, hepatocyte growth factor, hepatocyte growth factor receptor Examples include 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 suppressor factor, leukemia suppressor receptor α, nerve growth factor, nerve growth factor receptor, neurotrophin-3, neurotrophin-4, pre-B cell growth stimulant, stem cell factor, stem cell factor receptor, transforming growth factor α, transforming growth factor β, transforming growth factor β1, transforming growth factor β1.2, transforming growth factor β2, transforming growth factor β3, transforming growth factor β5, latent transforming growth factor β1, transforming growth factor β-binding protein I, transforming growth factor β-binding protein II, transforming growth factor β-binding protein III, tumor necrosis factor receptor I, tumor necrosis factor receptor II, urokinase-type plasminogen activator receptor, and chimeric proteins and their biologically or immunologically active fragments.

[0140] In some embodiments, the conjugate comprises an antigen-binding protein and a cytotoxic agent as described herein. The cytotoxic agent is any molecule (chemical or biochemical) that is toxic to cells. In some embodiments, the results obtained by conjugating the cytotoxic agent to the antigen-binding protein of this disclosure are synergistic. In other words, the combined therapeutic effect of the antigen-binding protein and cytotoxic agent is synergistic, meaning that the effect is greater than the effect that would be predicted from the individual additive effects. Therefore, the dose of the cytotoxic agent can be reduced, resulting in a simultaneous reduction in the risk of toxicity problems 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-coordinate compounds, topoisomerase inhibitors, antibiotics, antimitotic alkaloids, and difluoronucleosides as described in U.S. Patent No. 6,630,124.

[0141] In some embodiments, the chemotherapeutic agent is a platinum-coordinate compound. The term “platinum-coordinate compound” means any tumor cell proliferation inhibitory platinum-coordinate compound that yields platinum in ionic form. In some embodiments, cisplatin is the platinum-coordinate compound employed in the compositions and methods of this disclosure. In some embodiments, the chemotherapeutic agent is a topoisomerase inhibitor. In some embodiments, the topoisomerase inhibitor is camptothecin or a camptothecin analog. In other further embodiments of this 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. Generally, antimitotic alkaloids can be extracted from periwinkle and have been shown to be effective as anticancer chemotherapeutic agents. In other embodiments of this disclosure, the chemotherapeutic agent is a difluoronucleoside. It is well known in the art that 2'-deoxy-2',2'-difluoronucleosides possess antiviral activity. Such compounds are disclosed and taught in U.S. Patents 4,526,988 and 4,808,614. European Patent Application Publication 184,365 discloses that these same difluoronucleosides possess tumor-disrupting activity.

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

[0143] Linker In some embodiments, the conjugate is directly linked to the non-homologous portion. In alternative embodiments, the conjugate includes a linker that links the compound of the Disclosure to the non-homologous portion. In some embodiments, the linker includes a molecular chain of 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 in length. In some embodiments, all atoms of the molecular chain are carbon atoms. In some embodiments, the atoms of the molecular chain in the linker backbone 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 expected solubility (hydrophilicity) to result in a more soluble conjugate. In some embodiments, the linker provides a functional group that is susceptible to cleavage by enzymes or other catalytic or hydrolytic conditions present in the target tissue or target organ or target cell. In some embodiments, the linker length 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 approximately 1–50 amino acid long, 5–50, 3–5, 5–10, 5–15, or 10–30 amino acid long.

[0144] Compositions, pharmaceutical compositions and formulations This specification provides compositions comprising antigen-binding proteins, nucleic acids, vectors, host cells, or conjugates disclosed herein. In some embodiments, the composition comprises an isolated and / or purified form of the antigen-binding protein. In some embodiments, the composition comprises a single type (e.g., structure) of the antigen-binding protein of this disclosure, or a combination of two or more antigen-binding proteins of this disclosure, the combination comprising two or more antigen-binding proteins of different types (e.g., structures).

[0145] In some embodiments, the composition includes an agent that improves the chemical-physical properties of an antigen-binding protein, for example, by stabilizing the antigen-binding protein at a specific temperature, such as room temperature; extending its shelf life; inhibiting degradation, such as oxidative proteolytic enzyme-mediated degradation; or extending the half-life of the antigen-binding protein. In some embodiments, the composition optionally includes any of the agents disclosed herein as a non-homologous or conjugated portion, either in a mixture with the antigen-binding protein of the Disclosure or conjugated to the antigen-binding protein.

[0146] In various aspects of this disclosure, the composition further comprises a pharmaceutically acceptable carrier, diluent, or additive. In some embodiments, an antigen-binding protein, nucleic acid, vector, host cell, or conjugate disclosed herein (hereinafter referred to as the “Activator”) is formulated into a pharmaceutical composition comprising the Activator together with a pharmaceutically acceptable carrier, diluent, or additive. In this regard, this disclosure further provides pharmaceutical compositions comprising an Activator intended for administration to a target, e.g., a mammal.

[0147] In some embodiments, the activator is included in the pharmaceutical composition at a purity level suitable for administration to a patient. In some embodiments, the activator 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 has a pharmaceutically acceptable diluent, carrier, or additive. In some embodiments, the composition contains the activator at a concentration of about 0.001 to about 30.0 mg / ml.

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

[0149] Pharmaceutical compositions include, for example, acidifying agents, additives, adsorbents, aerosol propellants, exhaust agents, alkalizing agents, anti-solidification agents, anticoagulants, antimicrobial preservatives, antioxidants, preservatives, bases, binders, buffers, chelating agents, coating agents, colorants, drying agents, detergents, diluents, disinfectants, disintegrants, dispersants, dissolution accelerators, dyes, skin emollients, emulsifiers, emulsifying stabilizers, fillers, film-forming agents, flavor enhancers, flavoring agents, flow accelerators, gelling agents, granulating agents, humectants, lubricants, mucosal adhesives, ointment bases, ointments, oily media, organic bases, pastel bases, pigments, plasticizers, glossing agents, preservatives, metal ion sequestering agents, skin penetration agents, solubilizers, solvents, stabilizers, suppository bases, surfactants, suspending agents, sweeteners, therapeutic agents, and thickening agents. The formulation may contain any pharmaceutically acceptable ingredients, including agents, tonic agents, toxic agents, viscosity-increasing agents, water absorbents, water-miscible cosolvents, hard water softeners, or wetting agents. See, for example, the Handbook of Pharmaceutical Excipients, Third Edition, AH Kibbe (Pharmaceutical Press, London, UK, 2000) (the entire text is incorporated by reference); and Remington's Pharmaceutical Sciences, Sixteenth Edition, EW Martin (Mack Publishing Co., Easton, Pa., 1980) (the entire text is incorporated by reference).

[0150] In various embodiments, the pharmaceutical composition contains a formulation that is non-toxic to the recipient at the dose and concentration used. In certain embodiments, the pharmaceutical composition contains an activator and one or more pharmaceutically acceptable salts, polyols, surfactants, osmotic balancers, tonic agents, antioxidants, antibiotics, antifungals, fillers, freeze-drying protectants, defoamers, chelating agents, preservatives, colorants, analgesics, or further pharmaceuticals. In various embodiments, the pharmaceutical composition optionally contains one or more additives, including but not limited to pharmaceutically acceptable salts, osmotic balancers (tonic agents), antioxidants, antibiotics, antifungals, fillers, freeze-drying protectants, defoamers, chelating agents, preservatives, colorants, and analgesics, in addition to one or more polyols and / or one or more surfactants.

[0151] In certain embodiments, the pharmaceutical composition may contain, for example, compounding substances for regulating, maintaining, or preserving the composition's pH, volume molar osmotic pressure concentration, viscosity, clarity, color, isotonicity, aroma, sterility, stability, dissolution rate or release rate, adsorption, or osmosis. Suitable formulations in such embodiments include amino acids (e.g., glycine, glutamine, asparagine, arginine, or lysine), antimicrobial agents, antioxidants (e.g., ascorbic acid, sodium sulfite, or sodium bisulfite), buffers (e.g., borates, bicarbonates, tris-HCl, citrates, phosphates, or other organic acids), fillers (e.g., mannitol or glycine), chelating agents (e.g., ethylenediaminetetraacetic acid (EDTA)), complexing agents (e.g., caffeine, polyvinylpyrrolidone, β-cyclodextrin, or hydroxypropyl-β-cyclodextrin), fillers, monosaccharides, disaccharides, and other carbohydrates (e.g., glucose, mannose, or dextrin), proteins (e.g., serum albumin, gelatin, or immunoglobulin), colorants, flavoring agents, and diluents, emulsifiers, hydrophilic polymers (e.g., polyvinylpyrrolidone), low molecular weight polypeptides, and salt formation agents. Examples of additives include, but are not limited to, counterions (e.g., sodium), preservatives (e.g., benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid, or hydrogen peroxide), solvents (e.g., glycerin, propylene glycol, or polyethylene glycol), sugar alcohols (e.g., mannitol or sorbitol), suspending agents, surfactants or wetting agents (e.g., Pluronic®, PEG, sorbitan esters, polysorbates such as polysorbate 20, polysorbatec, Triton, tromethamine, lecithin, cholesterol, tyroxapol, etc.), stabilizing agents (e.g., sucrose or sorbitol), tonicity enhancers (e.g., alkali metal halides, preferably sodium chloride or potassium chloride, mannitol, sorbitol, etc.), delivery media, diluents, additives, and / or pharmaceutical adjuvants.See REMINGTON'S PHARMACEUTICAL SCIENCES, 18″ Edition, (ARGenrmo, 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] This disclosure provides a method for producing a pharmaceutical composition. In various embodiments, the method involves mixing an antigen-binding protein, conjugate, fusion protein, nucleic acid, vector, host cell, or combination thereof with a pharmaceutically acceptable carrier, diluent, or additive.

[0154] Route of administration In this disclosure, the activator, or a pharmaceutical composition containing the activator, may be administered to a subject via any preferred route of administration. For example, the activator may be administered parenterally, nasally, orally, pulmonaryly, topically, vagina, Alternatively, it may be administered to the subject via rectal administration. The following description of the route of administration is provided merely to illustrate various embodiments, but should not be construed as limiting the scope in any way.

[0155] Suitable formulations for parenteral administration include aqueous and non-aqueous isotonic sterile injection solutions that may contain antioxidants, buffers, bacteriostatic agents, and solutes that make the formulation isotonic with the blood of the target recipient, as well as aqueous and non-aqueous sterile suspensions that may contain suspending agents, solubilizers, thickeners, stabilizers, and preservatives. The term "parenteral" means administration that does not pass through the gastrointestinal tract, but rather through certain other routes such as subcutaneous, intramuscular, intraspinal, or intravenous. The activators of this disclosure may be administered together with a physiologically acceptable diluent, which may include a pharmaceutical carrier, such as a sterile solution or a mixture of liquids including water, physiological saline, aqueous glucose solution, and related sugar solutions; an alcohol, such as ethanol or hexadecyl alcohol; a glycol, such as propylene glycol or polyethylene glycol; dimethyl sulfoxide, glycerol, ketal, such as 2,2-dimethyl-1,3-dioxolane-4-methanol; an ether, poly(ethylene glycol) 400; an oil, fatty acid, fatty acid ester or glyceride, or acetylated fatty acid glyceride, with or without a pharmaceutically acceptable surfactant such as soap or detergent; a suspending agent, such as pectin, carbomer, methylcellulose, hydroxypropyl methylcellulose or carboxymethylcellulose; or an emulsifier; and other pharmaceutical adjuvants.

[0156] Oils that can be used in parenteral formulations include petroleum, animal oils, vegetable oils, and synthetic oils. Specific examples of oils include peanut, soybean, sesame, cottonseed, corn, olive, petrolatum, and minerals. 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 salts, ammonium salts, 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 sulfonates, aryl sulfonates, and olefin sulfonates, alkyl sulfates, olefin sulfates, ether sulfates, and monoglyceride sulfates, as well as alkyl sulfosuccinates, olefin sulfosuccinates, ether sulfosuccinates, and monoglyceride sulfosuccinates; (c) nonionic surfactants, such as aliphatic 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 activator of the Disclosure in the liquid. Preservatives and buffers may be used. To minimize or eliminate inflammation at the injection site, such compositions may contain one or more nonionic surfactants having a hydrophilic-lipophilic balance (HLB) of about 12 to about 17. The amount of surfactant in such formulations is typically in the range of about 5% to about 15% by weight. Suitable surfactants include polyethylene glycol sorbitan fatty acid esters such as sorbitan monooleate, and high molecular weight adducts of ethylene oxide containing hydrophobic bases, formed by concentrating propylene oxide with propylene glycol. In some embodiments, the parenteral formulation may be provided in single-dose or multi-dose sealed containers, such as ampoules and vials, and may be stored in a freeze-dried state requiring only the addition of a sterile liquid excipient for injection (e.g., water) immediately before use. In some embodiments, immediate injection solutions and immediate injection suspensions are prepared from the above-mentioned categories of sterile powders, granules, and tablets.

[0159] The requirements for injectable formulations are as described in this disclosure. The requirements for effective drug carriers for injectable compositions are well known to those skilled in the art (see, for example, Pharmaceuticals 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 activators of this disclosure are considered useful in other methods further described herein, including methods for treating or preventing cancer, in addition to methods for inhibiting tumor growth. For the purposes of this disclosure, the amount or dose of the activator administered needs to be sufficient to achieve, for example, a therapeutic or preventive effect in a subject, i.e., an animal, over an appropriate period of time. For example, the dose of the activator of this disclosure needs to be sufficient to treat the cancer described herein for a period of about 1 to 4 minutes, 1 to 4 hours, 1 to 4 weeks, or longer, from the time of administration, for example, 5 to 20 or more weeks. In certain embodiments, the period may be even longer. The dose is determined by the effect of the particular activator, as well as the symptoms of the animal being treated (e.g., human) 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, a starting dose to administer to mammals can be determined using an assay that includes comparing the degree of cancer treatment by administering a predetermined dose of the activator of this disclosure to mammals in sets of mammals, each set of which is administered different doses of the activator. The degree of cancer treatment by administering a predetermined dose can be expressed, for example, by the degree of tumor regression achieved with the activator in a mouse xenograft model. Methods for evaluating tumor regression are well known in the art and are described in the examples herein.

[0162] The dosage of the activators disclosed herein will also be determined by the presence, nature, and severity of any adverse events that may occur with the administration of a particular activator disclosed herein. Generally, the attending physician will determine the dosage of the activators disclosed herein for the treatment of each individual patient, taking into account various factors such as age, weight, overall health, nutrition, sex, the activator disclosed herein to be administered, the route of administration, and the severity of the condition being treated. For example, and not intended to limit the disclosure, the dosage of the activators disclosed herein may be approximately 0.0001 to approximately 1 g / kg (body weight of the patient) / day, approximately 0.0001 to approximately 0.001 g / kg (body weight) / day, or approximately 0.01 mg to approximately 1 g / kg (body weight) / day.

