WO2024035787A2 - Anticorps de claudine-6 et leurs conjugués et utilisations - Google Patents

Anticorps de claudine-6 et leurs conjugués et utilisations Download PDF

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WO2024035787A2
WO2024035787A2 PCT/US2023/029856 US2023029856W WO2024035787A2 WO 2024035787 A2 WO2024035787 A2 WO 2024035787A2 US 2023029856 W US2023029856 W US 2023029856W WO 2024035787 A2 WO2024035787 A2 WO 2024035787A2
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cldn6
antigen
seq
binding protein
amino acid
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PCT/US2023/029856
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English (en)
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Dennis Slamon
Martina MCDERMOTT
Neil A. O'BRIEN
Min Liang
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The Regents Of The University Of California
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • the present disclosure relates, in general, to antibodies specific for Claudin-6 (CLDN6) cytoplasmic domain and uses thereof to detect the presence of CLDN6 in a sample from a cancer patient, to determine the quantity of CLDN6 in the sample, to diagnose CLDN6-positive cancer in the patient.
  • CLDN6 Claudin-6
  • Antibodies constitute powerful therapeutic agents characterized by limited side effects due to their ability to specifically target and identify a distinct antigen on a cell, bacteria, virus, or toxin.
  • Orthoclone OKT3 the first therapeutic monoclonal antibody, Orthoclone OKT3 was introduced into the market. Since then, this class of biopharmaceutical products has significantly grown.
  • forty-seven monoclonal antibody products had received approval in the U.S. or Europe for the treatment of a variety of diseases, including cancer and inflammatory, cardiovascular, respiratory, and infectious diseases.
  • antigen-binding proteins which bind to Claudin-6 (CLDN6).
  • the antigen-binding protein of the present disclosure binds to a human CLDN6 and optionally binds to a mouse CLDN6.
  • the antigen-binding protein binds to the cytoplasmic domain of CLDN6.
  • the antigen binding protein binds selectively to CLDN6 over any other member of the Claudin family.
  • the antigen binding protein binds to CLDN6 and does not bind to any other member of the Claudin family.
  • the antigen binding protein binds to CLDN6 endogenously expressed by human ovarian cancer cells, e.g., OVCA429 cells.
  • an antigen-binding protein comprises (a) a heavy chain CDR1 amino acid sequence of SEQ ID NO: 18 or SEQ ID NO: 34, or a variant sequence thereof which differs by only one or two amino acids or which has at least or about 70% sequence identity; (b) a heavy chain CDR2 amino acid sequence of: SEQ ID NOs: 19 or SEQ ID NO: 35, or a variant sequence thereof which differs by only one or two amino acids or which has at least or about 70% sequence identity; (c) a heavy chain CDR3 amino acid sequence of SEQ ID NO: 20 or SEQ ID NO: 36, or a variant sequence thereof which differs by only one or two amino acids or which has at least or about 70% sequence identity; (d) a 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 which has at least or about 70% sequence identity; (e) a light chain CDR2 amino acid sequence of: SEQ ID NO: 18 or S
  • an antigen-binding protein comprises (a) a heavy chain variable region amino acid sequence of any one of SEQ ID NOs: 18-20, 34-36, or a variant sequence thereof which differs by only one or two amino acids or which has at least or about 70% sequence identity; or (b) a light chain variable region amino acid sequence of any one of SEQ ID NOs: 25-27, 41-43, or a variant sequence thereof which differs by only one or two amino acids or which has at least or about 70% sequence identity; or both (a) and (b).
  • antigen-binding proteins conjugated to a heterologous moiety e.g., a detectable label or a detection moiety
  • a heterologous moiety e.g., a detectable label or a detection moiety
  • the label or detection moiety may be a reporter protein.
  • 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, P-lactamase,or blue fluorescent protein (BFP).
  • GFP green fluorescent protein
  • RFP red fluorescent protein
  • GST glutathione-S-transferase
  • HRP horseradish peroxidase
  • AP alkaline phosphatase
  • CAT chloramphenicol acetyltransferase
  • beta-galactosidase glucose oxidase
  • GUS beta-glucuronidase
  • luciferase P-lactamase,or blue fluorescent protein
  • the antigen-binding protein binds to CLDN6 expressed by human cancer cells.
