Claudin-6 antibodies and conjugates thereof and uses thereof

Abstract

The present disclosure provides antigen binding proteins that bind to claudin-6 (CLDN6). In various aspects, the antigen binding proteins bind to the cytoplasmic domain of CLDN6. Related polypeptides, nucleic acids, vectors, host cells, and conjugates are further provided herein. Kits and pharmaceutical compositions comprising such entities are also provided. Methods for producing the antigen binding proteins, as well as methods for detecting and quantifying claudin-6 (CLDN6) in a sample from a subject and treating a subject with cancer, are also provided.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 63 / 396,383, filed August 9, 2022, the entire contents of which are incorporated herein by this reference in their entirety.

[0002] Incorporation by Reference of Electronically Submitted Materials The present disclosure generally relates to antibodies specific to the cytoplasmic domain of claudin-6 (CLDN6), and uses of the antibodies for detecting the presence of CLDN6 in samples from cancer patients, for determining the quantity of CLDN6 in a sample, and for diagnosing CLDN6-positive cancer in patients. [Background technology]

[0003] Antibodies are powerful therapeutic agents characterized by limited side effects due to their ability to specifically target and identify distinct antigens on cells, bacteria, viruses, or toxins. In 1986, the first therapeutic monoclonal antibody, Orthoclone OKT3, was introduced to the market. Since then, this class of biopharmaceutical products has grown significantly. As of the end of 2014, 47 monoclonal antibody products had been approved in the United States or Europe for the treatment of a variety of diseases, including cancer, as well as inflammatory, cardiovascular, respiratory, and infectious diseases.

[0004] Currently, more than 12 monoclonal antibodies have been approved by the U.S. Food and Drug Administration for the treatment or identification of cancer. These agents include blinatumomab (Blincyto), which is designed to recognize and bind to two different antigens. Despite the commercial availability of such antibody products, current cancer incidence and cancer mortality rates remain high. The cancer incidence rate is reported to be over 450 cases per 100,000 men and women per year, and the cancer mortality rate is just over 170 cases per 100,000 men and women per year. Additional antibodies for identifying and targeting cancer cells are important. Summary of the Invention

[0005] Provided herein are antigen binding proteins that bind to claudin-6 (CLDN6). In various embodiments, the antigen binding proteins of the present disclosure bind to human CLDN6, and optionally to mouse CLDN6. In various embodiments, the antigen binding proteins bind to the cytoplasmic domain of CLDN6. In various cases, the antigen binding proteins selectively bind to CLDN6 compared to any other members of the claudin family. In various cases, the antigen binding proteins bind to CLDN6 and do not bind to any other members of the claudin family. In various embodiments, the antigen binding proteins bind to CLDN6 endogenously expressed by human ovarian cancer cells, such as OVCA429 cells.

[0006] In various embodiments, the antigen binding protein has (a) the heavy chain CDR1 amino acid sequence of SEQ ID NO: 18 or SEQ ID NO: 34, or a variant sequence thereof that differs by only one or two amino acids or has at least or about 70% sequence identity; (b) the heavy chain CDR2 amino acid sequence of SEQ ID NO: 19 or SEQ ID NO: 35, or a variant sequence thereof that differs by only one or two amino acids or has at least or about 70% sequence identity; (c) the heavy chain CDR3 amino acid sequence of SEQ ID NO: 20 or SEQ ID NO: 36, or a variant sequence thereof that differs by only one or two amino acids or has at least or about 70% sequence identity; (d) the heavy chain CDR4 amino acid sequence of SEQ ID NO: 21 or SEQ ID NO: 22, or a variant sequence thereof that differs by only one or two amino acids or has at least or about 70% sequence identity; (e) the light chain CDR1 amino acid sequence of SEQ ID NO: 25 or SEQ ID NO: 41, or a variant sequence thereof which differs by only one or two amino acids or has at least or about 70% sequence identity; (f) the light chain CDR3 amino acid sequence of SEQ ID NO: 27 or SEQ ID NO: 43, or a variant sequence thereof which differs by only one or two amino acids or has at least or about 70% sequence identity; and / or (g) a combination of any two or more of (a) to (f).

[0007] In various embodiments, the antigen binding protein comprises (a) the heavy chain variable region amino acid sequence of any one of SEQ ID NOs: 18-20, 34-36, or a variant sequence thereof that differs by only one or two amino acids or has at least or about 70% sequence identity, or (b) the light chain variable region amino acid sequence of any one of SEQ ID NOs: 25-27, 41-43, or a variant sequence thereof that differs by only one or two amino acids or has at least or about 70% sequence identity, or both (a) and (b).

[0008] Further provided herein is an antigen binding protein conjugated to a heterologous moiety (e.g., a detectable label or detection moiety) for detecting CLDN6-positive tumors in a subject, e.g., a human. The label or detection moiety can be a reporter protein. Examples of reporter proteins include, but are not limited to, green fluorescent protein (GFP), red fluorescent protein (RFP), glutathione-S-transferase (GST), horseradish peroxidase (HRP), alkaline phosphatase (AP), chloramphenicol acetyltransferase (CAT), beta-galactosidase, glucose oxidase (GO), beta-glucuronidase (GUS), luciferase, beta-lactamase, or blue fluorescent protein (BFP). In various cases, the conjugated antigen binding protein is a monoclonal antibody conjugated to a heterologous moiety. In various embodiments, the conjugate is a homogeneous or heterogeneous conjugate. In various aspects, the heterologous moiety is conjugated to a specific site on the antigen binding protein.

[0009] In various embodiments, the antigen binding protein binds to CLDN6 expressed by human cancer cells. In various embodiments, the antigen binding protein inhibits the binding interaction between human CLDN6 and a reference anti-CLDN6 antibody. Without being bound by a particular theory, the inhibitory effect of the antigen binding proteins provided herein makes such entities useful in methods for detecting tumors or tumor growth. As further discussed herein, in various embodiments, the antigen binding protein is an antibody, an antigen-binding antibody fragment thereof, or an antibody protein preparation.

[0010] The present disclosure also provides antigen binding proteins comprising at least three, four, five, or all of the amino acid sequences of a particular group of amino acid sequences. In various embodiments, the antigen binding proteins comprise at least three, four, five, or six complementarity-determining region (CDR) amino acid sequences of a CLDN6 antibody disclosed herein.

[0011] The present disclosure further provides antigen binding proteins comprising the amino acid sequences detailed herein. In various embodiments, the antigen binding proteins comprise the amino acid sequence of any one of SEQ ID NOs: 16-47, or a combination thereof, as further described herein.

[0012] Related polypeptides, nucleic acids, vectors, host cells, and conjugates are further provided herein. Kits and pharmaceutical compositions containing such entities are further contemplated.

[0013] Methods for producing the antigen binding proteins are also provided, in various embodiments, the methods comprise culturing a host cell comprising nucleic acid encoding an antigen binding protein or polypeptide described herein to express the antigen binding protein or polypeptide.

[0014] Also provided are methods for producing conjugates with the antigen-binding proteins described herein. In various embodiments, the methods include culturing host cells containing nucleic acids encoding the antigen-binding proteins, fusion proteins, or polypeptides described herein to express the antigen-binding proteins, fusion proteins, or polypeptides, recovering the antigen-binding proteins, fusion proteins, or polypeptides from the cell culture medium, and then binding the antigen-binding proteins, fusion proteins, or polypeptides to a second moiety to produce the conjugate. In various aspects, the second moiety is a detectable label, drug, or moiety. In certain embodiments, the detectable label is a reporter protein, such as green fluorescent protein (GFP), red fluorescent protein (RFP), glutathione-S-transferase (GST), horseradish peroxidase (HRP), alkaline phosphatase (AP), chloramphenicol acetyltransferase (CAT), beta-galactosidase, glucose oxidase (GO), beta-glucuronidase (GUS), luciferase, β-lactamase, or blue fluorescent protein (BFP).

[0015] Methods for detecting or determining the quantity of claudin-6 (CLDN6) in a sample are provided herein. In various embodiments, the methods include contacting the sample with an antigen-binding protein and assaying for immune complexes comprising the antigen-binding protein bound to CLDN6, or quantifying the amount of immune complexes so assayed, wherein the immune complexes indicate the presence and quantity of CLDN6. Further contemplated are methods for diagnosing and monitoring the course of claudin-6 (CLDN6)-positive cancer in a subject, wherein the presence of immune complexes indicates the presence of CLDN6-positive cancer. Detecting the presence of CLDN6 can also be used in methods for classifying CLDN6-positive tumors in a subject.

[0016] In a further embodiment, the detection method comprises contacting a sample with a first antigen-binding protein specific to CLDN6 to form a first antigen-binding protein-CLDN6 immune complex, contacting the immune complex so formed with a second antigen-binding protein labeled with a detectable marker to form a second immune complex comprising the first antigen-binding protein-CLDN6-second antigen-binding protein, and detecting the second immune complex so formed, which indicates the presence of CLDN6 in the sample. In certain embodiments, the second antigen-binding protein is an antibody that recognizes the first antigen-binding protein.

[0017] Also provided herein are methods for determining whether a cancer is treatable by a cancer therapy targeting CLDN6 and for determining the prognosis of a subject with a CLDN6-positive cancer. The methods include quantitatively determining the presence of CLDN6 in a first sample from the subject and comparing the amount so determined with the amount present in a second sample from the subject, the samples being taken at various time points, and the amount of CLDN6 determined over time indicates that the CLDN6-positive cancer is treatable in the subject and the prognosis of the CLDN6-positive cancer in the subject. [Brief explanation of the drawings]

[0018] [Figure 1]

[0039] Figure 1 shows the results of epitope mapping for the CLDN6 antibodies 3H11 and 3D07, which bind to peptides in the cytoplasmic domain of claudin-6 (CLDN6). 3H11 bound to two peptides with an overlapping 10-amino acid region, APAISRGPSE (SEQ ID NO: 15). 3D07 bound to three peptides with an overlapping 6-amino acid region, SHYMAR (SEQ ID NO: 98). [Figure 2]

[0033] Figure 1 shows the results of epitope mapping for the CLDN6 antibody 3D07, which binds to peptides in the cytoplasmic domain of claudin-6 (CLDN6). 3D07 bound to one peptide having the amino acid sequence SHYMARYSTSAPAIS (SEQ ID NO: 99). [Figure 3]

[0023] Figure 1 shows the binding of antibodies 3H11 and 3D07 to HEK293 cell lines expressing CLDN6, CLDN3, CLDN4 or CLDN9. The results demonstrate that the antibodies specifically bind to CLDN6. [Figure 4] Binding of antibodies 3H11 and 3D07 to human ovarian cancer tissue sections is shown. The results show that the antibodies bind to CLDN6 in cancer tissue samples and identify cancer tissue sections with CLDN6-positive cells. DETAILED DESCRIPTION OF THE INVENTION

[0019] Sequence ID Number Table A [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 1-6]

[0020] Sequence Number Table B [Table 2-1] [Table 2-2]

[0021] Detailed Description 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 generally 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 cytoplasmic tail (domain) is publicly available on the National Center for Biotechnology Information (NCBI) website as NCBI reference sequence NP_067018.2 and is represented herein as SEQ ID NO: 1. The amino acid at position 143 of SEQ ID NO: 1 is Ile. In some cases, due to a single nucleotide polymorphism (SNP) in the DNA sequence encoding CLDN6, the amino acid at position 143 is Val. The amino acid sequence of human CLDN6 with Val at position 143 is represented herein as SEQ ID NO: 2.

[0022] definition In the context of describing this disclosure (particularly in the context of the claims that follow), the use of the terms "a," "an," and "the" and similar referents should be construed to encompass both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context.

[0023] The terms "comprising," "having," "including," and "containing" are to be construed as open-ended terms (i.e., meaning "including but not limited to") unless otherwise indicated.

[0024] As used herein, the term "conjugate" not only encompasses a polypeptide that is linked to a heterologous moiety via a chemical reaction, but also encompasses a polypeptide that is co-expressed to include a heterologous moiety as a fusion protein. Thus, as used herein, the term "conjugate" encompasses fusion proteins.

[0025] A molecule is "fixed" or "attached" to a substrate when it is covalently or non-covalently associated with the substrate such that the substrate can be rinsed with a fluid (e.g., standard citrate saline, pH 7.4) without a substantial portion of the molecule dissociating from the substrate.

[0026] The term "preventing" is art-recognized and, when used in the context of diseases such as cancer, is well understood in the art and includes the administration of a treatment, e.g., the administration of a composition, that reduces the frequency of or delays the onset of symptoms of a medical condition in a subject compared to subjects not receiving the treatment. Thus, preventing cancer includes, e.g., reducing, in a statistically and / or clinically significant amount, the number of detectable cancerous growths in a population of patients receiving prophylactic treatment compared to an untreated control population, and / or delaying the appearance of detectable cancerous growths in a treated population relative to an untreated control population.

[0027] The amount of a biomarker in a subject is "significantly" greater or less than the normal amount of the biomarker if the amount of the biomarker is greater or less, respectively, than the normal or control level by an amount that exceeds the standard error of the assay used to assess the amount, preferably at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 300%, 350%, 400%, 500%, 600%, 700%, 800%, 900%, 1000% or more. Alternatively, the amount of the biomarker in the subject may be at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 105%, 110%, 115%, 120%, 125%, 130%, 135%, 140%, 145%, 150%, 155%, 160%, 165%, 170%, 175%, 180%, 185%, 185%, 190%, 195%, 200%, 205%, 210%, 215%, 220%, 225%, 230%, 235%, 240%, 245%, 250%, 260%, 265%, 270%, 275%, 280%, 285%, 290%, 300%, 305%, 310%, 315%, 320%, 325%, 330%, 335%, 340%, 345%, 350%, 355%, 360%, 365%, 370%, 375%, 380%, 385%, 390%, 400%, 410%, 410%, 420%, 425%, 430%, 430%, 440%, 445%, 450%, 450%, 460%, 470%, 475%, 480%, 485%, 490%, 500%, 510%, 515%, 520%, 525%, 530%, 535%, 540%, 545%, A change of 130%, 135%, 140%, 145%, 150%, 155%, 160%, 165%, 170%, 175%, 180%, 185%, 190%, 195%, 2-fold, 3-fold, 4-fold, 5-fold, or more, or any range therebetween, e.g., 5% to 100% more or less, can be considered "significantly" greater or less than the normal and / or control amount. Such significant change can apply to any metric described herein, e.g., change in expression level, change in activity, change in hyperproliferative growth of cancer cells, change in cancer cell death, change in biomarker inhibition, change in test agent binding, etc.

[0028] The term "therapeutic effect" refers to a local or systemic effect in animals, particularly mammals, and more particularly humans, caused by a pharmacologically active substance. Thus, the term refers to any substance intended for use in the diagnosis, cure, mitigation, treatment, or prevention of disease, or the enhancement of desired physical or mental development and conditions, in animals or humans. The phrase "therapeutically effective amount" refers to that amount of such a substance that produces some desired local or systemic effect at a reasonable benefit / risk ratio applicable to any treatment. In certain embodiments, a therapeutically effective amount of a compound will depend on its therapeutic index, solubility, and the like. For example, certain compounds discovered by the methods of the present invention can be administered in an amount sufficient to provide a reasonable benefit / risk ratio applicable to such treatment.

[0029] The term "treating" includes prophylactic and / or therapeutic treatment. The term "prophylactic or therapeutic" treatment is art-recognized and includes administration to a host of one or more of the subject compositions or cancer therapies described herein. Treatment is prophylactic (i.e., the treatment protects the host from developing the undesirable condition) when administered prior to clinical manifestation of an undesirable condition (e.g., disease or other undesirable condition in the host animal), and therapeutic (i.e., the treatment is intended to reduce, ameliorate, or stabilize an existing undesirable condition or its side effects) when administered after the manifestation of the undesirable condition.

[0030] antigen-binding proteins Provided herein is an antigen-binding protein that binds to the cytoplasmic domain of claudin-6 (CLDN6). The antigen-binding protein of the present disclosure can take any one of the many forms of antigen-binding proteins known in the art. In various embodiments, the antigen-binding protein of the present disclosure takes the form of an antibody, an antigen-binding antibody fragment, or an antibody protein preparation.

[0031] In various embodiments of the present disclosure, the antigen-binding protein comprises, consists essentially of, or consists of an antibody. As used herein, the term "antibody" refers to a protein having a conventional immunoglobulin format, including heavy and light chains, and including a variable region and a constant region. For example, an antibody may be an IgG, which has a "Y-shaped" structure consisting of two identical pairs of polypeptide chains, each pair 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 generally about 100-110 amino acids or more, contains three complementarity-determining regions (CDRs), and is primarily responsible for antigen recognition, substantially differentiating it from 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 is composed of the N-terminal region of each light and heavy chain, and the constant region is composed of the C-terminal portion of each heavy and light chain (Janeway et al., “Structure of the Antibody Molecule and the Immunoglobulin Genes,” Immunobiology: The Immune System in Health and Disease, 4 th ed.Elsevier Science Ltd. / Garland Publishing, (1999)).

[0032] The general structure and properties of antibody CDRs have been described in the art. Briefly, in an antibody framework, CDRs are embedded within framework regions in the heavy and light chain variable regions, where they constitute the regions primarily responsible for antigen binding and recognition. A variable region typically contains at least three CDRs of the heavy or light chain (Kabat et al., 1991, Sequences of Proteins of Immunological Interest, Public Health Service NIH, Bethesda, Md.; see also Chothia and Lesk, 1987, J. Mol. Biol. 196:901-917; Chothia et al., 1989, Nature 342:877-883) within framework regions (framework regions 1-4 designated FR1, FR2, FR3, and FR4 by Kabat et al., 1991; see also Chothia and Lesk, 1987, supra).

[0033] Antibodies can include any constant region known in the art. Human light chains are classified as kappa and lambda light chains. Heavy chains are classified as mu, delta, gamma, alpha, or epsilon, defining the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively. IgG has several subclasses, including, but not limited to, IgG1, IgG2, IgG3, and IgG4. IgM has subclasses, including, but not limited to, IgM1 and IgM2. Embodiments of the present disclosure include all such classes or isotypes of antibodies. The light chain constant region can be, for example, a kappa- or lambda-type light chain constant region, e.g., a human kappa- or lambda-type light chain constant region. The heavy chain constant region can be, for example, an alpha-, delta-, epsilon-, gamma-, or mu-type heavy chain constant region, e.g., a human alpha-, delta-, epsilon-, gamma-, or mu-type heavy chain constant region. Thus, in various embodiments, the antibody is of the isotype IgA, IgD, IgE, IgG, or IgM, including any one of IgG1, IgG2, IgG3, or IgG4. In various aspects, the antibody comprises a constant region that contains one or more amino acid modifications relative to its naturally occurring counterpart to improve half-life / stability or to make the antibody more suitable for expression / manufacturability. In various cases, the antibody comprises a constant region in which the C-terminal Lys residue present in the naturally occurring counterpart has been removed or truncated.

[0034] The antibody may be a monoclonal antibody. In some embodiments, the antibody comprises a sequence substantially similar to a naturally occurring antibody produced by a vertebrate, e.g., a mammal, a bird, a reptile, or a fish. In some embodiments, the antibody comprises a sequence substantially similar to a naturally occurring antibody produced by a bird, e.g., a chicken, a duck, a quail, a pigeon, a turkey, or the like. In some embodiments, the antibody comprises a sequence substantially similar to a naturally occurring antibody produced by a mammal, e.g., a mouse, a rabbit, a goat, a horse, a hamster, a human, or the like. In this regard, the antibody can be considered a mammalian antibody, e.g., a mouse antibody, a rabbit antibody, a goat antibody, a horse antibody, a hamster antibody, a human antibody, or the like. In certain aspects, the antigen-binding protein is an antibody, e.g., a human antibody. In certain aspects, the antigen-binding protein is a chimeric antibody or a humanized antibody. The term "chimeric antibody" refers to an antibody that contains domains from two or more different antibodies. Chimeric antibodies may, for example, contain a constant domain from one species and a variable domain from a second species, or more commonly, may contain stretches of amino acid sequences from at least two species. Chimeric antibodies may also contain domains from two or more different antibodies of the same species. The term "humanized," when used in reference to antibodies, refers to an antibody having at least the CDR regions from a non-human source that has been engineered to have a structure and immunological function more similar to that of a true human antibody than the original source antibody. For example, humanization may involve transplanting CDRs from a non-human antibody, such as a murine antibody, into a human antibody. Humanization may also involve selective amino acid substitutions to make the non-human sequence more similar to a human sequence. Information, including sequence information for the heavy and light chain constant regions of human antibodies, is publicly available through the Uniprot database and other databases known to those skilled in the art of antibody engineering and generation. For example, the IgG2 constant region is available from the Uniprot database as Uniprot number P01859, which is incorporated herein by reference.

[0035] By way of example only, the sequence of the mouse IgG2a constant region includes: [Table 3] By way of example only, the sequence of the human immunoglobulin kappa constant (IGKC) domain includes: [Table 4]

[0036] Antibodies can be cleaved into fragments by enzymes such as papain and pepsin. Papain cleaves antibodies to generate two Fab fragments and a single Fc fragment. Pepsin cleaves antibodies to generate an F(ab')2 fragment and a pFc' fragment. In various aspects of the present disclosure, the antigen-binding proteins of the present disclosure are antigen-binding fragments of antibodies (also known as antigen-binding antibody fragments, antigen-binding fragments, or antigen-binding portions). In various cases, the antigen-binding antibody fragment is a Fab fragment or an F(ab')2 fragment.

[0037] Antibody architecture has been exploited to generate a diverse range of alternative antibody formats, spanning a molecular weight range of at least about 12-150 kDa, from monomers (n=1), to dimers (n=2), trimers (n=3), tetramers (n=4), and potentially even higher valencies (n). Such alternative antibody formats are referred to herein as "antibody protein formulations." Antibody protein formulations include those based on the complete antibody structure as well as antibody fragment mimics, such as scFv, Fab, and VHH / VH (discussed below), that retain full antigen-binding capacity. The smallest antigen-binding fragment that retains its complete antigen-binding site is the Fv fragment, which consists entirely of the variable (V) region. To stabilize the molecule, soluble and flexible amino acid peptide linkers are used to connect the V regions to form scFv (single-chain fragment variable) fragments, or constant (C) domains are added to the V regions to generate Fab fragments (fragment, antigen-binding). Both scFv and Fab fragments can be easily produced in host cells, e.g., prokaryotic host cells. Other antibody protein formulations include disulfide-stabilized scFv (ds-scFv), single-chain Fab (scFab), and dimeric and multimeric antibody formats, such as diabodies, triabodies, and tetrabodies, or minibodies (miniAbs), which include different formats consisting of scFv linked to an oligomerization domain. The smallest fragments are the VHH / VH of camelid heavy chain Abs and single-domain Abs (sdAbs). The building blocks most frequently used to generate novel antibody formats are single-chain variable (V) domain antibody fragments (scFv), which contain V domains (VH and VL domains) from the heavy and light chains linked by a peptide linker of approximately 15 amino acid residues. Peptibodies, or peptide-Fc fusions, are yet another antibody protein formulation. The peptibody structure consists of a biologically active peptide grafted onto an Fc domain. Peptibodies have been well described in the art.See, for example, Shimamoto et al., mAbs 4(5):586-591 (2012).

[0038] Other antibody protein formulations include single-chain antibodies (SCAs), diabodies, triabodies, tetrabodies, bispecific or trispecific antibodies, etc. Bispecific antibodies can be divided into five major classes: BsIgG, appended IgG, bispecific antibody (BsAb) fragments, bispecific fusion proteins, and BsAb conjugates. See, e.g., Spiess et al., Molecular Immunology 67(2) Part A:97-106(2015).

[0039] In various aspects, the antigen binding proteins of the present disclosure comprise, consist essentially of, or consist of any one of these antibody protein formulations. In various aspects, the antigen binding proteins of the present disclosure comprise, consist essentially of, or consist of any one of: scFv, Fab, VHH / VH, Fv fragment, ds-scFv, scFab, dimeric antibody, multimeric antibody (e.g., diabody, triabody, tetrabody), miniAb, peptibody, VHH / VH of camelid heavy chain antibody, sdAb, diabody; triabody; tetrabody; bispecific or trispecific antibody, BsIgG, adduct IgG, BsAb fragment, bispecific fusion protein, and BsAb conjugate.

[0040] In various cases, the antigen binding proteins of the present disclosure are antibody protein preparations in monomeric form, or in polymeric, oligomeric, or multimeric form. In certain embodiments, where an antibody comprises two or more distinct antigen-binding region fragments, the antibody is considered bispecific, trispecific, or multispecific, or bivalent, trivalent, or multivalent, depending on the number of distinct epitopes that the antibody recognizes and binds.

[0041] In various embodiments, the anti-CLDN6 antibody or antibody variant thereof is selected from the group consisting of a human antibody, a humanized antibody, a chimeric antibody, a monoclonal antibody, a recombinant antibody, an antigen-binding antibody fragment, a single-chain antibody, a monomeric antibody, a diabody, a triabody, a tetrabody, a Fab fragment, an IgG1 antibody, an IgG2 antibody, an IgG3 antibody, and an IgG4 antibody.

[0042] CLDN6 and epitopes The antigen binding proteins of the present disclosure bind to the cytoplasmic domain of CLDN6. In various embodiments, the CLDN6 is human CLDN6 having the following amino acid sequence: [ka]

[0043] In various embodiments, the cytoplasmic domain or tail of CLDN6 has the amino acid sequence CCTCPSGGSQGPSHYMARYSTSAPAISRGPSEYPTKNYV (SEQ ID NO: 3).

[0044] In various embodiments, human CLDN6 comprises the amino acid sequence of any one of SEQ ID NOs: 1-3.

[0045] In various aspects, the antigen-binding proteins of the present disclosure bind to an epitope within the amino acid sequence of the cytoplasmic domain of CLDN6. In various aspects, the CLDN6 is human CLDN6, and the antigen-binding proteins of the present disclosure bind to an epitope within the amino acid sequence of the cytoplasmic domain of human CLDN6, e.g., SEQ ID NOs: 3, 10, 15, and 95-99. "Epitope" refers to 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" refers to a region of CLDN6 or a region within CLDN6 to which the antigen-binding protein binds, which region is composed of consecutive amino acids in the amino acid sequence of CLDN6. The amino acids of a linear epitope are adjacent to each other in the primary structure of CLDN6. Thus, a linear epitope is an antigen, i.e., a fragment or portion of the amino acid sequence of CLDN6. In various other embodiments, the epitope is a conformational epitope or a structural epitope. "Conformational epitope" or "structural epitope" refers to an epitope composed of amino acids that are located adjacent to each other only when CLDN6 is in its properly folded state. Unlike a linear epitope, the amino acids of a conformational epitope or a structural epitope are not adjacent to each other in the primary structure (i.e., amino acid sequence) of CLDN6. A conformational epitope or a structural epitope is not composed of consecutive amino acids in the amino acid sequence of the antigen (CLDN6).

[0046] In various embodiments, the epitope is located within the cytoplasmic domain of CLDN6, e.g., human CLDN6. In various embodiments, the antigen binding protein binds to the cytoplasmic domain of CLDN6 having the amino acid sequence of CCTCPSGGSQGPSHYMARYSTSAPAISRGPSEYPTKNYV (SEQ ID NO: 3). In various embodiments, the antigen binding protein binds to the cytoplasmic domain of CLDN6 or a peptide having the amino acid sequence of RYSTSAPAISRGPSE (SEQ ID NO: 100). In various embodiments, the antigen binding protein binds to the cytoplasmic domain of CLDN6 or a peptide having the amino acid sequence of APAISRGPSEYPTKN (SEQ ID NO: 101).

[0047] In various embodiments, the epitope or peptide to which the antigen binding protein binds is within any of SEQ ID NOs: 1-3, 10, 15, 95-99.

[0048] In various aspects, the antigen binding protein binds to human CLDN6 and non-human CLDN6. In various cases, the non-human CLDN6 is CLDN6 from a chimpanzee, a rhesus monkey, a dog, a cow, a mouse, a rat, a zebrafish, or a frog. In various cases, the antigen binding protein binds to human CLDN6 and mouse CLDN6.