[0163] Controlled-release formulations In some embodiments, the activators described herein may be modified into depot formulations such that the release of the activator into the body to which the activator is administered is controlled according to time and location within the body (see, for example, U.S. Patent No. 4,450,150). The depot formulation of the activator of the disclosure may be, for example, an implantable composition comprising the activator and a porous or non-porous material such as a polymer, wherein the activator is encapsulated in or diffused within the porous material and / or diffused by the decomposition of the non-porous material. Therefore, when the depot formulation is implanted at a desired location within the body of the subject, the activator is released from the implant at a predetermined rate.

[0164] In certain embodiments, the pharmaceutical composition containing the activator is modified to have any type of in vivo release properties. In some embodiments, the pharmaceutical composition is an immediate-release formulation, a controlled-release formulation, a sustained-release formulation, a sustained-release formulation, a delayed-release formulation, or a two-phase 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 Application Publications WO2008 / 130158, WO2004 / 033036, WO2000 / 032218, and WO1999 / 040942.

[0165] The composition may further include, for example, micelles, liposomes, or some other encapsulation forms, or it may be administered in a sustained-release form to provide long-term storage and / or delivery effects.

[0166] use The antigen-binding proteins of this disclosure are useful in inhibiting tumor growth. While not bound by any particular theory, the inhibitory effect of the antigen-binding proteins provided herein makes such portions useful in methods for treating cancer.

[0167] Therefore, this specification provides methods for inhibiting tumor growth within a subject and methods for reducing the size of a tumor within a subject. In various embodiments, the methods include administering the pharmaceutical composition of this disclosure to a subject in an amount effective to inhibit tumor growth or reduce the size of a tumor within a subject. In various embodiments, the growth of ovarian tumors, melanoma tumors, bladder tumors, or endometrial tumors is inhibited. In various embodiments, the size of ovarian tumors, melanoma tumors, bladder tumors, or endometrial tumors is reduced.

[0168] As used herein, the terms “inhibit” or “reduce” and words derived therefrom do not necessarily mean 100%, i.e., complete inhibition or reduction. Rather, the degree of inhibition or reduction that a person skilled in the art would recognize as having a potential benefit or therapeutic effect varies. In this regard, the antigen-binding proteins of this disclosure may inhibit tumor growth or reduce tumor size to any amount or level. In various embodiments, the inhibition brought about by the methods of this 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 achieved by the methods of the present disclosure is at least 10% or about 10% (for example, at least 20% or about 20% reduction, at least 30% or about 30% reduction, at least 40% or about 40% reduction, at least 50% or about 50% reduction, at least 60% or about 60% reduction, at least 70% or about 70% reduction, at least 80% or about 80% reduction, at least 90% or about 90% reduction, at least 95% or about 95% reduction, at least 98% or about 98% reduction).

[0169] This specification further provides a method for treating a subject having cancer, for example, CLDN6-expressing cancer. In various embodiments, the method includes administering the pharmaceutical composition of this disclosure to a subject in an amount effective to treat the cancer within the subject.

[0170] For the purposes of this Specification, the cancer of the methods disclosed herein may be any cancer, for example, any malignant proliferation or malignant tumor resulting from abnormal and disordered cell division that can metastasize to another part of the body via the lymphatic system or the bloodstream. In some aspects, cancer includes acute lymphoblastic cancer, acute myeloid leukemia, alveolar rhabdomyosarcoma, bone cancer, brain cancer, breast cancer, cancer of the anus, anal canal or rectum, 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 lymphoma, hypopharyngeal cancer, kidney cancer, laryngeal cancer, liver cancer, lung cancer, malignant mesothelioma, melanoma, multiple myeloma, nasopharyngeal cancer, non-Hodgkin lymphoma, ovarian cancer, pancreatic cancer, cancer of the peritoneum, omentum and mesentery, pharyngeal cancer, prostate cancer, rectal cancer, and renal cancer. Cancer is selected from the group consisting of (e.g., renal cell carcinoma (RCC)), small intestine cancer, soft tissue cancer, gastric cancer, testicular cancer, thyroid cancer, ureteral cancer, and bladder cancer. In certain embodiments, cancer is selected from the group consisting of head and neck cancer, ovarian cancer, cervical cancer, bladder cancer and esophageal cancer, pancreatic cancer, gastrointestinal cancer, stomach cancer, breast cancer, endometrial cancer and colorectal cancer, hepatocellular carcinoma, glioblastoma, bladder cancer, lung cancer, e.g., non-small cell lung cancer (NSCLC), bronchioloalveolar carcinoma. In various embodiments, cancer is ovarian cancer, melanoma, bladder cancer, lung cancer, liver cancer, and endometrial cancer. In various embodiments, cancer is any cancer characterized by moderate to high expression of CLDN6. See, for example, Figures 1 to 3. In various aspects, 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, clear cell carcinoma of the kidney, head and neck cancer, sarcoma, unstained kidney cancer, 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 aspects, 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 “to treat” and related words do not necessarily mean 100%, or complete, treatment. Rather, the degree of treatment that a person skilled in the art would recognize as having potential benefits or therapeutic effects varies. In this regard, the methods for treating cancer of this disclosure may provide any amount or any level of treatment. Furthermore, the treatment provided by the methods of this disclosure may include treatment of one or more symptoms, signs, or indications of the cancer being treated. Also, the treatment provided by the methods of this disclosure may include slowing the progression of cancer. For example, the methods may treat cancer by improving T-cell activity or the immune response against cancer, inhibiting the growth of tumors or cancer, inhibiting the metastasis of tumor cells, or promoting cell death of tumor cells or cancer cells. In various embodiments, this method is used to treat patients with the aim of delaying the onset or recurrence of cancer for a period of 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 longer. In various embodiments, this method is used to treat patients with the aim of extending their survival time.

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

[0173] subject In some embodiments of this disclosure, the subject matter includes, but is not limited to, rodent mammals such as mice and hamsters, lagomorph mammals such as rabbits, carnivorous mammals including felines and canines, artiodactyla mammals including cats and pigs, or odd-toed ungulate mammals including horses. In some embodiments, the mammal is a primate, Ceboid, or Simoid (monkey) mammal, or an ape (human and ape) mammal. In some embodiments, the mammal is a human.

[0174] kit In some embodiments, the antigen-binding proteins of this disclosure are provided in a kit. In various embodiments, the kit comprises antigen-binding proteins as unit doses. For the purposes of this specification, “unit dose” means an individual amount dispersed in a suitable carrier. In various embodiments, a unit dose is an amount sufficient to produce a desired effect on a subject, e.g., inhibition of tumor growth, reduction of tumor size, or treatment of cancer. Therefore, this specification provides kits comprising antigen-binding proteins of this disclosure, provided optionally in unit doses. In various embodiments, the kit comprises several unit doses, e.g., a weekly or monthly supply, each optionally packaged individually or separated from other unit doses in another manner. In some embodiments, the components / unit doses of the kit are packaged together with instructions for administration to a patient. In some embodiments, the kit comprises one or more instruments for administration to a patient, e.g., a needle and a syringe. In some embodiments, the antigen-binding protein of this disclosure, a pharmaceutically acceptable salt thereof, a conjugate containing the antigen-binding protein, or a polymer or dimer containing the antigen-binding protein are pre-packaged in a ready-to-use form, such as a syringe or an intravenous infusion bag. In some embodiments, the kit further comprises other therapeutic or diagnostic agents or pharmaceutically acceptable carriers (e.g., solvents, buffers, diluents, etc.), including any of those described herein. In certain embodiments, the kit comprises the antigen-binding protein of this disclosure together with a drug, such as a therapeutic agent used for chemotherapy or radiotherapy.

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

[0176] In various embodiments, the antigen-binding protein of this disclosure inhibits the binding of a reference antibody to CLDN6 endogenously expressed by OVCA429 cells at a dose of less than approximately 1200 nM, wherein the reference antibody comprises the light chain variable sequence of SEQ ID NO: 181 and the heavy chain variable sequence of SEQ ID NO: 182, or the light chain variable sequence of SEQ ID NO: 185 and the heavy chain variable sequence of SEQ ID NO: 186. In various embodiments, the antigen-binding protein of this disclosure inhibits the binding of a reference antibody to CLDN6 endogenously expressed by OVCA429 cells at a dose of less than approximately 1000 nM or less than 750 nM (e.g., less than approximately 500 nM, less than approximately 250 nM, less than approximately 100 nM), wherein the reference antibody comprises the light chain variable sequence of SEQ ID NO: 181 and the heavy chain variable sequence of SEQ ID NO: 182, or the light chain variable sequence of SEQ ID NO: 185 and the heavy chain variable sequence of SEQ ID NO: 186.

[0177] In various embodiments, the antigen-binding protein is (a) a sequence selected from the group consisting of heavy chain CDR1 amino acid sequences listed in Table A or A1, i.e., 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 70% or about 70% (e.g., at least 85% or about 85%, at least 90% or about 90%) of the same amino acid sequence. (b) A variant sequence having column identity, (b) a heavy chain CDR2 amino acid sequence listed 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 sequence that differs by only one or two amino acids or is identical by at least 70% or about 70% (e.g., at least 85% or about 85%, at least 90% or about 90%). (c) A variant sequence having one identity, a heavy chain CDR3 amino acid 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 70% or about 70% (e.g., at least 85% or about 85%, at least 90% or about 90%) sequence identity. (d) A variant sequence having the following characteristics: (d) A sequence selected from the group consisting of the light chain CDR1 amino acid sequences listed in Table A or A1, i.e., 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 sequence having only one or two amino acids different or having at least 70% or about 70% (e.g., at least 85% or about 85%, at least 90% or about 90%) sequence identity.(e) Light chain CDR2 amino acid sequences listed in Table A or A1, i.e., sequences 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 variant sequences thereof that differ by only one or two amino acids or have at least 70% or about 70% (e.g., at least 85% or about 85%, at least 90% or about 90%) sequence identity, (f) As listed in Table A or A1 The light chain CDR3 amino acid sequence, 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, 130, 451, 454, and 460, or a variant thereof that differs by only one or two amino acids or has at least 70% or about 70% (e.g., at least 85% or about 85%, at least 90% or about 90%) sequence identity, including any two or more combinations of (g)(a) to (f).

[0178] In various embodiments, the antigen-binding protein comprises one or two of the light chain CDR1 amino acid sequences, light chain CDR2 amino acid sequences, and light chain CDR3 amino acid sequences described in Table A or A1, as well as one or two of the heavy chain CDR amino acid sequences described in Table A or A1. In some examples, the antigen-binding protein comprises one or two of the heavy chain CDR1 amino acid sequences, heavy chain CDR2 amino acid sequences, and heavy chain CDR3 amino acid sequences described in Table A or A1, as well as one or two of the light chain CDR amino acid sequences described in Table A or A1. In various aspects, antigen-binding proteins include (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) sequence It includes six CDR amino acid sequences selected from the group consisting of (q) SEQ ID NOs: 92-97, (r) SEQ ID NOs: 8-13, (s) SEQ ID NOs: 68-73, (t) SEQ ID NOs: 14-19, (u) SEQ ID NOs: 20-25, (v) SEQ ID NOs: 449-453 and 475, (w) SEQ ID NOs: 476-477, 454-457, (x) SEQ ID NOs: 458-463, (y) SEQ ID NOs: 57, 58, 464-467, (z) SEQ ID NOs: 68-71 and 468-469, and (aa) SEQ ID NOs: 112 and 470-474.In various embodiments, the antigen-binding protein is (a) a sequence selected from the group consisting of the heavy chain variable region amino acid sequences listed in Table B, namely, 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 sequence having only one or two amino acids different or having at least 70% or about 70% (e.g., at least 85% or about 85%, at least 90% or about 90%) sequence identity, (b ) A sequence selected from the group consisting of the light chain variable region amino acid sequences listed in Table B, namely sequence numbers: 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 that differs by only one or two amino acids or has at least 70% 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 embodiments, the antigen-binding protein is (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 1 67, comprising a pair of amino acid sequences selected from the group consisting of (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 is (a) a sequence selected from the group consisting of heavy chain variable region amino acid sequences listed in Table B1 or C, i.e., sequence numbers 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 that differs by only one or two amino acids, or has at least 70%, approximately 70%, approximately 80%, approximately 90%, or approximately 95% sequence identity. (b) A sequence comprising the light chain variable region amino acid sequence described 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 that differs by only one or two amino acids, or has at least 70% or about 70%, about 80%, about 90%, or about 95% sequence identity, or (c) both of (a) and (b). In various embodiments, the antigen-binding protein comprises a pair of amino acid sequences described in Table D.

[0180] This disclosure includes: (A) HC CDR1, which optionally includes the amino acid sequence of YTFTXYT (wherein X is T, V, D, or S) (Sequence ID: 452), which optionally includes the amino acid sequence of YTFTTYT (Sequence ID: 11); (B) HC CDR2, which optionally includes the amino acid sequence of IXPSSGYT (wherein X is Q, S, A, or N) (Sequence ID: 475), which optionally includes the amino acid sequence of INPSSGYT (Sequence ID: 12); and (C) HC CDR1, which optionally includes the amino acid sequence of AXGDYYVAY (wherein X is N, Q, H, or D) (Sequence ID: 453), which optionally includes the amino acid sequence of ANGDYYVAY (Sequence ID: 13). The present invention provides antigen-binding proteins comprising: CDR3, (D) optionally including the amino acid sequence SSVSSXY (wherein X is T, V, F, or D) (Sequence ID: 449), which includes the amino acid sequence SSVSSTY (Sequence ID: 8); LC CDR1, (E) optionally including the amino acid sequence XTX (wherein X at position 1 is S, T, Q, or A, and X at position 3 is S, T, D, or Q) (Sequence ID: 450), which includes the amino acid sequence STS (Sequence ID: 9); and LC CDR3, (F) optionally including the amino acid sequence HXYXRSPLT (wherein X at position 2 is Q, H, or S, and X at position 4 is H, Y, Q, or S) (Sequence ID: 451), which includes the amino acid sequence HQYHRSPLT (Sequence ID: 10).

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

[0182] The antigen-binding protein is (A) HC CDR1 containing the amino acid sequence YTXTXYT (wherein X at position 3 is F, Y, S, or T, and X at position 5 is S, T, Y, or D) (Sequence ID: 461), which optionally contains the amino acid sequence YTFTSYT (Sequence ID: 29), or (B) HC CDR1 containing the amino acid sequence INPSSTYT (Sequence ID: 30), which optionally contains the amino acid sequence IXPSSXYT (wherein X at position 2 is Q, S, A, or N, and X at position 6 is T, S, V, D, or G) (Sequence ID: 462), which optionally contains the amino acid sequence INPSSTYT (Sequence ID: 30), which optionally contains the amino acid sequence IXPSSXYT (wherein X at position 2 is Q, S, A, or N, and X at position 6 is T, S, V, D, or G). CDR2, (C) optionally contains 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) (Sequence ID: 463), which contains the amino acid sequence of SRGELGGFAY (Sequence ID: 31), HC CDR3, (D) optionally contains the amino acid sequence of QSLVHSDGNTY (Sequence ID: 26), which contains 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) (Sequence ID: 458), LC CDR1, (E) optionally including the 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) (Sequence ID: 459), which includes the amino acid sequence of KVS (Sequence ID: 27), and LC CDR2, (F) optionally including the 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) (Sequence ID: 460), which includes the amino acid sequence of SQSTHVPYT (Sequence ID: 28).