  • the antigen-binding protein inhibits a binding interaction between human CLDN6 and a reference anti-CLDN6 antibody.
  • the inhibiting action of the antigen-binding proteins provided herein allow such entities to be useful in methods of detecting tumors or tumor growth.
  • the antigen-binding protein is an antibody, antigen-binding antibody fragment thereof, or antibody protein product.
  • the present disclosure also provides antigen-binding proteins comprising at least 3, 4, 5, or all amino acid sequences of a specified group of amino acid sequences.
  • the antigen-binding proteins comprise at least 3, 4, 5, or 6 complementary determining region (CDR) amino acid sequences of CLDN6 antibodies disclosed herein.
  • the present disclosure further provides antigen-binding proteins comprising amino acid sequences as detailed herein.
  • the antigen-binding protein comprises an amino acid sequence of any one of SEQ ID NOs: 16-47, or a combination thereof, as further described herein.
  • kits and pharmaceutical compositions comprising such entities are moreover contemplated.
  • the method comprises culturing a host cell comprising a nucleic acid encoding an antigen-binding protein or a polypeptide as described herein so as to express the antigen-binding protein or polypeptide.
  • methods of making conjugate with the antigen-binding protein described herein are also provided.
  • the method comprises culturing a host cell comprising a nucleic acid encoding an antigen-binding protein, a fusion protein or a polypeptide as described herein so as to express the antigen-binding protein, fusion protein or polypeptide, harvesting the antigen-binding protein, fusion protein or the fusion protein from the cell culture medium then attaching the antigen binding protein, fusion protein or polypeptide to a second moiety so as to produce the conjugate.
  • the second moiety is a detectable label, agent or moiety.
  • 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, P-lactamase, or blue fluorescent protein (BFP).
  • GFP green fluorescent protein
  • RFP red fluorescent protein
  • GST glutathione-S-transferase
  • HRP horseradish peroxidase
  • AP alkaline phosphatase
  • CAT chloramphenicol acetyltransferase
  • beta-galactosidase beta-galactosidase
  • glucose oxidase GO
  • beta-glucuronidase GUS
  • Methods of detecting or determining the quantity of Claudin-6 (CLDN6) in a sample are provided herein.
  • the method comprises contacting the sample with an antigen-binding protein and assaying for an immunocomplex or quantifying an amount of the immunocomplexes so assayed comprising the antigenbinding protein bound to CLDN6, the immunocomplex indicative of the presence and quantity of CLDN6.
  • Further contemplated are methods diagnosing and monitoring the course of a Claudin-6 (CLDN6)-positive cancer in a subject wherein the presence of the immunocomplex is indicative of the presence of CLDN6-positive cancer.
  • the detection of the presence of CLDN6 can also be used in a method for typing CLDN6 positive tumors in subjects.
  • the method of detection comprises contacting a sample with a first antigen-binding protein which is specific for the CLDN6 to form a first- antigen-binding protein-CLDN6 immunocomplex, contacting the immunocomplex so formed with a second antigen-binding protein labeled with a detectable marker to form a second immunocomplex comprising first antigen-binding protein-CLDN6-second antigenbinding protein and detecting the second immunocomplex so formed indicative of the presence of CLDN6 in the sample.
  • the second antigen-binding protein is an antibody recognizing the first antigen-binding protein.
  • the methods comprise quantitatively determining in a first sample from the subject the presence of CLDN6, comparing the amount so determined with the amount present in a second sample from the subject, such samples being taken at different points in time, the quantity of CLDN6 determined over time indicative the CLDN6-positive cancer is treatable in the subject and the prognosis of the CLDN6- positive cancer in the subject.
  • FIG. 1 shows the results of epitope mapping for Claudin-6 (CLDN6) antibody 3H11 and 3D07 binding to peptides in the cytoplasmic domain of CLDN6.
  • 3H11 bound 2 peptides with an overlapping 10 amino acid region, APAISRGPSE (SEQ ID NO: 15).
  • 3D07 bound 3 peptides with an overlapping 6 amino acid region, SHYMAR (SEQ ID NO: 98).