[0049] Affinity and Avidity The antigen binding proteins provided herein bind to CLDN6 non-covalently and reversibly. In various embodiments, the binding strength of an antigen binding protein to CLDN6 can be expressed as its affinity, i.e., a measure of the strength of the interaction between the binding site of the antigen binding protein and the epitope. In various aspects, the antigen binding proteins provided herein have high affinity for CLDN6 and therefore bind to a greater amount of CLDN6 in a shorter period of time than low-affinity antigen binding proteins. In various aspects, the antigen binding protein has a binding affinity of at least 10 5 M -1 , at least 10 6 M -1 , at least 10 7M -1 , at least 10 8 M -1 , at least 10 9 M -1 , or at least 10 10 M -1 or at least 10 10 M -1 The equilibrium association constant K A As will be appreciated by those skilled in the art, K A can be affected by factors including pH, temperature and buffer composition.

[0050] In various embodiments, the binding strength of an antigen binding protein to CLDN6 can be expressed as its sensitivity. D is the equilibrium dissociation constant between the antigen-binding protein and CLDN6, i.e., k off / k on It is a ratio. K D and K. A is inversely proportional. D The value relates to the concentration of the antigen-binding protein (the amount of antigen-binding protein required for a particular experiment) and is therefore K D The lower the value (the lower the concentration), the higher the affinity of the antigen binding protein. In various embodiments, the binding strength of an antigen binding protein to CLDN6 is expressed as K D In various aspects, the K of the antigen binding proteins provided herein can be expressed as D is about 10 -1 M, about 10 -2 M, about 10 -3 M, about 10 -4 M, about 10 -5 M, about 10 -6 In various embodiments, the K of the antigen binding proteins provided herein is D is micromolar, nanomolar, picomolar, or femtomolar. In various embodiments, the K of the antigen binding proteins provided herein D is about 10 -4 ~10 -6 M or 10 -7 ~10 -9 M or 10 -10 ~10-12 M or 10 -13 ~10 -15 In various embodiments, the K of the antigen binding proteins provided herein is within the range of D is approximately 1.0 x 10 -12 M ~ approx. 1.0×10 -8 In various embodiments, the K of the antigen binding protein is within the range of D is approximately 1.0 x 10 -11 M ~ approx. 1.0×10 -9 It is within the range of M.

[0051] In various embodiments, the affinity of antigen-binding proteins is measured or ranked using flow cytometry or fluorescence-activated cell sorting (FACS)-based assays. Flow cytometry-based binding assays are 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 relative affinity of CLDN6 antibodies is determined via a FACS-based assay in which various concentrations of fluorophore-conjugated CLDN6 antibodies are incubated with purified cytoplasmic domain or peptides of CLDN6 coated on solid structures such as beads, and the emitted fluorescence, which is a direct measure of antibody-antigen binding, is determined. A curve is generated plotting fluorescence against each dose or concentration. The maximum is the lowest concentration at which fluorescence plateaus or reaches a maximum, which is when binding saturation occurs. Half of the maximum is considered to be the EC50 or IC50, and the antibody with the lowest EC50 / IC50 is considered to have the highest affinity relative to other antibodies similarly tested.

[0052] In various embodiments, the IC determined in a competitive binding inhibition assay 50 The value is the K DIn various embodiments, the antigen binding protein competes with the reference antibody for binding to human CLDN6, thereby reducing the amount of human CLDN6 that binds to the reference antibody as determined by an in vitro competitive binding assay. In various embodiments, the antigen binding protein of the present disclosure competes with the reference antibody for binding to human CLDN6, thereby reducing the amount of human CLDN6 that binds to the reference antibody as determined by an in vitro competitive binding assay. In various embodiments, the antigen binding protein of the present disclosure inhibits the binding interaction of human CLDN6 with the reference antibody, which inhibition is measured by an IC 50 In various embodiments, the antigen binding protein has an IC of less than about 2500 nM when inhibiting the binding interaction between human CLDN6 and the reference antibody. 50 In various embodiments, the antigen binding protein exhibits an IC of less than about 2000 nM, less than about 1500 nM, less than about 1000 nM, less than about 900 nM, less than about 800 nM, less than about 700 nM, less than about 600 nM, less than about 500 nM, less than about 400 nM, less than about 300 nM, less than about 200 nM, or less than about 100 nM. 50 In various embodiments, the antigen binding protein exhibits an IC of less than about 90 nM, less than about 80 nM, less than about 70 nM, less than about 60 nM, less than about 50 nM, less than about 40 nM, less than about 30 nM, less than about 20 nM, or less than about 10 nM. 50 In various cases, the antigen binding proteins of the present disclosure compete for binding to CLDN6 with a reference antibody known to bind to CLDN6 (wherein the reference antibody is different from any of the antigen binding proteins of the present disclosure). See further description below.

[0053] Competition assay Avidity provides a measure of the overall strength of the antibody-antigen complex. It depends on three main parameters: the affinity of the antigen-binding protein for the epitope, the valency of both the antigen-binding protein and CLDN6, and the structural arrangement of the interacting moieties. The greater the valency (number of antigen-binding sites) of the antigen-binding protein, the greater the amount of antigen (CLDN6) it can bind. In various embodiments, the antigen-binding protein has strong avidity for CLDN6. In various embodiments, the antigen-binding protein is multivalent. In various embodiments, the antigen-binding protein is bivalent. In various cases, the antigen-binding protein is monovalent.

[0054] Cross-reactivity In various embodiments, the antigen binding proteins of the present disclosure bind to CLDN6 and do not bind to any other members of the CLDN family, e.g., do not cross-react with any other members of the CLDN family. In various cases, the antigen binding proteins of the present disclosure are specific for CLDN6. In various embodiments, the antigen binding proteins of the present disclosure have selectivity for CLDN6 that is at least 10-fold, 5-fold, 4-fold, 3-fold, or 2-fold greater than the selectivity of the antigen binding protein for CLDN3, CLDN4, CLDN9, or a combination thereof. In various embodiments, the antigen binding proteins of the present disclosure have selectivity for CLDN6 that is at least 10-fold, 5-fold, 4-fold, 3-fold, or 2-fold greater than the selectivity of the antigen binding protein for each of CLDN3, CLDN4, and CLDN9. Selectivity is measured by the K D may be based on K D can be determined by techniques known in the art, for example, surface plasmon resonance, FACS-based affinity assays.

[0055] Competition assay In various embodiments, the antigen binding protein inhibits the binding interaction between human CLDN6 and a reference antibody, where the reference antibody is known to bind to CLDN6 but is not an antigen binding protein of the present disclosure. In various cases, the antigen binding protein of the present disclosure competes with the reference antibody for binding to human CLDN6, thereby reducing the amount of human CLDN6 that binds to the reference antibody as determined by an in vitro competitive binding assay. In various embodiments, the reference antibody binds to an epitope within the amino acid sequence of the cytoplasmic domain of human CLDN6. In various aspects, the antigen binding protein of the present disclosure inhibits the binding interaction between human CLDN6 and the reference antibody, where the inhibition is determined by an IC 50 In various embodiments, the antigen binding protein has an IC of less than about 2500 nM when inhibiting the binding interaction between human CLDN6 and the reference antibody. 50 In various embodiments, the antigen binding protein exhibits an IC of less than about 2000 nM, less than about 1500 nM, less than about 1000 nM, less than about 900 nM, less than about 800 nM, less than about 700 nM, less than about 600 nM, less than about 500 nM, less than about 400 nM, less than about 300 nM, less than about 200 nM, or less than about 100 nM. 50 In various embodiments, the antigen binding protein exhibits an IC of less than about 90 nM, less than about 80 nM, less than about 70 nM, less than about 60 nM, less than about 50 nM, less than about 40 nM, less than about 30 nM, less than about 20 nM, or less than about 10 nM. 50 Shows.

[0056] In various cases, the antigen binding protein of the present disclosure competes with a reference antibody for binding to human CLDN6, thereby reducing the amount of human CLDN6 that binds to the reference antibody as determined by 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 fluorophore-conjugated secondary antibody that binds to the Fc of the reference antibody in the absence or presence of a specific amount of an antigen binding protein of the present disclosure. In various embodiments, the FACS-based assay is performed using the reference antibody, the fluorophore-conjugated secondary antibody, and cells that express CLDN6. In various embodiments, the cells are genetically engineered to overexpress CLDN6. In some embodiments, the cells are HEK293T cells transduced with a viral vector to express CLDN6. In alternative embodiments, the cells endogenously express CLDN6. Prior to performing the FACS-based assay, in some embodiments, the cells that endogenously express CLDN6 are predetermined as CLDN6 low-expressing cells or CLDN6 high-expressing cells. In some aspects, the cell is a cancer or tumor cell. In various aspects, the cell is derived from a cell line, such as an ovarian cell line, an endometrial cell line, a bladder cell line, a lung cell line, a gastrointestinal (GI) cell line, a liver cell line, a lung cell line, etc. In various aspects, the cell that endogenously expresses CLDN6 is selected from the group consisting of OVCA429 ovarian cells, ARK2 endometrial cells, OAW28 ovarian cells, UMUC-4 bladder cells, PEO14 ovarian cells, OV177 ovarian cells, H1693 lung cells, MKN7 upper GI cells, OV-90 ovarian cells, HUH-7 liver cells, JHOS-4 ovarian cells, H1435 lung cells, and NUGC3 upper GI cells. In various cases, the antigen binding protein of the present disclosure binds with high affinity to CLDN6 endogenously expressed by one or more of ARK2 cells, OVCA429 cells, LS513 cells, or MCF7 cells. In various embodiments, the antigen binding protein has an IC of less than about 3000 nM as determined in a FACS-based competitive binding inhibition assay using one or more of ARK2 cells, OVCA429 cells, LS513 cells, or MCF7 cells. 50In various embodiments, the antigen binding protein exhibits an IC of less than about 2500 nM, less than about 2000 nM, less than about 1750 nM, less than about 1500 nM, less than about 1250 nM, less than about 1000 nM, less than about 750 nM, or less than about 500 nM as determined in a FACS-based competitive binding inhibition assay using one or more of ARK2 cells, OVCA429 cells, LS513 cells, or MCF7 cells. 50 In various embodiments, the antigen binding protein exhibits an IC of less than about 400 nM, less than about 300 nM, less than about 200 nM, less than about 100 nM, less than about 75 nM, less than about 50 nM, less than about 25 nM, or less than about 10 nM as determined in a FACS-based competitive binding inhibition assay using one or more of ARK2 cells, OVCA429 cells, LS513 cells, or MCF7 cells. 50 Shows.

[0057] Other binding assays, such as competitive binding assays or competition assays, that test the ability of an antibody to compete with a second antibody for binding to an antigen or its epitope are 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). See U.S. Patent Application Publication No. US20140178905; Chand et al., Biologicals 46:168-171 (2017); Liu et al., Anal Biochem 525:89-91 (2017); and Goolia et al., J Vet Diagn Invest 29(2):250-253 (2017). Other methods for comparing two antibodies are also known in the art, including, for example, surface plasmon resonance (SPR), which can be used to determine the binding constant of an antibody and a second antibody, and the two binding constants can be compared.

[0058] Antibody production methods and related methods Suitable methods for producing antigen-binding proteins (e.g., antibodies, antigen-binding antibody fragments, and antibody protein preparations) are known in the art. For example, standard hybridoma methods for producing antibodies are described, for example, in Harlow and Lane (eds.), Antibodies: A Laboratory Manual, CSH Press (1988), and CA. Janeway et al. (eds.), Immunobiology, 5 th Ed., Garland Publishing, New York, NY (2001). Various methods for preparing the CLDN6 monoclonal antibodies of the present disclosure are provided in the Examples herein.

[0059] Depending on the host species, various adjuvants can be used to enhance the immunological response and result in greater antibody production by the host. Such adjuvants include, but are not limited to, Freund's adjuvant, inorganic gels such as aluminum hydroxide, and surfactants such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, and dinitrophenol. BCG (bacilli Calmette-Guerin) and Corynebacterium parvum are potentially useful human adjuvants.

[0060] Other antibody production methods are summarized in Table 3. [Table 5]

[0061] Regardless of the method for generating an antibody, methods for testing the antibody for its ability to bind to an epitope of CLDN6 are known in the art and include any antibody-antigen binding assay, such as radioimmunoassay (RIA), ELISA, Western blot, immunoprecipitation, SPR, and competitive inhibition assay (see, e.g., Janeway et al., below, and U.S. Patent Application Publication No. 2002 / 0197266, and the above section on competitive assays). In one example, the binding assay was a bead-based flow assay by indirect flow cytometry using beads conjugated with BSA-peptide conjugates or formaldehyde-fixed GST fusion proteins (Press et al., Steroids, 67 (2002) 799-813).

[0062] Arrays / Structures (a) a heavy chain (HC) complementarity determining region (CDR) 1 amino acid sequence set forth in Table 4, or a variant sequence thereof that differs by only one or two amino acids or has at least or about 70% (e.g., at least about 80%, at least about 85%, at least about 90%, at least about 95%) sequence identity; (b) a HC CDR2 amino acid sequence set forth in Table 4, or a variant sequence thereof that differs by only one or two amino acids or has at least or about 70% (e.g., at least about 80%, at least about 85%, at least about 90%, at least about 95%) sequence identity; (c) a HC CDR2 amino acid sequence set forth in Table 4, or a variant sequence thereof that differs by only one or two amino acids or has at least or about 70% (e.g., at least about 80%, at least about 85%, at least about 90%, at least about 95%) sequence identity; (d) a light chain (LC) CDR1 amino acid sequence set forth in Table 4, or a variant sequence thereof which differs by only one or two amino acids or which has at least or about 70% (e.g., at least about 80%, at least about 85%, at least about 90%, at least about 95%) of sequence identity; (e) a LC CDR2 amino acid sequence set forth in Table 4, or a variant sequence thereof which differs by only one or two amino acids or which has at least or about 70% (e.g., at least about 80%, at least about 85%, at least about 90%, at least about 95%) of sequence identity; (f) a LC CDR3 amino acid sequence set forth in Table 4, or a variant sequence thereof which differs by only one or two amino acids or which has at least or about 70% (e.g., at least about 80%, at least about 85%, at least about 90%, at least about 95%) of sequence identity; Provided herein are antigen binding proteins comprising a CDR3 amino acid sequence, or a variant sequence thereof that differs by only one or two amino acids or has at least or about 70% (e.g., at least about 80%, at least about 85%, at least about 90%, at least about 95%) sequence identity, or a combination of any two or more of (g)(a)-(f). [Table 6] [Table 7] [Table 8] [Table 9]

[0063] In various aspects, the antigen binding protein comprises an LC CDR1 amino acid sequence, an LC CDR2 amino acid sequence, and an LC CDR3 amino acid sequence set forth in Table 4, and at least one or two of the HC CDR amino acid sequences set forth in Table 4. In various aspects, the antigen binding protein comprises an HC CDR1 amino acid sequence, an HC CDR2 amino acid sequence, and an HC CDR3 amino acid sequence set forth in Table 4, and at least one or two of the LC CDR amino acid sequences set forth in Table 4.

[0064] In various embodiments, the antigen binding protein comprises at least three, four, or five of the amino acid sequences set forth by the SEQ ID NOs in a row in Table 4. In various embodiments, the antigen binding protein comprises each of the LC CDR amino acid sequences set forth by the SEQ ID NOs in a row in Table 4 and at least one or two of the HC CDR amino acid sequences set forth by the SEQ ID NOs in a row in Table 4. In various embodiments, the antigen binding protein comprises each of the HC CDR amino acid sequences set forth by the SEQ ID NOs in a row in Table 4 and at least one or two of the LC CDR amino acid sequences set forth by the SEQ ID NOs in a row in Table 4. In various embodiments, the antigen binding protein comprises all six of the CDR amino acid sequences set forth by the SEQ ID NOs in a row in Table 4. In various embodiments, the antigen binding protein comprises six CDR amino acid sequences selected from the group consisting of (a) SEQ ID NOs: 18-20, 25-27, and (b) SEQ ID NOs: 34-36, 41-43.

[0065] In various cases, the amino acid sequences in Table 4 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 cases, there are about 10 to about 20 amino acids between the LC CDR1 and LC CDR2 sequences, and about 25 to about 40 amino acids between the LC CDR2 and LC CDR3 sequences. In various cases, there are about 14 to about 16 amino acids between the LC CDR1 and LC CDR2 sequences, and about 30 to about 35 amino acids between the LC CDR2 and LC CDR3 sequences. In various cases, there are about 10 to about 20 amino acids between the HC CDR1 and HC CDR2 sequences, and about 25 to about 40 amino acids between the HC CDR2 and HC CDR3 sequences. In various cases, there are about 14 to about 16 amino acids between the HC CDR1 sequence and the HC CDR2 sequence, and there are about 30 to about 35 amino acids between the HC CDR2 sequence and the HC CDR3 sequence.

[0066] In various embodiments, the antigen binding protein comprises (a) a heavy chain variable region amino acid sequence set forth in Table 5, or a sequence selected from the group consisting of SEQ ID NOs: 16, 32, or a variant sequence thereof that differs by only one or two amino acids or has at least or about 70% (e.g., at least about 80%, at least about 85%, at least about 90%, at least about 95%) sequence identity; or (b) a light chain variable region amino acid sequence set forth in Table 5, or a sequence selected from the group consisting of SEQ ID NOs: 17, 33, or a variant sequence thereof that differs by only one or two amino acids or has at least or about 70% (e.g., at least about 80%, at least about 85%, at least about 90%, at least about 95%) sequence identity; or (c) both (a) and (b). [Table 10]

[0067] In various embodiments, the antigen binding protein comprises a pair of amino acid sequences selected from the group consisting of: (a) SEQ ID NOs: 16-17, and (b) SEQ ID NOs: 32-33.

[0068] In various embodiments, the antigen binding protein comprises an amino acid sequence similar to those described above, but the antigen binding protein substantially retains its biological function, e.g., the ability to bind to human CLDN6, detect the presence or quantitate the presence of human CLDN6 in a sample.

[0069] In various embodiments, the antigen binding protein comprises an amino acid sequence that differs by only 1, 2, 3, 4, 5, 6, or more amino acids compared to the amino acid sequence(s) set forth above. In various embodiments, the antigen binding protein comprises a variant sequence of the referenced sequence, which variant sequence differs by only one or two amino acids compared to the referenced sequence. In various embodiments, the antigen binding protein comprises one or more amino acid substitutions that occur outside the CDRs, for example, one or more amino acid substitutions that occur within the framework region(s) of the heavy or light chain. In various embodiments, the antigen binding protein comprises one or more amino acid substitutions, but the antigen binding protein retains the amino acid sequence of the six CDRs. In various embodiments, the antigen binding protein comprises an amino acid sequence with only 1, 2, 3, 4, 5, 6, or more conservative amino acid substitutions compared to the amino acid sequence(s) set forth above. As used herein, the term "conservative amino acid substitution" refers to the replacement of an amino acid with another amino acid having similar properties, e.g., size, charge, hydrophobicity, hydrophilicity, and / or aromaticity, including exchanges within one of the following five groups: I. Small aliphatic, non-polar or slightly polar residues: Ala, Ser, Thr, Pro, Gly; II. Polar Negatively Charged Residues and Their Amides and Esters: Asp, Asn, Glu, Gln, cysteic acid and homocysteic acid; III. Polar positively charged residues: His, Arg, Lys; Ornithine (Orn) IV. Large aliphatic nonpolar residues: Met, Leu, Ile, Val, Cys, norleucine (Nle), homocysteine V. Large aromatic residues: Phe, Tyr, Trp, acetylphenylalanine.

[0070] In various embodiments, conservative amino acid substitutions are exchanges within one of the following groups of amino acids: I. Aliphatic amino acids: Gly, Ala, Val, Leu, Ile II. Non-aromatic amino acids containing side chain hydroxyl: Ser, Thr III. Amino acids containing sulfur side chains: Cys, Met IV: Amino acids containing aromatic rings in the side chain: Phe, Tyr, Trp V: Acidic amino acids: Glu; Asp VI: Basic amino acids: Arg; Lys VII: Amino acids containing side chain amides: Gln, Asn VIII: Amino acids containing side chain imidazole: His, alpha-dimethylimidiazoleacetic acid (DMIA) IX: Imino acids: Pro, 4-hydroxy-Pro, 4-amino-Pro.

[0071] In various embodiments, the antigen binding protein comprises an amino acid sequence having greater than or about 30%, greater than or about 50%, or greater than or about 70% sequence identity to the amino acid sequence set forth above. In various embodiments, the antigen binding protein comprises an amino acid sequence having at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, or greater than 90% sequence identity to the amino acid sequence set forth above. In various embodiments, the antigen binding protein comprises an amino acid sequence having at least 70%, at least 80%, at least 85%, at least 90%, or greater than 90% sequence identity to the full length of the amino acid sequence set forth above. In various embodiments, the antigen binding protein comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity to the full length of the amino acid sequence set forth above.

[0072] In various embodiments, the antigen binding proteins comprise variant sequences of the referenced sequences, which variant sequences have at least or about 70% sequence identity to the sequences described above. In various embodiments, the antigen binding proteins comprise variant sequences of the referenced sequences, which variant sequences have at least or about 80% sequence identity to the sequences described above. In various embodiments, the antigen binding proteins comprise variant sequences of the referenced sequences, which variant sequences have at least or about 90% sequence identity to the sequences described above. In various embodiments, the antigen binding proteins comprise variant sequences of the referenced sequences, which variant sequences have at least or about 95% sequence identity to the sequences described above.

[0073] In various embodiments, the antigen binding protein comprises one, two, three, four, or five of the sequences of SEQ ID NOs: 18-20, 25-27, 34-36, 41-43 in a row in Table 4, and at least one variant sequence having at least or about 70% (e.g., at least about 80%, at least about 85%, at least about 90%, at least about 95%) sequence identity to any of SEQ ID NOs: 18-20, 25-27, 34-36, 41-43. In various embodiments, the antigen binding protein comprises one, two, three, four, or five sequences of a set of sequences selected from (a) SEQ ID NOs: 18-20, 25-27, and (b) SEQ ID NOs: 34-36, 41-43, wherein the antigen binding protein further comprises at least one variant sequence having at least or about 70% (e.g., at least about 80%, at least about 85%, at least about 90%, at least about 95%) sequence identity to at least one of the sequences in said set.

[0074] In various embodiments, the antigen binding protein comprises a pair of variant sequences that share at least or about 70% (e.g., at least about 80%, at least about 85%, at least about 90%, at least about 95%) sequence identity to any of SEQ ID NOs: 16-17, 32-33. In various cases, the antigen binding protein comprises a pair of variant sequences that share at least or about 70% (e.g., at least about 80%, at least about 85%, at least about 90%, at least about 95%) sequence identity to (a) SEQ ID NOs: 16-17 and (b) SEQ ID NOs: 32-33. In various embodiments, the antigen binding protein comprises a pair of sequences: one sequence in Table 5 and another sequence that is a variant sequence that shares at least or about 70% (e.g., at least about 80%, at least about 85%, at least about 90%, at least about 95%) sequence identity to any of SEQ ID NOs: 16-17, 32-33. In various embodiments, the antigen binding protein comprises a pair of sequences: (a) one sequence selected from SEQ ID NOs: 16-17, and (b) SEQ ID NOs: 32-33, and another sequence that is a variant sequence having at least or about 70% (e.g., at least about 80%, at least about 85%, at least about 90%, at least about 95%) sequence identity to sequence (a)-(b). In various cases, the antigen binding protein comprises an amino acid sequence of the above-described amino acid sequence with one or more amino acid substitutions to reduce or eliminate reactive amino acids to reduce or prevent undesired side chain reactions. For example, the antigen binding protein comprises an amino acid sequence of the above-described amino acid sequence with one or more of: (i) a Trp residue substituted with His, Tyr, or Phe; (ii) an Asn residue substituted with Gln, Ser, Ala, or Asp; (iii) an Asp residue immediately preceding a Pro residue substituted with Ala, Ser, or Glu; (iv) an Asn residue substituted with Gln, Ser, or Ala; and / or (v) a Cys residue substituted with Tyr, Ser, or Ala. In various aspects, the antigen binding protein comprises an amino acid sequence of the above-described amino acid sequence with an amino acid substitution that is predicted to have greater binding affinity, greater stability, or other beneficial properties based on SHM events or based on statistical analysis of a large number of other similar antibody sequences.

[0075] In various cases, the amino acid sequences in Table 4 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 cases, there are about 10 to about 20 amino acids between the LC CDR1 and LC CDR2 sequences, and about 25 to about 40 amino acids between the LC CDR2 and LC CDR3 sequences. In various cases, there are about 14 to about 16 amino acids between the LC CDR1 and LC CDR2 sequences, and about 30 to about 35 amino acids between the LC CDR2 and LC CDR3 sequences. In various cases, there are about 10 to about 20 amino acids between the HC CDR1 and HC CDR2 sequences, and about 25 to about 40 amino acids between the HC CDR2 and HC CDR3 sequences. In various cases, there are about 14 to about 16 amino acids between the HC CDR1 sequence and the HC CDR2 sequence, and there are about 30 to about 35 amino acids between the HC CDR2 sequence and the HC CDR3 sequence.

[0076] Nucleic Acids The present disclosure further provides nucleic acids comprising nucleotide sequences encoding the antigen-binding proteins of the present disclosure. "Nucleic acid," as used herein, includes "polynucleotides," "oligonucleotides," and "nucleic acid molecules," and generally refers to a polymer of DNA or RNA, or modified forms thereof, which may be single- or double-stranded, may be synthesized or obtained (e.g., isolated and / or purified) from natural sources, may contain natural, non-natural, or modified nucleotides, and may contain natural, non-natural, or modified internucleotide linkages, such as phosphoramidate or phosphorothioate linkages, in place of the phosphodiesters found between nucleotides in unmodified oligonucleotides. A nucleic acid may include any nucleotide sequence that encodes any of the antigen-binding proteins of the present disclosure. In various embodiments, the nucleic acid is selected from the group consisting of: (a) a heavy chain (HC) complementarity determining region (CDR) 1 amino acid sequence set forth in Table 4, or a sequence selected from the group consisting of SEQ ID NOs: 18, 34, or a variant sequence thereof that differs by only one or two amino acids or has at least or about 70% (e.g., at least or about 80%, at least or about 85%, at least or about 90%, at least or about 95%) sequence identity; (b) a HC CDR2 amino acid sequence set forth in Table 4, or a sequence selected from the group consisting of SEQ ID NOs: 19, 35, or a variant sequence thereof that differs by only one or two amino acids or has at least or about 70% (e.g., at least or about 80%, at least or about 85%, at least or about 90%, at least or about 95%) sequence identity; (c) a HC CDR2 amino acid sequence set forth in Table 4, or a sequence selected from the group consisting of SEQ ID NOs: 19, 35, or a variant sequence thereof that differs by only one or two amino acids or has at least or about 70% (e.g., at least or about 80%, at least or about 85%, at least or about 90%, at least or about 95%) sequence identity;(d) a light chain (LC) CDR1 amino acid sequence set forth in Table 4, or a sequence selected from the group consisting of SEQ ID NOs: 25, 41, or a variant sequence thereof differing by only one or two amino acids or having at least or about 70% (e.g., at least or about 80%, at least or about 85%, at least or about 90%, at least or about 95%) sequence identity; (e) a light chain (LC) CDR2 amino acid sequence set forth in Table 4, or a sequence selected from the group consisting of SEQ ID NOs: 25, 41, or a variant sequence thereof differing by only one or two amino acids or having at least or about 70% (e.g., at least or about 80%, at least or about 85%, at least or about 90%, at least or about 95%) sequence identity; (f) a LC CDR2 amino acid sequence set forth in Table 4, or a sequence selected from the group consisting of SEQ ID NOs: 27, 43, or a variant sequence thereof that differs by only one or two amino acids or has at least or about 70% (e.g., at least or about 80%, at least or about 85%, at least or about 90%, at least or about 95%) sequence identity; (g) a nucleotide sequence encoding an antigen binding protein comprising a combination of any two or more of (a)-(f). In various aspects, the nucleic acid comprises a nucleotide sequence encoding an antigen binding protein comprising the LC CDR1 amino acid sequence, LC CDR2 amino acid sequence, and LC CDR3 amino acid sequence set forth in Table 4 and at least one or two of the HC CDR amino acid sequences set forth in Table 4. In various embodiments, the nucleic acid comprises a HC CDR1 amino acid sequence, a HC CDR2 amino acid sequence, and a HC CDR3 amino acid sequence set forth in Table 4, and a LC CDR1 amino acid sequence set forth in Table 4.In various embodiments, the nucleic acid comprises a nucleotide sequence encoding an antigen binding protein comprising at least one or two of the LC CDR amino acid sequences set forth in a row of SEQ ID NOs in Table 4 and at least three, four, or five of the amino acid sequences set forth in a row of SEQ ID NOs in Table 4, (b) each of the LC CDR amino acid sequences set forth in a row of SEQ ID NOs in Table 4 and at least one or two of the HC CDR amino acid sequences set forth in a row of SEQ ID NOs in Table 4, (c) each of the HC CDR amino acid sequences set forth in a row of SEQ ID NOs in Table 4 and at least one or two of the LC CDR amino acid sequences set forth in a row of SEQ ID NOs in Table 4.(d) all six of the CDR amino acid sequences set forth by the SEQ ID NOs in a horizontal row in Table 4, and / or (e) a nucleotide sequence encoding an antigen-binding protein comprising six CDR amino acid sequences selected from the group consisting of (a) SEQ ID NOs: 18 to 20, 25 to 27, and (b) SEQ ID NOs: 34 to 36, 41 to 43. In various embodiments, the nucleic acid comprises a nucleotide sequence encoding (a) a heavy chain variable region amino acid sequence set forth in Table 5, or a sequence selected from the group consisting of SEQ ID NOs: 16 and 32, or a variant sequence thereof that differs by only one or two amino acids or has at least or about 70% (e.g., at least or about 80%, at least or about 85%, at least or about 90%, at least or about 95%) sequence identity, or (b) a light chain variable region amino acid sequence set forth in Table 5, or a sequence selected from the group consisting of SEQ ID NOs: 17 and 33, or a variant sequence thereof that differs by only one or two amino acids or has at least or about 70% (e.g., at least or about 80%, at least or about 85%, at least or about 90%, at least or about 95%) sequence identity, or (c) an antigen binding protein comprising both (a) and (b). 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 (a) SEQ ID NOs: 16-17, and (b) SEQ ID NOs: 32-33. In various embodiments, the nucleic acid comprises a nucleotide sequence encoding an antigen binding protein comprising an amino acid sequence selected from the group set forth in Tables 4-5. In some embodiments, the nucleic acid does not contain any insertions, deletions, inversions, and / or substitutions. In other embodiments, the nucleic acid comprises one or more insertions, deletions, inversions, and / or substitutions.