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

[0184] This disclosure provides a conjugate comprising a non-homologous portion of an antigen-binding protein described herein. This disclosure also provides a fusion protein comprising an antigen-binding protein described herein. This disclosure further provides a nucleic acid comprising a nucleotide sequence encoding an antigen-binding protein, conjugate, or fusion protein of this disclosure. This disclosure provides a vector comprising a nucleic acid comprising a nucleotide sequence encoding an antigen-binding protein, conjugate, or fusion protein of this disclosure. This disclosure further provides a host cell comprising the nucleic acid or vector of this disclosure.

[0185] This disclosure provides a method for producing an antigen-binding protein that binds to a claudin 6 (CLDN6) protein, comprising (i) culturing host cells of this disclosure in a cell culture medium (the host cells comprising nucleic acids comprising a nucleotide sequence encoding the antigen-binding protein described in any one of the prior claims), and (ii) recovering the antigen-binding protein from the cell culture medium. This disclosure also provides a method for producing a fusion protein comprising an antigen-binding protein that binds to a claudin 6 (CLDN6) protein, comprising (i) culturing host cells of this disclosure in a cell culture medium (the host cells comprising nucleic acids comprising a nucleotide sequence encoding the fusion protein of this disclosure), and (ii) recovering the fusion protein from the cell culture medium.

[0186] This disclosure further provides a method for producing a pharmaceutical composition, comprising mixing the antigen-binding protein, conjugate, fusion protein, nucleic acid, vector, host cell, or combination thereof with a pharmaceutically acceptable carrier, diluent, or additive. The disclosure also provides a pharmaceutical composition comprising the antigen-binding protein, conjugate, fusion protein, nucleic acid, vector, host cell, or combination thereof with a pharmaceutically acceptable carrier, diluent, or additive.

[0187] This specification provides a method for treating a subject having CLDN6-expressing cancer, comprising administering the pharmaceutical composition described herein to the subject in an amount effective for treating cancer. It also provides a method for inhibiting tumor growth within a subject, comprising administering the pharmaceutical composition described herein to the subject in an amount effective for inhibiting tumor growth. This disclosure provides a method for reducing tumor size within a subject, comprising administering the pharmaceutical composition described herein to the subject in an amount effective for reducing tumor size. Furthermore, it provides a method for preventing cancer recurrence within a subject, comprising administering the pharmaceutical composition described herein to the subject in an amount effective for preventing cancer recurrence.

[0188] This 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 this disclosure, and assaying an immune complex comprising an antigen-binding protein, conjugate, or fusion protein bound to CLDN6. This specification also provides a method for diagnosing claudin 6 (CLDN6)-positive cancer in a subject, comprising contacting a biological sample, including cells or tissues taken from the subject, with an antigen-binding protein, conjugate, or fusion protein of this disclosure, and assaying an immune complex comprising an antigen-binding protein, conjugate, or fusion protein bound to CLDN6.

[0189] This disclosure also provides a method for treating cancer in subjects diagnosed with low overexpression of CLDN6. In various embodiments, the method involves administering the pharmaceutical composition disclosed herein to a subject in an amount effective to prevent cancer recurrence. In some embodiments, the administration induces apoptosis in tumor cells, and optionally, the administration induces apoptosis in cells expressing CLDN6. In various embodiments, the subjects have tumors, which are semi-quantitatively classified into one of four groups: high expression, moderate 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), where CLDN6 RNA is measured by RNASeq, or CLDN6 protein levels greater than 3+ as measured by immunohistochemistry (IHC). In various cases, moderate expression is defined as CLDN6 RNA greater than 10-log FPKM, where CLDN6 RNA is measured by RNASeq, or CLDN6 protein levels greater than 2+ as measured by IHC. In various cases, low expression is defined as CLDN6 RNA greater than 6-log FPKM, where CLDN6 RNA is measured by RNASeq, or CLDN6 protein levels greater than 1+ as measured by IHC. In various cases, non-expression is defined as CLDN6 RNA less than 6-log FPKM, where CLDN6 RNA is measured by RNASeq, or CLDN6 protein levels below the detection limit of IHC. In various embodiments, subjects with the above tumors are similarly described as having high, moderate, low, or non-expressing CLDN6.

[0190] The following embodiments are provided merely to illustrate the scope of this disclosure, but 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, specifically cells and tissues.

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

[0193] Using the information contained in the The Cancer Genome Atlas (TCGA) database managed by the National Cancer Institute (NCI), the levels of CLDN6 RNA in patient samples were measured. Using the information in the Genotype-Tissue Expression (GTEx) database managed by the Common Fund, the CLDN6 levels in normal tissues were measured. Analysis of tissues from the GTEx database revealed that CLDN6 was detectable in various sites, especially including the brain, pituitary gland, pancreas, kidney, lung, thyroid gland, and cervix among other tissues (Figure 1).

[0194] The Agilent 44K microarray (4×44K array chip, Agilent Technologies, Santa Clara, CA) and RNA sequencing (RNA-Seq) assay were used to measure the CLDN6 expression levels in the TORL cancer cell lines. At BGI Americas (Cambridge, MA), RNA-Seq was performed using BGI Americas' "Quantitative RNAseq" service. As shown in Figures 2 and 3, CLDN6 expression levels were detectable in breast cancer cells, kidney cancer cells, colon cancer cells, sarcoma cells, and liver cancer cells, but ovarian cancer cells, head and neck cancer cells, lung cancer cells, and bladder cancer cells expressed the highest levels of CLDN6.

[0195] [Example 2] In this example, the preparation of cells genetically engineered to overexpress CLDN6 will be described.

[0196] A model was generated that was genetically engineered to overexpress CLDN6. Using these models, the efficacy of the CLDN6 antibody described in Example 5 was confirmed. Briefly, the nucleotide sequence encoding CLDN6 was introduced into a bicistronic vector having a CMV promoter and an internal ribosome entry site (IRES) within the attenuated sequence of encephalomyocarditis virus (EMCV). An IRES was placed between the target gene (GOI) cDNA (CLDN6) and the puromycin cDNA. The woodchuck post-transcriptional regulatory element (WPRE) was placed downstream of the puromycin cDNA. The vector 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] The expression vector was transduced into HEK293T cells (for screening purposes) and NIH3T3 cells (for immunization). Clearly transduced cells were selected based on survival in medium containing puromycin (1 μg / ml). The clear cells were subcloned to obtain a stable and homogeneous clonal population of CLDN6-overexpressing cells.

[0198] BD Biosciences Accuri (商標) Subclonal CLDN6 expression was confirmed by flow cytometry using a reference CLDN6 monoclonal antibody (mAb) on a BD Biosciences Accuri flow cytometer (San Jose, CA). Secondary antibody and conjugate: Alexa Fluor® 6,47 goat anti-mouse IgG (minimal x-reactivity) antibody (Biolegend, San Diego, CA; catalog number 405322) was used to detect the binding activity between the reference CLDN6 mAb and the CLDN6 expressed by the subclone.

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

[0200] [Example 3] This embodiment describes the preparation of a reference antibody and a control antibody.

[0201] The heavy and light chain variable regions of the antibody are processed using ExpiCHO. (商標) A benchmark (reference) CLDN6-specific antibody and a control antibody were produced by cloning recombinant mouse IgG2A chimeric antibodies within an Expression System (ThermoFisher Scientific, Waltham, MA). These antibodies were tested together with the newly produced CLDN6-specific antibody described in Example 5.

[0202] In short, ExpiCHO follows the manufacturer's protocol. (商標) Plasmids containing control and benchmark antibody sequences were transfected using the Expression System (catalog number: A29133, ThermoFisher Scientific, USA). Cells were cultured in the kit medium at 37°C and 8% CO2 on day 1, and then at 32°C and 5% CO2 after transfection. ExpiCHO was performed by centrifugation at 1,000 g for 10 minutes followed by 5,000 g for 30 minutes. (商標)The antibody was purified by clearing the culture medium. Next, the supernatant was filtered using a 0.45 μm filter followed by a 0.22 μm filter. Subsequently, affinity purification was performed on the supernatant using Protein A / G resin (Life Technologies, Carlsbad, CA; catalog number 20424) according to the manufacturer's protocol. Prior to ELISA purification, the antibody titer in the culture medium was roughly measured to ensure that the amount of injected medium accounted for less than 80% of the resin's binding capacity. After incubation, the resin was washed with PBS and eluted with elution buffer (Life Technologies, catalog number 21004). The eluted fraction was immediately adjusted to physiological pH by adding Tris buffer (pH 8.0). Subsequently, buffer exchange and protein concentration were performed on the purified antibody using PBS buffer with an Amicon Ultra-15 Centrifugal Filter Unit (Life Technologies, catalog number UFC900324). The antibody concentration was measured by BCA protein assay. Antibody purity was tested using SDS-PAGE and Coomassie staining. The purified protein was aliquoted and stored at -80°C for long-term storage or at 4°C for immediate use.

[0203] When the integrity of the antibody was confirmed by Coomassie staining following SDS-PAGE under non-reducing and reducing conditions, one major band was observed per 150 kDa under non-reducing conditions, while two bands were observed at 50 kDa and 25 kDa under reducing conditions.

[0204] Antibodies specific to other CLDN family members with sequence similarity (Figure 5), namely CLDN3, CLDN4, and CLDN9, were produced using essentially the same method, except that the antibody sequence contained within the plasmid was specific to CLDN3, CLDN4, or CLDN9.

[0205] [Example 4] This example describes the characterization of cell lines that highly express endogenous CLDN6.

[0206] FACS and Western blotting were used to analyze the endogenous expression of CLDN6 in a panel of cancer cell lines. In short, 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 CLDN6 at high or low levels as measured in Example 1. CLDN6-expressing cells were incubated on ice for 30 minutes with a reference antibody or control antibody (described in Example 3), washed, and then incubated on ice for 30 minutes with Alexa Fluor® 647 conjugate goat anti-mouse IgG (minimal x-reactivity) antibody (Biolegend catalog number 405322). BD Biosciences Accuri (商標) The fluorescence was read using a flow cytometer (San Jose, CA).

[0207] Western blotting was performed on nitrocellulose using reference and control antibodies. Briefly, the sample, derived from cell lysates, was boiled to denature the protein components. Denatured proteins were separated by polypeptide length using SDS-PAGE (SDS-polyacrylamide gel electrophoresis). Next, the separated proteins were transferred from the acrylamide gel to a nitrocellulose membrane. The membrane was blocked with a 2% bovine serum albumin (BSA) solution 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 control and reference antibodies were identified using overexpression cells, and once identified, endogenous cell lines were characterized using the control and reference antibodies. Cells overexpressing CLDN6 were included in these assays as positive controls.

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

[0210] [Table 20]

[0211] [Example 5] This embodiment describes the immunization of mice to produce CLDN6-specific antibodies.

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

[0213] [Table 21]

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

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

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

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

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

[0219] For each cell type 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 a predetermined concentration in the dark on ice for 30 minutes. CLDN6 mAb was directly labeled with Alexa Fluor® 647NHS ester (succinimidyl ester). After washing, cells were read using a BD Accuri® Flow Cytometer C6, which detects antibody-antigen protein binding in the FL4H channel. Each antibody was tested at various concentrations to create dose-fluorescence curves. The EC50 / IC50 (antibody concentration at half the maximum value) of the antibody was calculated based on FL4H (gated to live singlet cells) values ​​using an extremely simple online IC50 toolkit that allows plotting biological dose-response data and fitting curve types to obtain EC50 / IC50. The maximum value was the lowest antibody concentration at which fluorescence was maximal. 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 tested antibody set. Cross-reactivity data were obtained using a similar method (but with cells having different expression profiles for CLDN6, CLDN3, CLDN4, and CLDN9).

[0220] The relative affinity and cross-reactivity data measured using this method are listed in Tables 4 and 5.

[0221] [Table 22]

[0222] [Table 23]

[0223] [Example 6] This example describes the characterization of chimeric mouse IgG mAbs.

[0224] The mAb described in Example 5 was further characterized by a soft agar 3D growth assay and a xenograft binding assay. Briefly, in each well of a 48-well plate, 250 μL of a top layer mixture containing 10,000 cells in 0.6% SeaPlaque agarose in 1×RPMI medium was seeded on top of the bottom layer of solidified 250 μL of 0.6% SeaPlaque agarose in 1×RPMI medium. A 250 μL liquid feeder layer containing 1×RPMI medium was placed on top of the solidified top layer. All three layers of the soft agar assay were prepared by diluting trastuzumab, CLDN6 mAb, or mouse IgG2a control (with or without) at a 1:10 ratio, starting at 150 ng / mL (1 μM) and ending at 1.5 ng / mL. Each test condition was performed using replication. Cells were allowed to colonize for three weeks, then stained with 0.05% neutral red and observed using an EVOS XL inverted optical microscope. Cell lines showing a ≥20% reduction in colony count compared to the treated-versus-control cell line were considered sensitive.

[0225] As shown in Table 6, treatment with the antibodies indicated resulted in a decrease in the number of colonies in most cell lines.

[0226] [Table 24]

[0227] In vivo binding studies were conducted using xenograft mice injected with human cancer cell lines. In short, a xenograft model of human cancer cell lines was created using 6-week-old CD-1 athymoid nude mice (Charles River Laboratories). Subcutaneous injection of each cell line was performed under the following conditions: ARK2 was injected at 0.75 × 10⁻⁶. 7 In the cells, UMUC4 has a size of 1.0 × 10 7 The cells, in OV90, are 1.0 × 10 7 In the cells, and in M202, 0.5 × 107 The cells (all containing 50% Matrigel (BD Biosciences)) were administered according to the instructions. A sufficient number of mice were injected, resulting in 8 mice per treatment group. Tumors were 150-300 mm. 3 When the average size was reached, the mice were randomized to the treatment group. For treatment, each therapeutic antibody (AB3, AB2, reference Ab1, reference Ab2, reference Ab3 (trastuzumab), and untargeted IgG2 control) was diluted in sterile saline to an effective concentration of 1 mg / ml for tail vein (IV) infusion. For the M202 study, trametinib (DMSO solvate, MedChem Express) was administered as a PO at 1.0 mg / kg (10% Cremaphor, 10% PEG400) in the first weekly cycle of 5 days of administration followed by 2 days of rest, and then reduced to 0.5 mg / kg for the remaining 2 weeks. Tumor xenografts were measured three times a week with calipers, and tumor volume (mm²) was calculated by multiplying height × width × length. 3 The researchers sought to determine the tumor volume. Mice were treated for 2–7 weeks. At the end of the study, the animals were euthanized, and the tumor tissue was excised and divided for preservation as rapidly frozen tissue or formalin-fixed paraffin-embedded (FFPE) tissue for biomarker analysis. All animal experiments were conducted under protocols approved by IACUC and the University of California's Los Angeles Animal Research Committee. Data were analyzed using StudyLog software from StudyDirector (San Francisco, CA). Results are shown as the mean volume for each group. Error bars indicate the standard error (SE) of the mean.