  • FIG. 2 shows the results of epitope mapping for Claudin-6 (CLDN6) antibody 3D07 binding to peptides in the cytoplasmic domain of CLDN6.
  • 3D07 bound 1 peptide with the amino acid sequence SHYMARYSTSAPAIS (SEQ ID NO: 99).
  • FIG. 3 shows the binding of antibodies 3H11 and 3D07 to HEK293 cell lines expressing CLDN6, CLDN3, CLDN4 or CLDN9. The results show that the antibodies specifically bind CLDN6.
  • FIG. 4 shows the binding of antibodies 3H11 and 3D07 to tissues sections of human ovarian cancer. The results show that the antibodies bind CLDN6 in the cancer tissue samples and identifies the sections of cancer tissue having CLDN6-positive cells.
  • Claudin-6 is a member of the CLDN family.
  • the gene encoding the human CLDN6 protein is located on the p arm of human chromosome 16 at 16pl3.3 and is conserved in chimpanzee, Rhesus monkey, dog, cow, mouse, rat, zebrafish, and frog.
  • CLDN6 is generally expressed in humans as a 220-amino acid precursor protein; the first 21 amino acids of which constitute the signal peptide.
  • the amino acid sequence of the CLDN6 cytoplasmic tail (domain) is publicly available at the National Center for Biotechnology Information (NCBI) website as NCBI Reference Sequence NP_067018.2 and is provided herein as SEQ ID NO: 1.
  • the amino acid at position 143 of SEQ ID NO: 1 is He.
  • the amino acid at position 143 is a Vai.
  • the amino acid sequence of human CLDN6 having a Vai at position 143 is provided herein as SEQ ID NO: 2.
  • conjugate not only encompasses a polypeptide that is linked to a heterologous moiety via a chemical reaction, but also encompasses a polypeptide that is coexpressed to comprise a heterologous moiety as a fusion protein. Accordingly, as used herein, the term “conjugate” encompasses a fusion protein.
  • a molecule is “fixed” or “affixed” to a substrate if it is covalently or non- covalently associated with the substrate such the substrate can be rinsed with a fluid (e.g, standard saline citrate, pH 7.4) without a substantial fraction of the molecule dissociating from the substrate.
  • a fluid e.g, standard saline citrate, pH 7.4
  • prevention of cancer includes, for example, reducing the number of detectable cancerous growths in a population of patients receiving a prophylactic treatment relative to an untreated control population, and/or delaying the appearance of detectable cancerous growths in a treated population versus an untreated control population, e.g., by a statistically and/or clinically significant amount.
  • the amount of a biomarker in a subject is “significantly” higher or lower 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 greater than the standard error of the assay employed to assess amount, and preferably at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 300%, 350%, 400%, 500%, 600%, 700%, 800%, 900%, 1000% or than that amount.
  • the amount of the biomarker in the subject can be considered “significantly” higher or lower than the normal and/or control amount if the amount is 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%, 190%, 195%, two times, three times, four times, five times, or more, or any range in between, such as 5%-100%, higher or lower, respectively, than the normal and/or control amount of the biomarker.
  • Such significant modulation values can be applied to any metric described herein, such as altered level of expression, altered activity, changes in cancer cell hyperproliferative growth, changes in cancer cell death, changes in biomarker inhibition, changes in test agent binding, and the like.
  • 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.
  • the term thus means any substance intended for use in the diagnosis, cure, mitigation, treatment or prevention of disease or in the enhancement of desirable physical or mental development and conditions in an animal or human.
  • the phrase “therapeutically - effective amount” means that amount of such a substance that produces some desired local or systemic effect at a reasonable benefit/risk ratio applicable to any treatment.
  • a therapeutically effective amount of a compound will depend on its therapeutic index, solubility, and the like.
  • certain compounds discovered by the methods of the present invention may be administered in a sufficient amount to produce a reasonable benefit/risk ratio applicable to such treatment.
  • the term “treating” includes prophylactic and/or therapeutic treatments.