[0077] In various embodiments, the nucleic acid comprises a nucleotide sequence encoding an antigen binding protein as set forth in Tables 4-5 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 the heavy chain (HC) variable region or the light chain (LC) variable region, or both. In exemplary embodiments, the nucleic acid comprises a nucleotide sequence encoding an antigen binding protein comprising a HC of SEQ ID NO: 16 having one, two, three, four, or five amino acid substitutions. In exemplary embodiments, the nucleic acid comprises a nucleotide sequence encoding an antigen binding protein comprising a HC CDR1 of SEQ ID NO: 18, a HC CDR2 of SEQ ID NO: 19, a HC CDR3 of SEQ ID NO: 20, or a combination thereof. In exemplary cases, the nucleic acid comprises a nucleotide sequence encoding an antigen binding protein comprising an HC of SEQ ID NO: 16. In some embodiments, the nucleic acid comprises a nucleotide sequence encoding an antigen binding protein comprising an HC of any one of SEQ ID NOs: 18-20. In exemplary embodiments, the nucleic acid comprises a nucleotide sequence encoding an antigen binding protein comprising an LC of SEQ ID NO: 17 with one, two, three, four, or five amino acid substitutions. In various cases, the nucleic acid comprises a nucleotide sequence encoding a light chain variable region comprising an LC CDR1 of SEQ ID NO: 25, an LC CDR2 of SEQ ID NO: 26, an LC CDR3 of SEQ ID NO: 27, or a combination thereof. In some embodiments, the nucleic acid comprises a nucleotide sequence encoding an antigen binding protein comprising an LC of any one of SEQ ID NOs: 25-27. In exemplary embodiments, the nucleic acid comprises a nucleotide sequence encoding an antigen binding protein comprising an HC of SEQ ID NO: 32 with one, two, three, four, five, or six amino acid substitutions. In exemplary embodiments, the nucleic acid comprises a nucleotide sequence encoding an antigen binding protein comprising an HC CDR1 of SEQ ID NO: 34, an HC CDR2 of SEQ ID NO: 35, an HC CDR3 of SEQ ID NO: 36, or a combination thereof. In an exemplary case, the nucleic acid comprises a nucleotide sequence encoding an antigen binding protein comprising the HC of SEQ ID NO:32.In some embodiments, the nucleic acid comprises a nucleotide sequence encoding an antigen binding protein comprising an HC of any one of SEQ ID NOs: 34-26. In exemplary embodiments, the nucleic acid comprises a nucleotide sequence encoding an antigen binding protein comprising an LC of SEQ ID NO: 33 with 1, 2, 3, 4, or 5 amino acid substitutions. In various cases, the nucleic acid comprises a nucleotide sequence encoding a light chain variable region comprising an LC CDR1 of SEQ ID NO: 41, an LC CDR2 of SEQ ID NO: 42, an LC CDR3 of SEQ ID NO: 43, or a combination thereof. In some embodiments, the nucleic acid comprises a nucleotide sequence encoding an antigen binding protein comprising an LC of any one of SEQ ID NOs: 41-43.

[0078] In some embodiments, the nucleic acids of the present disclosure are recombinant. As used herein, the term "recombinant" refers to (i) a molecule constructed outside a living cell by combining natural or synthetic nucleic acid segments into a nucleic acid molecule capable of replicating in the living cell, or (ii) a molecule resulting from replication of the molecule described in (i) above. For purposes of this specification, replication may be in vitro or in vivo replication.

[0079] In some embodiments, nucleic acids are constructed based on chemical synthesis and / or enzymatic ligation reactions using procedures known in the art. See, e.g., Sambrook et al., supra, and Ausubel et al., supra. For example, nucleic acids can be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides (e.g., phosphorothioate derivatives and acridine-substituted nucleotides) designed to increase the biological stability of the molecule or to increase the physical stability of the duplex formed upon hybridization. Examples of modified nucleotides that can be used to generate nucleic acids include, but are not limited to, 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxymethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N-acetyl-3-methyl-4-methyl-5-methyl-1-methyl-2 ... 6 -Isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N-substituted adenines, 7-methylguanine, 5-methylammomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueuosine, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N 6 -isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxocine, pseudouracil, queusine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methyl ester, 3-(3-amino-3-N-2-carboxypropyl)uracil, and 2,6-diaminopurine. Alternatively, one or more of the nucleic acids of the present disclosure can be purchased from companies such as Macromolecular Resources (Fort Collins, CO) and Synthegen (Houston, TX).

[0080] vector In some embodiments, the nucleic acids of the present disclosure are incorporated into vectors. In this regard, the present disclosure provides vectors comprising any of the nucleic acids of the present disclosure. In various embodiments, the vector is a recombinant expression vector. For purposes of this specification, the term "recombinant expression vector" refers to a genetically engineered oligonucleotide or polynucleotide construct that allows expression of an mRNA, protein, polypeptide, or peptide by a host cell when the construct contains a nucleotide sequence encoding the mRNA, protein, polypeptide, or peptide and the vector contacts the cell under conditions sufficient for the mRNA, protein, polypeptide, or peptide to be expressed in the cell. The vectors of the present disclosure are not naturally occurring in their entirety. However, portions of the vector may be naturally occurring. The vectors of the present disclosure may be single-stranded or double-stranded, may be partially synthesized or obtained from natural sources, and may contain any type of nucleotide, including, but not limited to, DNA and RNA, which may contain natural, non-natural, or altered nucleotides. The vector may contain naturally occurring or non-naturally occurring internucleotide linkages, or both types of linkages. In some aspects, the modified nucleotides or non-naturally occurring internucleotide linkages do not interfere with the transcription or replication of the vector.

[0081] The vectors of the present disclosure can be any suitable vector and can be used to transduce, transform, or transfect any suitable host. Suitable vectors include those designed for propagation and propagation, or for expression, or both, such as plasmids and viruses. The vector can be a plasmid-based expression vector. In various embodiments, the vector is selected from the group consisting of the pUC series (Fermentas Life Sciences), pBluescript series (Stratagene, LaJolla, CA), pET series (Novagen, Madison, WI), pGEX series (Pharmacia Biotech, Uppsala, Sweden), and pEX series (Clontech, Palo Alto, CA). Bacteriophage vectors such as λGTIO, λGTl1, λZapII (Stratagene), λEMBL4, and λNMl149 can also be used. Examples of plant expression vectors include pBI01, pBI101.2, pBI101.3, pBI121, and pBIN19 (Clontech). Examples of animal expression vectors include pEUK-Cl, pMAM, and pMAMneo (Clontech). In some embodiments, the vector is a viral vector, e.g., a retroviral vector. In various embodiments, the vector is an adenoviral vector, an adeno-associated viral (AAV) vector, a herpes simplex virus (HSV) vector, a vesicular stomatitis virus (VSV) vector, a vaccinia viral vector, or a lentiviral vector. See, e.g., Howarth et al., Cell Biol. Toxicol. 26(1):1-20 (2010). In various embodiments, the vector is a baculoviral vector that infects arthropods, e.g., insects. In various embodiments, the baculovirus vector is Autographa californica polynuclear virus (AcMNPV) or Bombyx mori nuclear polyhedrosis virus (BmNPV).See, e.g., Khan, Adv Pharm Bull 3(2):257-263 (2013), Miller, Bioessays 11(4):91-96 (1989), Atkinson et al., Pestic Sci 28:215-224 (1990).

[0082] Table 6: Expression vectors used to generate anti-CLDN6 antibodies [Table 11]

[0083] The vectors of the present disclosure can be prepared using standard recombinant DNA techniques, for example, as described in Sambrook et al., supra, and Ausubel et al., supra. Circular or linear expression vector constructs can be prepared to contain a replication system that functions in prokaryotic or eukaryotic host cells. Replication systems can be derived, for example, from CoIE1, 2μ plasmid, λ, SV40, bovine papilloma virus, etc.

[0084] In some aspects, vectors include regulatory sequences, such as transcriptional and translational initiation and termination codons, that are specific to the type of host (e.g., bacterial, fungal, plant, or animal) into which the vector will be introduced, as appropriate and taking into account whether the vector is DNA- or RNA-based.

[0085] Vectors can contain one or more marker genes to allow for the selection of transformed or transfected hosts. Marker genes include biocide resistance, resistance to, e.g., antibiotics, heavy metals, etc., complementation in auxotrophic hosts to provide prototrophy, etc. Suitable marker genes for expression vectors of the present disclosure include, for example, neomycin / G418 resistance genes, hygromycin resistance genes, histidinol resistance genes, tetracycline resistance genes, and ampicillin resistance genes.

[0086] A vector can include a native or normative promoter operably linked to a nucleotide sequence encoding a polypeptide (including functional portions and functional variants thereof) or a nucleotide sequence complementary to or hybridizing with the nucleotide sequence encoding the polypeptide. Selection of a promoter, such as a strong, weak, inducible, tissue-specific, or developmentally specific promoter, is within the ordinary skill of one of ordinary skill in the art. Similarly, combining a nucleotide sequence with a promoter is also within the ordinary skill of one of ordinary skill in the art. The promoter can be a non-viral promoter or a viral promoter, such as a cytomegalovirus (CMV) promoter, an SV40 promoter, an RSV promoter, or a promoter found in the long terminal repeat of murine stem cell virus.

[0087] host cell Host cells comprising the nucleic acid or vector of the present disclosure are provided herein.As used herein, the term "host cell" refers to any type of cell that can contain the vector of the present disclosure and produce the expression product (e.g., mRNA, protein) encoded by the nucleic acid.In some embodiments, host cells are adherent cells or suspension cells, i.e., cells that grow in suspension.In various embodiments, host cells are cultured cells or primary cells, i.e., cells directly isolated from an organism, such as a human.Host cells can be any cell type, originate from any type of tissue, and be at any developmental stage.

[0088] In various embodiments, the antigen-binding protein is a glycosylated protein and the host cell is a cell capable of glycosylation. In various embodiments, the cell capable of glycosylation is a eukaryotic cell, including, but not limited to, a yeast cell, a filamentous fungal cell, a protozoan cell, an algae cell, an insect cell, or a mammalian cell. Such host cells are described in the art. See, for example, Frenzel, et al., Front Immunol 4:217 (2013). In various embodiments, the eukaryotic cell is a mammalian cell. In various embodiments, the mammalian cell is a non-human mammalian cell. In some embodiments, the cells are selected from the group consisting of Chinese hamster ovary (CHO) cells and derivatives thereof (e.g., CHO-K1, CHO pro-3), mouse myeloma cells (e.g., NS0, GS-NS0, Sp2 / 0), cells engineered to lack dihydrofolate reductase (DHFR) activity (e.g., DUKX-X11, DG44), human embryonic kidney 293 (HEK293) cells or derivatives thereof (e.g., HEK293T, HEK293-EBNA), African green monkey kidney cells (e.g., COS cells, VERO cells), human cervical carcinoma cells (e.g., HeLa), human osteosarcoma epithelial cells U2-OS, adenocarcinoma human alveolar basal epithelial cells A549, human fibrosarcoma cells HT1080, mouse brain tumor cells CAD, embryonic carcinoma cells P19, mouse embryonic fibroblast cells NIH These cells include 3T3, mouse fibroblast L929, mouse neuroblastoma N2a, human breast cancer MCF-7, retinoblastoma Y79, human retinoblastoma SO-Rb50, human hepatoma Hep G2, mouse myeloma B J558L, and baby hamster kidney (BHK) cells (Gaillet et al. 2007; Khan, Adv Pharm Bull 3(2):257-263(2013)).

[0089] For purposes of amplifying or replicating the vector, the host cell is in some embodiments a prokaryotic cell, such as a bacterial cell.

[0090] Also provided by the present disclosure are populations of cells comprising at least one host cell described herein. The population of cells, in some aspects, is a heterogeneous population comprising host cells comprising the described vectors and at least one other cell that does not comprise any of the vectors. Alternatively, in some aspects, the population of cells is a substantially homogeneous population primarily comprising (e.g., consisting essentially of) host cells comprising the vector. In some aspects, the population is a clonal population of cells, in which all cells in the population are clones of a single host cell comprising the vector, and thus all cells in the population comprise the vector. In various embodiments of the present disclosure, the population of cells is a clonal population comprising host cells comprising the vectors described herein.

[0091] Manufacturing method Also provided herein are methods for producing an antigen binding protein that binds to CLDN6. In various embodiments, the method comprises culturing host cells comprising 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 cells may be any of the host cells described herein. In various aspects, the host cells are selected from the group consisting of CHO cells, NS0 cells, COS cells, VERO cells, and BHK cells. In various aspects, culturing the host cells comprises culturing the host cells in a growth medium that supports the growth and proliferation of the host cells. In various aspects, the growth medium increases cell density, culture viability, and productivity in a timely manner. In various aspects, the growth medium comprises amino acids, vitamins, inorganic salts, glucose, and serum as a source of growth factors, hormones, and attachment factors. In various aspects, the growth medium is a synthetic medium consisting of amino acids, vitamins, trace elements, inorganic salts, lipids, and insulin or insulin-like growth factors. In addition to nutrients, the growth medium also serves to maintain pH and osmolality. Several growth media are commercially available and described in the art, see, e.g., Arora, "Cell Culture Media: A Review," MATER METHODS 3:175 (2013).

[0092] In various embodiments, the method comprises culturing the host cells in a feed medium. In various embodiments, the method comprises fed-batch culturing in a feed medium. Methods for recombinant protein production are known in the art. See, e.g., Li et al., "Cell culture processes for monoclonal antibody production" MAbs 2(5):466-477 (2010).

[0093] Methods for producing antigen-binding proteins can include one or more steps for purifying the protein from a cell culture or its supernatant, and preferably recovering the purified protein. In various embodiments, the methods include one or more chromatography steps, such as affinity chromatography (e.g., Protein A affinity chromatography), ion exchange chromatography, or hydrophobic interaction chromatography. In various embodiments, the methods include purifying the protein using a Protein A affinity chromatography resin.

[0094] In various embodiments, the method further includes a step of formulating the purified protein, etc., thereby obtaining a formulation comprising the purified protein. Such steps are described in Formulation and Process Development Strategies for Manufacturing, eds. Jameel and Hershenson, John Wiley & Sons, Inc. (Hoboken, NJ), 2010.

[0095] In various aspects, the antigen binding protein linked to the polypeptide and the antigen binding protein are part of a fusion protein. Accordingly, the present disclosure further provides methods of making a fusion protein comprising an antigen binding protein that binds to CLDN6. In various embodiments, the method comprises culturing in a cell culture medium a host cell comprising a nucleic acid comprising a nucleotide sequence encoding a fusion protein described herein, and harvesting the fusion protein from the cell culture medium.

[0096] Conjugates The present disclosure also provides antigen-binding proteins bound, linked, or conjugated to a second moiety (e.g., a heterologous moiety, a conjugate moiety). Accordingly, the present disclosure provides conjugates comprising an antigen-binding protein and a heterologous moiety. As used herein, the term "heterologous moiety" is synonymous with "conjugate moiety" and refers to any molecule (naturally occurring or non-encoded chemical or biochemical) that is distinct from the antigen-binding protein of the present disclosure. Various heterologous moieties include, but are not limited to, polymers, carbohydrates, lipids, nucleic acids, oligonucleotides, DNA or RNA, amino acids, peptides, polypeptides, proteins, detectable labels, therapeutic agents (e.g., cytotoxic agents, cytokines), or diagnostic agents.

[0097] In some embodiments, the heterologous moiety is a detectable label (as used herein, "detectable label" is synonymous with "detectable marker"). Various detectable labels include, but are not limited to, a fluorophore, a radioactive label, a colorimetric label, or the detectable label is an enzyme. In some embodiments, the detectable label is horseradish peroxidase (HRP). In some embodiments, the product of the horseradish peroxidase (HRP) enzymatic reaction is a chromogenic substance. In some embodiments, the chromogenic substance is detected after an oxidation reaction catalyzed by HRP. In one embodiment, the oxidation reaction catalyzed by HRP comprises HRP, an HRP chromogenic substrate, and an oxidizing agent. In one embodiment, the oxidation reaction catalyzed by HRP further comprises a metal and / or imidazole. In one embodiment, the oxidizing agent is hydrogen peroxide (H2O2). In one embodiment, the metal is nickel (Ni 2+ ), copper (Cu 2+ ), silver (Ag 2+ ) and cobalt (Co 2+ In a preferred embodiment, the metal is selected from the group consisting of nickel (Ni 2+ ) and cobalt (Co 2+In one embodiment, the HRP chromogenic substrate is selected from the group consisting of 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), aminoethylcarbazole (AEC), 4,4'-diaminobiphenyl (benzidine), 3,3',5,5'-tetramethylbenzidine (TMB), 3,3'-diaminobenzidine (DAB), o-phenylenediamine (OPD), 3-methyl-2-benzothiazolinone (MBTH), 2-methoxyphenol (guaiacol), 1,2,3-trihydroxybenzene (pyrogallol), or 1,2,3-trihydroxybenzene (pyrogallol). 1,4-Dihydroxybenzene (hydroquinone), 4-amino-2,3-dimethyl-1-phenyl-3-pyrazolinone (4-aminoantipyrene), 5-aminosalicylic acid (5-AS), and 4-chloro-1-naphthol (4-CN). In a preferred embodiment, the chromogenic HRP substrate is selected from the group consisting of 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), aminoethylcarbazole (AEC), 3,3',5,5'-tetramethylbenzidine (TMB), 3,3'-diaminobenzidine (DAB), o-phenylenediamine (OPD), and 4-chloro-1-naphthol (4-CN).

[0098] In one embodiment, brown colored substances are detected after oxidation of DAB (3,3'-diaminobenzidine) by HRP. In one embodiment, copper (Cu) 2+ In one embodiment, the detection of a gray-blue colored product is achieved by oxidation of DAB (3,3'-diaminobenzidine) in the presence of nickel (Ni 2+ In one embodiment, the detection of a gray-black, deep blue, or purple-blue colored substance is achieved by oxidation of DAB (3,3'-diaminobenzidine) in the presence of cobalt (Co 2+In one embodiment, a deep blue or blue-black colored substance is detected after oxidation of DAB (3,3'-diaminobenzidine) in the presence of HCl. In one embodiment, a blue / dark blue colored substance is detected after oxidation of TMB (3,3',5,5'-tetramethylbenzidine) by HRP. In one embodiment, a red colored substance is detected after oxidation of AEC (aminoethylcarbazole) by HRP. In one embodiment, a deep blue / purple colored substance is detected after oxidation of 4-chloro-1-naphthol (4-CN) by HRP.

[0099] In one embodiment, the product of a horseradish peroxidase (HRP) enzymatic reaction is a fluorogenic label. In one embodiment, the detectable label is a fluorogenic label or fluorophore following an oxidation reaction catalyzed by HRP. In one embodiment, the fluorogenic label or fluorophore is the oxidized compound following an oxidation reaction catalyzed by HRP. In one embodiment, the oxidized compound is a fluorescent oxidized compound. In one embodiment, the oxidized compound has a fluorescence emission and / or absorbance that is substantially different from the non-oxidized compound from which the oxidized compound is derived. In one embodiment, the oxidation reaction catalyzed by HRP comprises HRP, an HRP fluorogenic substrate, and an oxidizing agent. In one embodiment, the oxidation reaction catalyzed by HRP further comprises a metal and / or imidazole. In one embodiment, the oxidizing agent is hydrogen peroxide (H2O2). In one embodiment, the metal is nickel (Ni 2+ ), copper, silver and cobalt (Co 2+In one embodiment, the HRP fluorogenic substrate is selected from the group consisting of 10-acetyl-3,7-dihydroxyphenoxazine (ADHP or AmplexRed), homovanillic acid (HVA), and 2,7-dichloro-9-(2-(hydroxymethyl)phenyl)-9H-xanthene-3,6-diol (DCFH-1). In one embodiment, the detectable label is 7-hydroxy-3H-phenoxazin-3-one (resorufin) after oxidation of 10-acetyl-3,7-dihydroxyphenoxazine (ADHP or AmplexRed) by HRP. In one embodiment, the detectable label is 2,2'-(6,6'-dihydroxy-5-methoxy-[1,1'-biphenyl]-3,3'-diyl)diacetic acid (HVA dimer) after oxidation of 2-(4-hydroxy-3-methoxyphenyl)acetic acid (homovanillic acid or HVA monomer) by HRP. In one embodiment, the detectable label is 2,7-dichloro-6-hydroxy-9-(2-(hydroxymethyl)phenyl)-3H-xanthen-3-one following oxidation of 2,7-dichloro-9-(2-(hydroxymethyl)phenyl)-9H-xanthen-3,6-diol (DCFH-1) by HRP.

[0100] In various embodiments, commercial kits for detecting HRP enzyme activity are known in the art and readily available. HRP color development kits include, but are not limited to, nickel-containing DAB Peroxidase (HRP) Substrate Kit (Vector Labs, Catalog No. SK-4100), ImmPACT® DAB Peroxidase (HRP) Substrate Kit (Vector Labs, Catalog No. SK-4105), ImmPACT® DAB EqV Peroxidase (HRP) Substrate Kit (Vector Labs, Catalog No. SK-4103), Vector® VIP Peroxidase (HRP) Substrate Kit (Vector Labs, Catalog No. SK-4600), ImmPACT® VIP Peroxidase (HRP) Substrate Kit (Vector Labs, Catalog No. SK-4605), Vector® SG Peroxidase (HRP) Substrate Kit (Vector Labs, Catalog No. SK-4606), and the like. Labs, Catalog No. SK-4700), ImmPACT® SG Peroxidase (HRP) Substrate Kit (Vector Labs, Catalog No. SK-4705), Vector NovaRED® Peroxidase (HRP) Substrate Kit (Vector Labs, Catalog No. SK-4800), ImmPACT NovaRED® Peroxidase (HRP) Substrate Kit (Vector Labs, Catalog No. SK-4805), AEC Peroxidase (HRP) Substrate Kit (Vector Labs, Catalog No. SK-4200), ImmPACT® AEC Peroxidase (HRP) Substrate Kit (Vector Labs, Catalog No. SK-4205), ImmPACT® AMEC Red Peroxidase (HRP) Substrate Kit (Vector Labs, Catalog No. SK-4285), TMB Peroxidase (HRP) Substrate Kit (Vector Labs, Catalog Number: SK-4400), Betazoid DAB Chromogen Kit (BioCare Medical, Catalog Number: BDB2004L), intelliPATH™ DAB Chromogen Kit (BioCareMedical, Catalog Number: IPK5010G80), Cardassian DAB (BioCare Medical, Catalog Number: DBC859L10), DAB Chromogen Kit (BioCare Medical, Catalog Number: DB801L), Vina Green™ Chromogen Kit (BioCare Medical, Catalog Number: BRR807AS), Romulin AEC (BioCare Medical, Catalog Number: RAEC810L), Bajoran Purple Chromogen Kit (BioCare Medical, catalog number BJP811L or control number 902-811A-083011), HIGHDEF® DAB chromogen / substrate set (Enzo, catalog number ENZ-ACC105), HIGHDEF® Yellow IHC chromogen (HRP) (Enzo, catalog number ADI-950-170), HIGHDEF® Red IHC chromogen (Enzo, catalog number ADI-950-210), HIGHDEF® Blue IHC chromogen (HRP) (Enzo, catalog number ADI-950-151), and HIGHDEF® Black IHC chromogen (HRP) (Enzo, catalog number ADI-950-171).

[0101] In one embodiment, the detectable label is a product of an alkaline phosphatase (AP) enzymatic reaction. In one embodiment, the product of the alkaline phosphatase (AP) enzymatic reaction is a chromogenic substance. In one embodiment, the detectable label is a chromogenic substance following a dephosphorylation reaction catalyzed by AP. In one embodiment, the dephosphorylation reaction catalyzed by AP comprises AP and an AP substrate. In one embodiment, the dephosphorylation reaction catalyzed by AP further comprises a diazonium salt. In one embodiment, the dephosphorylation reaction catalyzed by AP comprises AP, an AP substrate, and a diazonium salt. In one embodiment, the product of the alkaline phosphatase (AP) enzymatic reaction reacts with the diazonium salt to produce a chromogenic substance.

[0102] In one embodiment, the dephosphorylation reaction catalyzed by alkaline phosphatase (AP) is a hydrolysis reaction resulting in the release of a phosphate group. In one embodiment, the detectable label is a color-forming substance after the AP-catalyzed hydrolysis reaction. In one embodiment, the AP-catalyzed hydrolysis reaction comprises AP, a substrate, and a diazonium salt. In one embodiment, the AP substrate is selected from the group consisting of 5-bromo-4-chloro-3-indolyl phosphate (BCIP), 3-hydroxy-2-naphthoic acid 2,4-dimethylanilide phosphate (naphthol AS-MX phosphate), 6-chloro-3-indolyl phosphate-toluidine salt (salmon phosphate), and 5-bromo-6-chloro-3-indolyl phosphate p-toluidine salt (magenta phosphate). In one embodiment, the detectable label is a blue color-forming substance produced after hydrolysis of BCIP (5-bromo-4-chloro-3-indolyl phosphate) by AP (alkaline phosphatase) in the presence of NBT (p-nitro blue tetrazolium chloride). In one embodiment, the detectable label is a purple color-forming substance produced after hydrolysis of BCIP (5-bromo-4-chloro-3-indolyl phosphate) by AP in the presence of TNBT (tetranitro blue tetrazolium). In one embodiment, the detectable label is a blue color-forming substance produced after hydrolysis of naphthol AS-MX phosphate (3-hydroxy-2-naphthoic acid 2,4-dimethylanilide phosphate) by AP in the presence of Fast Blue BB. In one embodiment, the detectable label is a red color-forming substance produced after hydrolysis of naphthol AS-MX phosphate by AP in the presence of Red TR. In one embodiment, the detectable label is a red color-forming substance produced after hydrolysis of naphthol AS-MX phosphate by AP in the presence of New Fuchsin. In one embodiment, the detectable label is a green chromogen using a green AP chromogen / substrate system such as HIGHDEF Green AP chromogen / substrate (Enzo, catalog number: ENZ-ACC130) or STAYGREEN / AP Plus (Abcam, catalog number: ab156428).