[0228] The results of the xenograft assay are shown in Figures 6-10. As shown in Figures 6A and 6B, AB2 and AB3, respectively, resulted in a significantly larger mean change in tumor volume at day 14 in mice with endometrial tumors compared to the control IgG2 antibody. As shown in Figures 7A and 7B, AB3 resulted in a significantly larger mean change in tumor volume at day 35 in mice with bladder tumors compared to the control IgG2 antibody. Figures 8A and 8B show that AB2 and AB3, respectively, resulted in a significantly larger mean change in tumor volume at day 20 in mice with ovarian tumors compared to the control IgG2 antibody. Figures 9A and 9B show that AB3 acted in a CLDN-specific manner, as the models used in Figures 9A and 9B did not express any of CLDN6, CLDN3, CLDN4, and CLDN9, and were therefore used as negative controls. 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 from Figures 6 to 9. As shown in Figure 10A, AB3 significantly suppressed tumor growth in mice with endometrial tumors, bladder tumors, and ovarian tumors that expressed CLDN6, respectively, but did not suppress tumor growth in mice with melanoma tumors that did not express CLDN6 (Figure 10B). As shown in Figure 11, the mean change in body weight of the treated mice did not change significantly, suggesting the safety of the treatment.

[0229] A second set of experiments was conducted using a xenograft model of the human ovarian cancer cell line OV90. 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 solvent control. There were 8 mice per group, and each animal received an intravenous injection of 10 mg / kg of antibody every 4 days. As shown in Figures 12A and 12B, several antibodies described in Example 5 reduced tumor volume in mice with ovarian tumors. All antibodies tested compared to the solvent control reduced tumor volume, but AB3, AB4, AB7, and AB10 showed the best performance. 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] This example describes further characterization of chimeric mouse IgG mAb.

[0231] A quantitative assay for internal translocation was performed. Briefly, using a positive control (a ubiquitously expressed transferrin receptor (TfR), a well-known cell surface receptor that undergoes internal translocation after antibody binding), the assay examined the internal translocation of the CLDN6 protein induced by the binding of reference Ab1, AB3, or AB4.

[0232] TfR and CLDN6 antibodies are used in Texas Red (商標)Cells were labeled with -X, succinimidyl ester, mixed isomers, catalog number T6134 (ThermoFisher Scientific). Cells were seeded in μ-Slide 8-Well chambers (catalog number 80826, ibidi Cells In Focus Inc.) one day prior to antibody treatment for cell attachment and proliferation. Cells were incubated with the labeled antibody in the dark on ice for 30 minutes. Next, the chambers containing CLDN6 or TfR-labeled cells were read with an Echo Lab fluorescence microscope to collect images before internal migration. The chambers were then incubated at 37°C for 40 minutes to allow internal migration, and images were collected again with an Echo Lab fluorescence microscope. AB3 and AB4 showed a greater degree of internal migration of CLDN6 compared to the degree of internal migration of CLDN6 obtained with reference Ab1 (data omitted).

[0233] [Example 8] This example describes further characterization of chimeric mouse IgG mAb.

[0234] A two-dimensional (2D) proliferation assay was performed using the selective antibody described in Example 5 as follows. Cells were replicated and seeded in 24-well plates at concentrations of 5,000 to 20,000 cells per well. The following day, cells were treated with 1 to 5 dilutions of six mAbs (starting at 100 nM of either trastuzumab, CLDN6 mAb, or mouse IgG2a control) and monomethyl auristatin E (MMAE) conjugate anti-mouse secondary (Moradec, LLC) at a fixed concentration of 1 ng / μL to create dose-response curves. Cells in the untreated wells were quantified on day 1, the day of antibody treatment, and the range of cell proliferation was measured later on day 6. Wells treated with mAbs were quantified on day 6, and proliferation under each treatment condition was determined as a normalized percentage ratio to the proliferation of untreated cells. Quantification was performed using 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 effects. The IC50 for each of these antibodies was 0.1 nM to 1 nM. AB7, AB10, AB11, and AB15 also showed growth inhibitory activity in this assay, albeit to a relatively lower degree than AB2, AB3, AB4, and AB5.

[0236] [Example 9] This embodiment will explain the humanization of the antibody of this 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 with libraries of known human germline sequences derived from the human VH and human VLκ genes (IMGT®, the international ImMunoGeneTics information system®, www.imgt.org; founders and administrators: Marie-Paule Lefranc, Montpellier, France). The databases used were the IMGT human VH gene (F+ORF, 273 germline sequences) and the IMGT human VLκ gene (F+ORF, 74 germline sequences). The receptor human germline was selected from those most closely related to the parental antibody sequence.

[0238] Table 7 describes the information regarding the human germline sequence and the selected human heavy chain binding region (J gene) for each antibody in its respective VH and VL regions. The binding region (J gene) was selected from the human binding region sequences stored in IMGT (registered trademark), the international ImMunoGeneTics information system (registered trademark), www.imgt.org (founder and administrator: Marie-Paule Lefranc, Montpellier, France).

[0239] [Table 25]

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

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

[0242] For AB1, Asn52 (sequential number) of HC's CDR2 and Asn54 of LC's CDR2 were selected based on their sequence and three-dimensional structure to have a low probability of amide degradation.

[0243] For AB3, Asn31 (sequential number) of HC's CDR1, Asn57 of HC's CDR2, Asn28 of LC's CDR1, and Asn50 of LC's CDR2 were determined to have a low probability of amide degradation based on their sequence and three-dimensional structure. Trp33 of HC's CDR1 was determined to be susceptible to oxidation, particularly under stress conditions, due to the possibility of exposure to solvents. For HC's CDR3, it was determined that free Cys106, which could be problematic during antibody production due to its potential exposure to solvents, would be present in the CDR. This Cys residue was recommended for modification to Tyr, Ser, or Ala. The maintenance of binding of these modified antibodies was tested. Ile53 of LC's CDR2 was determined to be susceptible to exposure to solvents, leading to nonspecific binding. It was suggested that this Ile residue be modified to Ser. The maintenance of binding of this modified antibody was tested.

[0244] For AB4, Asn52 (sequence number) of HC's CDR2 and Asn58 of LC's CDR2 were determined to have a low probability of amide degradation based on their sequence and three-dimensional structure. The sequence DGNT within LC's CDR1 was determined to be as problematic as the sequence NT, in addition to having a high probability of isoaspartate formation (in sequence DG). This sequence was recommended for modification.

[0245] For AB9, Asn33 (sequential number) in HC's CDR1, and Asn52 and Asn59 in HC's CDR2 were determined to have a low probability of amide degradation based on their sequence and conformation. Asn54 was determined to have a moderate probability of amide degradation based on its sequence and conformation. The NGG sequence in HC's CDR2 was determined to have a high / moderate probability of isoaspartate formation following amide degradation. Therefore, modification of this amino acid sequence was recommended. Free Cys106 in HC's CDR3 is determined to be potentially exposed to solvents, which could be a problem during antibody production. 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 HC's CDR1 is not frequently present in human antibodies. This residue was modified to Thr, and the maintenance of binding was tested. For AB9, Trp32 (sequentially numbered) within LC's CDR1 was determined to be potentially exposed to solvents and susceptible to oxidation, particularly under stress conditions. Leu24 in the same CDR is not frequently present in human antibodies. This residue was modified to Arg, and testing was conducted to maintain binding.

[0246] For AB11, the Asp54-Ser55 (sequential numbering) within the CDR2 of HC was determined to have a low probability of isoaspartate formation. For the Asn57 within the CDR2 of LC, it was determined to have a low probability of amide degradation based on its sequence and stereochemistry.

[0247] For AB18, Asn33 in HC's CDR1 and CDR-H2 Asn50 (sequentially numbered) were determined to have a low probability of amide degradation based on their sequence and stereostructure. The Asp-Pro(DP) sequence in HC's CDR2 was determined to be susceptible to fragmentation under acidic conditions. Within the VL domain, Asn34 and Asn37 in LC's CDR1 were determined to have a low probability of amide degradation based on their sequence and stereostructure. Trp56 in LC's CDR3 was determined to be susceptible to exposure to solvents and oxidation, particularly under stress conditions.

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

[0249] [Table 26] TIFF0007869831000030.tif239157

[0250] Humanized antibodies as described in Table 8 were constructed and expressed essentially as described in Example 5. A FACS assay was performed essentially as described in Example 5 to measure the relative antigen-binding affinity of the humanized antibodies. Binding of two doses (1.5 μg or 0.3 μg) of the humanized antibodies to either human CLDN6 or mouse CLDN6 (the protein expressed by the genetically modified 293T clone) was tested. The assay results are shown in Table 9.

[0251] [Table 27]

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

[0253] [Table 28]

[0254] Based on 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 forms AB1, AB3, and AB4 were performed using xenograft mice injected with the bladder cancer cell line UMUC4, as described in Example 6. Briefly, a xenograft model of UMUC4 was created using 6-week-old CD-1 athymoid nude mice (Charles River Laboratories). The tumor size was 150-300 mm. 3 After reaching an average size, mice were randomized to the treatment group. Humanized antibodies were diluted in sterile saline to an effective concentration of 1 mg / ml for tail vein (IV) injection. Tumor xenografts were measured three times a week with calipers, and the tumor volume (mm²) was calculated by multiplying the volume by height × width × length. 3 The researchers sought to obtain the tumor. Mice were treated for 2 to 7 weeks. At the end of the study, the animals were euthanized, and the tumor tissue was excised and divided and preserved as rapidly frozen tissue or formalin-fixed paraffin-embedded (FFPE) tissue for biomarker analysis.

[0256] The results of the xenograft assays are shown in Figures 15 to 21. Figure 15 shows the xenograft assay results for humanized AB3 type 2 (AB3-2 and AB3-4), with treatment including 10 mg / kg administered via Q4D. Controls included a solvent control (PBS), human IgG (10 mg / kg Q4D), and mouse-type AB3 (10 mg / kg Q4D). As shown in this figure, humanized AB3-4 showed a reduction in tumor volume over a 35-day treatment period. Figure 16 shows the xenograft assay results for the same treatment as in Figure 15, except that two additional controls (64A MSE and its chimeric form (64A-CHIM)) were used. 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 with humanized AB1-5. The controls included a solvent control (PBS), human IgG (10 mg / kg Q4D), and mouse-type 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 with humanized AB4-3. Controls included a solvent control (PBS), human IgG (10 mg / kg Q4D), and mouse-type 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 xenograft assays testing all four humanized antibodies shown in Figures 15-18. Mouse 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 the period up to day 55. The body weight of the mice in the assay is shown in Figure 21.

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

[0261] [Table 29]

[0262] Basically, multiple antibodies having sequences defined by these consensus sequences were prepared as described in Example 5, and antigen binding was tested in vitro by FACS (basically as described in Example 5), and the ability to reduce tumor volume in mice was tested in vivo (basically as described in Example 6).

[0263] All references cited herein, including publications, patent applications, and patents, are incorporated herein by reference to the same extent as if each reference were incorporated individually and explicitly by reference, and as if the entirety of each reference were included herein.

[0264] In the context describing this disclosure (in particular in the context of the following claims), the use of the terms “a,” “an,” “the,” and similar referents should be interpreted as including both singular and plural forms unless otherwise specifically stated herein or unless the context particularly clearly contradicts this interpretation. The terms “comprising,” “having,” “including,” and “containing” should be interpreted as open-ended terms (i.e., including but not limited to these) unless otherwise specifically stated.

[0265] The enumeration of value ranges in this specification is intended solely as a shorthand notation to mean each independent value and each endpoint within the range, unless otherwise specified herein, and each independent value and endpoint is incorporated herein as if they were individually cited herein.

[0266] All methods described herein may be carried out in any preferred order unless otherwise specified herein or unless it is particularly clearly inconsistent in the context. All examples provided herein or the use of various terms (e.g., "etc.") are solely for the purpose of making this disclosure more clearly evident and, unless specifically requested, do not imply any limitation to the scope of this disclosure. No terms herein should be construed as indicating any unclaimed element essential to carrying out this disclosure.