  • the term “prophylactic or therapeutic” treatment is art-recognized and includes administration to the host of one or more of the subject compositions or a cancer therapy described herein. If it is administered prior to clinical manifestation of the unwanted condition (e.g., disease or other unwanted state of the host animal), then the treatment is prophylactic (i.e., it protects the host against developing the unwanted condition); whereas, if it is administered after manifestation of the unwanted condition, the treatment is therapeutic (i.e., it is intended to diminish, ameliorate, or stabilize the existing unwanted condition or side effects thereol).
  • the unwanted condition e.g., disease or other unwanted state of the host animal
  • antigen-binding proteins that bind to the cytoplasmic domain of Claudin-6 (CLDN6).
  • CLDN6 Claudin-6
  • the antigen-binding proteins of the present disclosure can take any one of many forms of antigen-binding proteins known in the art.
  • the antigen-binding proteins of the present disclosure take the form of an antibody, or antigen-binding antibody fragment, or an antibody protein product.
  • the antigen-binding protein comprises, consists essentially of, or consists of an antibody.
  • antibody refers to a protein having a conventional immunoglobulin format, comprising heavy and light chains, and comprising variable and constant regions.
  • an antibody may be an IgG which is a “Y-shaped” structure of two identical pairs of polypeptide chains, each pair having one “light” (typically having a molecular weight of about 25 kDa) and one “heavy” chain (typically having a molecular weight of about 50-70 kDa).
  • An antibody has a variable region and a constant region.
  • variable region is generally about 100-110 or more amino acids, comprises three complementarity determining regions (CDRs), is primarily responsible for antigen recognition, and substantially varies among other antibodies that bind to different antigens.
  • the constant region allows the antibody to recruit cells and molecules of the immune system.
  • the variable region is made of the N-terminal regions of each light chain and heavy chain, while the constant region is made of the C-terminal portions of each of the heavy and light chains.
  • CDRs of antibodies have been described in the art. Briefly, in an antibody scaffold, the CDRs are embedded within a framework in the heavy and light chain variable region where they constitute the regions largely responsible for antigen binding and recognition.
  • a variable region typically comprises at least three heavy or light chain CDRs (Kabat et al., 1991, Sequences of Proteins of Immunological Interest, Public Health Service N.I.H., Bethesda, Md.; see also Chothia and Lesk, 1987, J. Mol. Biol.
  • framework region designated framework regions 1-4, FR1, FR2, FR3, and FR4, by Kabat et al., 1991; see also Chothia and Lesk, 1987, supra).
  • Antibodies can comprise 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, and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively.
  • IgG has several subclasses, including, but not limited to IgGl, IgG2, IgG3, and IgG4.
  • IgM has subclasses, including, but not limited to, IgMl 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 regions, e.g., a human alpha-, delta-, epsilon-, gamma-, or mu-type heavy chain constant region.
  • the antibody is an antibody of isotype IgA, IgD, IgE, IgG, or IgM, including any one of IgGl, IgG2, IgG3 or IgG4.
  • the antibody comprises a constant region comprising one or more amino acid modifications, relative to the naturally occurring counterpart, in order to improve half-life/stability or to render the antibody more suitable for expression/manufacturability.
  • the antibody comprises a constant region wherein the C-terminal Lys residue that is present in the naturally occurring counterpart is removed or clipped.
  • the antibody can be a monoclonal antibody.
  • the antibody comprises a sequence that is substantially similar to a naturally occurring antibody produced by a vertebrate animal, e.g., mammal, bird, reptile, and fish.
  • the antibody comprises a sequence that is substantially similar to a naturally occurring antibody produced by a bird, e.g., chicken, duck, quail, pigeon, turkey, and the like.
  • the antibody comprises a sequence that is substantially similar to a naturally occurring antibody produced by a mammal, e.g., mouse, rabbit, goat, horse, hamster, human, and the like.
  • the antibody can be considered as a mammalian antibody, e.g., a mouse antibody, rabbit antibody, goat antibody, horse antibody, hamster antibody, human antibody, and the like.
  • the antigenbinding protein is an antibody, such as a human antibody.
  • the antigenbinding protein is a chimeric antibody or a humanized antibody.
  • the term "chimeric antibody" refers to an antibody containing domains from two or more different antibodies.
  • a chimeric antibody can, for example, contain the constant domains from one species and the variable domains from a second, or more generally, can contain stretches of amino acid sequence from at least two species.