[0103] In one embodiment, the product of the alkaline phosphatase (AP) enzymatic reaction is a fluorogenic label. In one embodiment, the detectable label is a fluorogenic label or fluorophore after the AP-catalyzed hydrolysis reaction. In one embodiment, the hydrolysis compound is a fluorescent hydrolysis compound. In one embodiment, the AP fluorogenic substrate is selected from the group consisting of 2-(benzothiazol-2-yl)-4-bromophenol phosphate (BT3-Phos), Vector Red (Vector Labs, Catalog No. SK-5100), ImmPACT Vector Red (Vector Labs, Catalog No. SK5105), and Vector Blue (Vector Labs, Catalog No. SK-5300).

[0104] In various embodiments, commercial kits for detecting AP enzyme activity are known in the art and readily available. AP color development kits include, but are not limited to, the ImmPRESS® Duet Dual Stain Polymer Kit (Vector Labs, Catalog No. MP-7714), the ImmPRESS® Duet Dual Stain Polymer Kit (Vector Labs, Catalog No. MP-7724), and the Human Tissue-Human Antibody IHC Kit (AP / Permanent Immunosorbent Assay Kit). These include the M&R double-stain IHC kit for human tissue (DAB, AP / Red&HRP / Green; Abcam, catalog number: ab214753), the M&R double-stain IHC kit for human tissue (DAB, AP / Red&HRP / Green; Abcam, catalog number: ab210059), the M&R&G triple-stain IHC kit for human tissue (DAB, AP / Red&HRP / Green; Abcam, catalog number: ab183290), the M&R&Rt triple-stain IHC kit for rodent tissue (DAB, DAB / Ni&AP / Red; Abcam, catalog number: ab183298), and the R&R&M triple-stain IHC kit for human tissue (DAB, AP / Red&Green / HRP; Abcam, catalog number: ab183288).

[0105] In some embodiments, the heterologous moiety is a polymer. The polymer may be branched or unbranched. The polymer may be of any molecular weight. In some embodiments, the polymer has an average molecular weight of about 2 kDa to about 100 kDa (the term "about" indicates that in preparing water-soluble polymers, some molecules may be larger and some molecules may be smaller than the stated molecular weight). The average molecular weight of the polymer, in some aspects, is about 5 kDa to about 50 kDa, about 12 kDa to about 40 kDa, or about 20 kDa to about 35 kDa.

[0106] In some embodiments, the polymer is modified to have a single reactive group, such as an active ester for acylation or an aldehyde for alkylation, so that the degree of polymerization can be controlled. In some embodiments, the polymer is water-soluble so that the protein to which the polymer is attached does not precipitate in an aqueous environment, such as a physiological environment. In some embodiments, for example, when the composition is used for therapeutic purposes, the polymer is a pharmaceutically acceptable polymer. In addition, in some aspects, the polymer is a mixture of polymers, such as a copolymer or block copolymer.

[0107] In some embodiments, the polymer is selected from the group consisting of polyamides, polycarbonates, polyalkylenes and their derivatives, such as polyalkylene glycols, polyalkylene oxides, polyalkylene terephthalates, polymers of acrylic and methacrylic esters, such as poly(methyl methacrylate), poly(ethyl methacrylate), poly(butyl methacrylate), poly(isobutyl methacrylate), poly(hexyl methacrylate), poly(isodecyl methacrylate), poly(lauryl methacrylate), poly(phenyl methacrylate), poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutyl acrylate), and poly(octadecyl acrylate), polyvinyl polymers, such as polyvinyl alcohol, polyvinyl ethers, polyvinyl esters, polyhalogenated vinyl esters, and the like. The polymer may be selected from the group consisting of vinyl, poly(vinyl acetate), and polyvinylpyrrolidone, polyglycolide, polysiloxane, polyurethane and copolymers thereof, cellulose such as alkyl cellulose, hydroxyalkyl cellulose, cellulose ether, cellulose ester, nitrocellulose, methyl cellulose, ethyl cellulose, hydroxypropyl cellulose, hydroxypropylmethyl cellulose, hydroxybutylmethyl cellulose, cellulose acetate, cellulose propionate, cellulose acetate butyrate, cellulose acetate phthalate, carboxyethyl cellulose, cellulose triacetate, and sodium cellulose sulfate, polypropylene, polyethylene such as poly(ethylene glycol), poly(ethylene oxide), and poly(ethylene terephthalate), and polystyrene.

[0108] A particularly preferred water-soluble polymer for use herein is polyethylene glycol (PEG). As used herein, polyethylene glycol is intended to encompass 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 that is soluble in water and most organic solvents.

[0109] In some embodiments, the heterologous moiety is a carbohydrate. In some embodiments, the carbohydrate is a monosaccharide (e.g., glucose, galactose, fructose), a disaccharide (e.g., sucrose, lactose, maltose), an oligosaccharide (e.g., raffinose, stachyose), or a polysaccharide (starch, amylase, amylopectin, cellulose, chitin, callose, laminarin, xylan, mannan, fucoidan, galactomannan).

[0110] In some embodiments, the heterologous moiety is a lipid, which in some embodiments is a fatty acid, eicosanoid, prostaglandin, leukotriene, thromboxane, N-acylethanolamine, glycerolipid (e.g., mono-, di-, or tri-substituted glycerol), glycerophospholipid (e.g., phosphatidylcholine, phosphatidylinositol, phosphatidylethanolamine, phosphatidylserine), sphingolipid (e.g., sphingosine, ceramide), sterol lipid (e.g., steroid, cholesterol), prenol lipid, glycolipid, or polyketide, oil, wax, cholesterol, sterol, fat-soluble vitamin, monoglyceride, diglyceride, triglyceride, or phospholipid.

[0111] The present disclosure also provides conjugates comprising an antigen binding protein conjugated to a heterologous moiety (e.g., conjugated to a detectable label or detection moiety) for detecting CLDN6-positive tumors in a subject, e.g., a human. In various cases, the conjugated antigen binding protein is a monoclonal antibody conjugated to a heterologous moiety. The heterologous moiety can be a polypeptide. A polypeptide heterologous moiety can be chemically conjugated to, for example, a monoclonal antibody, although the polypeptide heterologous moiety may also be co-expressed as a fusion protein of the monoclonal antibody. Thus, the present disclosure also provides conjugates comprising an antigen binding protein of the present disclosure linked to a polypeptide, thereby being a fusion protein. Thus, the present disclosure provides fusion proteins comprising an antigen binding protein of the present disclosure linked to a polypeptide. In various cases, the polypeptide can be a reporter protein. Examples of reporter proteins include, but are not limited to, green fluorescent protein (GFP), red fluorescent protein (RFP), glutathione-S-transferase (GST), horseradish peroxidase (HRP), alkaline phosphatase (AP), chloramphenicol acetyltransferase (CAT), beta-galactosidase, glucose oxidase (GO), beta-glucuronidase (GUS), luciferase, β-lactamase, or blue fluorescent protein (BFP). In various embodiments, the polypeptide is a diagnostic label, such as a fluorescent protein, e.g., green fluorescent protein, red fluorescent protein, and blue fluorescent protein. In various embodiments, the polypeptide is an epitope tag, e.g., a myc tag, a hemagglutinin (HA) tag, a hexahistidine tag, a FLAG™ tag, a VSV-G tag, and a V5 tag.

[0112] The present disclosure also provides a conjugate comprising an antigen-binding protein of the present disclosure linked to a small molecule, in various embodiments, the small molecule is, but is not limited to, biotin, digoxigenin (DIG), fluorescein isothiocyanate (FITC), or allophycocyanin (APC).

[0113] In various embodiments, the antigen-binding protein or conjugate thereof (e.g., an epitope-tagged conjugate) may be detected by a secondary antibody linked to a label or detection moiety, where the secondary antibody binds to the antigen-binding protein or conjugate thereof. The label or detection moiety may be a reporter protein. Examples of reporter proteins include, but are not limited to, green fluorescent protein (GFP), red fluorescent protein (RFP), glutathione-S-transferase (GST), horseradish peroxidase (HRP), alkaline phosphatase (AP), chloramphenicol acetyltransferase (CAT), beta-galactosidase, glucose oxidase (GO), beta-glucuronidase (GUS), luciferase, beta-lactamase, or blue fluorescent protein (BFP). In various embodiments, the label or detection moiety may be an epitope tag or a small molecule. In various embodiments, epitope tags include, but are not limited to, myc tags, hemagglutinin (HA) tags, hexahistidine tags, FLAG™ tags, VSV-G tags, and V5 tags. In various embodiments, small molecules include, but are not limited to, biotin, DIG, FITC, and APC.

[0114] In various aspects, the antigen-binding proteins of the present invention are bound by a secondary antibody conjugated to an enzyme, and the detectable label is the product of the enzymatic reaction. The antigen-binding proteins of the present invention may be monoclonal antibodies or fragments thereof. The antigen-binding proteins may be free of heterologous moieties or may not be conjugated to heterologous moieties. The enzyme may be horseradish peroxidase (HRP), alkaline phosphatase (AP), chloramphenicol acetyltransferase (CAT), beta-galactosidase, glucose oxidase (GO), beta-glucuronidase (GUS), luciferase, or beta-lactamase. In preferred embodiments, the enzyme is HRP or AP. In various aspects, the product of the enzymatic reaction is a chromogenic substance, a fluorogenic label, or a fluorophore. In one embodiment, the antigen-binding proteins of the present invention are bound by a secondary antibody conjugated to HRP, and the detectable label is the product of the HRP enzymatic reaction. In various aspects, the product of the HRP enzymatic reaction is a chromogenic substance, a fluorogenic label, or a fluorophore after oxidation of the HRP substrate by HRP. In a preferred embodiment, the product of the HRP enzymatic reaction is a chromogenic substance. The HRP substrate can be an HRP chromogenic substrate or an HRP fluorogenic substrate. In a preferred embodiment, the HRP substrate is an HRP chromogenic substrate. In one embodiment, the antigen binding protein of the present invention is bound by a secondary antibody conjugated to AP, and the detectable label is the product of the AP enzymatic reaction. In various aspects, the product of the AP enzymatic reaction is a chromogenic substance, a fluorogenic label, or a fluorophore. In a preferred embodiment, the product of the AP enzymatic reaction is a chromogenic substance. The AP substrate can be an AP chromogenic substrate or an AP fluorogenic substrate. In a preferred embodiment, the AP substrate is an AP chromogenic substrate.

[0115] In various aspects, the antigen binding proteins of the present invention are bound by a secondary antibody conjugated to biotin (i.e., a biotinylated secondary antibody). In various aspects, a label or detection moiety is conjugated to biotin (i.e., a biotinylated label or detection moiety). In one embodiment, the label or detection moiety is a reporter protein. In various aspects, the reporter protein can be an enzyme. In one embodiment, the enzyme is conjugated to biotin. In one embodiment, the enzyme conjugated to biotin (i.e., a biotinylated enzyme) can be horseradish peroxidase (HRP), alkaline phosphatase (AP), chloramphenicol acetyltransferase (CAT), beta-galactosidase, glucose oxidase (GO), beta-glucuronidase (GUS), luciferase, or beta-lactamase. In preferred embodiments, the enzyme conjugated to biotin (i.e., the biotinylated enzyme) is horseradish peroxidase (HRP) or alkaline phosphatase (AP). In various aspects, immunodetection of CLDN6 involves an immunoconjugate comprising an antigen binding protein of the present invention, a biotinylated secondary antibody, a biotinylated enzyme, and a biotin-binding protein selected from the group including avidin, streptavidin, and NeutrAvidin. In various aspects, immunodetection further comprises an enzyme substrate that generates an enzyme product that is a detectable label. In one embodiment, immunodetection of CLDN6 involves an immunoconjugate comprising an antigen binding protein of the present invention, a biotinylated secondary antibody, biotinylated HRP or AP, and a biotin-binding protein selected from the group including avidin, streptavidin, and NeutrAvidin (deglycosylated avidin). In various aspects, immunodetection further comprises an HRP or AP enzyme substrate that generates an enzyme product that is a chromogenic substance, a fluorogenic label, or a fluorophore. In a preferred embodiment, the enzyme product is a chromogenic substance.

[0116] In various aspects, the antigen binding proteins of the present invention are bound by a secondary antibody conjugated to biotin (i.e., a biotinylated secondary antibody). In various aspects, the label or detection moiety is conjugated to biotin (i.e., a biotinylated label or detection moiety). In one embodiment, the label or detection moiety is a reporter protein. In various aspects, the reporter protein can be an enzyme. In one embodiment, the enzyme is conjugated to avidin, streptavidin, or NeutrAvidin. In one embodiment, the enzyme conjugated to avidin, streptavidin, or NeutrAvidin (i.e., the avidin-, streptavidin-, or NeutrAvidin-conjugated enzyme) can be horseradish peroxidase (HRP), alkaline phosphatase (AP), chloramphenicol acetyltransferase (CAT), beta-galactosidase, glucose oxidase (GO), beta-glucuronidase (GUS), luciferase, or β-lactamase. In a preferred embodiment, the enzyme conjugated to avidin, streptavidin, or NeutrAvidin is horseradish peroxidase (HRP) or alkaline phosphatase (AP). In various aspects, immunodetection of CLDN6 involves an immune complex comprising an antigen-binding protein of the present invention, a biotinylated secondary antibody, and an avidin-, streptavidin-, or NeutrAvidin-conjugated enzyme. In various aspects, the immunodetection further comprises an enzyme substrate that generates an enzyme product that is a detectable label. In one embodiment, the immunodetection of CLDN6 comprises an immunoconjugate comprising an antigen binding protein of the present invention, a biotinylated secondary antibody, and an avidin-, streptavidin-, or NeutrAvidin-conjugated HRP or AP. In various aspects, the immunodetection further comprises an HRP or AP enzyme substrate that generates an enzyme product that is a chromogenic substance, a fluorogenic label, or a fluorophore. In a preferred embodiment, the enzyme product is a chromogenic substance. Linker

[0117] In some embodiments, a protein of the present disclosure (e.g., an antigen-binding protein, a fusion protein, etc.) is directly linked to a heterologous moiety. In alternative embodiments, the conjugate comprises a linker connecting the compound of the present disclosure to the heterologous moiety. In some aspects, the linker comprises a chain length of 1 to about 60 atoms, or 1 to 30 or more atoms, 2 to 5 atoms, 2 to 10 atoms, 5 to 10 atoms, or 10 to 20 atoms. In some embodiments, the chain atoms are all carbon atoms. In some embodiments, the chain atoms in the backbone of the linker are selected from the group consisting of C, O, N, and S. The chain atoms and linker can be selected to provide a more soluble conjugate depending on their expected solubility (hydrophilicity). In some embodiments, the linker provides a functional group that is subject to cleavage by an enzyme or other catalyst or to hydrolysis conditions found in the target tissue, organ, or cell. In some embodiments, the length of the linker is long enough to reduce the possibility of steric hindrance. In some embodiments, the linker is an amino acid or peptidyl linker. Such peptidyl linkers can be of any length, with various linkers being about 1-50 amino acids in length, 5-50, 3-5, 5-10, 5-15, or 10-30 amino acids in length.

[0118] A variety of suitable linkers are known in the art. The linker may be cleavable (cleavable linker).

[0119] The linker may be attached to a chemically reactive group of the antibody moiety, such as a free amino, imino, hydroxyl, thiol, or carboxyl group (e.g., the N- or C-terminus, the epsilon amino group of one or more lysine residues, the free carboxylic acid groups of one or more glutamic or aspartic acid residues, the sulfhydryl groups of one or more cysteinyl residues, or the hydroxyl groups of one or more serine or threonine residues). In some embodiments, the linker or heterologous moiety is attached to the interchain disulfide of the antibody (see, e.g., ADCETRIS®; see also Jain et al. (2015) Pharmaceutical Research 32:3526-3540, incorporated herein by reference). The linker attachment site may be a natural residue in the amino acid sequence of the antibody moiety, or may be introduced into the antibody moiety, for example, by recombinant DNA techniques (e.g., by introducing a cysteine ​​or protease cleavage site into the amino acid sequence) or by protein biochemistry (e.g., reduction, pH adjustment, or proteolysis). The linker attachment site may also be a non-natural amino acid. The linker attachment site may also be a glycan of the antibody.

[0120] Typically, a linker is substantially inert under the conditions under which the two groups it connects are linked. The terms "bifunctional crosslinker," "bifunctional linker," or "crosslinker" refer to a modifying agent having two reactive groups at each end of the linker, such that one reactive group can first react with a cytotoxic compound to provide a compound having a linker moiety and a second reactive group, and the second reactive group can then react with an antibody. Alternatively, one end of the bifunctional crosslinker can first react with an antibody to provide an antibody having a linker moiety and a second reactive group, and the second reactive group can then react with a cytotoxic compound. The linking moiety can contain a chemical bond that allows for release of the cytotoxic moiety at a specific site. Suitable chemical bonds are well known in the art and include disulfide bonds, thioether bonds, acid-labile bonds, photolabile bonds, protease / peptidase-labile bonds, and esterase-labile bonds. See, e.g., U.S. Patent Nos. 5,208,020, 5,475,092, 6,441,163, 6,716,821, 6,913,748, 7,276,497, 7,276,499, 7,368,565, 7,388,026, and 7,414,073. In some embodiments, the linkage is a disulfide bond, a thioether, and / or a protease / peptidase labile bond. Other linkers that can be used in the present invention include non-cleavable linkers such as those described in detail in US20050169933, charged linkers such as those described in US2009 / 0274713, US2010 / 0129314, and WO2009 / 134976, or hydrophilic linkers, each of which is expressly incorporated herein by reference.

[0121] In some embodiments, the linker is a hydrophilic linker that confers hydrophilicity to the conjugate. In some embodiments, the hydrophilic linker comprises polyethylene glycol (PEG). In some embodiments, the hydrophilic linker is CL2A. In some embodiments, the CL2A linker has the following structure: [ka] CL2A is described in U.S. Patent Nos. 8,080,250, 8,759,496, and 10,195,288.

[0122] In some embodiments, the hydrophilic linker is CL2E. In some embodiments, CL2E has the following structure: [ka] CL2E is described in U.S. Patent Nos. 8,080,250, 8,759,496, and 10,195,288.

[0123] In some embodiments, the linker is cleavable by a cleaving agent present in the intracellular environment (e.g., within a lysosome, endosome, or caveolae). The linker can be, for example, a peptide linker that is cleaved by an intracellular or extracellular peptidase or protease enzyme, including, but not limited to, a lysosomal or endosomal protease. In some embodiments, the peptide linker is at least 2, at least 3, at least 4, or at least 5 amino acids in length.

[0124] In some embodiments, the peptide linker is VC-PAB, which comprises valine and citrulline residues. In some such embodiments, the peptide linker is MC-VC-PAB. In some embodiments, the VC-PAB linker has the following structure: [ka] MC-VC-PAB is described in U.S. Patent Nos. 7,659,241, 7,829,531, 6,884,869, 6,214,345, and 6,214,345.

[0125] In some embodiments, the peptide linker is glycine-glycine-phenylalanine-glycine (GGFG). In some such embodiments, the peptide linker is maleimidocaproylglycine-glycine-phenylalanine-glycine (MC-GGFG). In some embodiments, the MC-GGFG linker has the following structure: [ka] MC-GGFG is described in U.S. Patent Nos. 9,808,537 and 10,195,288.

[0126] In other embodiments, the cleavable linker is pH-sensitive, i.e., sensitive to hydrolysis at a certain pH value.In some embodiments, the pH-sensitive linker is hydrolyzable under acidic conditions.For example, acid-labile linkers (such as hydrazones, semicarbazones, thiosemicarbazones, cis-aconitic amides, orthoesters, acetals, ketals, etc.) that are hydrolyzable in lysosomes can be used (see, for example, U.S. Patent Nos. 5,122,368, 5,824,805, 5,622,929; Dubowchik and Walker, 1999, Pharm.Therapeutics 83:67-123; Neville et al., 1989, Biol.Chem.264:14653-14661).Such linkers are relatively stable under neutral pH conditions, such as in blood, but are unstable below pH 5.5 or 5.0, which is the approximate pH of lysosomes. In certain embodiments, the hydrolyzable linker is a thioether linker, such as a thioether that is attached to the therapeutic agent via an acylhydrazone bond (see, eg, US Pat. No. 5,622,929).

[0127] In other embodiments, the linker is cleavable under reducing conditions (e.g., disulfide linker). Bifunctional crosslinkers that allow the antibody to be linked to a cytotoxic compound via a disulfide bond include, but are not limited to, N-succinimidyl-4-(4-nitropyridyl-2-dithio) butanoate, N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP), N-succinimidyl-4-(2-pyridyldithio) pentanoate (SPP), N-succinimidyl-4-(2-pyridyldithio) butanoate (SPDB), and N-succinimidyl-4-(2-pyridyldithio)-2-sulfobutanoate (sulfo-SPDB). Sulfo-SPDB is described, for example, in U.S. Patent No. 8,236,319, which is incorporated herein by reference. Alternatively, cross-linkers that introduce thiol groups, such as 2-iminothiolane, homocysteine ​​thiolactone, or S-acetylsuccinic anhydride, may be used. In other embodiments, the linker may contain a combination of one or more of the peptide linkers, pH-sensitive linkers, or disulfide linkers described above.

[0128] A "heterobifunctional crosslinker" is a bifunctional crosslinker having two different reactive groups. Heterobifunctional crosslinkers containing both an amine-reactive N-hydroxysuccinimide group (NHS group) and a carbonyl-reactive hydrazine group can also be used to link cytotoxic compounds to antibodies. Examples of such commercially available heterobifunctional crosslinkers include succinimidyl 6-hydrazinonicotinamide acetone hydrazone (SANH), succinimidyl 4-hydrazide terephthalate hydrochloride (SHTH), and succinimidyl hydrazinium nicotinate hydrochloride (SHNH). Conjugates with acid-labile linkages can also be prepared using the hydrazine-containing benzodiazepine derivatives of the present invention. Examples of bifunctional crosslinkers that can be used include succinimidyl-p-formylbenzoate (SFB) and succinimidyl-p-formylphenoxyacetate (SFPA).

[0129] The linkers described herein can be used in any combination with the heterologous moieties described herein. In addition, the linkers described herein can have any chemically reactive moiety (e.g., maleimide, cysteine, sulfhydryl, etc.) that can react with any portion of the antigen-binding proteins of the disclosure (e.g., amino acids, disulfide bonds, carbohydrates (e.g., from post-translational modifications), etc.). All of the above-listed linkers and heterologous moieties described herein are commercially available and / or can be prepared by conventional techniques, including those described in the references listed above.

[0130] Conjugation The heterologous moiety-to-antigen-binding protein ratio (HAR) represents the number of heterologous moieties linked per antigen-binding molecule. In some embodiments, the HAR is in the range of 1 to 15, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 to 2. In some embodiments, the HAR is in the range of 2 to 10, 2 to 9, 2 to 8, 2 to 7, 2 to 6, 2 to 5, 2 to 4, or 2 to 3. In other embodiments, the HAR is about 2, about 2.5, about 3, about 4, about 5, or about 6. In some embodiments, the HAR is in the range of about 2 to about 4. The HAR can be characterized by conventional means, such as mass spectrometry, UV / Vis spectroscopy, ELISA assay, and / or HPLC.

[0131] In some embodiments, the conjugates are heterogeneous conjugates (also referred to as "conventional") in which antigen binding proteins are conjugated to different numbers of heterologous moieties. In some embodiments, the heterogeneous conjugates follow a Gaussian or quasi-Gaussian distribution of conjugates, where the distribution is centered around the mean heterologous moiety loading value, with some antigen binding proteins being more conjugated than the mean and some antigen binding proteins being less conjugated than the mean.

[0132] In some embodiments, the conjugate is a homogeneous conjugate in which a substantial proportion of the antigen binding protein is conjugated to a defined number of heterologous moieties. In some embodiments, the homogeneous conjugate has an HAR of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, the homogeneous conjugate has an HAR of 2, 4, 6, or 8. In preferred embodiments, the homogeneous conjugate has an HAR of 4. In other preferred embodiments, the homogeneous conjugate has an HAR of 2. In some embodiments, the homogeneous conjugate has a defined HAR of 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 percent or more, or 100 percent of the conjugates. In some embodiments, homogeneous conjugates have about 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 percent of the conjugates have the defined HAR. In some embodiments, homogeneous conjugates have at least 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 percent of the conjugates have the defined HAR. In some embodiments, the homogeneous conjugate comprises a HAR distribution that is neither Gaussian nor quasi-Gaussian.In some embodiments, the homogeneity of the homogeneous conjugate is determined by chromatogram, for example, HPLC or any suitable chromatography.In some embodiments, the chromatogram is HIC chromatogram.The homogeneous conjugate can be produced by site-specific conjugation.

[0133] In some embodiments, the heterologous moiety is site-specifically conjugated to an antigen binding protein (eg, an antibody). Various site-specific conjugation methods, such as conjugation at thiomab or TDC or unpaired cysteine ​​residues (Junutula et al. (2008) Nat. Biotechnol. 26:925-932, Dimasi et al. (2017) Mol. Pharm. 14:1501-1516, Shen et al. (2012) Nat. Biotechnol. 30:184-9), thiol cross-linkers (Behrens et al. (2015) Mol. Pharm. 12:3986-98), conjugation at glutamine using transglutaminase (Dennler et al. (2013) Methods Mol. Bio. 1045:205-15, Dennler et al. (2014) Bioconjug Chem. 25:569-78), conjugation at engineered unnatural amino acid residues (Axup et al. (2012) Proc Natl Acad Sci USA 104-16101-6, Tian et al. (2014) Proc Natl Acad Sci USA 111:1766-71, VanBrunt et al. (2015) Bioconjug Chem 26:2249-60, Zimmerman et al. (2014) Bioconjug Chem 25:351-61), selenocysteine ​​conjugation (Li et al. (2017) Cell Chem Biol 24:433-442), and glycan-mediated conjugation (Okeley et al. (2013) Bioconjug Chem 24:1650-5), conjugation with galactose or GalNAc analogues (Ramakrishnan and Qasba (2002) J Biol Chem 277:20833-9, van Geel et al. (2015) Bioconjug Chem 26:2233-42), or via glycan engineering (Zhou et al. (2014) Bioconjug Chem 25:510-20, Tang et al.(2017) Nat Protoc 12:1702-1721), via short peptide tags, such as glutamine tag engineering or sortase A-mediated transpeptidation (Strop et al. (2013) Chem Biol 20:161-7, Beerli et al. (2015) PLoS One 10:e0131177), and via aldehyde tags (Wu et al. (2009) Proc Natl Acad Sci USA 106:3000-5), are known in the art.

[0134] Compositions and Formulations Provided herein are compositions comprising the antigen-binding proteins, nucleic acids, vectors, host cells, or conjugates of the present disclosure. In some embodiments, the compositions comprise the antigen-binding proteins in isolated and / or purified form. In some embodiments, the compositions comprise a single type (e.g., structure) of the antigen-binding proteins of the present disclosure, or a combination of two or more antigen-binding proteins of the present disclosure, wherein the combination comprises two or more antigen-binding proteins of different types (e.g., structures).

[0135] In some aspects, the composition comprises an agent that enhances the chemical or physical characteristics of the antigen binding protein, e.g., by stabilizing the antigen binding protein at a particular temperature, e.g., room temperature, extending its shelf life, inhibiting degradation, e.g., oxidative protease-mediated degradation, extending the half-life of the antigen binding protein, etc. In some aspects, the composition comprises any of the agents disclosed herein as a heterologous or conjugated moiety, optionally in admixture with or conjugated to an antigen binding protein of the present disclosure.