[0267] Preferred embodiments of the Disclosure are described herein and include the best mode for carrying out the Disclosure as known to the inventors. Variations of these preferred embodiments may become apparent to those skilled in the art by reading the above description. The inventors anticipate that those skilled in the art will adopt such variations as needed, and the inventors intend that the Disclosure may be carried out in ways other than those expressly described herein. Therefore, the Disclosure includes all modifications and equivalents of the subject matter enumerated in the claims appended herein. Furthermore, any combination of the above elements, including all possible variations, is incorporated herein unless otherwise specifically stated herein or unless it is particularly clearly inconsistent with the 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 the extracellular loop 2 (EL2) of the extracellular domain (ECD) of CLDN6, but does not bind to the extracellular loop 1 (EL1) of the ECD of CLDN6. b. A reference antibody that does not bind to any of claudin 3 (CLDN3), claudin 4 (CLDN4), or claudin 9 (CLDN9), and inhibits the binding of a reference antibody to CLDN6, which is endogenously expressed in OVCA429 cells, at a dose of less than approximately 1200 nM, or c. These are combinations, The aforementioned antigen-binding protein. 2. The antigen-binding protein described in 1 above, which binds to an epitope in the amino acid sequence of WTAHAIIRDFYNPLVAEAQKREL (Sequence ID: 2). 3. The antigen-binding protein described in item 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 items 1 to 3 above, which does not bind to one or more of claudin 3 (CLDN3), claudin 4 (CLDN4), and claudin 9 (CLDN9). 5. An antigen-binding protein as described in item 4 above, which does not bind to CLDN3. 6. The antigen-binding protein described in item 5 above, which binds to CLDN6, CLDN4, and CLDN9. 7. An antigen-binding protein as described in item 5 above, which does not bind to CLDN9. 8. The antigen-binding protein described in item 7 above, which binds to CLDN6 and CLDN4. 9. An antigen-binding protein as described in item 5 above, which does not bind to CLDN4. 10. The antigen-binding protein described in 9 above, which binds to CLDN6 and CLDN9. 11. The reference antibody is an antigen-binding protein according to any one of the above, comprising the light chain variable sequence of SEQ ID NO: 181 and the heavy chain variable sequence of SEQ ID NO: 182, or the light chain variable sequence of SEQ ID NO: 185 and the heavy chain variable sequence of SEQ ID NO: 186. 12. a. A sequence selected from the group consisting of heavy chain CDR1 amino acid sequences listed in Table A, i.e., sequence numbers 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 sequence having at least one or two different amino acids or having at least 70% or approximately 70% sequence identity. b. A sequence selected from the group consisting of heavy chain CDR2 amino acid sequences listed in Table A, i.e., sequence numbers 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 sequence having at least one or two different amino acids or having at least 70% or approximately 70% sequence identity. c. A sequence selected from the group consisting of heavy chain CDR3 amino acid sequences listed in Table A, i.e., sequence numbers 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 sequence having only one or two amino acids different or having at least 70% or approximately 70% sequence identity. d. A sequence selected from the group consisting of the light chain CDR1 amino acid sequences listed in Table A, i.e., sequence numbers 8, 14, 20, 32, 38, 44, 50, 56, 62, 68, 74, 80, 86, 92, 98, 104, 110, 116, 122, and 128, or a variant sequence having at least one or two different amino acids or having at least 70% or approximately 70% sequence identity. e. A sequence selected from the group consisting of light chain CDR2 amino acid sequences listed in Table A, i.e., sequence numbers 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 sequence having at least one or two different amino acids or having at least 70% or approximately 70% sequence identity. f. A sequence selected from the group consisting of light chain CDR3 amino acid sequences listed in Table A, i.e., sequence numbers: 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 sequence having at least one or two different amino acids or having at least 70% or approximately 70% sequence identity. g. Any two or more combinations of (a) to (f), An antigen-binding protein as described above, including any of the above. 13. The antigen-binding protein according to 12, wherein the mutant sequence has at least about 80% or at least 85% or about 85% sequence identity. 14. The antigen-binding protein according to 13, wherein the mutant sequence has at least about 90% sequence identity or at least 95% or about 95% sequence identity. 15. The antigen-binding protein described in 12 above, comprising one or two of the light chain CDR1 amino acid sequence, light chain CDR2 amino acid sequence, and light chain CDR3 amino acid sequence described in Table A, as well as the heavy chain CDR amino acid sequence described in Table A. 16. The antigen-binding protein described in 12 above, comprising one or two of the heavy chain CDR1 amino acid sequence, heavy chain CDR2 amino acid sequence, and heavy chain CDR3 amino acid sequence described in Table A, as well as the light chain CDR amino acid sequence described in Table A. 17. a. Sequence IDs: 74-79, b. Sequence IDs: 50-55, c. Sequence IDs: 122~127, d. Sequence numbers: 26-31, e. Sequence numbers: 128-133, f. Sequence numbers: 38-43, g. Sequence numbers: 62-67, h. Sequence IDs: 80-85, i. Sequence IDs: 44-49, j. Sequence IDs: 86-91, k. Sequence IDs: 104-109, l. Sequence IDs: 56-61, m. Sequence IDs: 32-37, n. Sequence IDs: 110-115, o. Sequence IDs: 98-103, p. Sequence IDs: 92-97, q. Sequence numbers: 116~121, r. Sequence numbers: 8-13, s. Sequence numbers: 68-73, t. Sequence IDs: 14-19, and, u. Sequence IDs: 20-25, An antigen-binding protein according to any one of the above 12 to 16, comprising six CDR amino acid sequences selected from the group consisting of the above. 18. a. A sequence selected from the group consisting of heavy chain variable region amino acid sequences listed in Table B, i.e., sequence numbers: 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 sequence having only one or two different amino acids or having at least 70% or approximately 70% sequence identity. b. A sequence selected from the group consisting of light chain variable region amino acid sequences listed in Table B, i.e., sequence numbers 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 sequence having only one or two amino acids different or having at least 70% or approximately 70% sequence identity, or c. Both (a) and (b), An antigen-binding protein according to any one of items 12 to 19 above, including the above. 19. The antigen-binding protein according to 18, wherein the mutant sequence has at least about 80% or at least about 85% sequence identity. 20. The antigen-binding protein according to 19, wherein the mutant sequence has at least about 90% or at least about 95% sequence identity. 21. a. Sequence IDs: 156 and 157, b. Sequence IDs: 148 and 149, c. Sequence IDs: 172 and 173, d. Sequence IDs: 140 and 141, e. Sequence IDs: 174 and 175, f. Sequence IDs: 144 and 145, g. Sequence IDs: 152 and 153, h. Sequence IDs: 158 and 159, i. Sequence IDs: 146 and 147, j. Sequence IDs: 160 and 161, k. Sequence IDs: 166 and 167, l. Sequence IDs: 150 and 151, m. Sequence IDs: 142 and 143, n. Sequence IDs: 168 and 169, o. Sequence IDs: 164 and 165, p. Sequence IDs: 162 and 163, q. Sequence IDs: 170 and 171, r. Sequence IDs: 134 and 135, s. Sequence IDs: 154 and 155, t. Sequence IDs: 136 and 137, and, u. Sequence IDs: 138 and 139, The antigen-binding protein described in 18 above, comprising a pair of amino acid sequences selected from the group consisting of the following. 22. An antibody, which is an antigen-binding protein as described above. 23. A monoclonal antibody, which is the antigen-binding protein described in item 22 above. 24. An antigen-binding protein, which is IgG, as described in item 22 or 23 above. 25. An antigen-binding protein according to any of the above, which inhibits colony growth by at least approximately 50% in a soft agar 3D growth assay. 26. An antigen-binding protein according to any of the above, which inhibits tumor growth in xenograft mice injected with human cancer cells. 27. The antigen-binding protein described in 26 above, which inhibits tumor growth in xenograft mice injected with ovarian cancer cells, melanoma cancer cells, bladder cancer cells, or endometrial cancer cells. 28. The antigen-binding protein described in 27 above, 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) HC CDR1 amino acid sequences listed in Table A or A1, i.e., sequences 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 variant sequences thereof that differ by only one or two amino acids or have at least 70% or approximately 70% sequence identity, (b) HC listed in Table A or A1 (c) A sequence selected from the group consisting of CDR2 amino acid sequences, i.e., sequence numbers: 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 that differs by only one or two amino acids or has at least 70% or approximately 70% sequence identity, (c) HC as described in Table A or A1 (d) LC sequences listed in Table A or A1, which are CDR3 amino acid sequences, i.e., sequences 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 variant sequences thereof that differ by only one or two amino acids or have at least 70% or approximately 70% sequence identity, (d) LC sequences listed in Table A or A1 A sequence selected from the group consisting of CDR1 amino acid sequences: 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 that differs by only one or two amino acids or has at least 70% or approximately 70% sequence identity, (e) LC as listed in Table A or A1(f) LC sequences listed in Table A or A1, which are CDR2 amino acid sequences, i.e., sequences 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 variant sequences thereof that differ by only one or two amino acids or have at least 70% or approximately 70% sequence identity, (f) LC sequences listed in Table A or A1 An antigen-binding protein comprising a CDR3 amino acid sequence, 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, 130, 451, 454, and 460; or a variant thereof that differs by only one or two amino acids or has at least 70% or approximately 70% sequence identity; or a combination of any two or more of (g)(a) to (f). 30. a. Sequence IDs: 74-79, b. Sequence IDs: 50-55, c. Sequence IDs: 122~127, d. Sequence numbers: 26-31, e. Sequence numbers: 128-133, f. Sequence numbers: 38-43, g. Sequence numbers: 62-67, h. Sequence IDs: 80-85, i. Sequence IDs: 44-49, j. Sequence IDs: 86-91, k. Sequence IDs: 104-109, l. Sequence IDs: 56-61, m. Sequence IDs: 32-37, n. Sequence IDs: 110-115, o. Sequence IDs: 98-103, p. Sequence IDs: 92-97, q. Sequence numbers: 116~121, r. Sequence numbers: 8-13, s. Sequence numbers: 68-73, t. Sequence IDs: 14-19, u. Sequence IDs: 20-25, v. Sequence IDs: 449-453 and 475, w. Sequence IDs: 476-477, 454-457, x. Sequence IDs: 458~463, y. Sequence numbers: 57, 58, 464~467, z. Sequence IDs: 68-71 and 468-469, and, aa. Sequence IDs: 112 and 470-474, An antigen-binding protein containing six CDR amino acid sequences selected from the group consisting of the following. 31. a. A sequence selected from the group consisting of heavy chain variable region amino acid sequences listed in Table B, i.e., sequence numbers: 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 sequence having only one or two different amino acids or having at least 70% or approximately 70% sequence identity. b. A sequence selected from the group consisting of light chain variable region amino acid sequences listed in Table B, i.e., sequence numbers 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 sequence having only one or two amino acids different or having at least 70% or approximately 70% sequence identity, or c. Both (a) and (b), Antigen-binding proteins, including those mentioned above. 32. The antigen-binding protein according to 31, wherein the mutant sequence has at least about 85% sequence identity or about 90% or about 95% sequence identity. 33. a. Sequence IDs: 156 and 157, b. Sequence IDs: 148 and 149, c. Sequence IDs: 172 and 173, d. Sequence IDs: 140 and 141, e. Sequence IDs: 174 and 175, f. Sequence IDs: 144 and 145, g. Sequence IDs: 152 and 153, h. Sequence IDs: 158 and 159, i. Sequence IDs: 146 and 147, j. Sequence IDs: 160 and 161, k. Sequence IDs: 166 and 167, l. Sequence IDs: 150 and 151, m. Sequence IDs: 142 and 143, n. Sequence IDs: 168 and 169, o. Sequence IDs: 164 and 165, p. Sequence IDs: 162 and 163, q. Sequence IDs: 170 and 171, r. Sequence IDs: 134 and 135, s. Sequence IDs: 154 and 155, t. Sequence IDs: 136 and 137, and, u. Sequence IDs: 138 and 139, An antigen-binding protein comprising a pair of amino acid sequences selected from the group consisting of the following. 34. a. A sequence selected from the group consisting of heavy chain variable region amino acid sequences listed in Table C, i.e., sequence numbers 376-379, 384-387, 391-396, 403-408, 412-413, 416-419, and 422-427, or a variant sequence having at least one or two different amino acids or having at least 70% or approximately 70% sequence identity. b. Light chain variable region amino acid sequences listed in Table C, i.e., sequences selected from the group consisting of SEQ ID NOs: 380-383, 388-390, 397-402, 409-411, 414-415, and 420-421, or variant sequences having only one or two amino acids different or having at least 70% or approximately 70% sequence identity, or c. Both (a) and (b), Antigen-binding proteins, including those mentioned above. 35. The antigen-binding protein according to item 6 above, wherein the mutated sequence has at least approximately 85% sequence identity. 36. The antigen-binding protein according to item 7 above, wherein the mutant sequence has at least about 90% or about 95% sequence identity. 37. a. Sequence IDs: 376 and 380, b. Sequence IDs: 377 and 380, c. Sequence IDs: 377 and 381, d. Sequence IDs: 377 and 382, e. Sequence IDs: 377 and 383, f. Sequence numbers: 378 and 381, g. Sequence IDs: 378 and 382, h. Sequence IDs: 378 and 383, i. Sequence IDs: 379 and 381, j. Sequence IDs: 379 and 382, k. Sequence IDs: 379 and 383, l. Sequence numbers: 384 and 388, m. Sequence IDs: 385 and 388, n. Sequence IDs: 385 and 389, o. Sequence IDs: 386 and 388, p. Sequence IDs: 386 and 389, q. Sequence IDs: 387 and 389, r. Sequence IDs: 422 and 389, s. Sequence IDs: 391 and 397, t. Sequence IDs: 392 and 397, u. Sequence IDs: 393 and 398, v. Sequence IDs: 394 and 398, w. Sequence IDs: 395 and 398, x. Sequence IDs: 396 and 398, y. Sequence IDs: 423 and 398, z. Sequence IDs: 424 and 398, aa. Sequence IDs: 425 and 398, bb. Sequence IDs: 426 and 398, cc. Sequence IDs: 427 and 398, dd. Sequence IDs: 403 and 409, ee. Sequence IDs: 404 and 409, ff. Sequence IDs: 405 and 410, gg. Sequence IDs: 405 and 411, hh. Sequence IDs: 406 and 410, ii. Sequence IDs: 406 and 411, jj. Sequence IDs: 407 and 410, kk. Sequence IDs: 407 and 411, ll. Sequence IDs: 408 and 410, mm. Sequence IDs: 408 and 411, nn. Sequence IDs: 412 and 414, oo. Sequence IDs: 413 and 414, pp. Sequence IDs: 416 and 420, qq. Sequence IDs: 417 and 420, rr. Sequence IDs: 417 and 421, ss. Sequence IDs: 418 and 420, tt. Sequence IDs: 418 and 421, uu. Sequence IDs: 419 and 420, and, vv. Sequence IDs: 419 and 421, An antigen-binding protein comprising a pair of amino acid sequences selected from the group consisting of the following. 38. (A) Optionally, HC CDR1 containing the amino acid sequence YTFTTYT (SEQ ID NO: 11), YTFTXYT (wherein X is T, V, D, or S) (SEQ ID NO: 452), (B) Optionally, HC CDR2 containing the amino acid sequence of INPSSGYT (SEQ ID NO: 12) or IXPSSGYT (wherein X is Q, S, A, or N) (SEQ ID NO: 475), (C) Optionally, HC CDR3 containing the amino acid sequence of AXGDYYVAY (wherein X is N, Q, H, or D) (Sequence ID: 453), which contains the amino acid sequence of ANGDYYVAY (Sequence ID: 13), (D) Optionally, LC CDR1 containing the amino acid sequence of SSVSSTY (SEQ ID NO: 8) or the amino acid sequence of SSVSXY (wherein X is T, V, F, or D) (SEQ ID NO: 449), (E) Optionally, LC CDR2 containing the amino acid sequence of XTX (wherein the formula, X at position 1 is S, T, Q, or A, and X at position 3 is S, T, D, or Q) (Sequence ID: 450), which contains the amino acid sequence of STS (Sequence ID: 9), and (F) LC CDR3 containing the amino acid sequence of HXYXRSPLT (wherein the formula, X at position 2 is Q, H, or S, and X at position 4 is H, Y, Q, or S) (Sequence ID: 451), which optionally contains the amino acid sequence of HQYHRSPLT (Sequence ID: 10). Antigen-binding proteins, including those mentioned above. 39. (A) Optionally, HC CDR1 containing the amino acid sequence of FTFSNYW (SEQ ID NO: 23), or 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), (B) Optionally, HC CDR2 containing 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) (Sequence ID: 456), which contains the amino acid sequence of IRLKSDNYAT (Sequence ID: 24), (C) Optionally, HC CDR3 containing 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) (Sequence ID: 457), which contains the amino acid sequence of NDGPPSGC (Sequence ID: 25), (D) Optionally, LC CDR1 containing the amino acid sequence of ENIYSY (SEQ ID NO: 20) or EXIYSY (wherein X is Q, S, A, D, or N) (SEQ ID NO: 476), (E) LC CDR2 containing the amino acid sequence of NAK (SEQ ID NO: 21), the amino acid sequence of XAK (wherein the formula, X at position 1 is Q, S, A, D, or N) (SEQ ID NO: 477), and (F) LC CDR3 containing 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) (Sequence ID: 454), which optionally contains the amino acid sequence of QHHYTVPWT (Sequence ID: 22), Antigen-binding proteins, including those mentioned above. 40. (A) HC CDR1 containing the amino acid sequence YTXTXYT (wherein X at position 3 is F, Y, S, or T, and X at position 5 is S, T, Y, or D) (Sequence ID: 461), which optionally contains the amino acid sequence YTFTSYT (Sequence ID: 29), (B) HC CDR2 containing the amino acid sequence INPSSTYT (Sequence ID: 30), which optionally contains the amino acid sequence IXPSSXYT (wherein X at position 2 is Q, S, A, or N, and X at position 6 is T, S, V, D, or G) (Sequence ID: 462), (C) Optionally, HC CDR3 containing 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) (Sequence ID: 463), which contains the amino acid sequence of SRGELGGFAY (Sequence ID: 31), (D) Optionally, LC CDR1 containing the amino acid sequence QSLVHSDGNTY (SEQ ID NO: 26) or 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), (E) Optionally, LC CDR2 containing the 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) (Sequence ID: 459), which contains the amino acid sequence of KVS (Sequence ID: 27), and (F) Optionally, LC CDR3 containing the amino acid sequence of SXXTHVPYT (Sequence ID: 460) containing the amino acid sequence of SQSTHVPYT (Sequence ID: 28), where X at position 2 is Q, H, or T, and X at position 3 is S, G, T, or D. Antigen-binding proteins, including those mentioned above. 41. A conjugate containing an antigen-binding protein as described above. 42. A fusion protein comprising any of the antigen-binding proteins described above. 43. A nucleic acid comprising a nucleotide sequence encoding an antigen-binding protein as described in any of the above, a conjugate as described in 41 above, or a fusion protein as described in 42 above. 44. A vector containing the nucleic acid described in 43 above. 45. A host cell containing the nucleic acid described in 43 above or the vector described in 44 above. 46. ​​A method for producing an antigen-binding protein that binds to claudin 6 (CLDN6) protein, comprising: (i) culturing the host cells described in 45 above in a cell culture medium, wherein the host cells contain nucleic acids comprising a nucleotide sequence encoding the antigen-binding protein described in any of the 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 a claudin 6 (CLDN6) protein, comprising: (i) culturing the host cells described in 45 above in a cell culture medium, wherein the host cells comprise nucleic acids comprising a nucleotide sequence encoding the fusion protein described in 30 above; 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 described in any of items 1 to 40 above, a conjugate described in item 41 above, a fusion protein described in item 42 above, a nucleic acid described in item 43 above, a vector described in item 44 above, a host cell described in item 45 above, or a combination thereof, with a pharmaceutically acceptable carrier, diluent, or additive. 49. A pharmaceutical composition comprising an antigen-binding protein as described in any of items 1 to 40 above, a conjugate as described in item 41 above, a fusion protein as described in item 42 above, a nucleic acid as described in item 43 above, a vector as described in item 44 above, a host cell as described in item 45 above, or a combination thereof, and a pharmaceutically acceptable carrier, diluent, or additive. 50. A method for treating a subject having CLDN6-expressing cancer, comprising administering to the subject the pharmaceutical composition described in 49 above in an amount effective for treating the cancer. 51. A method for inhibiting tumor growth within a subject, comprising administering the pharmaceutical composition described in 49 above to the subject in an amount effective for inhibiting tumor growth. 52. A method for reducing the size of a tumor within a subject, comprising administering the pharmaceutical composition described in 49 above to the subject in an amount effective for reducing the size of the tumor. 53. A method for preventing cancer recurrence within a subject, comprising administering the pharmaceutical composition described in 49 above to the subject in an amount effective for preventing cancer recurrence. 54. A method for treating cancer in a subject diagnosed with low overexpression of CLDN6, comprising administering the pharmaceutical composition described in 49 above to the subject in an amount effective in preventing cancer recurrence. 55. The method according to any one of the above 50 to 54, wherein the administration induces apoptosis in tumor cells. 56. The method according to any one of 50 to 55, wherein the administration 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 of items 1 to 40 above, a conjugate described in item 41 above, or a fusion protein described in item 42 above; 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 within a subject, comprising: contacting a biological sample, including cells or tissues collected from the subject, with an antigen-binding protein according to any one of the above 1 to 40, a conjugate according to 41, or a fusion protein according to 42; and assaying an immune complex containing the antigen-binding protein, conjugate, or fusion protein bound to CLDN6.