  • a chimeric antibody also can contain domains of two or more different antibodies within the same species.
  • humanized when used in relation to antibodies refers to antibodies having at least CDR regions from a non-human source which are engineered to have a structure and immunological function more similar to true human antibodies than the original source antibodies.
  • humanizing can involve grafting a CDR from a non-human antibody, such as a mouse antibody, into a human antibody.
  • Humanizing also can involve select amino acid substitutions to make a non-human sequence more similar to a human sequence.
  • Information, including sequence information for human antibody heavy and light chain constant regions is publicly available through the Uniprot database as well as other databases well-known to those in the field of antibody engineering and production.
  • the IgG2 constant region is available from the Uniprot database as Uniprot number P01859, incorporated herein by reference.
  • sequence for a murine IgG2a constant region includes the following.
  • IGKC domains include the following.
  • an antibody can be cleaved into fragments by enzymes, such as, e.g., papain and pepsin.
  • Papain cleaves an antibody to produce two Fab fragments and a single Fc fragment.
  • Pepsin cleaves an antibody to produce a F(ab’)2 fragment and a pFc’ fragment.
  • the antigen-binding protein of the present disclosure is an antigen-binding fragment of an antibody (a.k.a., antigen-binding antibody fragment, antigen-binding fragment, antigen-binding portion).
  • the antigenbinding antibody fragment is a Fab fragment or a F(ab’)2 fragment.
  • antibody protein products include those based on the full antibody structure and those that mimic antibody fragments which retain full antigen-binding capacity, e.g., scFvs, Fabs and VHH/VH (discussed below).
  • the smallest antigen-binding fragment that retains its complete antigen binding site is the Fv fragment, which consists entirely of variable (V) regions.
  • a soluble, flexible amino acid peptide linker is used to connect the V regions to a scFv (single chain fragment variable) fragment for stabilization of the molecule, or the constant (C) domains are added to the V regions to generate a Fab fragment (fragment, antigen-binding).
  • scFv and Fab fragments can be easily produced in host cells, e.g., prokaryotic host cells.
  • ds-scFv disulfide-bond stabilized scFv
  • scFab single chain Fab
  • minibodies minibodies that comprise different formats consisting of scFvs linked to oligomerization domains.
  • the smallest fragments are VHH/VH of camelid heavy chain Abs as well as single domain Abs (sdAb).
  • the building block that is most frequently used to create novel antibody formats is the single-chain variable (V)-domain antibody fragment (scFv), which comprises V domains from the heavy and light chain (VH and VL domain) linked by a peptide linker of ⁇ 15 amino acid residues.
  • a peptibody or peptide-Fc fusion is yet another antibody protein product.
  • the structure of a peptibody consists of a biologically active peptide grafted onto an Fc domain.
  • Peptibodies are well-described in the art. See, e.g., Shimamoto et al., mAbs 4(5): 586-591 (2012).
  • 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).
  • the antigen-binding protein of the present disclosure comprises, consists essentially of, or consists of any one of these antibody protein products.
  • the antigen-binding protein of the present disclosure comprises, consists essentially of, or consists of any one of an scFv, Fab VHH/VH, Fv fragment, ds-scFv, scFab, dimeric antibody, multimeric antibody (e.g., a diabody,, triabody, tetrabody), miniAb, peptibody VHH/VH of camelid heavy chain antibody, sdAb, diabody; atriabody; a tetrabody; a bispecific or trispecific antibody, BsIgG, appended IgG, BsAb fragment, bispecific fusion protein, and BsAb conjugate.
  • the antigen-binding protein of the present disclosure is an antibody protein product in monomeric form, or polymeric, oligomeric, or multimeric form.
  • the antibody comprises two or more distinct antigen binding regions fragments, the antibody is considered bispecific, trispecific, or multispecific, or bivalent, trivalent, or multivalent, depending on the number of distinct epitopes that are recognized and bound by the antibody.
  • an 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 IgGl antibody, an IgG2 antibody, an IgG3 antibody, and an IgG4 antibody.
  • the antigen-binding proteins of the present disclosure bind to the cytoplasmic domain of a CLDN6.