[0136] In various aspects of the disclosure, the composition further comprises a pharmaceutically acceptable carrier, diluent, or excipient. In some embodiments, the antigen binding protein, nucleic acid, vector, host cell, or conjugate of the disclosure (hereinafter referred to as the "active agent") is formulated into a pharmaceutical composition comprising the active agent together with a pharmaceutically acceptable carrier, diluent, or excipient.

[0137] In some embodiments, the active agent is present in the composition and in a pharmaceutically acceptable diluent, carrier, or excipient at 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%. In some embodiments, the composition contains the active agent at a concentration of about 0.001 to about 30.0 mg / ml.

[0138] In various embodiments, the composition comprises a pharmaceutically acceptable carrier. As used herein, the term "pharmaceutically acceptable carrier" includes any of the standard pharmaceutical carriers, such as phosphate buffered saline solution, water, emulsions such as oil-in-water or water-in-oil emulsions, and various types of wetting agents. The term also encompasses any of the agents approved by a regulatory agency of the U.S. federal government or listed in the U.S. Pharmacopoeia for use in animals, including humans.

[0139] The composition may contain, for example, an acidifying agent, an additive, an adsorbent, an aerosol propellant, an air displacing agent, an alkalizing agent, an anti-caking agent, an anticoagulant, an antimicrobial preservative, an antioxidant, a preservative, a base, a binder, a buffer, a chelating agent, a coating agent, a colorant, a drying agent, a detergent, a diluent, a disinfectant, a disintegrant, a dispersant, a dissolution enhancer, a pigment, an emollient, an emulsifier, an emulsion stabilizer, a filler, a film former, a flavor enhancer, a flavoring agent, a flow improver, a gelling agent, a soluble solid ... Any pharmaceutically acceptable ingredient may be included, including: granulating agent, humectant, lubricant, mucoadhesive agent, ointment base, ointment, oily vehicle, organic base, lozenge base, pigment, plasticizer, abrasive, preservative, sequestering agent, skin penetration agent, solubilizer, solvent, stabilizer, suppository base, surface active agent, surfactant, suspending agent, sweetener, therapeutic agent, viscosity agent, tonicity agent, toxicity agent, thickener, water absorption agent, water-miscible cosolvent, water softener or humectant.See, for example, Handbook of Pharmaceutical Excipients, Third Edition, AH Kibbe (Pharmaceutical Press, London, UK, 2000) (incorporated in its entirety by reference), Remington's Pharmaceutical Sciences, Sixteenth Edition, EW Martin (Mack Publishing Co., Easton, Pa., 1980) (incorporated in its entirety by reference).

[0140] In various aspects, the compositions comprise formulation materials that are non-toxic to recipients at the dosages and concentrations employed. In specific embodiments, the compositions comprise an active agent and one or more pharmaceutically acceptable salts, polyols, surfactants, osmotic agents, tonicity agents, antioxidants, antibiotics, antifungals, bulking agents, lyoprotectants, antifoaming agents, chelating agents, preservatives, coloring agents, analgesics, or additional pharmaceutical agents. In various aspects, the compositions comprise one or more polyols and / or one or more surfactants, optionally in addition to one or more excipients, including, but not limited to, pharmaceutically acceptable salts, osmotic agents (tonicity agents), antioxidants, antibiotics, antifungals, bulking agents, lyoprotectants, antifoaming agents, chelating agents, preservatives, coloring agents, and analgesics.

[0141] In certain embodiments, the compositions can contain formulation materials to alter, maintain, or preserve, for example, the pH, osmolality, viscosity, clarity, color, isotonicity, flavor, sterility, stability, rate of dissolution or release, adsorption, or penetration of the composition. In such embodiments, suitable formulation materials include, but are not limited to, 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., borate, bicarbonate, Tris-HCl, citrate, phosphate, or other organic acids); bulking agents (e.g., mannitol or glycine); chelating agents (e.g., ethylenediaminetetraacetic acid (EDTA)); complexing agents (e.g., caffeine, polyvinylpyrrolidone, beta-cyclodextrin, or hydroxypropyl-beta-cyclodextrin); fillers; monosaccharides; disaccharides; and other carbohydrates (e.g., glucose, mannose, or dextrin); proteins (e.g., serum albumin, gelatin, or immunoglobulins); colorants, flavoring agents, and diluents; emulsifiers; hydrophilic polymers (e.g., polyvinylpyrrolidone); low molecular weight polypeptides; salt-forming agents. 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, polysorbate (polysorbate), triton, tromethamine, lecithin, cholesterol, tyloxapol); stability enhancers (e.g., sucrose or sorbitol); tonicity enhancers (e.g., alkali metal halides, preferably sodium or potassium chloride, mannitol, sorbitol); delivery vehicles; diluents; excipients, and / or pharmaceutical adjuvants.See REMINGTON'S PHARMACEUTICAL SCIENCES, 18th Edition, (AR Genrmo, ed.), 1990, Mack Publishing Company.

[0142] The composition may be formulated to achieve a physiologically compatible pH. In some embodiments, the pH of the composition may be, for example, about 4, or about 5 to about 8.0, or about 4.5 to about 7.5, or about 5.0 to about 7.5. In various embodiments, the pH of the composition is 5.5 to 7.5.

[0143] The present disclosure provides methods of making a composition. In various aspects, the method comprises combining an antigen binding protein, a conjugate, a fusion protein, a nucleic acid, a vector, a host cell, or a combination thereof with a pharmaceutically acceptable carrier, diluent, or excipient.

[0144] use The antigen binding proteins of the present disclosure are useful for detecting or monitoring tumors or tumor growth. Thus, the antigen binding proteins are useful in methods or assays for detecting or monitoring tumors or tumor growth. In some aspects, the methods or assays include, but are not limited to, immunohistochemistry (IHC), immunofluorescence (IF), flow cytometry, surface plasmon resonance (SPR), localized SPR, radioimmunoassay (RIA), enzyme immunoassay (EIA) (such as enzyme-linked immunosorbent assay, ELISA), fluorescent immunoassay (FIA), chemiluminescent immunoassay (CLIA), counting immunoassay (CIA), and immunoblotting (e.g., Western blot), in which the antigen binding proteins of the present disclosure are used to detect the presence of CLDN6 protein in a sample. In one embodiment, the method or assay is immunohistochemistry (IHC). In various aspects, the sample can be a sample freshly excised or extracted from a subject, a fresh sample, a frozen sample, a flash-frozen sample, a formalin-fixed sample, a paraffin-embedded sample, or a lysate. In various aspects, the sample may be a bodily fluid from the subject, such as blood, plasma, saliva, secretions or excretions, or tissue from the subject. In various aspects, the tumor may be benign or cancerous. In various aspects, the antigen binding proteins of the present disclosure may be used in conjunction with a therapeutic agent to treat benign or cancerous tumors, where the antigen binding proteins of the present disclosure are used to monitor the tumor or tumor growth.

[0145] In addition, provided herein is a method for treating a subject diagnosed with cancer, for example, a CLDN6-expressing cancer. In various embodiments, the method comprises administering to the subject an amount of the pharmaceutical composition of the present disclosure or any cancer therapy known in the art that is effective to treat the cancer in the subject.

[0146] For purposes herein, the cancer of the methods disclosed herein can be any cancer, for example, any malignant growth or tumor caused by abnormal and uncontrolled cell division that can spread to other parts of the body via the lymphatic system or bloodstream. Cancer, in some aspects, includes acute lymphocytic cancer, acute myeloid leukemia, alveolar rhabdomyosarcoma, bone cancer, brain cancer, breast cancer, cancer of the anus, anal canal, or anorectum, eye cancer, cancer of the intrahepatic bile duct, cancer of the joints, cancer of the neck, gallbladder, or pleura, cancer of the nose, nasal cavity, or middle ear, cancer of the oral cavity, cancer of the vulva, chronic lymphocytic leukemia, chronic myeloid cancer, colon cancer, esophageal cancer, cervical cancer, gastrointestinal carcinoid tumor, Hodgkin's disease, and the like. In certain embodiments, the cancer is selected from the group consisting of head and neck cancer, ovarian cancer, ovarian cancer, cervical cancer, bladder and esophageal cancer, pancreatic cancer, gastric 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, ovarian cancer, rectal cancer, renal cancer (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, the cancer is selected from the group consisting of head and neck cancer, ovarian cancer, cervical cancer, bladder and esophageal cancer, pancreatic cancer, gastrointestinal cancer, gastric cancer, breast cancer, endometrial cancer and colorectal cancer, hepatocellular carcinoma, glioblastoma, bladder cancer, lung cancer, e.g., non-small cell lung cancer (NSCLC), bronchioloalveolar carcinoma. In various embodiments, the cancer is ovarian cancer, melanoma, bladder cancer, lung cancer, liver cancer, or endometrial cancer. In various embodiments, the cancer is any cancer characterized by moderate to high expression of CLDN6. See, e.g., Figure 4. In various embodiments, the cancer is acute myeloid leukemia, large B-cell lymphoma, gastric cancer, prostate cancer, melanoma, colon cancer, rectal cancer, bladder cancer, cervical cancer, liver cancer, breast cancer, renal clear cell carcinoma, head and neck cancer, sarcoma, chromophobe renal carcinoma, low-grade glioma, adrenocortical carcinoma, glioblastoma, papillary renal cell carcinoma, lung squamous cell carcinoma, thyroid cancer, lung adenocarcinoma, pancreatic cancer, endometrioid carcinoma, uterine carcinosarcoma, or ovarian cancer. In various embodiments, the cancer is selected from ovarian cancer, endometrioid carcinoma, uterine cancer, lung cancer, gastric cancer, breast cancer, head and neck squamous cell carcinoma (HNSCC), cervical cancer, and bladder cancer.

[0147] As used herein, the term "treat" and related terms do not necessarily mean 100% or complete treatment. Rather, there are various degrees of treatment that those skilled in the art will recognize as having potential benefit or therapeutic effect. In this regard, the methods of treating cancer disclosed herein may provide any amount or level of treatment. Furthermore, the treatment provided by the methods of the present disclosure may include treatment of one or more conditions, symptoms, or signs of the cancer being treated. The treatment provided by the methods of the present disclosure may also include slowing the progression of the cancer. For example, the methods may treat cancer by enhancing T cell activity or immune response against the cancer, suppressing tumor or cancer growth, suppressing tumor cell metastasis, increasing tumor or cancer cell cell death, and the like. In various embodiments, the methods treat to delay the onset or recurrence of cancer for at least 1 day, 2 days, 4 days, 6 days, 8 days, 10 days, 15 days, 30 days, 2 months, 3 months, 4 months, 6 months, 1 year, 2 years, 3 years, or 4 years or more. In various embodiments, the methods treat to prolong survival of a subject.

[0148] The antigen binding proteins of the present disclosure can also be used to detect or determine the quantity of claudin-6 (CLDN6) in a sample, diagnose claudin-6 (CLDN6)-positive cancer in a subject, monitor the course of claudin-6 (CLDN6)-positive cancer in a subject, classify tumors in a subject, determine whether a cancer is treatable by a cancer therapy targeting claudin-6 (CLDN6)-positive cancer in a subject, and / or determine the prognosis of a subject with claudin-6 (CLDN6)-positive cancer. Accordingly, the present disclosure provides methods for detecting or quantifying claudin-6 (CLDN6) in a sample. In various embodiments, the method comprises contacting the sample with an antigen binding protein, conjugate, or fusion protein described herein and assaying for immune complexes comprising the antigen binding protein, conjugate, or fusion protein bound to CLDN6. The present disclosure also provides methods for monitoring, classifying, or diagnosing claudin-6 (CLDN6)-positive cancer in a subject. In various embodiments, the method comprises contacting a biological sample comprising cells or tissue obtained from a subject with an antigen binding protein, conjugate, or fusion protein described herein, and assaying for immune complexes comprising the antigen binding protein, conjugate, or fusion protein bound to CLDN6. The present disclosure also provides methods for determining whether a cancer is treatable by a cancer therapy that targets CLDN6-positive cancer in a subject, or for determining the prognosis of a subject with claudin-6 (CLDN6)-positive cancer in a subject. In various embodiments, the method comprises contacting a biological sample comprising cells or tissue obtained from a subject with an antigen binding protein, conjugate, or fusion protein described herein, and assaying for immune complexes comprising the antigen binding protein, conjugate, or fusion protein bound to CLDN6.

[0149] The present disclosure also provides assays for detecting or determining the quantity of claudin-6 (CLDN6) in a sample, assays for diagnosing claudin-6 (CLDN6)-positive cancer in a subject, assays for monitoring the course of claudin-6 (CLDN6)-positive cancer in a subject, assays for classifying tumors in a subject, assays for determining whether a cancer in a subject is treatable by a cancer therapy that targets claudin-6 (CLDN6)-positive cancer, and / or assays for determining the prognosis of a subject with claudin-6 (CLDN6)-positive cancer, the assays comprising contacting the sample with a first antigen binding protein specific for CLDN6 to form a first antigen binding protein-CLDN6 immune complex, contacting the immune complex so formed with a second antigen binding protein labeled with a detectable label to form a second immune complex comprising the first antigen binding protein-CLDN6-second antigen binding protein, and detecting the second immune complex so formed, which indicates the presence of CLDN6 in the sample. In certain embodiments, the second antigen binding protein is an antibody that recognizes the first antigen binding protein.

[0150] Diagnostic and prognostic methods The present invention, in part, provides methods, systems, and code for accurately classifying whether a biological sample contains CLDN6 and / or whether the level of CLDN6 is altered (e.g., upregulated or downregulated), thereby indicating the status of a disorder of interest, such as cancer. In some embodiments, the present invention is useful for classifying a sample (e.g., from a subject) as associated with or at risk for cancer or a subtype thereof using statistical algorithms and / or empirical data (e.g., the presence, absence, or level of CLDN6).

[0151] An exemplary method for detecting the level of CLDN6, and thus useful for classifying whether a sample is associated with cancer or its clinical subtypes or various stages of cancer, involves obtaining a biological sample from a test subject and contacting the biological sample with an antigen binding protein of the present invention capable of detecting CLDN6, such that the level of CLDN6 in the biological sample is detected. In some embodiments, at least one antigen binding protein is used, and two, three, four, five, six, seven, eight, nine, ten, or more such antibodies or antibody fragments may be used in combination (e.g., sandwich ELISA) or sequentially. In certain cases, the statistical algorithm is a single learning statistical classifier system. For example, a single learning statistical classifier system can be used to classify a sample as a cancer sample based on a predictive value or probability value and the presence or level of CLDN6. Use of a single learning statistical classifier system typically classifies a sample as a cancer sample with a sensitivity, specificity, positive predictive value, negative predictive value, and / or overall accuracy of at least or about 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.

[0152] Other suitable statistical algorithms are well known to those skilled in the art.For example, learning statistical classifier system includes machine learning algorithm technology, which can adapt to complex data sets (for example, a panel of target markers) and make decisions based on such data sets.In some embodiments, a single learning statistical classifier system is used, such as classification tree (for example, random forest).In other embodiments, a combination of 2, 3, 4, 5, 6, 7, 8, 9, 10 or more learning statistical classifier systems is preferably used in tandem. Examples of learning statistical classifier systems include, but are not limited to, those that use inductive learning (e.g., decision / classification trees, e.g., random forests, classification and regression trees (C&RT), boosted trees, etc.), probabilistic and approximately correct (PAC) learning, connectionist learning (e.g., neural networks (NNs), artificial neural networks (ANNs), neuro-fuzzy networks (NFNs), network structures, perceptrons such as multi-layer perceptrons, multi-layer feed-forward networks, applications of neural networks, Bayesian learning in belief networks, etc.), reinforcement learning (e.g., passive learning in a known environment, e.g., naive learning, adaptive dynamic learning, and time-lag learning; passive learning in an unknown environment; active learning in an unknown environment; learning action-value functions; applications of reinforcement learning, etc.), and genetic algorithms and evolutionary programming. Other learning statistical classifier systems include support vector machines (e.g., kernel methods), multivariate adaptive regression splines (MARS), the Levenberg-Marquardt algorithm, the Gauss-Newton algorithm, Gaussian mixtures, gradient descent algorithms, and learning vector quantization (LVQ). In certain embodiments, the methods of the invention further comprise sending the sample classification results to a clinician (a non-specialist, e.g., a primary care physician; and / or a specialist, e.g., a histopathologist or oncologist).

[0153] In some embodiments, the methods of the present disclosure further provide a diagnosis in the form of a probability that an individual has cancer. For example, an individual may have a probability of having cancer of about 0%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more. In yet another embodiment, the methods of the present disclosure further provide a prognosis of cancer for the individual. In some cases, classifying a sample as a cancer sample may be further based on symptoms (e.g., clinical factors) of the individual from whom the sample was obtained. The symptom or symptoms may be, for example, lymphocyte count, white blood cell count, erythrocyte sedimentation rate, diarrhea, abdominal pain, abdominal distension, pelvic pain, lower back pain, cramps, fever, anemia, weight loss, anxiety, depression, and combinations thereof. In some cases, the method of classifying a sample as a cancer sample may be further based on genetic mutations and / or predisposition to cancer, regardless of symptoms. In some embodiments, diagnosing an individual as having cancer is followed by administering a therapeutically effective amount of a cancer therapy (e.g., a chemotherapeutic agent) to the individual.

[0154] An exemplary method for detecting the presence or absence of CLDN6 includes using an antigen-binding protein of the present disclosure capable of binding to CLDN6, preferably an antigen-binding protein having a detectable label. The antigen-binding protein can be an antibody. The antibody can be a polyclonal antibody, or more preferably, a monoclonal antibody. Such agents can be labeled. With respect to antibodies, the term "labeled" is intended to encompass both direct labeling of the probe or antibody by coupling (i.e., physically linking) a detectable substance to the probe or antibody, and indirect labeling of the probe or antibody by reactivity with another directly labeled reagent. An example of indirect labeling is detection of a primary antibody using a fluorescently labeled secondary antibody. The term "biological sample" is intended to include tissues, cells, and biological fluids isolated from a subject, such as serum and blood, as well as tissues, cells, and fluids present within a subject. That is, the detection method of the present disclosure can be used to detect CLDN6 in a biological sample in vitro and / or ex vivo. In vitro techniques for detection of CLDN6 include enzyme-linked immunosorbent assay (ELISA), immunoprecipitation, immunohistochemistry (IHC), flow cytometry and related techniques, and immunofluorescence.

[0155] In some embodiments, the method further involves obtaining a control biological sample (e.g., a biological sample from a subject without cancer), a biological sample from a subject in remission or before the onset of cancer, or a biological sample from a subject being treated for the onset of cancer.

[0156] In some embodiments, the method includes contacting the control sample with a compound or agent capable of detecting CLDN6 so as to detect the presence and / or level of CLDN6 in the biological sample, and comparing the presence or level of CLDN6 in the control sample with the presence or level of CLDN6 in the test sample.

[0157] In some embodiments, the biological sample is serum, blood, saliva, tissue, tumor microenvironment, peritumoral sample, or intratumoral sample isolated by conventional means from a subject.

[0158] In some embodiments, the antigen-binding protein may be associated with a component or device for using the antibody in an ELISA or RIA. Non-limiting examples include antigen-binding proteins immobilized on a solid surface for use in these assays (e.g., linked and / or conjugated to a label detectable based on luminescence or radiation emission, as described above). In other embodiments, the antigen-binding protein is associated with a device or strip for detection of CLDN6 by use of immunochromatographic or immunochemical assays, such as "sandwich" or competitive assays, immunohistochemistry, immunofluorescence microscopy, etc. Further examples of such devices or strips are those designed for home testing or rapid point-of-care testing. Further examples include those designed for simultaneous analysis of multiple analytes in a single sample. For example, an unlabeled antibody of the present invention may be applied to "capture" CLDN6 in a biological sample, and the captured (or immobilized) CLDN6 may be bound to an antigen-binding protein of the present disclosure in labeled form for detection. Other embodiments of immunoassays are well known to those skilled in the art, including, for example, immunodiffusion, immunoelectrophoresis, immunohistopathology, immunohistochemistry, and histopathology-based assays.

[0159] In some embodiments, the compositions and methods of the present disclosure can be used to determine the grade of a cancer based on the level of CLDN6 determined as described herein. Cancer grade refers to how abnormal the appearance of cancer cells and tissues is compared to healthy cells under a microscope. Cancer cells whose appearance and organization most closely resemble healthy cells and tissues are low-grade tumors. Physicians describe these cancers as well-differentiated. Typically, the lower the grade of a cancer, the less aggressive it is and the better the prognosis. The more abnormal the appearance and organization of the cells, the higher the grade of the cancer. Cancer cells with a higher grade tend to be more aggressive. These are called poorly differentiated or undifferentiated. Some cancers have their own systems for assessing tumor grade. Many other cancers use a standard 1-4 grading scale.

[0160] Grade 1: The tumor cells and tissues look most similar to healthy cells and tissues. These are called well-differentiated tumors and are considered low grade.

[0161] Grade 2: The cells and tissue are slightly abnormal and are called moderately differentiated. These are intermediate-grade tumors.

[0162] Grade 3: The cancer cells and tissue look very abnormal. These cancers are considered poorly differentiated because they no longer have an architectural structure or pattern.

[0163] Grade 3 tumors are considered high grade.

[0164] Grade 4: These undifferentiated cancers have the most abnormal-looking cells. They are the highest grade and typically grow and spread more quickly than lower-grade tumors.

[0165] As used herein, low-grade cancer refers to grade I cancer, and high-grade cancer refers to grade 2-4 cancer.

[0166] In some embodiments, the compositions and methods of the present disclosure can be used to determine the grade of a cancer based on the level of CLDN6 determined as described herein.

[0167] Cancer stages describe how large the primary tumor is and how far the cancer has spread in the patient's body. Several different staging systems exist. Many of these are created for specific types of cancer. Other systems can be used to describe several types of cancer. One common system that many people are aware of rates cancer on a scale of 0 to IV.

[0168] Stage 0 is for abnormal cells that have not spread and are not considered cancerous, but have the potential to become cancerous cells in the future. This stage is also called "in-situ."

[0169] Stages I through III refer to cancer that has not spread beyond the primary tumor site or has only spread to nearby tissues. The higher the stage number, the larger and more widespread the tumor.

[0170] Stage IV cancer has spread to distant areas of the body.

[0171] As used herein, early / low stage cancer refers to stage I cancer, and late / high / advanced stage cancer includes stage II through stage IV cancer.

[0172] In some embodiments, the compositions and methods of the present disclosure can be used to determine a subject's tumor burden based on the level of CLDN6 determined as described herein. Tumor burden (or tumor burden) is defined as the total amount (cells / mass) of tumor distributed throughout a patient's body, including the bone marrow. In Response Evaluation Criteria in Solid Tumors (RECIST) analysis, tumor burden is considered to be the sum of the longest diameters of all measurable lesions. Various methods can be used to determine tumor burden in a subject. For example, computed tomography (CT) and magnetic resonance (MR) imaging have been used to evaluate tumor response based on morphological (size, location) criteria, specifically by using RECIST. RECIST classification describes the size of the lesion and distinguishes between four types of treatment response: stable disease (SD), partial response (PR), complete response (CR), or progressive disease (PD).

[0173] The assays described herein can be used for prognosis determination methods. The term "prognosis" includes the prediction of the probable course and outcome of cancer, or the likelihood of recovery from the disease. In some embodiments, the use of statistical algorithms provides a prognosis for cancer in an individual. For example, the prognosis can be surgical treatment, the occurrence of a clinical subtype of cancer (e.g., solid tumors such as lung cancer, melanoma, and renal cell carcinoma), the occurrence of one or more clinical factors, the occurrence of intestinal cancer, or recovery from the disease.

[0174] The assays described herein can be used to determine whether a subject can be administered an agent (e.g., an agonist, antagonist, peptidomimetic, polypeptide, peptide, nucleic acid, small molecule, immunotherapy, immune checkpoint inhibitor therapy, or other drug candidate) for treating cancer. For example, such methods can be used to determine whether a subject can be effectively treated with one or a combination of agents. Thus, the present disclosure provides a method for determining whether a subject can be effectively treated with one or more agents for treating cancer, wherein a test sample is obtained and CLDN6 is detected.

[0175] The methods described herein may be practiced, for example, by utilizing pre-packaged diagnostic kits containing at least one antibody reagent described herein, which can be conveniently used, for example, in a clinical setting, to diagnose patients who exhibit symptoms or a family history of cancer.

[0176] Other aspects of the present disclosure include the use of the compositions and methods described herein for association and / or stratification analyses that analyze CLDN6 in biological samples from individuals with cancer and compare the information to that of controls, preferably of similar age and race (e.g., individuals without cancer; controls may also be referred to as "healthy" or "normal" individuals or individuals at an earlier time point in a given time course study). Appropriate selection of patients and controls can be critical to the success of association and / or stratification studies. Therefore, a population of individuals with a well-characterized phenotype is highly desirable. Criteria for cancer diagnosis, cancer predisposition screening, prediction of clinical outcome, cancer prognosis, determination of drug response (pharmacogenomics), drug toxicity screening, and the like are described herein.

[0177] Various study designs can be used for genetic association and / or stratification studies (Modern Epidemiology, Lippincott Williams & Wilkins (1998), 609-622). Observational studies, which do not interfere with patient responses, are the most frequently performed. The first type of observational study involves identifying a sample from people with a suspected cause of disease and another sample from people without the suspected cause, and then comparing the frequency of the disease in the two samples. These sampled populations are called cohorts, and the study is prospective. Other types of observational studies are case-control or retrospective studies. In a typical case-control study, samples are collected from individuals with a phenotype of interest (such as a specific finding of disease) and individuals without the phenotype (controls) in a population from which conclusions are drawn (the target population). The possible causes of the disease are then investigated retrospectively. Because the time and cost of collecting samples in a case-control study are significantly less than those in a prospective study, case-control studies are a more commonly used study design for genetic association studies, at least in the discovery and discovery stages.

[0178] After all relevant phenotypic and / or genotypic information is obtained, statistical analysis is performed to determine whether there is any significant correlation between the presence of a certain allele or genotype and an individual's phenotypic traits. Preferably, data inspection and cleaning is performed first, followed by statistical testing of genetic association. Epidemiological and clinical data of the samples can be summarized by descriptive statistics using tables and graphs well known in the art. Preferably, data validation is performed to check for completeness, inconsistent entries, and outliers. Chi-square tests and t-tests (or Wilcoxon rank-sum tests if the distribution is not normal) can then be used to identify significant differences between cases and controls for discrete and continuous variables, respectively.

[0179] An important decision in conducting gene association testing is determining the significance level at which the p-value of the test can indicate a significant association when it reaches that level. In exploratory analysis, where positive hits are followed up in subsequent confirmatory testing, for example, an unadjusted p-value of less than 0.2 (a generous significance level) can be used to hypothesize a significant association between CLDN6 levels and certain phenotypic traits of cancer. In order for CLDN6 levels to be considered to be associated with cancer, it is preferable to achieve a p-value of less than 0.05 (the significance level traditionally used in the art). When hits in more samples from the same source or in various samples from different sources are followed up in confirmatory analysis, multiple testing adjustments are performed to avoid excessive hit numbers while maintaining an error rate per experiment of 0.05. There are various methods for adjusting for multiple testing to control various types of error rates, but a commonly used but fairly conservative method is the Bonferroni correction to control the error rate per experiment or family-wise error rate (Multiple comparisons and multiple tests, Westfall et al., SAS Institute (1999)). Permutation tests to control the false positive rate (FDR) may have greater power (Benjamini and Hochberg, Journal of the Royal Statistical Society, Series B57, 1289-1300, 1995; Resampling-based Multiple Testing, Westfall and Young, Wiley (1993)). Such methods for controlling multiplicity are considered preferable when the test is dependent and controlling the false positive rate is sufficient, rather than controlling the error rate per experiment.

[0180] Once genetic or non-genetic individual risk factors for disease predisposition are identified, a classification / prediction scheme can be constructed to predict the category (e.g., disease or non-disease) to which an individual belongs according to the individual's phenotype and / or genotype and other non-genetic risk factors. Logistic regression for discrete traits and linear regression for continuous traits are standard techniques for such tasks (Applied Regression Analysis, Draper and Smith, Wiley (1998)). In addition, other techniques can also be used to establish classification. Such techniques include, but are not limited to, MART, CART, neural networks, and discriminant analysis, which are suitable for comparing the performance of various methods (The Elements of Statistical Learning, Hastie, Tibshirani & Friedman, Springer (2002)).