[0268] SEQUENCE LISTING <110> THE REGENTS OF THE UNIVERSITY OF CALIFORNIA <120> CLAUDIN6 ANTIBODIES AND METHODS OF TREATING CANCER <130> PA24-320 <150> US 62 / 560,143 <151> 2017-09-18 <160> 489 <170> PatentIn version 3.5 <210> 1 <211> 220 <212> PRT <213> Homo sapiens <300> <308> NCBI / NP_067018.2 <309> 2017-06-04 <313> (1)..(220) <400> 1 Met Ala Ser Ala Gly Met Gln Ile Leu Gly Val Val Leu Thr Leu Leu 1 5 10 15 Gly Trp Val Asn Gly Leu Val Ser Cys Ala Leu Pro Met Trp Lys Val 20 25 30 Thr Ala Phe Ile Gly Asn Ser Ile Val Val Ala Gln Val Val Trp Glu 35 40 45 Gly Leu Trp Met Ser Cys Val Val Gln Ser Thr Gly Gln Met Gln Cys 50 55 60 Lys Val Tyr Asp Ser Leu Leu Ala Leu Pro Gln Asp Leu Gln Ala Ala 65 70 75 80 Arg Ala Leu Cys Val Ile Ala Leu Leu Val Ala Leu Phe Gly Leu Leu 85 90 95 Val Tyr Leu Ala Gly Ala Lys Cys Thr Thr Cys Val Glu Glu Lys Asp 100 105 110 Ser Lys Ala Arg Leu Val Leu Thr Ser Gly Ile Val Phe Val Ile Ser 115 120 125 Gly Val Leu Thr Leu Ile Pro Val Cys Trp Thr Ala His Ala Ile Ile 130 135 140 Arg Asp Phe Tyr Asn Pro Leu Val Ala Glu Ala Gln Lys Arg Glu Leu 145 150 155 160 Gly Ala Ser Leu Tyr Leu Gly Trp Ala Ala Ser Gly Leu Leu Leu Leu 165 170 175 Gly Gly Gly Leu Leu Cys Cys Thr Cys Pro Ser Gly Gly Ser Gln Gly 180 185 190 Pro Ser His Tyr Met Ala Arg Tyr Ser Thr Ser Ala Pro Ala Ile Ser 195 200 205 Arg Gly Pro Ser Glu Tyr Pro Thr Lys Asn Tyr Val 210 215 220 <210> 2 <211> 23 <212> PRT <213> Homo sapiens <400> 2 Trp Thr Ala His Ala Ile Ile Arg Asp Phe Tyr Asn Pro Leu Val Ala 1 5 10 15 Glu Ala Gln Lys Arg Glu Leu 20 <210> 3 <211> 13 <212> PRT <213> Homo sapiens <400> 3 Thr Ala His Ala Ile Ile Arg Asp Phe Tyr Asn Pro Leu 1 5 10 <210> 4 <211> 10 <212> PRT <213> Homo sapiens <400> 4 Leu Val Ala Glu Ala Gln Lys Arg Glu Leu 1 5 10 <210> 5 <211> 220 <212> PRT <213> Homo sapiens <300> <308> NCBI / NP_001297.1 <309> 2017-07-10 <313> (1)..(220) <400> 5 Met Ser Met Gly Leu Glu Ile Thr Gly Thr Ala Leu Ala Val Leu Gly 1 5 10 15 Trp Leu Gly Thr Ile Val Cys Cys Ala Leu Pro Met Trp Arg Val Ser 20 25 30 Ala Phe Ile Gly Ser Asn Ile Ile Thr Ser Gln Asn Ile Trp Glu Gly 35 40 45 Leu Trp Met Asn Cys Val Val Gln Ser Thr Gly Gln Met Gln Cys Lys 50 55 60 Val Tyr Asp Ser Leu Leu Ala Leu Pro Gln Asp Leu Gln Ala Ala Arg 65 70 75 80 Ala Leu Ile Val Val Ala Ile Leu Leu Ala Ala Phe Gly Leu Leu Val 85 90 95 Ala Leu Val Gly Ala Gln Cys Thr Asn Cys Val Gln Asp Asp Thr Ala 100 105 110 Lys Ala Lys Ile Thr Ile Val Ala Gly Val Leu Phe Leu Leu Ala Ala 115 120 125 Leu Leu Thr Leu Val Pro Val Ser Trp Ser Ala Asn Thr Ile Ile Arg 130 135 140 Asp Phe Tyr Asn Pro Val Val Pro Glu Ala Gln Lys Arg Glu Met Gly 145 150 155 160 Ala Gly Leu Tyr Val Gly Trp Ala Ala Ala Ala Leu Gln Leu Leu Gly 165 170 175 Gly Ala Leu Leu Cys Cys Ser Cys Pro Pro Arg Glu Lys Lys Tyr Thr 180 185 190 Ala Thr Lys Val Val Tyr Ser Ala Pro Arg Ser Thr Gly Pro Gly Ala 195 200 205 Ser Leu Gly Thr Gly Tyr Asp Arg Lys Asp Tyr Val 210 215 220 <210> 6 <211> 209 <212> PRT <213> Homo sapiens <300> <308> NCBI / NP_001296.1 <309> 2017-07-10 <313> (1)..(209) <400> 6 Met Ala Ser Met Gly Leu Gln Val Met Gly Ile Ala Leu Ala Val Leu 1 5 10 15 Gly Trp Leu Ala Val Met Leu Cys Cys Ala Leu Pro Met Trp Arg Val 20 25 30 Thr Ala Phe Ile Gly Ser Asn Ile Val Thr Ser Gln Thr Ile Trp Glu 35 40 45 Gly Leu Trp Met Asn Cys Val Val Gln Ser Thr Gly Gln Met Gln Cys 50 55 60 Lys Val Tyr Asp Ser Leu Leu Ala Leu Pro Gln Asp Leu Gln Ala Ala 65 70 75 80 Arg Ala Leu Val Ile Ile Ser Ile Ile Val Ala Ala Leu Gly Val Leu 85 90 95 Leu Ser Val Val Gly Gly Lys Cys Thr Asn Cys Leu Glu Asp Glu Ser 100 105 110 Ala Lys Ala Lys Thr Met Ile Val Ala Gly Val Val Phe Leu Leu Ala 115 120 125 Gly Leu Met Val Ile Val Pro Val Ser Trp Thr Ala His Asn Ile Ile 130 135 140 Gln Asp Phe Tyr Asn Pro Leu Val Ala Ser Gly Gln Lys Arg Glu Met 145 150 155 160 Gly Ala Ser Leu Tyr Val Gly Trp Ala Ala Ser Gly Leu Leu Leu Leu 165 170 175 Gly Gly Gly Leu Leu Cys Cys Asn Cys Pro Pro Arg Thr Asp Lys Pro 180 185 190 Tyr Ser Ala Lys Tyr Ser Ala Ala Arg Ser Ala Ala Ala Ser Asn Tyr 195 200 205 Wave <210> 7 <211> 217 <212> PRT <213> Homo sapiens <300> <308> NCBI / NP_066192.1 <309> 2017-04-15 <313> (1)..(217) <400> 7 Met Ala Ser Thr Gly Leu Glu Leu Leu Gly Met Thr Leu Ala Val Leu 1 5 10 15 Gly Trp Leu Gly Thr Leu Val Ser Cys Ala Leu Pro Leu Trp Lys Val 20 25 30 Thr Ala Phe Ile Gly Asn Ser Ile Val Val Ala Gln Val Val Trp Glu 35 40 45 Gly Leu Trp Met Ser Cys Val Val Gln Ser Thr Gly Gln Met Gln Cys 50 55 60 Lys Val Tyr Asp Ser Leu Leu Ala Leu Pro Gln Asp Leu Gln Ala Ala 65 70 75 80 Arg Ala Leu Cys Val Ile Ala Leu Leu Leu Ala Leu Leu Gly Leu Leu 85 90 95 Val Ala Ile Thr Gly Ala Gln Cys Thr Thr Cys Val Glu Asp Glu Gly 100 105 110 Ala Lys Ala Arg Ile Val Leu Thr Ala Gly Val Ile Leu Leu Leu Ala 115 120 125 Gly Ile Leu Val Leu Ile Pro Val Cys Trp Thr Ala His Ala Ile Ile 130 135 140 Gln Asp Phe Tyr Asn Pro Leu Val Ala Glu Ala Leu Lys Arg Glu Leu 145 150 155 160 Gly Ala Ser Leu Tyr Leu Gly Trp Ala Ala Ala Ala Leu Leu Met Leu 165 170 175 Gly Gly Gly Leu Leu Cys Cys Thr Cys Pro Pro Pro Gln Val Glu Arg 180 185 190 Pro Arg Gly Pro Arg Leu Gly Tyr Ser Ile Pro Ser Arg Ser Gly Ala 195 200 205 Ser Gly Leu Asp Lys Arg Asp Tyr Val 210 215 <210> 8 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 8 Ser Ser Val Ser Ser Thr Tyr 1 5 <210> 9 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 9 Ser Thr Ser 1 <210> 10 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 10 His Gln Tyr His Arg Ser Pro Leu Thr 1 5 <210> 11 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 11 Gly Tyr Thr Phe Thr Thr Tyr Thr 1 5 <210> 12 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 12 Ile Asn Pro Ser Ser Gly Tyr Thr 1 5 <210> 13 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 13 Ala Asn Gly Asp Tyr Tyr Val Ala Tyr 1 5 <210> 14 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 14 Glu Asn Ile Tyr Ser Tyr 1 5 <210> 15 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 15 Asn Ala Lys 1 <210> 16 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 16 Gln His His Tyr Thr Val Pro Trp Thr 1 5 <210> 17 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 17 Gly Phe Thr Phe Ser Asp Tyr Trp 1 5 <210> 18 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 18 Ile Arg Leu Lys Ser Asp Asn Tyr Ala Thr 1 5 10 <210> 19 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 19 Asn Asp Gly Pro Pro Ser Gly Cys 1 5 <210> 20 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 20 Glu Asn Ile Tyr Ser Tyr 1 5 <210> 21 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 21 Asn Ala Lys 1 <210> 22 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 22 Gln His His Tyr Thr Val Pro Trp Thr 1 5 <210> 23 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 23 Gly Phe Thr Phe Ser Asn Tyr Trp 1 5 <210> 24 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 24 Ile Arg Leu Lys Ser Asp Asn Tyr Ala Thr 1 5 10 <210> 25 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 25 Asn Asp Gly Pro Pro Ser Gly Cys 1 5 <210> 26 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 26 Gln Ser Leu Val His Ser Asp Gly Asn Thr Tyr 1 5 10 <210> 27 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 27 Lys Val Ser 1 <210> 28 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 28 Ser Gln Ser Thr His Val Pro Tyr Thr 1 5 <210> 29 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 29 Gly Tyr Thr Phe Thr Ser Tyr Thr 1 5 <210> 30 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 30 Ile Asn Pro Ser Ser Thr Tyr Thr 1 5 <210> 31 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 31 Ser Arg Gly Glu Leu Gly Gly Phe Ala Tyr 1 5 10 <210> 32 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 32 Gln Ser Ile Val His Ser Asn Gly Asn Thr Tyr 1 5 10 <210> 33 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 33 Lys Val Ser 1 <210> 34 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 34 Phe Gln Gly Ser His Val Pro Phe Thr 1 5 <210> 35 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 35 Gly Tyr Ile Phe Thr His Tyr Ile 1 5 <210> 36 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 36 Ile Asn Pro Tyr Asn Asp Gly Thr 1 5 <210> 37 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 37 Ala Arg Tyr Tyr Gly Tyr Pro Tyr Tyr Ser Met Asp Tyr 1 5 10 <210> 38 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 38 Gln Ser Leu Leu Asn Ser Arg Thr Arg Lys Asn Tyr 1 5 10 <210> 39 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 39 Trp Ala Ser 1 <210> 40 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 40 Lys Gln Ser Tyr Tyr Leu Tyr Thr 1 5 <210> 41 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 41 Gly Tyr Ser Ile Thr Ser Gly Tyr Tyr 1 5 <210> 42 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 42 Ile Ser Tyr Asp Gly Gly Ile 1 5 <210> 43 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 43 Ala Arg Phe Gly Lys Gly Ala Met Asp Tyr 1 5 10 <210> 44 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 44 Ser Ser Val Ser Ser Ser Tyr 1 5 <210> 45 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 45 Ser Thr Ser 1 <210> 46 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 46 His Gln Tyr His Arg Ser Pro Pro Thr 1 5 <210> 47 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 47 Gly Tyr Ser Phe Thr Gly Tyr Thr 1 5 <210> 48 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 48 Ile Asn Pro Tyr Asn Gly Gly Thr 1 5 <210> 49 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 49 Ala Arg Gly Val Tyr Asp Tyr Asp Gly Phe Thr Tyr 1 5 10 <210> 50 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 50 Gln Ser Leu Val His Ser Asp Gly Asn Thr Tyr 1 5 10 <210> 51 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 51 Lys Val Ser 1 <210> 52 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 52 Ser Gln Ser Thr His Val Pro Tyr Thr 1 5 <210> 53 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 53 Gly Tyr Thr Phe Thr Thr Tyr Thr 1 5 <210> 54 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 54 Ile Asn Pro Arg Ser Gly Tyr Ser 1 5 <210> 55 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 55 Ser Arg Gly Glu Leu Gly Gly Phe Ala Tyr 1 5 10 <210> 56 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 56 Gln Thr Ile Gly Thr Trp 1 5 <210> 57 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 57 Ala Ala Ala 1 <210> 58 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 58 Gln Gln Leu Tyr Ser Ile Pro Arg Thr 1 5 <210> 59 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 59 Gly Tyr Arg Phe Thr Asp Tyr Asn 1 5 <210> 60 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 60 Ile Asn Pro Asn Asn Gly Gly Thr 1 5 <210> 61 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 61 Ala Arg Asp Tyr Leu Tyr Phe Phe Asp Cys 1 5 10 <210> 62 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 62 Gln Ser Leu Val His Ser Asn Gly Asn Thr Tyr 1 5 10 <210> 63 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 63 Lys Val Ser 1 <210> 64 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 64 Ser Gln Ile Thr His Val Pro Tyr Thr 1 5 <210> 65 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 65 Gly Tyr Thr Phe Thr Asp Tyr Ser 1 5 <210> 66 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 66 Ile Ser Thr Glu Thr Gly Glu Pro 1 5 <210> 67 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 67 Thr Arg Gly Leu Trp Ser Ser Phe Ala Tyr 1 5 10 <210> 68 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 68 Lys Ser Val Ser Thr Ser Gly Tyr Ser Tyr 1 5 10 <210> 69 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 69 Leu Ala Ser 1 <210> 70 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 70 Gln His Ser Arg Glu Leu Pro Leu Thr 1 5 <210> 71 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 71 Gly Phe Thr Phe Ser Ser Phe Gly 1 5 <210> 72 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 72 Ile Ser Ser Asp Ser Arg Thr Ile 1 5 <210> 73 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 73 Ala Arg Asp Tyr Gly Arg Thr Tyr Glu Ala Tyr 1 5 10 <210> 74 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 74 Gln Asp Ile Gly Gly Asn 1 5 <210> 75 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 75 Ser Thr Ser 1 <210> 76 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 76 Leu Gln Arg Asn Ala Tyr Pro Leu Thr 1 5 <210> 77 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 77 Gly Phe Thr Phe Ser Ser Tyr Ala 1 5 <210> 78 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 78 Ile Arg Ser Gly Gly Thr Thr 1 5 <210> 79 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 79 Ala Lys Val Gly Gly Asn Pro Tyr Pro Met Asp Tyr 1 5 10 <210> 80 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 80 Ser Ser Ile Ser Ser Asn Tyr 1 5 <210> 81 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 81 Arg Thr Ser 1 <210> 82 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 82 Gln Gln Gly Ser Ser Ile Pro Leu Thr 1 5 <210> 83 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 83 Gly Tyr Ala Phe Ser Asn Tyr Leu 1 5 <210> 84 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 84 Ile Asn Pro Gly Ser Gly Gly Thr 1 5 <210> 85 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 85 Ala Arg Ser Tyr Phe Gly Arg Ser Tyr Pro Tyr Thr Met Asp Tyr 1 5 10 15 <210> 86 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 86 Gln Ser Val Asp Tyr Asp Gly Asp Asn Tyr 1 5 10 <210> 87 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 87 Ala Ala Ser 1 <210> 88 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 88 Gln Gln Ser Asn Glu Asp Pro Phe Thr 1 5 <210> 89 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 89 Gly Tyr Thr Phe Thr Asp Tyr Ala 1 5 <210> 90 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 90 Ile Ser Thr Tyr Ser Gly Asn Thr 1 5 <210> 91 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 91 Ala Arg Arg Gly Asp Tyr Ser Leu Tyr Ala Met Asp Tyr 1 5 10 <210> 92 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 92 Gln Ser Val Leu Phe Ser Ser Asn Gln Lys Asn Tyr 1 5 10 <210> 93 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 93 Trp Ala Ser 1 <210> 94 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 94 His Gln Tyr Leu Ser Ser Arg Thr 1 5 <210> 95 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 95 Gly Phe Thr Phe Ser Ser Phe Gly 1 5 <210> 96 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 96 Ile Ser Ser Asp Ser Arg Thr Ile 1 5 <210> 97 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 97 Ala Arg Asp Tyr Gly Arg Thr Tyr Glu Ala Tyr 1 5 10 <210> 98 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 98 Glu Ser Val Asp Asn Tyr Gly Ile Ser Phe 1 5 10 <210> 99 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 99 Ala Ala Ser 1 <210> 100 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 100 Gln Gln Ser Lys Glu Val Pro Leu Thr 1 5 <210> 101 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 101 Gly Phe Pro Phe Ser Ser Ser Ala 1 5 <210> 102 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 102 Ile Asn Ser Asp Gly Asn Thr 1 5 <210> 103 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 103 Thr Arg Asn Gly Asp Tyr Arg Tyr Asp Glu Phe Ala Tyr 1 5 10 <210> 104 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 104 Ser Ser Val Ser Ser Ser Tyr 1 5 <210> 105 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 105 Ser Thr Ser 1 <210> 106 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 106 His Gln Tyr His Arg Ser Pro Pro Thr 1 5 <210> 107 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 107 Gly Tyr Thr Phe Thr Gly Tyr Trp 1 5 <210> 108 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 108 Ile Asn Pro Ser Thr Gly Tyr Thr 1 5 <210> 109 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 109 Ala Arg Glu Gly Ile Thr Thr Val Leu Val Asp Tyr 1 5 10 <210> 110 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 110 Gln Ser Val Leu Phe Ser Ser Asn Gln Lys Asn Tyr 1 5 10 <210> 111 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 111 Trp Ala Ser 1 <210> 112 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 112 His Gln Tyr Leu Ser Ser Arg Thr 1 5 <210> 113 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 113 Gly Tyr Ser Phe Thr Gly Tyr Asn 1 5 <210> 114 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 114 Ile Asp Pro Tyr Tyr Gly Gly Ser 1 5 <210> 115 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 115 Ala Arg Glu Arg Ser Gly Tyr Val Phe Ser Ala Met Asp Tyr 1 5 10 <210> 116 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 116 Gln Ser Val Leu Phe Ser Ser Asn Gln Lys Asn Tyr 1 5 10 <210> 117 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 117 Trp Ala Ser 1 <210> 118 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 118 His Gln Tyr Leu Ser Ser Arg Thr 1 5 <210> 119 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 119 Gly Tyr Ser Phe Thr Gly Tyr Thr 1 5 <210> 120 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 120 Ile Asn Pro Tyr Asn