  • the CLDN6 is a human CLDN6 having the amino acid sequence of:
  • the CLDN6 cytoplasmic domain or tail has the amino acid sequence of: CCTCPSGGSQGPSHYMARYSTSAPAISRGPSEYPTKNYV (SEQ ID NO: 3).
  • the human CLDN6 comprises the amino acid sequence of any one of SEQ ID NOs: 1-3.
  • the antigen-binding proteins of the present disclosure bind to an epitope within an amino acid sequence of the cytoplasmic domain of CLDN6.
  • CLDN6 is a human CLDN6 and the antigen-binding proteins of the present disclosure bind to an epitope within an amino acid sequence of the cytoplasmic domain of human CLDN6, e.g., SEQ ID NOs: 3, 10,15, 95-99.
  • epitope is meant the region of or within CLDN6 which is bound by the antigen-binding protein.
  • the epitope is a linear epitope.
  • Linear epitope refers to the region of or within the CLDN6 which is bound by the antigen-binding protein and which region is composed of contiguous amino acids of the amino acid sequence of the CLDN6.
  • the amino acids of a linear epitope are adjacent to each other in the primary structure of the CLDN6. Accordingly, a linear epitope is a fragment or portion of the amino acid sequence of the antigen, i.e., CLDN6.
  • the epitope is a conformational or structural epitope.
  • conformational epitope or “structural epitope” is meant an epitope which is composed of amino acids which are located in close proximity to one another only when the CLDN6 is in its properly folded state.
  • amino acids of a conformational or structural epitope are not adjacent to each other in the primary structure (i.e., amino acid sequence) of the CLDN6.
  • a conformational or structural epitope is not made of contiguous amino acids of the amino acid sequence of the antigen (CLDN6).
  • the epitope is located within the cytoplasmic domain of CLDN6, e.g., human CLDN6.
  • the antigen binding protein binds to the cytoplasmic domain of CLDN6 having the amino acid sequence of CCTCPSGGSQGPSHYMARYSTSAPAISRGPSEYPTKNYV (SEQ ID NO: 3).
  • 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).
  • 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).
  • the epitope or a peptide to which the antigen-binding protein binds is within any of SEQ ID NO: 1-3, 10,15, 95-99.
  • the antigen-binding proteins bind to human CLDN6 and a nonhuman CLDN6.
  • the non-human CLDN6 is a CLDN6 of chimpanzee, Rhesus monkey, dog, cow, mouse, rat, zebrafish, or frog.
  • the antigenbinding proteins bind to human CLDN6 and mouse CLDN6.
  • the antigen-binding proteins provided herein bind to CLDN6 in a non-covalent and reversible manner.
  • the binding strength of the antigenbinding protein to CLDN6 may be described in terms of its affinity, a measure of the strength of interaction between the binding site of the antigen-binding protein and the epitope.
  • the antigen-binding proteins provided herein have high-affinity for CLDN6 and thus will bind a greater amount of CLDN6 in a shorter period of time than low-affinity antigen-binding proteins.
  • the antigen-binding protein has an equilibrium association constant, KA, which is at least 10 5 M’ 1 , at least 10 6 M 4 , at least 10 7 M 4 , at least 10 8 M 4 , at least 10 9 M 4 , or at least IO 10 M or at least IO 10 M 4 .
  • KA can be influenced by factors including pH, temperature and buffer composition.
  • the binding strength of the antigen-binding protein to CLDN6 may be described in terms of its sensitivity.
  • KD is the equilibrium dissociation constant, a ratio of k O ff/k O n, between the antigen-binding protein and CLDN6.
  • KD and KA are inversely related.
  • the KD value relates to the concentration of the antigen-binding protein (the amount of antigen-binding protein needed for a particular experiment) and so the lower the KD value (lower concentration) the higher the affinity of the antigen-binding protein.
  • the binding strength of the antigen-binding protein to CLDN6 may be described in terms of KD.