[0181] Cancer therapy The therapeutic agents of the present invention can be used alone or in combination with, for example, chemotherapeutic agents, hormones, antiangiogenic agents (antiangiogens), radiolabeled compounds, or surgical procedures, cryotherapy, immunotherapy, cancer vaccines, immune cell manipulation (e.g., CAR-T), and / or radiation therapy. The aforementioned treatment methods can be used in conjunction with other forms of conventional therapy (e.g., standard of care treatments for cancer known to those skilled in the art), administered sequentially with, prior to, or after conventional therapy. For example, the agents of the present invention can be administered with a therapeutically effective dose of a chemotherapeutic agent. In other embodiments, the agents of the present invention are administered in combination with chemotherapy to enhance the activity and effectiveness of the chemotherapeutic agent. The Physicians' Desk Reference (PDR) discloses dosages of chemotherapeutic agents used in the treatment of various cancers. The therapeutically effective dosing regimens and dosages of these aforementioned chemotherapeutic agents depend on the particular cancer being treated, the extent of the disease, and other factors well known to physicians skilled in the art, and can be determined by a physician.

[0182] Immunotherapy is a targeted therapy that may involve, for example, the use of cancer vaccines and / or sensitized antigen-presenting cells. For example, oncolytic viruses are viruses that can infect and lyse cancer cells while leaving normal cells unharmed, making them potentially useful in cancer therapy. Oncolytic virus replication promotes tumor cell destruction and also results in dose amplification at the tumor site. They can also act as vectors for anti-cancer genes, allowing them to be delivered specifically to the tumor site. Immunotherapy may involve passive immunity for short-term host protection, achieved by administering preformed antibodies against cancer or disease antigens (e.g., administering monoclonal antibodies to tumor antigens, optionally linked to chemotherapeutic agents or toxins). For example, anti-VEGF is known to be effective in treating renal cell carcinoma. Immunotherapy can also focus on the use of epitopes of cancer cell lines that are recognized by cytotoxic lymphocytes. Alternatively, antisense polynucleotides, ribozymes, RNA interference molecules, triple helix polynucleotides, and the like can be used to selectively modulate biomolecules associated with tumor or cancer initiation, progression, and / or pathology.

[0183] Immunotherapy also encompasses immune checkpoint modulators, which are groups of molecules on the cell surface of CD4+ and / or CD8+ T cells that fine-tune the immune response by downregulating or inhibiting anti-tumor immune responses. Immune checkpoint proteins are well known in the art and include, but are not limited to, CTLA-4, PD-1, VISTA, B7-H2, B7-H3, PD-L1, B7-H4, B7-H6, 2B4, ICOS, HVEM, PD-L2, CD160, gp49B, PIR-B, KIR family receptors, TIM-1, TIM-3, TIM-4, LAG-3, BTLA, SIRP alpha (CD47), CD48, 2B4 (CD244), B7.1, B7.2, ILT-2, ILT-4, TIGIT, HHLA2, TMIDG2, KIR3DL3, and A2aR (see, e.g., WO2012 / 177624). Inhibition of one or more immune checkpoint inhibitors can block or otherwise neutralize inhibitory signaling, thereby upregulating the immune response, to more effectively treat cancer. In some embodiments, the cancer vaccine is administered in combination with an inhibitor of one or more immune checkpoints (immune checkpoint blockade therapy), such as a PD1, PD-L1, and / or CD47 inhibitor.

[0184] Adoptive cell-based immunotherapy can be combined with the therapies of the present invention. Known adoptive cell-based immunotherapy modalities include, but are not limited to, irradiated autologous or allogeneic tumor cells, tumor lysates or apoptotic tumor cells, antigen-presenting cell-based immunotherapy, dendritic cell-based immunotherapy, adoptive T cell transfer, adoptive CART cell therapy, autologous immune enhancement therapy (AIET), cancer vaccines, and / or antigen-presenting cells. Such cell-based immunotherapies can be further modified to express one or more gene products that further modulate the immune response, such as expression of cytokines like GM-CSF, and / or to express antigens of tumor-associated antigens (TAA), e.g., Mage-1, gp-100, etc.

[0185] The term "chimeric antigen receptor" or "CAR" refers to an engineered T cell receptor (TCR) with a desired antigen specificity. T lymphocytes recognize specific antigens through the interaction of the T cell receptor (TCR) with short peptides presented by major histocompatibility complex (MHC) class I or II molecules. For initial activation and clonal expansion, naive T cells depend on professional antigen-presenting cells (APCs) that provide additional costimulatory signals. Activation of the TCR without costimulation can result in unresponsiveness and clonal anergy. To avoid immunization, different approaches have been developed to induce cytotoxic effector cells with the transplanted recognition specificity. CARs have been constructed, consisting of binding domains derived from natural ligands or antibodies specific for cell surface components of the CD3 complex associated with the TCR. Upon antigen binding, such chimeric antigen receptors engage endogenous signaling pathways in the effector cell, generating activation signals similar to those initiated by the TCR complex. Since the first report of chimeric antigen receptors, this concept has been steadily refined, and the molecular design of chimeric receptors has been optimized and now routinely uses many well-known binding domains, such as scFVs and other protein-binding fragments described herein.

[0186] In other embodiments, the immunotherapy comprises non-cell-based immunotherapy. In some embodiments, a composition comprising an antigen, with or without a vaccine-enhancing adjuvant, is used. Such compositions exist in many well-known forms, such as peptide compositions, oncolytic viruses, recombinant antigens including fusion proteins, and the like. In some embodiments, immunomodulatory cytokines, such as interferons, G-CSF, imiquimod, TNF-alpha, and the like, and their modulators (e.g., blocking antibodies or more potent or longer-lasting forms), are used. In some embodiments, immunomodulatory interleukins, such as IL-2, IL-6, IL-7, IL-12, IL-17, IL-23, and the like, and their modulators (e.g., blocking antibodies or more potent or longer-lasting forms), are used. In some embodiments, immunomodulatory chemokines, such as CCL3, CCL26, and CXCL7, and their modulators (e.g., blocking antibodies or more potent or longer-lasting forms), are used. In some embodiments, immunomodulatory molecule targeted immunosuppressants are used, such as STAT3 signaling modulators, NF-kappa B signaling modulators, and immune checkpoint modulators.

[0187] In yet other embodiments, immunomodulatory agents, such as immune cytostatic agents, glucocorticoids, cytostatic agents, immunophilins and their modulators (e.g., rapamycin, calcineurin inhibitors, tacrolimus, cyclosporine, pimecrolimus, avetimus, gusperimus, ridaforolimus, everolimus, temsirolimus, zotarolimus, etc.), hydrocortisone (cortisol), cortisone acetate, prednisone, prednisolone, methyl Ruprednisolone, dexamethasone, betamethasone, triamcinolone, beclomethasone, fludrocortisone acetate, deoxycorticosterone acetate (doca), aldosterone, non-glucocorticoid steroids, pyrimidine synthesis inhibitors, leflunomide, teriflunomide, folic acid analogues, methotrexate, antithymocyte globulin, antilymphocyte globulin, thalidomide, lenalidomide, pentoxifylline, bupropion, curcumin, catechin, opioids , IMPDH inhibitors, mycophenolic acid, myriocin, fingolimod, NF-xB inhibitors, raloxifene, drotrecogin alfa, denosumab, NF-xB signaling cascade inhibitors, disulfiram, olmesartan, dithiocarbamates, proteasome inhibitors, bortezomib, MG132, Prol, NPI-0052, curcumin, genistein, resveratrol, parthenolide, thalidomide, lenalidomide, flavopiridol, Nonsteroidal anti-inflammatory drugs (NSAIDs), arsenic trioxide, dehydroxymethylepoxyquinomycin (DHMEQ), I3C (indole-3-carbinol) / DIM (di-indolemethane) (I3C / DIM), Bay11-7082, luteolin, cell-penetrating peptide SN-50, IKBa.-superrepressor overexpression, NFKB decoy oligodeoxynucleotide (ODN), or any derivative or analog thereof, are used. In yet other embodiments, immunomodulatory antibodies or proteins are used.For example, antibodies that bind to CD40, Toll-like receptors (TLRs), OX40, GITR, CD27, or 4-1BB, T cell bispecific antibodies, anti-IL-2 receptor antibodies, anti-CD3 antibodies, OKT3 (muromonab), otelixizumab, teplizumab, visilizumab, anti-CD4 antibodies, clenoliximab, keliximab, zanolimumab, anti-CD11 antibodies, efalizumab, anti-CD18 antibodies, erulizumab, rovelizumab, anti-CD20 antibodies, afutuzumab, and Clerizumab, ofatumumab, pascolizumab, rituximab, anti-CD23 antibody, lumiliximab, anti-CD40 antibody, teneliximab, toralizumab, anti-CD40L antibody, ruplizumab, anti-CD62L antibody, aselizumab, anti-CD80 antibody, galiximab, anti-CD147 antibody, gavilimomab, B-lymphocyte stimulator (BLyS) inhibitor antibody, belimumab, CTLA4-Ig fusion protein, abatacept, belatacept, anti-CTLA4 antibody, ipilimumab , tremelimumab, anti-eotaxin 1 antibody, bertilimumab, anti-a4-integrin antibody, natalizumab, anti-IL-6R antibody, tocilizumab, anti-LFA-1 antibody, ozlimomab, anti-CD25 antibody, basiliximab, daclizumab, inolimomab, anti-CD5 antibody, zolimomab, anti-CD2 antibody, siplizumab, nerelimomab, faralimomab, atlizumab, atolimumab, cedelizumab, dorlimomab-allitoxin, dorlixizumab, phontri Ibuprofen, gantenerumab, gomiliximab, levrilizumab, maslimomab, morolimumab, pexelizumab, reslizumab, rovelizumab, talizumab, terimomab alitox, bapaliximab, beparimomab, aflibercept, alefacept, rilonacept, IL-1 receptor antagonists, anakinra, anti-IL-5 antibodies, mepolizumab, IgE inhibitors, omalizumab, talizumab, IL12 inhibitors, IL23 inhibitors, ustekinumab, etc.

[0188] Nutritional supplements that enhance immune responses, such as vitamin A, vitamin E, vitamin C, and the like, are known in the art (see, e.g., U.S. Pat. Nos. 4,981,844 and 5,230,902 and PCT Publication No. WO2004 / 004483), and can be used in the methods described herein.

[0189] Similarly, various agents or combinations thereof can be used to treat cancer, such as chemotherapy, radiation, epigenetic modifiers (e.g., histone deacetylase (HDAC) modifiers, methylation modifiers, phosphorylation modifiers, etc.), targeted therapies, etc., are well known in the art.

[0190] In some embodiments, chemotherapy is used. Chemotherapy includes the administration of a chemotherapeutic agent. Such chemotherapeutic agents may be selected from the group of compounds, but are not limited to, platinum compounds, cytotoxic antibiotics, antimetabolites, antimitotic agents, alkylating agents, arsenic compounds, DNA topoisomerase inhibitors, taxanes, nucleoside analogs, plant alkaloids, and toxins; and synthetic derivatives thereof. Exemplary compounds include, but are not limited to, alkylating agents: cisplatin, treosulfan, and trophosphamide; plant alkaloids: vinblastine, paclitaxel, docetaxel; DNA topoisomerase inhibitors: teniposide, crisnatol, and mitomycin; antifolates: methotrexate, mycophenolic acid, and hydroxyurea; pyrimidine analogs: 5-fluorouracil, doxifluridine, and cytosine arabinoside; purine analogs: mercaptopurine and thioguanine; DNA antimetabolites: 2'-deoxy-5-fluorouridine, aphidicolin glycinate, and pyrazoloimidazole; and antimitotic agents: halichondrin, colchicine, and rhizoxin. Compositions containing one or more chemotherapeutic agents (e.g., FLAG, CHOP) can also be used. FLAG includes fludarabine, cytosine arabinoside (Ara-C), and G-CSF. CHOP includes cyclophosphamide, vincristine, doxorubicin, and prednisone. In another embodiment, a PARP (e.g., PARP-1 and / or PARP-2) inhibitor is used, and such inhibitors are well known in the art (e.g., olaparib, ABT-888, BSI-201, BGP-15 (N-Gene Research Laboratories, Inc.); INO-1001 (Inotek Pharmaceuticals Inc.); PJ34 (Soriano et al., 2001; Pacher et al., 2002b); 3-aminobenzamide (Trevigen); 4-amino-1,8-naphthalimide (Trevigen); 6(5H)-phenanthridinone (Trevigen); benzamide (U.S. Reissue Patent No. 36,397); and NU1025 (Bowman et al.).The mechanism of action is generally related to the ability of PARP inhibitors to bind to and reduce the activity of PARP. PARP catalyzes the conversion of beta-nicotinamide adenine dinucleotide (NAD) to nicotinamide and poly-ADP-ribose (PAR). Both poly(ADP-ribose) and PARP are involved in the regulation of transcription, cell proliferation, genome stability, and carcinogenesis (Bouchard VJ et al., Experimental Hematology, Volume 31, Number 6, June 2003, pp. 446-454(9); Herceg Z.; Wang Z.-Q. Mutation Research / Fundamental and Molecular Mechanisms of Mutagenesis, Volume 477, Number 1, 2 June 2001, pp. 97-110(14)). Poly(ADP-ribose) polymerase 1 (PARP1) is a key molecule in the repair of DNA single-strand breaks (SSBs) (de Murcia J. et al. 1997. Proc Natl Acad Sci USA 94:7303-7307; Schreiber V, Dantzer F, Ame JC, de Murcia G (2006) Nat Rev Mol Cell Biol 7:517-528; Wang ZQ, et al. (1997) Genes Dev 11:2347-2358). Knockout of SSB repair by inhibiting PARP1 function induces DNA double-strand breaks (DSBs), which can induce synthetic lethality in cancer cells with defective homology-directed DSB repair (Bryant HE, et al. (2005) Nature 434:913-917, Farmer H, et al. (2005) Nature 434:917-921). The above examples of chemotherapeutic agents are illustrative and are not intended to be limiting.

[0191] In other embodiments, radiation therapy is used. The radiation used in radiation therapy can be ionizing radiation. Radiation therapy can also be gamma rays, X-rays, or protons. Examples of radiation therapy include, but are not limited to, external beam radiation therapy, interstitial implantation of radioisotopes (I-125, palladium, iridium), radioisotopes such as strontium-89, thoracic radiation therapy, intraperitoneal P-32 radiation therapy, and / or whole abdominal and pelvic radiation therapy. For a general overview of radiation therapy, see Hellman, Chapter 16: Principles of Cancer Management: Radiation Therapy, 6th edition, 2001, DeVita et al., eds., J.B. Lippencott Company, Philadelphia. Radiation therapy can be administered as external beam radiation or teletherapy, in which radiation is directed from a remote source. Radiation treatment can also be administered as internal therapy or brachytherapy, in which a radiation source is placed inside the body in close proximity to the cancer cells or tumor mass. Also encompassed is the use of photodynamic therapy, which involves the administration of photosensitizers such as hematoporphyrin and its derivatives, vertoporphine (BPD-MA), phthalocyanines, the photosensitizer Pc4, demethoxyhypocrelin A, and 2BA-2-DMHA.

[0192] In other embodiments, hormone therapy is used. Hormone therapy treatments can include, for example, hormone agonists, hormone antagonists (e.g., flutamide, bicalutamide, tamoxifen, raloxifene, leuprolide acetate (LUPRON), LH-RH antagonists), hormone biosynthesis and processing inhibitors, and steroids (e.g., dexamethasone, retinoids, deltoids, betamethasone, cortisol, cortisone, prednisone, dehydrotestosterone, glucocorticoids, mineralocorticoids, estrogens, testosterone, progestins), vitamin A derivatives (e.g., all-trans retinoic acid (ATRA)), vitamin D3 analogs, antigestagens (e.g., mifepristone, onapristone), or antiandrogens (e.g., cyproterone acetate).

[0193] In other embodiments, photodynamic therapy (PDT, also called photoradiotherapy, phototherapy, or photochemotherapy) is used to treat some types of cancer. It is based on the discovery that certain chemicals, known as photosensitizing agents, can kill single-celled organisms when exposed to certain types of light.

[0194] In yet other embodiments, laser therapy is used to destroy cancer cells using high-intensity light. This technique is often used to relieve cancer symptoms such as bleeding or blockages, especially when other treatments are unable to cure the cancer. It can also be used to treat cancer by shrinking or destroying tumors.

[0195] subject In some embodiments of the present disclosure, the subject is a mammal, including, but not limited to, mammals of the order Rodentia, such as mice and hamsters, and mammals of the order Logomorpha, such as rabbits, mammals of the order Carnivora, including Felines (cats) and Canines (dogs), mammals of the order Artiodactyla, including Bovines (cattle) and Swines (pigs), or mammals of the order Perssodactyla, including Equines (horses). In some aspects, the mammal is a mammal of the order Primates, Ceboids, or Simoids (monkeys), or a mammal of the order Anthropoids (humans and apes). In some aspects, the mammal is a human.

[0196] kit In some embodiments, the antigen binding proteins of the present disclosure are provided in a kit. In various aspects, the kit includes the antigen binding protein(s) as a unit assay. For purposes herein, "unit assay" refers to a discrete amount for use in a single assay. In various aspects, each unit assay may include multiple reagents, each separated from one another as needed in the assay. Thus, kits are provided herein that include the antigen binding proteins of the present disclosure, optionally provided in unit assays. In various aspects, the kit includes multiple unit assays, e.g., 5, 10, 25, 50, 100, 500, or 1,000 unit assays, optionally each individually packaged or otherwise separated from the other unit assays. Alternatively, the kit includes multiple unit assays, with each reagent packaged in a single vessel or container for multiple assays. In some embodiments, the components of the kit / unit dose are packaged with instructions for use. In some embodiments, the kit includes one or more devices for use, such as a needle and syringe. In some embodiments, the antigen-binding protein of the present disclosure, its pharmaceutically acceptable salt, conjugate comprising the antigen-binding protein, or multimer or dimer comprising the antigen-binding protein is pre-packaged in a ready-to-use form, e.g., all reagents are in solution, avoiding the need to resuspend or add additional components. In some embodiments, the kit further comprises a therapeutic or additional diagnostic agent or a pharmaceutically acceptable carrier (e.g., solvent, buffer, diluent, etc.), including any of those described herein. In certain embodiments, the kit comprises the antigen-binding protein of the present disclosure together with an agent used in chemotherapy or radiation therapy, e.g., a therapeutic agent. In some embodiments, the kit comprises the antigen-binding protein of the present disclosure together with a second antigen-binding protein labeled with a detectable marker. In some embodiments, the kit comprises the antigen-binding protein of the present disclosure together with a second antigen-binding protein labeled with an epitope or a small molecule.In some embodiments, the kit comprises an antigen binding protein of the present disclosure together with a second antigen binding protein that is labeled with an epitope or small molecule, and a label or detection moiety that binds to the epitope or small molecule. In some embodiments, the kit comprises an antigen binding protein of the present disclosure together with a second antigen binding protein that is labeled with an epitope or small molecule, and a label or detection moiety linked to a binding partner of the epitope or small molecule. In various embodiments, the second antigen binding protein binds to the antigen binding protein of the present disclosure.

[0197] Various embodiments In various embodiments of the present disclosure, the antigen binding protein binds to human claudin-6 (CLDN6) protein (SEQ ID NOS: 1-2), but not to claudin 3 (CLDN3), claudin 4 (CLDN4), or claudin 9 (CLDN9). In various cases, the antigen binding protein binds to an epitope within the amino acid sequence of CCTCPSGGSQGPSHYMARYSTSAPAISRGPSEYPTKNYV (SEQ ID NO: 3), or the amino acid sequence of CLDN6, MASAGMQILGVVLTLLGWVNGLVSCALPMWKVTAFIGNSIVVAQVVWEGLWMSCVVQSTGQMQCKVYDSLLALPQDLQAARALCVIALLVALFGLLVYLAGAKCTTCVEEKDSKARLVLTSGIVFVISGVLTLIPVCWTAHAIIRDFYNPLVAEAQKRELGASLYLGWAASGLLLLGGGLLCCTCPS GGSQGPSHYMARYSTSAPAISRGPSEYPTKNYV (SEQ ID NO: 1) or MASAGMQILGVVLTLLGWVNGLVSCALPMWKVTAFIGNSIVVAQVVWEGLWMSCVVQSTGQMQCKVYDSLLALPQDLQAARALCVIALLVALFGLLVYLAGAKCTTCVEEKDSKARLVLTSGIVFVISGVLTLIPVCWTAHAVIRDFYNPLVAEAQKRELGASLYLGWAASGLLLLGGGLLCCTCPSGGSQGPSHYMARYSTSAPAISRGPSEYPTKNYV (SEQ ID NO: 2). In various aspects, the antigen binding protein does not bind to any one or more of claudin 3 (CLDN3), claudin 4 (CLDN4), and claudin 9 (CLDN9).

[0198] In various embodiments of the present disclosure, the antigen binding protein comprises (a) CDRs 1-3 derived from a heavy chain variable region comprising the amino acid sequence: EIQLQQSGAELVRPGALVRLSCKPSGFNIKDYYIHWVKERPEQGLEWIGWIDPDNGDTLYDSKFQGKASLTADTSSNTAYLQLTSLTSEDTAVYYCATYRYSFAYWGQGTLVTVSA (SEQ ID NO: 16), or a variant sequence thereof differing by only one or two amino acids or having at least or about 80% sequence identity, and / or (b) CDRs 1-3 derived from a light chain variable region comprising the amino acid sequence: DIQMNQSPSSLSASLGDTITITCHASQNINVWLNWYQQKPGNIPKLLIFKASNLYTGVPSRFSGGGSGTGFTLTISSLQPEDIATYYCQQGQTYPLTFGGGTKLEIK (SEQ ID NO: 17), or a variant sequence thereof differing by only one or two amino acids or having at least or about 80% sequence identity. In certain embodiments, the antigen binding protein (a) binds to human claudin-6 (CLDN6), wherein the antigen binding protein binds to CLDN6 by binding at least to an epitope within CLDN6 having the amino acid sequence CCTCPSGGSQGPSHYMARYSTSAPAISRGPSEYPTKNYV (SEQ ID NO: 3); (b) binds to human claudin-6 (CLDN6), wherein the antigen binding protein binds to CLDN6 by binding at least to an epitope within CLDN6 having the amino acid sequence RYSTSAPAISRGPSEYPTKN (SEQ ID NO: 10); (c) binds to human claudin-6 (CLDN6), wherein the antigen binding protein binds to CLDN6 by binding at least to an epitope within CLDN6 having the amino acid sequence RYSTSAPAISRGPSEYPTKN (SEQ ID NO: 100); and / or (d) binds to human claudin-6 (CLDN6), wherein the antigen binding protein binds to CLDN6 by binding at least to an epitope within CLDN6 having the amino acid sequence APAISRGPSEYPTKN (SEQ ID NO: 101).In certain embodiments, the antigen binding protein binds to one or more of the following peptides having the amino acid sequence of CCTCPSGGSQGPSHYMARYSTSAPAISRGPSEYPTKNYV (SEQ ID NO: 3), RYSTSAPAISRGPSEYPTKN (SEQ ID NO: 10), RYSTSAPAISRGPSE (SEQ ID NO: 100), or APAISRGPSEYPTKN (SEQ ID NO: 101).

[0199] In various embodiments of the present disclosure, an antigen binding protein that specifically binds to the cytoplasmic domain of CLDN6 comprises: (a) a heavy chain CDR1 comprising the amino acid sequence: GFNIKDYY (SEQ ID NO: 18) or a variant sequence thereof that differs by only one or two amino acids or has at least or about 70% sequence identity; (b) a heavy chain CDR2 comprising the amino acid sequence: IDPDNGDT (SEQ ID NO: 19) or a variant sequence thereof that differs by only one or two amino acids or has at least or about 70% sequence identity; (c) an amino acid sequence: ATYRYSFAY (SEQ ID NO: 20) or a variant sequence thereof that differs by only one or two amino acids or has at least or about 70% sequence identity; (d) a light chain CDR1 comprising the amino acid sequence: QNINVW (SEQ ID NO: 25) or a variant sequence thereof which differs by only one or two amino acids or has at least or about 70% sequence identity; (e) a light chain CDR2 comprising the amino acid sequence: KAS (SEQ ID NO: 26) or a variant sequence thereof which differs by only one or two amino acids or has at least or about 70% sequence identity; (f) a light chain CDR3 comprising the amino acid sequence: QQGQTYPLT (SEQ ID NO: 27) or a variant sequence thereof which differs by only one or two amino acids or has at least or about 70% sequence identity; or (g) a combination of any two or more of (a) to (f).In certain embodiments, the antigen binding protein (a) binds to human claudin-6 (CLDN6), wherein the antigen binding protein binds to CLDN6 by binding at least to an epitope within CLDN6 having the amino acid sequence CCTCPSGGSQGPSHYMARYSTSAPAISRGPSEYPTKNYV (SEQ ID NO: 3); (b) binds to human claudin-6 (CLDN6), wherein the antigen binding protein binds to CLDN6 by binding at least to an epitope within CLDN6 having the amino acid sequence RYSTSAPAISRGPSEYPTKN (SEQ ID NO: 10); (c) binds to human claudin-6 (CLDN6), wherein the antigen binding protein binds to CLDN6 by binding at least to an epitope within CLDN6 having the amino acid sequence RYSTSAPAISRGPSEYPTKN (SEQ ID NO: 100); and / or (d) binds to human claudin-6 (CLDN6), wherein the antigen binding protein binds to CLDN6 by binding at least to an epitope within CLDN6 having the amino acid sequence APAISRGPSEYPTKN (SEQ ID NO: 101). In certain embodiments, the antigen binding protein binds to one or more of the following peptides having the amino acid sequence of CCTCPSGGSQGPSHYMARYSTSAPAISRGPSEYPTKNYV (SEQ ID NO: 3), RYSTSAPAISRGPSEYPTKN (SEQ ID NO: 10), RYSTSAPAISRGPSE (SEQ ID NO: 100), or APAISRGPSEYPTKN (SEQ ID NO: 101).

[0200] In various embodiments of the present disclosure, an antigen binding protein that specifically binds to the cytoplasm of CLDN6 comprises: (a) a heavy chain CDR1 comprising the amino acid sequence: GFNIKDYY (SEQ ID NO: 18); (b) a heavy chain CDR2 comprising the amino acid sequence: IDPDNGDT (SEQ ID NO: 19); (c) a heavy chain CDR3 comprising the amino acid sequence: ATYRYSFAY (SEQ ID NO: 20); (d) a light chain CDR1 comprising the amino acid sequence: QNINVW (SEQ ID NO: 25); (e) a light chain CDR2 comprising the amino acid sequence: KAS (SEQ ID NO: 26); and (f) a light chain CDR3 comprising the amino acid sequence: QQGQTYPLT (SEQ ID NO: 27), and (a) binds to human claudin-6 (CLDN6), wherein the antigen binding protein binds to an epitope in CLDN6 having the amino acid sequence CCTCPSGGSQGPSHYMARYSTSAPAISRGPSEYPTKNYV (SEQ ID NO: 3). (b) binds to human claudin-6 (CLDN6), wherein the antigen binding protein binds to CLDN6 by at least binding to an epitope within CLDN6 having the amino acid sequence RYSTSAPAISRGPSEYPTKN (SEQ ID NO: 10); (c) binds to human claudin-6 (CLDN6), wherein the antigen binding protein binds to CLDN6 by at least binding to an epitope within CLDN6 having the amino acid sequence RYSTSAPAISRGPSE (SEQ ID NO: 100); and / or (d) binds to human claudin-6 (CLDN6), wherein the antigen binding protein binds to CLDN6 by at least binding to an epitope within CLDN6 having the amino acid sequence APAISRGPSEYPTKN (SEQ ID NO: 101). In certain embodiments, the antigen binding protein binds to one or more of the peptides having the amino acid sequence of any of the following: CCTCPSGGSQGPSHYMARYSTSAPAISRGPSEYPTKNYV (SEQ ID NO: 3), RYSTSAPAISRGPSEYPTKN (SEQ ID NO: 10), RYSTSAPAISRGPSE (SEQ ID NO: 100), or APAISRGPSEYPTKN (SEQ ID NO: 101).