Gly Val Thr 1 5 <210> 121 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 121 Thr Arg Asp Pro Leu Tyr Tyr Gly Tyr Arg Asp Ser Thr Met Asp Tyr 1 5 10 15 <210> 122 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 122 Gln Ser Leu Val His Ser Asp Gly Asn Thr Tyr 1 5 10 <210> 123 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 123 Lys Val Ser 1 <210> 124 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 124 Ser Gln Ser Thr His Val Pro Tyr Thr 1 5 <210> 125 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 125 Gly Tyr Thr Phe Thr Ser Tyr Thr 1 5 <210> 126 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 126 Ile Asn Pro Ser Ser Thr Tyr Thr 1 5 <210> 127 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 127 Ser Arg Gly Glu Leu Gly Gly Phe Ala Tyr 1 5 10 <210> 128 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 128 Gln Gly Ile Arg Gly Asn 1 5 <210> 129 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 129 Ser Thr Ser 1 <210> 130 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 130 Leu Gln Arg Asn Ala Tyr Pro Leu Thr 1 5 <210> 131 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 131 Gly Phe Thr Phe Ser Ser Phe Ala 1 5 <210> 132 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 132 Ile Arg Ser Gly Gly Ile Thr 1 5 <210> 133 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 133 Ala Arg Val Ser Thr Ala Thr Tyr Tyr Gly Met Asp Tyr 1 5 10 <210> 134 <211> 108 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 134 Gln Ile Val Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Leu Gly 1 5 10 15 Glu Arg Val Thr Met Thr Cys Thr Ala Ser Ser Ser Val Ser Ser Thr 20 25 30 Tyr Phe His Trp Tyr Gln Gln Lys Pro Gly Ser Ser Pro Lys Leu Trp 35 40 45 Ile Tyr Ser Thr Ser Asn Leu Ala Ser Gly Val Pro Arg Arg Phe Ser 50 55 60 Gly Ser Ala Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu 65 70 75 80 Ala Glu Asp Ala Ala Thr Tyr Tyr Cys His Gln Tyr His Arg Ser Pro 85 90 95 Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys 100 105 <210> 135 <211> 116 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 135 Gln Val Gln Leu Gln Gln Ser Ala Ala Glu Leu Ala Arg Pro Gly Ala 1 5 10 15 Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Thr Tyr 20 25 30 Thr Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Phe Ile Asn Pro Ser Ser Gly Tyr Thr Asp Tyr Asn Gln Lys Phe 50 55 60 Lys Asp Arg Thr Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Val Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Asn Gly Asp Tyr Tyr Val Ala Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ala 115 <210> 136 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 136 Asp Ile Gln Met Thr Gln Ser Pro Ala Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Glu Thr Val Thr Ile Thr Cys Arg Ile Ser Glu Asn Ile Tyr Ser Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Gln Gly Lys Ser Pro Gln Leu Leu Val 35 40 45 Tyr Asn Ala Lys Ile Leu Val Glu Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Gln Phe Ser Leu Lys Ile Asn Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Gly Asn Tyr Tyr Cys Gln His His Tyr Thr Val Pro Trp 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 137 <211> 117 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 137 Glu Val Lys Leu Glu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Met Lys Leu Ser Cys Val Ala Ser Gly Phe Thr Phe Ser Asp Tyr 20 25 30 Trp Met Asn Trp Val Arg Gln Ser Pro Glu Lys Gly Leu Glu Trp Val 35 40 45 Ala Gln Ile Arg Leu Lys Ser Asp Asn Tyr Ala Thr His Tyr Ala Glu 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Arg Ser 65 70 75 80 Val Tyr Leu Gln Met Asn Asn Leu Arg Ala Glu Asp Thr Gly Thr Tyr 85 90 95 Tyr Cys Asn Asp Gly Pro Pro Ser Gly Cys Trp Gly Gln Gly Thr Thr 100 105 110 Leu Ile Val Ser Ser 115 <210> 138 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 138 Asp Ile Gln Met Thr Gln Ser Pro Ala Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Glu Thr Val Thr Ile Thr Cys Arg Ile Ser Glu Asn Ile Tyr Ser Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Gln Gly Lys Ser Pro Gln Leu Leu Val 35 40 45 Tyr Asn Ala Lys Ile Leu Val Glu Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Gln Phe Ser Leu Lys Ile Asn Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Gly Asn Tyr Tyr Cys Gln His His Tyr Thr Val Pro Trp 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 139 <211> 117 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 139 Glu Val Lys Leu Glu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Met Lys Leu Ser Cys Val Ala Ser Gly Phe Thr Phe Ser Asn Tyr 20 25 30 Trp Met Asn Trp Val Arg Gln Ser Pro Glu Lys Gly Leu Glu Trp Val 35 40 45 Ala Gln Ile Arg Leu Lys Ser Asp Asn Tyr Ala Thr His Tyr Ala Glu 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Arg Ser 65 70 75 80 Val Tyr Leu Gln Met Asn Asn Leu Arg Ala Glu Asp Thr Gly Thr Tyr 85 90 95 Tyr Cys Asn Asp Gly Pro Pro Ser Gly Cys Trp Gly Gln Gly Thr Thr 100 105 110 Leu Ile Val Ser Ser 115 <210> 140 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 140 Asp Val Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly 1 5 10 15 Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser 20 25 30 Asp Gly Asn Thr Tyr Leu Asn Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Arg Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Phe Cys Ser Gln Ser 85 90 95 Thr His Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 141 <211> 117 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 141 Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala 1 5 10 15 Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Thr Met His Trp Ile Lys Gln Arg Pro Gly Gln Gly Gln Glu Trp Ile 35 40 45 Gly Tyr Ile Asn Pro Ser Ser Thr Tyr Thr His Tyr Ile Lys Lys Phe 50 55 60 Lys Asp Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Arg Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ser Arg Gly Glu Leu Gly Gly Phe Ala Tyr Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ala 115 <210> 142 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 142 Asp Val Leu Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly 1 5 10 15 Asp Gln Pro Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Val His Ser 20 25 30 Asn Gly Asn Thr Tyr Leu Asp Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Phe Gln Gly 85 90 95 Ser His Val Pro Phe Thr Phe Gly Ser Gly Thr Arg Leu Glu Ile Lys 100 105 110 <210> 143 <211> 120 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 143 Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Ile Phe Thr His Tyr 20 25 30 Ile Met His Trp Val Lys Gln Lys Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Cys Ile Asn Pro Tyr Asn Asp Gly Thr Lys Tyr Asn Glu Lys Phe 50 55 60 Lys Gly Lys Ala Thr Leu Thr Ser Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Tyr Tyr Gly Tyr Pro Tyr Tyr Ser Met Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Ser Val Thr Val Ser Ser 115 120 <210> 144 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 144 Ala Ile Val Met Phe Gln Ser Pro Ser Ser Leu Val Val Ser Ala Gly 1 5 10 15 Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser 20 25 30 Arg Thr Arg Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 Ser Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Val Tyr Tyr Cys Lys Gln 85 90 95 Ser Tyr Tyr Leu Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 145 <211> 117 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 145 Asp Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Ser Ser Gln 1 5 10 15 Ser Leu Ser Leu Thr Cys Ser Val Thr Gly Tyr Ser Ile Thr Ser Gly 20 25 30 Tyr Tyr Trp Lys Trp Ile Arg Gln Phe Pro Gly Asn Lys Leu Glu Trp 35 40 45 Met Gly Tyr Ile Ser Tyr Asp Gly Gly Ile Asn Tyr Asn Pro Ser Leu 50 55 60 Lys Asn Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln Phe Phe 65 70 75 80 Leu Lys Leu Asn Ser Val Thr Thr Glu Asp Thr Ala Lys Tyr Tyr Cys 85 90 95 Ala Arg Phe Gly Lys Gly Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser 100 105 110 Val Thr Val Ser Ser 115 <210> 146 <211> 108 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 146 Gln Ile Val Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Leu Gly 1 5 10 15 Asp Arg Val Thr Met Thr Cys Thr Ala Ser Ser Ser Val Ser Ser Ser 20 25 30 Tyr Leu His Trp Tyr Gln Gln Lys Pro Gly Ser Ser Pro Lys Leu Trp 35 40 45 Ile Tyr Ser Thr Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu 65 70 75 80 Ala Glu Asp Ala Ala Thr Tyr Tyr Cys His Gln Tyr His Arg Ser Pro 85 90 95 Pro Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 147 <211> 119 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 147 Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Met Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Gly Tyr 20 25 30 Thr Met Asn Trp Val Lys Gln Ser His Gly Lys Asn Leu Glu Trp Ile 35 40 45 Gly Leu Ile Asn Pro Tyr Asn Gly Gly Thr Asn Tyr Asn Gln Lys Phe 50 55 60 Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Leu Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Val Tyr Asp Tyr Asp Gly Phe Thr Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ala 115 <210> 148 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 148 Asp Val Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly 1 5 10 15 Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser 20 25 30 Asp Gly Asn Thr Tyr Leu Tyr Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Phe Cys Ser Gln Ser 85 90 95 Thr His Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 149 <211> 117 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 149 Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala 1 5 10 15 Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Thr Tyr 20 25 30 Thr Met His Trp Leu Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Asn Pro Arg Ser Gly Tyr Ser Asn Tyr Asn Gln Lys Phe 50 55 60 Lys Asp Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Asn Thr Ala Tyr 65 70 75 80 Met Gln Leu Asn Thr Leu Thr Ser Glu Asp Ser Lys Val Tyr Tyr Cys 85 90 95 Ser Arg Gly Glu Leu Gly Gly Phe Ala Tyr Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ala 115 <210> 150 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 150 Asp Ile Gln Met Thr Gln Ser Pro Ala Ser Gln Ser Ala Ser Leu Gly 1 5 10 15 Glu Ser Val Thr Ile Thr Cys Leu Ala Ser Gln Thr Ile Gly Thr Trp 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ser Pro Gln Leu Leu Ile 35 40 45 Tyr Ala Ala Ala Ser Leu Ala Asp Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Arg Phe Ser Phe Lys Ile Ser Ser Leu Gln Ala 65 70 75 80 Glu Asp Phe Val Ser Tyr Tyr Cys Gln Gln Leu Tyr Ser Ile Pro Arg 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 151 <211> 117 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 151 Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Arg Phe Thr Asp Tyr 20 25 30 Asn Met His Trp Val Lys Gln Ser His Gly Lys Ser Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Asn Pro Asn Asn Gly Gly Thr Asn Tyr Asn Gln Asn Phe 50 55 60 Lys Gly Lys Ala Thr Leu Thr Val Asn Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Arg Ser Leu Thr Ser Glu Asp Ser Ala Ala Tyr Tyr Cys 85 90 95 Ala Arg Asp Tyr Leu Tyr Phe Phe Asp Cys Trp Gly Gln Gly Thr Thr 100 105 110 Leu Thr Val Ser Ser 115 <210> 152 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 152 Asp Val Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly 1 5 10 15 Asp Gln Ala Ser Ile Ser Cys Arg Ala Ser Gln Ser Leu Val His Ser 20 25 30 Asn Gly Asn Thr Tyr Leu His Trp Phe Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Arg Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Phe Cys Ser Gln Ile 85 90 95 Thr His Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 153 <211> 117 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 153 Gln Ile Gln Leu Val Gln Ser Gly Pro Ala Leu Lys Lys Pro Gly Glu 1 5 10 15 Thr Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Ser Met His Trp Ile Lys Gln Ala Pro Gly Lys Gly Leu Lys Trp Met 35 40 45 Gly Trp Ile Ser Thr Glu Thr Gly Glu Pro Thr Tyr Ala Asp Gly Phe 50 55 60 Lys Gly Arg Phe Asp Phe Ser Leu Glu Thr Ser Ala Asp Thr Ala Tyr 65 70 75 80 Leu Ser Ile Asn Asn Leu Thr Asn Glu Asp Thr Ala Thr Tyr Phe Cys 85 90 95 Thr Arg Gly Leu Trp Ser Ser Phe Ala Tyr Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ala 115 <210> 154 <211> 111 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 154 Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Lys Ser Val Ser Thr Ser 20 25 30 Gly Tyr Ser Tyr Ile His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro 35 40 45 Lys Leu Leu Ile Tyr Leu Ala Ser Asn Leu Glu Ser Gly Val Pro Ala 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile His 65 70 75 80 Pro Val Glu Glu Glu Asp Ala Ala Thr Tyr Tyr Cys Gln His Ser Arg 85 90 95 Glu Leu Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys 100 105 110 <210> 155 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 155 Asp Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Arg Gly 1 5 10 15 Ser Arg Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Phe 20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Glu Lys Gly Leu Glu Trp Val 35 40 45 Ala Tyr Ile Ser Ser Asp Ser Arg Thr Ile Tyr Tyr Ala Asp Thr Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Pro Thr Asn Thr Leu Phe 65 70 75 80 Leu Gln Met Thr Ser Leu Arg Ser Glu Asp Thr Ala Met Tyr Tyr Cys 85 90 95 Ala Arg Asp Tyr Gly Arg Thr Tyr Glu Ala Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ala 115 <210> 156 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 156 Asp Ile Gln Met Ile Gln Ser Pro Ser Ser Met Phe Ala Ser Leu Gly 1 5 10 15 Asp Arg Val Ser Leu Ser Cys Arg Ala Ser Gln Asp Ile Gly Gly Asn 20 25 30 Leu Asp Trp Tyr Gln Gln Lys Pro Gly Gly Thr Ile Lys Leu Leu Ile 35 40 45 Tyr Ser Thr Ser Asn Leu Asn Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Ser Asp Tyr Ser Leu Thr Ile Thr Ser Leu Glu Ser 65 70 75 80 Glu Asp Phe Ala Asp Tyr Tyr Cys Leu Gln Arg Asn Ala Tyr Pro Leu 85 90 95 Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys 100 105 <210> 157 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 157 Glu Val Lys Leu Met Glu Ser Gly Gly Asp Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln Thr Pro Glu Lys Arg Leu Glu Trp Val 35 40 45 Ala Ser Ile Arg Ser Gly Gly Thr Thr Tyr Tyr Pro Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Arg Asn Ile Leu Tyr Leu 65 70 75 80 Arg Met Ser Ser Leu Arg Ser Glu Asp Thr Ala Ile Tyr Tyr Cys Ala 85 90 95 Lys Val Gly Gly Asn Pro Tyr Pro Met Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Ser Val Thr Val Ser Ser 115 <210> 158 <211> 108 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 158 Glu Ile Val Leu Thr Gln Ser Pro Thr Thr Met Ala Ala Ser Pro Gly 1 5 10 15 Glu Lys Ile Thr Ile Thr Cys Ser Ala Ser Ser Ser Ile Ser Ser Asn 20 25 30 Tyr Leu His Trp Tyr Gln Gln Lys Pro Gly Phe Ser Pro Lys Leu Leu 35 40 45 Ile Tyr Arg Thr Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Gly Thr Met Glu 65 70 75 80 Ala Glu Asp Val Ala Thr Tyr Tyr Cys Gln Gln Gly Ser Ser Ile Pro 85 90 95 Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys 100 105 <210> 159 <211> 122 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 159 Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Arg Pro Gly Thr 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ala Phe Ser Asn Tyr 20 25 30 Leu Ile Glu Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Val Ile Asn Pro Gly Ser Gly Gly Thr Asn Tyr Asn Glu Lys Phe 50 55 60 Lys Gly Lys Ala Thr Met Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met His Leu Ser Asn Leu Thr Ser Glu Asp Ser Val Val Tyr Phe Cys 85 90 95 Ala Arg Ser Tyr Phe Gly Arg Ser Tyr Pro Tyr Thr Met Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Ser Val Thr Val Ser Ser 115 120 <210> 160 <211> 111 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 160 Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Gln Arg Ala Thr Ile Ser Cys Lys Ala Ser Gln Ser Val Asp Tyr Asp 20 25 30 Gly Asp Asn Tyr Val Asn Trp Tyr Gln Gln Lys Val Gly Gln Pro Pro 35 40 45 Lys Leu Leu Ile Ser Ala Ala Ser Asn Leu Glu Ser Gly Ile Pro Ala 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile His 65 70 75 80 Pro Val Glu Glu Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Ser Asn 85 90 95 Glu Asp Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 161 <211> 120 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 161 Gln Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Arg Pro Gly Val 1 5 10 15 Ser Val Lys Ile Ser Cys Lys Gly Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Ala Met His Trp Val Lys Gln Ser His Ala Lys Ser Leu Glu Trp Ile 35 40 45 Gly Val Ile Ser Thr Tyr Ser Gly Asn Thr Asn Tyr Asn Gln Lys Phe 50 55 60 Gln Asp Lys Ala Thr Met Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Ala Leu Ala Arg Leu Thr Ser Asp Asp Ser Ala Ile Tyr Tyr Cys 85 90 95 Ala Arg Arg Gly Asp Tyr Ser Leu Tyr Ala Met Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Ser Val Thr Val Ser Ser 115 120 <210...