[0201] In various embodiments of the disclosure, the antigen binding protein comprises: (a) a heavy chain variable region comprising the amino acid sequence: EVQLQQFGAELVKPGASVKISCRTSGYTFTDYNIDWVRQSHGKSLEWIGDINPNSENTDYNQKFKGKATLTVDKSSSTAYLELRSLTSEDTAVYYCARSPYGNYVGYLMDYWGQGTSVTVSS (SEQ ID NO: 32), or a variant sequence thereof that differs by only one or two amino acids or has at least or about 80% sequence identity; and / or (b) CDRs 1-3 derived from a light chain variable region comprising the amino acid sequence: DVVLTQTPLSLPVNIGDQASISCRSTKSLLNSDGFTYLDWYLQKPGQSPQVLIYLISNRFSGVPDRFSGNGSGTDFTLKISRVEAEDLGVYYCFQSNYIPLTFGAGTKLELK (SEQ ID NO: 33), or a variant sequence thereof differing by only one or two amino acids or having at least or about 80% sequence identity.In certain embodiments, the antigen binding protein (a) binds to human claudin-6 (CLDN6), wherein the antigen binding protein binds to CLDN6 by binding at least to an epitope within CLDN6 having the amino acid sequence CCTCPSGGSQGPSHYMARYSTSAPAISRGPSEYPTKNYV (SEQ ID NO: 3); (b) binds to human claudin-6 (CLDN6), wherein the antigen binding protein binds to CLDN6 by binding at least to an epitope within CLDN6 having the amino acid sequence CPSGGSQGPSHYMARYSTSAPAIS (SEQ ID NO: 95); or (c) binds to human claudin-6 (CLDN6), wherein the antigen binding protein binds to an epitope within CLDN6 having the amino acid sequence CPSGGSQGPSHYMARYSTS (SEQ ID NO: 96). (d) binds to human claudin-6 (CLDN6), wherein the antigen binding protein binds to CLDN6 by binding at least to an epitope within CLDN6 having the amino acid sequence SQGPSHYMARYSTSAPAIS (SEQ ID NO: 97); (e) binds to human claudin-6 (CLDN6), wherein the antigen binding protein binds to CLDN6 by binding at least to an epitope within CLDN6 having the amino acid sequence SHYMAR (SEQ ID NO: 98); and / or (f) binds to human claudin-6 (CLDN6), wherein the antigen binding protein binds to CLDN6 by binding at least to an epitope within CLDN6 having the amino acid sequence SHYMARYSTSAPAIS (SEQ ID NO: 99). In certain embodiments, the antigen binding protein binds to one or more of the following peptides having the amino acid sequence of any of the following: CCTCPSGGSQGPSHYMARYSTSAPAIS (SEQ ID NO: 3), CPSGGSQGPSHYMARYSTSAPAIS (SEQ ID NO: 95), CPSGGSQGPSHYMARYSTS (SEQ ID NO: 96), SQGPSHYMARYSTSAPAIS (SEQ ID NO: 97), SHYMAR (SEQ ID NO: 98), and / or SHYMARYSTSAPAIS (SEQ ID NO: 99).

[0202] In various embodiments of the present disclosure, an antigen binding protein that specifically binds to the cytoplasmic domain of CLDN6 comprises: (a) a heavy chain CDR1 comprising the amino acid sequence: GYTFTDYN (SEQ ID NO: 34) or a variant sequence thereof that differs by only one or two amino acids or has at least or about 70% sequence identity; (b) a heavy chain CDR2 comprising the amino acid sequence: INPNSENT (SEQ ID NO: 35) or a variant sequence thereof that differs by only one or two amino acids or has at least or about 70% sequence identity; (c) an amino acid sequence: ARSPYGNYVGYLMDY (SEQ ID NO: 36) or a variant sequence thereof that differs by only one or two amino acids or has at least or about 70% sequence identity; (d) a light chain CDR1 comprising the amino acid sequence: KSLLNSDGFTY (SEQ ID NO: 41) or a variant sequence thereof which differs by only one or two amino acids or has at least or about 70% sequence identity; (e) a light chain CDR2 comprising the amino acid sequence: LIS (SEQ ID NO: 42) or a variant sequence thereof which differs by only one or two amino acids or has at least or about 70% sequence identity; (f) a light chain CDR3 comprising the amino acid sequence: FQSNYIPLT (SEQ ID NO: 43) or a variant sequence thereof which differs by only one or two amino acids or has at least or about 70% sequence identity; or (g) a combination of any two or more of (a) to (f).In certain embodiments, the antigen binding protein (a) binds to human claudin-6 (CLDN6), wherein the antigen binding protein binds to CLDN6 by binding at least to an epitope within CLDN6 having the amino acid sequence CCTCPSGGSQGPSHYMARYSTSAPAISRGPSEYPTKNYV (SEQ ID NO: 3); (b) binds to human claudin-6 (CLDN6), wherein the antigen binding protein binds to CLDN6 by binding at least to an epitope within CLDN6 having the amino acid sequence CPSGGSQGPSHYMARYSTSAPAIS (SEQ ID NO: 95); or (c) binds to human claudin-6 (CLDN6), wherein the antigen binding protein binds to an epitope within CLDN6 having the amino acid sequence CPSGGSQGPSHYMARYSTS (SEQ ID NO: 96). (d) binds to human claudin-6 (CLDN6), wherein the antigen binding protein binds to CLDN6 by binding at least to an epitope within CLDN6 having the amino acid sequence SQGPSHYMARYSTSAPAIS (SEQ ID NO: 97); (e) binds to human claudin-6 (CLDN6), wherein the antigen binding protein binds to CLDN6 by binding at least to an epitope within CLDN6 having the amino acid sequence SHYMAR (SEQ ID NO: 98); and / or (f) binds to human claudin-6 (CLDN6), wherein the antigen binding protein binds to CLDN6 by binding at least to an epitope within CLDN6 having the amino acid sequence SHYMARYSTSAPAIS (SEQ ID NO: 99). In certain embodiments, the antigen binding protein binds to one or more of the following peptides having the amino acid sequence of any of the following: CCTCPSGGSQGPSHYMARYSTSAPAIS (SEQ ID NO: 3), CPSGGSQGPSHYMARYSTSAPAIS (SEQ ID NO: 95), CPSGGSQGPSHYMARYSTS (SEQ ID NO: 96), SQGPSHYMARYSTSAPAIS (SEQ ID NO: 97), SHYMAR (SEQ ID NO: 98), and / or SHYMARYSTSAPAIS (SEQ ID NO: 99).

[0203] In various embodiments of the present disclosure, an antigen binding protein that specifically binds to the cytoplasmic domain of CLDN6 comprises (a) a heavy chain CDR1 comprising the amino acid sequence: GYTFTDYN (SEQ ID NO: 34), (b) a heavy chain CDR2 comprising the amino acid sequence: INPNSENT (SEQ ID NO: 35), (c) a heavy chain CDR3 comprising the amino acid sequence: ARSPYGNYVGYLMDY (SEQ ID NO: 36), (d) a light chain CDR1 comprising the amino acid sequence: KSLLNSDGFTY (SEQ ID NO: 41), (e) a light chain CDR2 comprising the amino acid sequence: LIS (SEQ ID NO: 42), and (f) a light chain CDR3 comprising the amino acid sequence: FQSNYIPLT (SEQ ID NO: 43). In certain embodiments, the antigen binding protein (a) binds to human claudin-6 (CLDN6), wherein the antigen binding protein binds to CLDN6 by binding at least to an epitope within CLDN6 having the amino acid sequence CCTCPSGGSQGPSHYMARYSTSAPAISRGPSEYPTKNYV (SEQ ID NO: 3); (b) binds to human claudin-6 (CLDN6), wherein the antigen binding protein binds to CLDN6 by binding at least to an epitope within CLDN6 having the amino acid sequence CPSGGSQGPSHYMARYSTSAPAIS (SEQ ID NO: 95); or (c) binds to human claudin-6 (CLDN6), wherein the antigen binding protein binds to an epitope within CLDN6 having the amino acid sequence CPSGGSQGPSHYMARYSTS (SEQ ID NO: 96). (d) binds to human claudin-6 (CLDN6), wherein the antigen binding protein binds to CLDN6 by binding at least to an epitope within CLDN6 having the amino acid sequence SQGPSHYMARYSTSAPAIS (SEQ ID NO: 97); (e) binds to human claudin-6 (CLDN6), wherein the antigen binding protein binds to CLDN6 by binding at least to an epitope within CLDN6 having the amino acid sequence SHYMAR (SEQ ID NO: 98); and / or (b) binds to human claudin-6 (CLDN6), wherein the antigen binding protein binds to CLDN6 by binding at least to an epitope within CLDN6 having the amino acid sequence SHYMARYSTSAPAIS (SEQ ID NO: 99).In certain embodiments, the antigen binding protein binds to one or more of the following peptides having the amino acid sequence of any of the following: CCTCPSGGSQGPSHYMARYSTSAPAIS (SEQ ID NO: 3), CPSGGSQGPSHYMARYSTSAPAIS (SEQ ID NO: 95), CPSGGSQGPSHYMARYSTS (SEQ ID NO: 96), SQGPSHYMARYSTSAPAIS (SEQ ID NO: 97), SHYMAR (SEQ ID NO: 98), and / or SHYMARYSTSAPAIS (SEQ ID NO: 99).

[0204] Optionally, the variant sequence has at least about 80%, at least about 85%, at least about 90%, at least about 95% sequence identity, at least about 97%, at least about 98% sequence identity or at least 99% sequence identity.

[0205] In certain embodiments, the antigen-binding protein is a fusion protein, an antibody, or an antigen-binding antibody fragment. In certain aspects, the antibody is a monoclonal antibody, a fragment thereof, a human antibody, a humanized antibody, or a chimeric antibody. In certain embodiments, the antigen-binding protein is an IgG. The IgG can be selected from the group consisting of IgG1, IgG2, IgG3, and IgG4. In certain embodiments, the antigen-binding protein is an antibody fragment selected from the group consisting of scFv, F(ab')2, Fab, Fab', and Fv.

[0206] In certain embodiments, the antigen binding proteins disclosed herein inhibit tumor growth in xenograft mice injected with human cancer cells.

[0207] In certain embodiments, the antigen binding proteins disclosed herein comprise an Fc polypeptide comprising non-fucosylated glycans.

[0208] In certain embodiments, the antigen-binding proteins disclosed herein are conjugated with a detectable label. In various aspects of the present invention, the detectable label is at least one detectable label that is coupled to or co-expressed with the antigen-binding protein. In certain embodiments, the detectable label comprises a fluorophore, a radioactive label, a colorimetric label, or the detectable label is the product of an enzymatic reaction. In certain embodiments, the label or drug is conjugated to the antigen-binding protein via a cleavable linker, such as VC-PAB. In certain aspects, the label or drug is conjugated to a specific site on the antigen-binding protein, for example, the specific site is an unpaired cysteine ​​residue.

[0209] In certain embodiments, the antigen-binding proteins disclosed herein are conjugated to a detectable label, and the antigen-binding protein is an antibody. In certain embodiments, the conjugated antigen-binding protein is a conjugated fusion protein. In certain embodiments, the conjugated antigen-binding protein is a conjugated antibody. In certain embodiments, the conjugated antibody is a conjugated monoclonal antibody, a conjugated antibody fragment, a conjugated human antibody, a conjugated humanized antibody, or a conjugated chimeric antibody. In certain embodiments, the conjugated antigen-binding protein is an IgG. The conjugated IgG can be selected from the group consisting of IgG1, IgG2, IgG3, and IgG4. In certain embodiments, the conjugated antigen-binding protein is an antibody fragment selected from the group consisting of scFv, F(ab')2, Fab, Fab', and Fv.

[0210] In certain embodiments, the conjugated antigen binding proteins are conjugated to an average number of units of label or drug per antigen binding protein in the range of 1-8, preferably the average number of units of label or drug conjugated per antigen binding protein is in the range of 3-8.

[0211] In various aspects of the invention, the conjugate is a heterogeneous or homogeneous conjugate.

[0212] In certain embodiments, the conjugated antigen binding protein comprises a polypeptide comprising the amino acid sequences set forth in SEQ ID NO: 16 and SEQ ID NO: 17 conjugated to horseradish peroxidase (HRP).

[0213] In certain embodiments, the conjugated antigen binding protein comprises a polypeptide comprising the amino acid sequences set forth in SEQ ID NO: 32 and SEQ ID NO: 33 conjugated to horseradish peroxidase (HRP).

[0214] In certain embodiments, the nucleic acid molecule comprising the nucleotide sequence encodes an antigen-binding protein or a conjugated antigen-binding protein. In one embodiment, the nucleic acid is a cDNA. In some embodiments, the nucleic acid molecule is inserted into a vector (e.g., an expression vector). In addition, the vector may comprise an internal ribosome entry site (IRES).

[0215] In certain embodiments, a vector containing a nucleic acid encoding an antigen-binding protein or a conjugate thereof is transfected or transformed into a host cell. Optionally, the host cell is a bacterial cell or a eukaryotic cell. In a specific aspect, the eukaryotic cell is a mammalian cell, such as a Chinese hamster ovary (CHO) cell.

[0216] Certain embodiments of the present invention provide methods for producing an antigen binding protein or a conjugate thereof that binds to claudin-6 (CLDN6) protein, the method comprising: (i) culturing a host cell in a cell culture medium; and (ii) harvesting the antigen binding protein or a conjugate thereof from the cell culture medium.

[0217] Certain embodiments of the present invention provide methods for making a fusion protein comprising an antigen binding protein that binds to claudin-6 (CLDN6) protein, the method comprising: (i) culturing a host cell in a cell culture medium; and (ii) harvesting the fusion protein from the cell culture medium.

[0218] Certain embodiments of the present invention provide methods of making a composition, the method comprising combining (a) an antigen binding protein, a conjugate thereof, a fusion protein, a nucleic acid, a vector and / or a host cell, or any combination thereof, each as described herein, and (b) a pharmaceutically acceptable carrier, diluent and / or excipient.

[0219] In certain embodiments, a composition comprising (a) an antigen binding protein, a conjugate thereof, a fusion protein, a nucleic acid, a vector, and / or a host cell, or any combination thereof, each as described herein, and (b) a pharmaceutically acceptable carrier, diluent, and / or excipient.

[0220] Certain embodiments of the present invention provide methods of treating a subject having a CLDN6-expressing cancer, comprising administering to the subject a therapeutic amount of a composition described herein effective to treat the cancer.

[0221] Certain embodiments of the present invention provide methods for detecting or determining the quantity of claudin-6 (CLDN6) in a sample, comprising: (a) contacting the sample with an antigen binding protein described herein; (b) assaying for immune complexes comprising the antigen binding protein bound to CLDN6, or quantifying the amount of immune complexes so assayed, wherein the immune complexes indicate the presence and quantity of CLDN6; and (c) thereby detecting or determining the quantity of CLDN6 in the sample. In one embodiment, the method further comprises contacting the immune complexes so formed with an antibody labeled with a detectable marker to form a second immune complex comprising the antigen binding protein-CLDN6-labeled antibody, and detecting the second immune complexes so formed.

[0222] Certain embodiments of the present invention provide methods for diagnosing claudin-6 (CLDN6)-positive cancer in a subject, the method comprising: (a) contacting a biological sample comprising cells or tissue obtained from the subject with an antigen binding protein described herein; (a) assaying for immune complexes comprising the antigen binding protein or quantifying the immune complexes so assayed, wherein the presence of the immune complex indicates the presence of CLDN6-positive cancer; and (b) thereby diagnosing CLDN6-positive cancer in the subject. In one embodiment, the method further comprises contacting the immune complexes so formed with an antibody labeled with a detectable marker to form a second immune complex comprising the antigen binding protein-CLDN6-labeled antibody, and detecting the second immune complexes so formed.

[0223] In certain embodiments, a method for monitoring the progression of claudin-6 (CLDN6)-positive cancer in a subject is provided. The monitoring method includes (a) quantitatively determining the presence of CLDN6 in a first sample from the subject by forming an immune complex as described herein, (b) comparing the amount so determined with the amount present in a second sample from the subject, where such samples are taken at various time points, and (c) determining that the difference in the determined amounts indicates the progression of the cancer. In one embodiment, the method further includes contacting the immune complex thus formed with an antibody labeled with a detectable marker to form a second immune complex comprising the antigen-binding protein-CLDN6-labeled antibody, and detecting the second immune complex thus formed.

[0224] In certain embodiments, a method for classifying a tumor in a subject is provided. The tumor classification method includes (a) detecting the presence of claudin-6 (CLDN6) in a sample from the subject by the formation of an immune complex described herein, and (b) the presence of the immune complex indicates a specific claudin-6 (CLDN6) tumor type. In one embodiment, the method further includes contacting the immune complex thus formed with an antibody labeled with a detectable marker to form a second immune complex comprising the antigen-binding protein-CLDN6-labeled antibody, and detecting the second immune complex thus formed.

[0225] In certain embodiments, a method for determining whether a cancer in a subject is treatable by a cancer therapy targeting claudin-6 (CLDN6)-positive cancer is provided. The method includes (a) quantitatively determining the presence of CLDN6 in a first sample from the subject by forming an immune complex as described herein, and (b) comparing the amount so determined with the amount present in a second sample from the subject, the samples being taken at various time points. A decrease in the determined amount of CLDN6 over time indicates that the CLDN6-positive cancer is treatable in the subject. In one embodiment, the method further includes contacting the thus-formed immune complex with an antibody labeled with a detectable marker to form a second immune complex comprising the antigen-binding protein-CLDN6-labeled antibody, and detecting the thus-formed second immune complex.

[0226] In certain embodiments, a method for determining the prognosis of a subject with claudin-6 (CLDN6)-positive cancer is provided. The method includes (a) quantitatively determining the presence of CLDN6 in a first sample from the subject by forming an immune complex as described herein, and (b) comparing the amount so determined with the amount present in a second sample from the subject, where the samples are taken at various time points. The amount of CLDN6 determined over time indicates the prognosis of the CLDN6-positive cancer in the subject. In one embodiment, the method further includes contacting the immune complex thus formed with an antibody labeled with a detectable marker to form a second immune complex comprising the antigen-binding protein-CLDN6-labeled antibody, and detecting the second immune complex thus formed.

[0227] Certain embodiments of the present invention provide methods for detecting or quantifying the presence of CLDN6 in a sample, the method comprising: (a) contacting the sample under suitable conditions with a first antigen binding protein specific for claudin-6 (CLDN6) to form a first antigen binding protein-CLDN6 immune complex; (b) contacting the immune complex so formed with a second antigen binding protein labeled with a detectable marker to form a second immune complex comprising the first antigen binding protein-CLDN6-second antigen binding protein, and detecting the second immune complex so formed; and (c) thereby detecting or quantifying the presence of CLDN6 in the sample.

[0228] Certain embodiments of the present invention provide methods for making a conjugate comprising an antigen binding protein or fusion protein thereof that binds to claudin-6 (CLDN6) protein, the method comprising: (a) culturing host cells in cell culture medium, wherein the host cells comprise a nucleotide sequence encoding the antigen binding protein or fusion protein thereof; (b) harvesting the antigen binding protein or fusion protein from the cell culture medium; and (c) attaching the antigen binding protein or fusion protein to a second moiety, where the second moiety is a detectable label, to make the conjugate.

[0229] Certain embodiments of the present invention provide methods of using the antigen binding proteins described herein or conjugates thereof for the assays and assay methods described herein.

[0230] In certain embodiments of the invention, an antigen-binding protein is used that is an antibody or an antigen-binding antibody fragment. In certain embodiments, the antibody is a monoclonal antibody. In certain embodiments, the antibody is a human or humanized antibody, or a chimeric antibody. In certain embodiments, the antigen-binding protein is an IgG. In certain embodiments, the IgG is selected from the group consisting of IgG1, IgG2, IgG3, and IgG4. In certain embodiments, the antigen-binding antibody fragment is selected from the group consisting of scFv, F(ab')2, Fab, Fab', and Fv. In certain embodiments, the antibody is one of a selected combination of antibodies described herein that are specific for claudin-6 (CLDN6).

[0231] In certain embodiments of the present invention, antigen-binding proteins labeled with a detectable label are used. In certain embodiments, the detectable label comprises a fluorophore, a radioactive label, a colorimetric label, or the detectable label is the product of an enzymatic reaction. In certain aspects, the detectable label comprises horseradish peroxidase (HRP).

[0232] Certain embodiments of the present invention provide diagnostic kits comprising (a) an antigen binding protein described herein, and (b) a conjugate of a detectable label and a specific binding partner of the antigen binding protein of (a). In certain embodiments of the diagnostic kit, the label is selected from the group consisting of an enzyme, a radiolabel, a chromophore, and a fluorophore.

[0233] Also provided are methods for producing conjugates with the antigen-binding proteins described herein. In various embodiments, the methods include culturing host cells containing nucleic acids encoding the antigen-binding proteins, fusion proteins, or polypeptides described herein to express the antigen-binding proteins, fusion proteins, or polypeptides, recovering the antigen-binding proteins, fusion proteins, or polypeptides from the cell culture medium, and then binding the antigen-binding proteins, fusion proteins, or polypeptides to a second moiety to produce the conjugate. In various aspects, the second moiety is a detectable label. In certain embodiments, the detectable label is horseradish peroxidase (HRP).

[0234] The following examples are offered merely to illustrate the present disclosure and in no way limit its scope. [Example]

[0235] Example 1 This example demonstrates the generation of a CLDN6-specific antibody.

[0236] Mice were immunized with a mixture of peptides and fusion proteins (Table 7) using standard methods described below. [Table 12]

[0237] Spleen cells were harvested from immunized mice and fused with a myeloma line by electrofusion to generate hybridomas. Approximately 2,000 primary hybridoma cultures were generated and cultured in 96-well plates. BSA-conjugated peptide and GST fusion proteins (fusion proteins were formalin-fixed using published methods; see Press et al., 2002) were conjugated to beads and used to identify antibody-binding epitopes and map them to the CLDN6 cytoplasmic domain using bead-based indirect flow cytometry and ELISA (Figures 1 and 2).

[0238] These nucleic acid sequences were used to generate the CLDN6 antibody formatted as a full-length human IgG antibody (e.g., mouse IgG2a) using ExpiCHO™ expression. The heavy and light chain variable regions of the antibody were cloned into the antibody expression vector pcDNA™3.4-TOPO® Vector (Cat. No.: A14697, ThermoFisher Scientific, USA) and designated phlgG1-K-GG. Transfection of phlgG1-K-GG into CHO cells according to the protocol provided in the kit (ExpiCHO™ Expression System, Cat. No.: A29133, ThermoFisher Scientific, USA) resulted in the production of a bicistronic mRNA in which the IRES drives expression of the second immunoglobulin chain.

[0239] Two CLDN6-specific antibodies, 3D07 and 3H11, were selected for further study. These antibodies were subcloned, and the variable heavy and light chain sequences were determined. CDRs were defined according to the IMGT definition (IMGT®, the international ImMunoGeneTics information system®, www.imgt.org, Founder and Director: Marie-Paule Lefranc, Montpellier, France). See Tables 8-9 and the Sequence Listing below. [Table 13] [Table 14]

[0240] The CLDN6 antibody was formatted as a full-length IgG antibody using ExpiCHO™ expression. The heavy and light chain variable regions of the antibody were cloned into a laboratory-engineered antibody expression vector based on the pcDNA™ 3.4-TOPO® vector (catalog number: A14697, ThermoFisher Scientific, USA) and transfected into CHO cells according to the protocol provided in the kit (ExpiCHO™ Expression System, catalog number: A29133, ThermoFisher Scientific, USA). The antibody was purified, and its binding to CLDN6 was evaluated as described in Examples 3 and 4 below.

[0241] Example 2 This example describes the mapping of antibody binding epitopes of CLDN6 to antibodies 3H11 and 3D07.

[0242] Peptides were designed across the cytoplasmic domain of CLDN6 (SEQ ID NO: 3). The peptides were staggered by several amino acids, i.e., each peptide overlapped with another peptide before and / or after it by several amino acids, as shown in Figures 1-2 and Table 7 above.

[0243] Binding assays were performed using a bead-based flow assay with indirect flow cytometry. BSA-conjugated peptides and GST fusion proteins (formalin-fixed fusion proteins using published methods (Press et al., Steroids, 67 (2002) 799-813) were conjugated to beads and used to identify antibody-binding epitopes and map them to the CLDN6 cytoplasmic domain using bead-based indirect flow cytometry.

[0244] For the 3H11 antibody, binding assays identified overlapping peptides 4 and 5 with a 10 amino acid (APAISRGPSE, SEQ ID NO: 15) core region for binding to the cytoplasmic domain of CLDN6 (Figure 1).

[0245] For the 3D07 antibody, binding assays identified overlapping peptides 1R, 2, and 3 with a 6-amino acid (SHYMAR, SEQ ID NO: 98) core region for binding to the cytoplasmic domain of CLDN6 (Figure 1). The binding data in Figure 2 show that 3D07 does not bind to SHYMA, and therefore requires the R of SHYMAR.

[0246] Example 3 In this example, two antibodies, 3H11 and 3D07, described in Example 1, were characterized for binding to CLDN6 in HEK293T cells. CLDN6-expressing cells were used in assays to determine the ability of each CLDN6 antibody to bind to CLDN6 on the cell surface and to cross-react with other CLDN family members (CLDN3, CLDN4, or CLDN9). HEK293T cells engineered to express human CLDN6 fused to GFP, CLDN9-GFP, CLDN4-GFP, or CLDN3-GFP were used as an artificial model of CLDN6 expression.

[0247] Two clones, 3H11 and 3D07, were characterized for binding to CLDN6 in formalin-fixed, paraffin-embedded (FFPE) HEK293_CLDN6 cells, with HEK293_CLDN3, HEK293_4, and HEK293_9 cells included as counterstains (Figure 3).

[0248] Samples of CLDN6, 3, 4, and 9-engineered HEK293 cells were prepared as follows: Formalin-fixed, paraffin-embedded cells were cut into 4 mm thin sections and mounted on slides. Cell-fixed slides were dried overnight, heated at 60°C for 1 hour, passed through xylene three times for 5 minutes each, treated twice with 100%, 95%, 70%, and 50% ethanol for 2 minutes each, washed twice in dH2O for 2 minutes each, immersed in 0.3% H2O2 for 15 minutes, rinsed with dH2O, and finally washed twice in 1x TBS for 2 minutes each. Slides were then placed in a slide chamber containing 1x Tris-EDTA buffer, pH 9 (Abcam, #ab93684) and incubated at 110°C for 30 minutes in a Biocare Decloaking Chamber (Biocare Medical, #DC2012).

[0249] Binding of antibodies 3H11 and 3D07 to slide-fixed HEK293_CLDN6 cells was assessed as follows: Slides were washed twice for 2 minutes in 1x TBS, blocked with background suppressor (Biocare Medical, #BP974L) for 15 minutes at room temperature, rinsed with 1x TBS, and incubated with 3H11 or 3D07 antibody overnight at 40°C. The next day, slides were washed for 15 minutes in 1x TBST (1x TBS + 0.1% Tween 20) and twice for 2 minutes in TBS, then incubated with goat polyclonal antibody (pAb) against mouse IgG HRP polymer (Abcam, #ab214879) for 30 minutes at room temperature, washed for 20 minutes in 1x TBST, rinsed twice for 2 minutes in 1x TBS, and analyzed by ImmPACT® DAB. The slides were stained with EqV peroxidase (HRP) substrate (Vectorlab, #SK-4103-400) for 6 minutes and transferred to a large container of dH2O. Finally, the slides were removed, stained with hematoxylin (Abcam, ab220365) for 15 seconds, rinsed under running tap water for 3 minutes, washed twice in dH2O for 2 minutes each, dehydrated by treatment with 95% and 100% ethanol twice for 2 minutes each and xylene three times for 5 minutes each, and fixed in Permount.

[0250] The results of the assay show that antibodies 3H11 and 3D07 specifically bound to CLDN6 without cross-reacting with any of CLDN3, CLDN4, or CLDN9.

[0251] Example 4 Two clones, 3H11 and 3D07, were further characterized for binding to CLDN6 in formalin-fixed, paraffin-embedded (FFPE) human ovarian cancer cells (Figure 4).

[0252] Human ovarian cancer tissue samples were prepared as follows: Formalin-fixed, paraffin-embedded tissues were cut into 4 μm sections and mounted on slides. The fixed slides were dried overnight, heated at 60°C for 1 hour, passed through xylene three times for 5 minutes each, treated twice with 100%, 95%, 70%, and 50% ethanol for 2 minutes each, washed twice in dH2O for 2 minutes each, immersed in 0.3% H2O2 for 15 minutes, rinsed with dH2O, and finally washed twice in 1x TBS for 2 minutes each. The slides were then placed in a slide chamber containing 1x Tris-EDTA buffer, pH 9 (Abcam, #ab93684) and incubated at 110°C for 30 minutes in a Biocare Decloaking Chamber (Biocare Medical, #DC2012).

[0253] The binding of antibodies 3H11 and 3D07 to slide-mounted CLDN6-positive human ovarian cancer tissue samples was assessed as follows: Slides were washed twice for 2 minutes in 1x TBS, blocked with background suppressor (Biocare Medical, #BP974L) for 15 minutes at room temperature, rinsed with 1x TBS, and incubated with 3H11 or 3D07 antibody overnight at 40°C. The next day, slides were washed for 15 minutes in 1x TBST (1x TBS + 0.1% Tween 20) and twice for 2 minutes in TBS, then incubated with goat polyclonal antibody (pAb) against mouse IgG HRP polymer (Abcam, #ab214879) for 30 minutes at room temperature, washed for 20 minutes in 1x TBST, rinsed twice for 2 minutes in 1x TBS, and analyzed by ImmPACT® DAB. The slides were stained with EqV peroxidase (HRP) substrate (Vectorlab, #SK-4103-400) for 6 minutes and transferred to a large container of dH2O. Finally, the slides were removed, stained with hematoxylin (Abcam, ab220365) for 15 seconds, rinsed under running tap water for 3 minutes, washed twice in dH2O for 2 minutes each, dehydrated by treatment with 95% and 100% ethanol twice for 2 minutes each and xylene three times for 5 minutes each, and fixed in Permount® fixative medium.

[0254] Figure 4 shows the binding of antibodies 3H11 and 3D07 to human ovarian cancer tissue sections. The results show that the antibodies stained human CLDN6-positive ovarian cancer tissue but not CLDN6-negative ovarian cancer tissue. Antibodies 3H11 and 3D07 identified ovarian cancer tissue sections containing CLDN6-positive cells. Example 5 The amino acid sequences of the heavy and light chains of antibody 3H11 (SEQ ID NOs: 16-17) and 3D07 (SEQ ID NOs: 32-33) were submitted to abYsis (http: / / www.abysis.org / abysis / index.html) for analysis. The amino acid sequences were annotated by the IMGT, KABAT, CHOTHIA, and AbM methods, and the results are shown in Tables 10-13 below. [Table 15] Table 16 Table 17 Table 18 Table 19 Table 20 Table 21 Table 22 Table 23 Table 24 Table 25 Table 26 Table 27 Table 28 Table 29 Table 30

[0255] All references cited herein, including publications, patent applications, and patents, are incorporated by reference herein to the same extent as if each reference was individually and specifically indicated to be incorporated by reference and was set forth in its entirety herein.

[0256] The recitation of ranges of values ​​herein, unless otherwise indicated herein, is merely intended to serve as a shorthand method of referring individually to each separate value and each endpoint falling within that range, and each separate value and endpoint is incorporated herein as if it were individually recited herein.

[0257] All methods described herein can be performed in any suitable order unless otherwise indicated herein or clearly contradicted by context. Any examples provided herein or the use of various terms (e.g., "such as") are merely intended to better illustrate the disclosure and no limitation on the scope of the disclosure is asserted unless specifically claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the disclosure.

[0258] Preferred embodiments of the present disclosure are described herein, including the best mode known to the inventors for carrying out the disclosure. Variations of these preferred embodiments will become apparent to those skilled in the art upon reading the foregoing description. The inventors anticipate that those skilled in the art will employ such variations as appropriate, and intend for the present disclosure to be practiced otherwise than as specifically described herein. Accordingly, this disclosure includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the present disclosure unless otherwise indicated herein or otherwise clearly contradicted by context.

Claims

1. a. CDRs 1-3 from a heavy chain variable region comprising the amino acid sequence: EIQLQQSGAELVRPGALVRLSCKPSGFNIKDYYIHWVKERPEQGLEWIGWIDPDNGDTLYDSKFQGKASLTADTSSNTAYLQLTSLTSEDTAVYYCATYRYSFAYWGQGTLVTVSA (SEQ ID NO: 16), or a variant sequence thereof that differs by only one or two amino acids or has at least or about 80% sequence identity; and / or b. CDRs 1-3 from a light chain variable region comprising the amino acid sequence: DIQMNQSPSSLSASLGDTITITCHASQNINVWLNWYQQKPGNIPKLLIFKASNLYTGVPSRFSGGGSGTGFTLTISSLQPEDIATYYCQQGQTYPLTFGGGTKLEIK (SEQ ID NO: 17), or a variant sequence thereof that differs by only one or two amino acids or has at least or about 80% sequence identity.

1. An antigen-binding protein comprising:

2. a. a heavy chain CDR1 comprising the amino acid sequence: GFNIKDYY (SEQ ID NO: 18) or a variant sequence thereof differing by only one or two amino acids or having at least or about 70% sequence identity; b. A heavy chain CDR2 comprising the amino acid sequence: IDPDNGDT (SEQ ID NO: 19) or a variant sequence thereof that differs by only one or two amino acids or has at least or about 70% sequence identity; c. A heavy chain CDR3 comprising the amino acid sequence: ATYRYSFAY (SEQ ID NO: 20) or a variant sequence thereof that differs by only one or two amino acids or has at least or about 70% sequence identity; d. A light chain CDR1 comprising the amino acid sequence: QNINVW (SEQ ID NO: 25) or a variant sequence thereof differing by only one or two amino acids or having at least or about 70% sequence identity; e. A light chain CDR2 comprising the amino acid sequence: KAS (SEQ ID NO: 26) or a variant sequence thereof that differs by only one or two amino acids or has at least or about 70% sequence identity; f. a light chain CDR3 comprising the amino acid sequence: QQGQTYPLT (SEQ ID NO: 27) or a variant sequence thereof that differs by only one or two amino acids or has at least or about 70% sequence identity; or g. A combination of two or more of (a) to (f) An antigen-binding protein that specifically binds to the cytoplasmic domain of CLDN6, comprising:

3. a. Binds to human claudin-6 (CLDN6), wherein the antigen binding protein binds to CLDN6 by binding at least to an epitope within CLDN6 having the amino acid sequence CCTCPSGGSQGPSHYMARYSTSAPAISRGPSEYPTKNYV (SEQ ID NO: 3); b. Binds to human claudin-6 (CLDN6), wherein the antigen binding protein binds to CLDN6 by binding at least to an epitope within CLDN6 having the amino acid sequence RYSTSAPAISRGPSEYPTKN (SEQ ID NO: 10); c. Binds to human claudin-6 (CLDN6), wherein the antigen binding protein binds to CLDN6 by at least binding to an epitope within CLDN6 having the amino acid sequence RYSTSAPAISRGPSE (SEQ ID NO: 100); d. Binds to human claudin-6 (CLDN6), wherein the antigen binding protein binds to CLDN6 by binding at least to an epitope within CLDN6 having the amino acid sequence APAISRGPSEYPTKN (SEQ ID NO: 101); and / or e. binds to one or more of the following peptides having the amino acid sequence of: CCTCPSGGSQGPSHYMARYSTSAPAISRGPSEYPTKNYV (SEQ ID NO: 3), RYSTSAPAISRGPSEYPTKN (SEQ ID NO: 10), RYSTSAPAISRGPSE (SEQ ID NO: 100), or APAISRGPSEYPTKN (SEQ ID NO: 101); 3. The antigen-binding protein of claim 1 or 2.

4. a. a heavy chain CDR1 comprising the amino acid sequence: GFNIKDYY (SEQ ID NO: 18); b. a heavy chain CDR2 comprising the amino acid sequence: IDPDNGDT (SEQ ID NO: 19); c. a heavy chain CDR3 comprising the amino acid sequence: ATYRYSFAY (SEQ ID NO: 20); d. a light chain CDR1 comprising the amino acid sequence: QNINVW (SEQ ID NO: 25); e. a light chain CDR2 comprising the amino acid sequence: KAS (SEQ ID NO: 26), and f. Light chain CDR3 comprising the amino acid sequence: QQGQTYPLT (SEQ ID NO: 27) An antigen-binding protein that specifically binds to the cytoplasm of CLDN6, comprising: g. Binds to human claudin-6 (CLDN6), wherein the antigen binding protein binds to CLDN6 by binding at least to an epitope within CLDN6 having the amino acid sequence CCTCPSGGSQGPSHYMARYSTSAPAISRGPSEYPTKNYV (SEQ ID NO: 3); h. Binds to human claudin-6 (CLDN6), wherein the antigen binding protein binds to CLDN6 by at least binding to an epitope within CLDN6 having the amino acid sequence RYSTSAPAISRGPSEYPTKN (SEQ ID NO: 10); i. binds to human claudin-6 (CLDN6), wherein the antigen binding protein binds to CLDN6 by at least binding to an epitope within CLDN6 having the amino acid sequence RYSTSAPAISRGPSE (SEQ ID NO: 100); j. binds to human claudin-6 (CLDN6), wherein the antigen binding protein binds to CLDN6 by at least binding to an epitope within CLDN6 having the amino acid sequence APAISRGPSEYPTKN (SEQ ID NO: 101); and / or k. binds to one or more of the following peptides having the amino acid sequence of any of the following: CCTCPSGGSQGPSHYMARYSTSAPAISRGPSEYPTKNYV (SEQ ID NO: 3), RYSTSAPAISRGPSEYPTKN (SEQ ID NO: 10), RYSTSAPAISRGPSE (SEQ ID NO: 100), or APAISRGPSEYPTKN (SEQ ID NO: 101); The antigen-binding protein.

5. CDRs 1-3 from a heavy chain variable region comprising the amino acid sequence: EVQLQQFGAELVKPGASVKISCRTSGYTFTDYNIDWVRQSHGKSLEWIGDINPNSENTDYNQKFKGKATLTVDKSSSTAYLELRSLTSEDTAVYYCARSPYGNYVGYLMDYWGQGTSVTVSS (SEQ ID NO: 32), or a variant sequence thereof differing by only one or two amino acids or having at least or about 80% sequence identity; and / or b. CDRs 1-3 from a light chain variable region comprising the amino acid sequence: DVVLTQTPLSLPVNIGDQASISCRSTKSLLNSDGFTYLDWYLQKPGQSPQVLIYLISNRFSGVPDRFSGNGSGTDFTLKISRVEAEDLGVYYCFQSNYIPLTFGAGTKLELK (SEQ ID NO: 33), or a variant sequence thereof that differs by only one or two amino acids or has at least or about 80% sequence identity.

1. An antigen-binding protein comprising:

6. a. a heavy chain CDR1 comprising the amino acid sequence: GYTFTDYN (SEQ ID NO: 34) or a variant sequence thereof that differs by only one or two amino acids or has at least or about 70% sequence identity; b. A heavy chain CDR2 comprising the amino acid sequence: INPNSENT (SEQ ID NO: 35) or a variant sequence thereof that differs by only one or two amino acids or has at least or about 70% sequence identity; c. A heavy chain CDR3 comprising the amino acid sequence: ARSPYGNYVGYLMDY (SEQ ID NO: 36) or a variant sequence thereof differing by only one or two amino acids or having at least or about 70% sequence identity; d. A light chain CDR1 comprising the amino acid sequence: KSLLNSDGFTY (SEQ ID NO: 41) or a variant sequence thereof that differs by only one or two amino acids or has at least or about 70% sequence identity; e. A light chain CDR2 comprising the amino acid sequence: LIS (SEQ ID NO: 42) or a variant sequence thereof that differs by only one or two amino acids or has at least or about 70% sequence identity; f. a light chain CDR3 comprising the amino acid sequence: FQSNYIPLT (SEQ ID NO: 43) or a variant sequence thereof that differs by only one or two amino acids or has at least or about 70% sequence identity; or g. A combination of two or more of (a) to (f) An antigen-binding protein that specifically binds to the cytoplasmic domain of CLDN6, comprising:

7. a. Binds to human claudin-6 (CLDN6), wherein the antigen binding protein binds to CLDN6 by binding at least to an epitope within CLDN6 having the amino acid sequence CCTCPSGGSQGPSHYMARYSTSAPAISRGPSEYPTKNYV (SEQ ID NO: 3); b. Binds to human claudin-6 (CLDN6), wherein the antigen binding protein binds to CLDN6 by binding at least to an epitope within CLDN6 having the amino acid sequence CPSGGSQGPSHYMARYSTSAPAIS (SEQ ID NO: 95); c. Binds to human claudin-6 (CLDN6), wherein the antigen binding protein binds to CLDN6 by at least binding to an epitope within CLDN6 having the amino acid sequence CPSGGSQGPSHYMARYSTS (SEQ ID NO: 96); d. Binds to human claudin-6 (CLDN6), wherein the antigen binding protein binds to CLDN6 by binding at least to an epitope within CLDN6 having the amino acid sequence SQGPSHYMARYSTSAPAIS (SEQ ID NO: 97); e. Binds to human claudin-6 (CLDN6), wherein the antigen binding protein binds to CLDN6 by binding at least to an epitope within CLDN6 having the amino acid sequence SHYMAR (SEQ ID NO: 98); f. binds to human claudin-6 (CLDN6), wherein the antigen binding protein binds to CLDN6 by at least binding to an epitope within CLDN6 having the amino acid sequence SHYMARYSTSAPAIS (SEQ ID NO: 99); and / or g. binds to one or more of the peptides having the amino acid sequence of any of the following: CCTCPSGGQSGPSHYMARYSTSAPAISRGPSEYPTKNYV (SEQ ID NO: 3), CPSGGSQGPSHYMARYSTSAPAIS (SEQ ID NO: 95), CPSGGSQGPSHYMARYSTS (SEQ ID NO: 96), SQGPSHYMARYSTSAPAIS (SEQ ID NO: 97), SHYMAR (SEQ ID NO: 98), or SHYMARYSTSAPAIS (SEQ ID NO: 99); 7. An antigen-binding protein according to claim 5 or 6.

8. a. a heavy chain CDR1 comprising the amino acid sequence: GYTFTDYN (SEQ ID NO: 34); b. a heavy chain CDR2 comprising the amino acid sequence: INPNSENT (SEQ ID NO: 35); c. a heavy chain CDR3 comprising the amino acid sequence: ARSPYGNYVGYLMDY (SEQ ID NO: 36); d. a light chain CDR1 comprising the amino acid sequence: KSLLNSDGFTY (SEQ ID NO: 41); e. a light chain CDR2 comprising the amino acid sequence: LIS (SEQ ID NO: 42), and f. Light chain CDR3 comprising the amino acid sequence: FQSNYIPLT (SEQ ID NO: 43) An antigen-binding protein that specifically binds to the cytoplasmic domain of CLDN6, comprising: g. Binds to human claudin-6 (CLDN6), wherein the antigen binding protein binds to CLDN6 by binding at least to an epitope within CLDN6 having the amino acid sequence CCTCPSGGSQGPSHYMARYSTSAPAISRGPSEYPTKNYV (SEQ ID NO: 3); h. Binds to human claudin-6 (CLDN6), wherein the antigen binding protein binds to CLDN6 by at least binding to an epitope within CLDN6 having the amino acid sequence CPSGGSQGPSHYMARYSTSAPAIS (SEQ ID NO: 95); i. binds to human claudin-6 (CLDN6), wherein the antigen binding protein binds to CLDN6 by at least binding to an epitope within CLDN6 having the amino acid sequence CPSGGSQGPSHYMARYSTS (SEQ ID NO: 96); j. binds to human claudin-6 (CLDN6), wherein the antigen binding protein binds to CLDN6 by at least binding to an epitope within CLDN6 having the amino acid sequence SQGPSHYMARYSTSAPAIS (SEQ ID NO: 97); k. Binds to human claudin-6 (CLDN6), wherein the antigen binding protein binds to CLDN6 by binding at least to an epitope within CLDN6 having the amino acid sequence SHYMAR (SEQ ID NO: 98); l. binds to human claudin-6 (CLDN6), wherein the antigen binding protein binds to CLDN6 by at least binding to an epitope within CLDN6 having the amino acid sequence SHYMARYSTSAPAIS (SEQ ID NO: 99); and / or m. binds to one or more of the peptides having the amino acid sequence of any of the following: CCTCPSGGSQGPSHYMARYSTSAPAISRGPSEYPTKNYV (SEQ ID NO: 3), CPSGGSQGPSHYMARYSTSAPAIS (SEQ ID NO: 95), CPSGGSQGPSHYMARYSTS (SEQ ID NO: 96), SQGPSHYMARYSTSAPAIS (SEQ ID NO: 97), SHYMAR (SEQ ID NO: 98), or SHYMARYSTSAPAIS (SEQ ID NO: 99); The antigen-binding protein.

9. 10. The antigen binding protein of any one of the preceding claims, wherein said variant sequence has at least about 80%, or at least or about 85% sequence identity.

10. 10. The antigen binding protein of any one of the preceding claims, wherein said variant sequence has at least about 90% sequence identity, or at least or about 95% sequence identity.

11. 10. The antigen binding protein of any one of the preceding claims, wherein said variant sequence has at least about 97% sequence identity, 98% sequence identity or at least 99% sequence identity.

12. 10. An antigen-binding protein according to any one of the preceding claims which is an antibody or an antigen-binding antibody fragment.

13. 13. The antigen-binding protein of claim 12, wherein the antibody is a monoclonal antibody.

14. 13. The antigen-binding protein of claim 12, wherein the antibody is a human or humanized antibody, or a chimeric antibody.

15. 13. The antigen-binding protein of claim 12, which is an IgG.

16. 16. The antigen binding protein of claim 15, wherein the IgG is selected from the group consisting of IgG1, IgG2, IgG3 and IgG4.

17. 17. The antigen-binding protein of claim 15 or 16, wherein the IgG is IgG1.

18. The antigen-binding antibody fragment is an scFv, F(ab') 2 13. The antigen-binding protein of claim 12, wherein the antigen-binding protein is selected from the group consisting of Fab, Fab', and Fv.

19. 10. A conjugate comprising an antigen-binding protein according to any one of the preceding claims or as described herein and a detectable label.

20. 20. The conjugate of claim 19, comprising at least one detectable label coupled (e.g., post-translationally, e.g., chemically coupled) to or co-expressed with said antigen binding protein.

21. 20. The conjugate of claim 19, wherein the detectable label comprises a fluorophore, a radioactive label, a colorimetric label, or a reporter protein.

22. 22. The conjugate of claim 21, wherein the reporter protein comprises an enzyme.

23. 23. The conjugate of claim 21 or 22, wherein the enzyme comprises horseradish peroxidase (HRP).

24. 24. The conjugate of any one of claims 19 to 23, wherein the antigen-binding protein is an antibody.

25. 25. The conjugate of claim 24, wherein the antibody is a monoclonal antibody.

26. 25. The conjugate of claim 24, wherein the antibody is a human antibody, a humanized antibody, or a chimeric antibody.

27. 27. The conjugate of any one of claims 24 to 26, wherein the antibody is an IgG antibody.

28. 28. The antigen binding protein of claim 27, wherein the IgG is selected from the group consisting of IgG1, IgG2, IgG3 and IgG4.

29. 29. The conjugate of any one of claims 19 to 28, wherein the average number of units of label or agent conjugated per antigen-binding protein is at least 1, 2, 3, 4, 5, 6, 7, or 8.

30. 30. The conjugate of any one of claims 19 to 29, which is a heterogeneous conjugate.

31. 30. The conjugate of any one of claims 19 to 29, which is a homogeneous conjugate.

32. 32. The conjugate of any one of claims 19 to 31, wherein the label or drug is conjugated to a specific site on the antigen-binding protein.

33. 33. The conjugate of claim 32, wherein the specific site is an unpaired cysteine ​​residue.

34. 34. A conjugate according to any one of claims 19 to 33, comprising a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 16 and / or SEQ ID NO: 17 conjugated to horseradish peroxidase (HRP).

35. 35. A conjugate according to any one of claims 19 to 34, comprising a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 32 and / or SEQ ID NO: 33 conjugated to horseradish peroxidase (HRP).

36. A fusion protein comprising an antigen-binding protein according to any one of the preceding claims.

37. 37. A nucleic acid comprising a nucleotide sequence encoding an antigen-binding protein according to any one of claims 1 to 18, a conjugate according to claims 19 to 35, or a fusion protein according to claim 36.

38. 38. The nucleic acid of claim 37, which is a cDNA.

39. A vector (e.g., an expression vector) comprising the nucleic acid of claim 37 or 38.

40. 40. The vector of claim 39, further comprising an internal ribosome entry site (IRES).

41. 41. A host cell comprising a nucleic acid according to claim 37 or 38 or a vector according to claim 39 or 40.

42. 42. The host cell of claim 41, which is a bacterial cell.

43. 42. The host cell of claim 41, which is a eukaryotic cell.

44. 44. The host cell of claim 43, wherein the eukaryotic cell is a mammalian cell.

45. 45. The host cell of claim 44, wherein the mammalian cell is a Chinese hamster ovary (CHO) cell.

46. 46. ​​A method of producing an antigen binding protein that binds to claudin-6 (CLDN6) protein, the method comprising: (i) culturing a host cell of any one of claims 41 to 45 in a cell culture medium; and (ii) recovering the antigen binding protein from the cell culture medium.

47. 46. ​​A method of making a fusion protein comprising an antigen-binding protein that binds to claudin-6 (CLDN6) protein, the method comprising: (i) culturing a host cell of any one of claims 41 to 45 in a cell culture medium; and (ii) recovering the fusion protein from the cell culture medium.

48. 48. A method of making the antigen binding protein of claim 46 or the fusion protein of claim 47, further comprising chemically linking a label to said protein.

49. 49. The method of claim 48, wherein the detectable label is a colorimetric marker.

50. 49. The method of claim 48, wherein the detectable label is a fluorescent marker.

51. 49. The method of claim 48, wherein the detectable label is an enzyme.

52. A method for detecting or determining the amount of claudin-6 (CLDN6) in a sample, comprising: contacting the sample with an antigen binding protein or conjugate according to any one of the preceding claims; b. Assaying for the presence of an immune complex comprising the antigen-binding protein or the conjugate bound to CLDN6, or determining the amount of the immune complex so assayed, wherein the immune complex indicates the presence and amount of CLDN6; c. Thereby detecting or determining the amount of CLDN6 in said sample. The method comprising:

53. 1. A method for diagnosing claudin-6 (CLDN6)-positive cancer in a subject, comprising: contacting a biological sample comprising cells or tissue obtained from said subject with an antigen binding protein or conjugate according to any one of the preceding claims; b. Assaying for the presence of an immune complex comprising the antigen-binding protein or conjugate, or determining the amount of the immune complex so assayed, wherein the presence of the immune complex indicates the presence of a CLDN6-positive cancer; c. Thereby diagnosing said CLDN6-positive cancer in said subject. The method comprising:

54. 54. The method of claim 53, further comprising treating the subject diagnosed with CLDN6-positive cancer by administering a cancer therapy to the subject diagnosed with CLDN6-positive cancer, thereby treating the subject diagnosed with CLDN6-positive cancer.

55. 1. A method for monitoring the progress of claudin-6 (CLDN6)-positive cancer in a subject, comprising: a. Determining the amount of CLDN6 in a first sample from the subject by the method of claim 52; b. comparing the amount so determined with the amount present in a second sample from the subject, the samples being taken at different time points; c. the difference in the determined amounts indicates the progression of the cancer. The method comprising:

56. 1. A method for classifying a tumor in a subject, comprising: a. Detecting the presence of claudin-6 (CLDN6) in a sample from the subject by the method of claim 52; b. Its presence indicates a claudin-6 (CLDN6) tumor. The method comprising:

57. 1. A method for determining the efficacy of a cancer therapy targeting claudin-6 (CLDN6)-positive cancer in a subject, comprising: a. In a first sample from the subject, determining the amount of CLDN6 in the sample by the method of claim 52; b. comparing the amount so determined with the amount present in a second sample from the subject, wherein the sample is taken after administration of the cancer therapy; c. A significant decrease in the amount of CLDN6 after the administration of the cancer therapy indicates that the cancer therapy is effective. The method comprising:

58. 1. A method for determining the prognosis of a subject with claudin-6 (CLDN6)-positive cancer, comprising: a. In a first sample from the subject, determining the amount of CLDN6 in the sample by the method of claim 52; b. comparing the amount so determined with the amount present in a second sample from the subject, the samples being taken at different time points; c. The amount of CLDN6 determined over time indicates a prognosis for the CLDN6-positive cancer in the subject. The method comprising:

59. 59. The method of any one of claims 52 to 58, wherein the immune complex comprising the antigen-binding protein bound to CLDN6 is detected using a secondary antibody that detects the antigen-binding protein, wherein the secondary antibody is labeled with a detectable marker.

60. A method for determining the presence or amount of CLDN6 in a sample, comprising: a. Contacting the sample with a first antigen binding protein of any one of the preceding claims specific for claudin-6 (CLDN6) to form a first antigen binding protein-CLDN6 immune complex; b. Contacting the immune complex thus formed with a second antigen binding protein labeled with a detectable marker to form a second immune complex comprising the first antigen binding protein-CLDN6-second antigen binding protein, and detecting the second immune complex thus formed; c. thereby detecting or quantifying the presence of CLDN6 in said sample; The method comprising:

61. 10. A method of making a conjugate according to any of the preceding claims comprising an antigen binding protein that binds to claudin-6 (CLDN6) protein or a fusion protein thereof, comprising: a. Culturing a host cell in a cell culture medium, wherein the host cell of any one of claims 41 to 45 comprises a nucleic acid encoding an antigen binding protein or a fusion protein thereof; b. harvesting the antigen binding protein or the fusion protein from the cell culture medium; c. binding the antigen binding protein or fusion protein to a second moiety, said second moiety being a detectable label, to form the conjugate; The method comprising:

62. 10. The method of any one of the preceding claims, wherein the antigen-binding protein is an antibody or an antigen-binding antibody fragment.

63. 63. The method of claim 62, wherein the antibody is a monoclonal antibody.

64. 63. The method of claim 62, wherein the antibody is a human or humanized antibody, or a chimeric antibody.

65. 63. The method of claim 62, wherein the antigen binding protein is an IgG.

66. 66. The method of claim 65, wherein the IgG is selected from the group consisting of IgG1, IgG2, IgG3, and IgG4.

67. 63. The method of claim 62, wherein the antigen-binding antibody fragment is selected from the group consisting of scFv, F(ab')2, Fab, Fab', and Fv.

68. 63. The method of claim 62, wherein the antibody is a combination of antibodies according to any one of the preceding claims specific for claudin-6 (CLDN6).

69. 62. The method of claim 61, wherein the detectable label comprises a fluorophore, a radioactive label, a colorimetric label, or an enzyme.

70. 70. The method of claim 69, wherein the detectable label comprises horseradish peroxidase (HRP).

71. a. an antigen-binding protein according to any of the preceding claims, and / or b. A conjugate of a detectable label and a specific binding partner of the antigen-binding protein of (a) above. A diagnostic kit comprising:

72. 72. The diagnostic kit of claim 71, wherein the label is selected from the group consisting of an enzyme, a radiolabel, a chromophore, and a fluorophore.

73. 61. The sample or biological sample of any one of claims 52 to 60, wherein the sample comprises fresh, frozen, flash-frozen, formalin-fixed, and / or paraffin-embedded cells or tissues.

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