Claims

1. An antigen-binding protein that binds to human claudin 6 (CLDN6) protein (SEQ ID NO: 200), (i) HC CDR1 containing FTFSNYW (Sequence ID: 455), (ii) HC CDR2 containing IRLKSDNYAT (Sequence ID: 24), (iii) HC CDR3 containing XDGPPSGX (Sequence ID: 457), where the first X is N and the eighth X is T, A, or Y. (iv) LC CDR1 containing ENIYSY (Sequence ID: 20), (v) LC CDR2 containing NAK (Sequence ID: 21), (vi) LC CDR3 containing QHHYTVPWT (Sequence ID: 22), Antigen-binding proteins, including those mentioned above.

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

3. The antigen-binding protein according to claim 1, wherein the X at position 8 is T.

4. The antigen-binding protein according to claim 1, wherein the X at position 8 is A.

5. The antigen-binding protein according to claim 1, wherein the X at position 8 is Y.

6. The antigen-binding protein according to claim 1, wherein the antigen-binding protein is an antigen-binding antibody fragment.

7. The antigen-binding antibody fragment is scFv, F(ab') ２ The antigen-binding protein according to claim 6, selected from the group consisting of Fab, Fab', and Fv.

8. The antigen-binding protein according to claim 1, wherein the antigen-binding protein is an antibody.

9. The antigen-binding protein according to claim 8, wherein the antibody is (i) a monoclonal antibody, (ii) a humanized antibody, or a chimeric antibody, or (iii) IgG.

10. The antigen-binding protein according to claim 9, wherein IgG is selected from the group consisting of IgG1, IgG2, IgG3, and IgG4.

11. The antigen-binding protein according to claim 9, wherein IgG is IgG1.

12. A conjugate comprising an antigen-binding protein according to any one of claims 1 to 11 and a cytotoxic agent or chemotherapeutic agent.

13. The conjugate according to claim 12, further comprising a linker.

14. The conjugate according to claim 13, wherein the linker is a severable linker.

15. The conjugate according to claim 12, wherein a cytotoxic agent or chemotherapeutic agent is conjugated to an antigen-binding protein via a linker.

16. The conjugate according to claim 12, wherein the chemotherapeutic agent is an antimitotic agent.

17. The conjugate according to claim 16, wherein the antimitotic agent is auristatin.

18. The conjugate according to claim 17, wherein auristatin is an MMAE.

19. The conjugate according to claim 12, wherein the chemotherapeutic agent is conjugated to an antigen-binding protein via a linker in an MMAE.

20. A fusion protein comprising the antigen-binding protein described in claim 1 or 2.

21. A nucleic acid encoding an antigen-binding protein according to claim 1 or 2, or a fusion protein according to claim 20.

22. A vector comprising the nucleic acid described in Claim 21.

23. A host cell comprising the nucleic acid described in Claim 21.

24. (a) an antigen-binding protein according to any one of claims 1 to 11 or a conjugate according to any one of claims 12 to 19, a fusion protein according to claim 20, a nucleic acid according to claim 21, a vector according to claim 22, or a host cell according to claim 23; and (b) a pharmaceutical composition comprising a pharmaceutically acceptable carrier, diluent, and / or excipient.

25. An antigen-binding protein according to any one of claims 1 to 11 or a conjugate according to any one of claims 12 to 19, or a pharmaceutical composition according to claim 24, for use in the treatment of cancer.

26. Use of an antigen-binding protein according to any one of claims 1 to 11 or a conjugate according to any one of claims 12 to 19, or a pharmaceutical composition according to claim 24, in the manufacture of a pharmaceutical for treating cancer.

27. An in vitro method for detecting claudin 6 (CLDN6) in a sample, comprising (a) contacting the sample with an antigen-binding protein according to any one of claims 1 to 11, and (b) assaying an immune complex comprising an antibody bound to CLDN6 or an antigen-binding fragment thereof.

28. A pharmaceutical composition comprising an antigen-binding protein according to any one of claims 1 to 11, for use in a method for diagnosing claudin 6 (CLDN6)-positive cancer in a subject in vivo.

29. An antigen-binding protein that binds to human claudin 6 (CLDN6) protein (SEQ ID NO: 200), (i) HC CDR1 containing FTFSNYW (Sequence ID: 455), (ii) HC CDR2 containing IRLKSDNYAT (Sequence ID: 24), (iii) HC CDR3 containing XDGPPSGX (Sequence ID: 457), where the first X is N and the eighth X is S, T, A, C, or Y. (iv) LC CDR1 containing ENIYSY (Sequence ID: 20), (v) LC CDR2 containing NAK (Sequence ID: 21), (vi) LC CDR3 containing QHHYTVPWT (Sequence ID: 22), A pharmaceutical composition comprising an antigen-binding protein, The pharmaceutical composition, administered via parenteral, nasal, oral, pulmonary, topical, vaginal, or rectal route.

30. The pharmaceutical composition according to claim 29, wherein HC CDR1 comprises the amino acid sequence GFTFSNYW (SEQ ID NO: 23).

31. The pharmaceutical composition according to claim 29, wherein the X at position 8 is S.

32. The pharmaceutical composition according to claim 29, wherein the 8th position X is T.

33. The pharmaceutical composition according to claim 29, wherein the 8th position X is A.

34. The pharmaceutical composition according to claim 29, wherein X at position 8 is C.

35. The pharmaceutical composition according to claim 29, wherein the 8th position X is Y.

36. The pharmaceutical composition according to claim 29, wherein parenteral administration is selected from the group consisting of subcutaneous, intramuscular, intraspinal, or intravenous administration.

37. The pharmaceutical composition according to claim 29, for use in the treatment of cancer.

38. Use of the pharmaceutical composition according to claim 29 in the manufacture of a pharmaceutical for treating cancer.

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