  • the KD of the antigen-binding proteins provided herein is about 10 4 M, about 10' 2 M, about 10 4 M, about 10 M, about 10' 5 M, about 10' 6 M, or less. In various aspects, the KD of the antigen-binding proteins provided herein is micromolar, nanomolar, picomolar or femtomolar. In various aspects, the KD of the antigen-binding proteins provided herein is within a range of about 10 4 to 10' 6 M or I O' 7 to 10' 9 M or 10 0 to 10 42 M or 10 3 to 10 5 M.
  • the KD of the antigenbinding proteins provided herein is within a range of about 1.0 x 10 42 M to about 1.0 x 10' 8 M. In various aspects, the KD of the antigen-binding proteins is within a range of about 1.0 x 10 41 M to about 1.0 x 10' 9 M.
  • the affinity of the antigen-binding proteins is measured or ranked using a flow cytometry- or Fluorescence- Activated Cell Sorting (FACS)-based assay.
  • FACS Fluorescence- Activated Cell Sorting
  • the relative affinity of a CLDN6 antibody is determined via a FACS-based assay in which different concentrations of a CLDN6 antibody conjugated to a fluorophore are incubated with purified cytoplasmic domain or peptides of CLDN6 which are coated on a solid structure such as a bead and the fluorescence emitted (which is a direct measure of antibody-antigen binding) is determined.
  • a curve plotting the fluorescence for each dose or concentration is made.
  • the max value is the lowest concentration at which the fluorescence plateaus or reaches a maximum, which is when binding saturation occurs.
  • Half of the max value is considered an EC50 or an IC50 and the antibody with the lowest EC50/IC50 is considered as having the highest affinity relative to other antibodies tested in the same manner.
  • the ICso value as determined in a competitive binding inhibition assay, approximates the KD of the antigen-binding protein.
  • the competition assay is a FACS-based assay carried out with a reference antibody, fluorophore-conjugated secondary antibody, and purified cytoplasmic domain or peptides of CLDN6 which are coated on a solid structure such as a bead.
  • the antigen-binding protein inhibits the binding interaction between human CLDN6 and the reference antibody, which reference antibody is known to bind to CLDN6 but is not an antigen-binding protein of the present disclosure.
  • the antigen-binding proteins of the present disclosure compete with the reference antibody for binding to human CLDN6 and thereby reduce the amount of human CLDN6 bound to the reference antibody as determined by an in vitro competitive binding assay.
  • the antigenbinding proteins of the present disclosure inhibit the binding interaction between human CLDN6 and the reference antibody and the inhibition is characterized by an ICso.
  • the antigen-binding proteins exhibit an ICso of less than about 2500 nM for inhibiting the binding interaction between human CLDN6 and the reference antibody.
  • the antigen-binding proteins exhibit an ICso 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.
  • the antigen-binding proteins exhibit an ICso 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.
  • the antigen binding proteins of the present disclosure compete against a reference antibody known to bind to CLDN6 (which reference antibody is different from any of the antigen-binding proteins of the present disclosure) for binding to CLDN6. See further description under
  • Avidity gives a measure of the overall strength of an antibody-antigen complex. It is dependent on three major parameters: affinity of the antigen-binding protein for the epitope, valency of both the antigen-binding protein and CLDN6, and structural arrangement of the parts that interact. The greater an antigen-binding protein’s valency (number of antigen binding sites), the greater the amount of antigen (CLDN6) it can bind.
  • the antigen-binding proteins have a strong avidity for CLDN6.
  • the antigen-binding proteins are multivalent. In various aspects, the antigen-binding proteins are bivalent. In various instances, the antigen antigen-binding proteins are monovalent.
  • the antigen-binding proteins of the present disclosure bind to CLDN6 and do not bind to any other member of the CLDN family, e.g., do not cross-react with any other member of the CLDN family.
  • the antigenbinding proteins of the present disclosure are CLDN-6 specific.
  • the antigen-binding proteins of the present disclosure have a selectivity for CLDN6 which is at least 10-fold, 5-fold, 4-fold, 3-fold, 2-fold greater than the selectivity of the antigenbinding protein for CLDN3, CLDN4, CLDN9, or a combination thereof.
  • the antigen-binding proteins of the present disclosure have a selectivity for CLDN6 which is at least 10-fold, 5-fold, 4-fold, 3-fold, 2-fold greater than the selectivity of the antigen-binding protein for each of CLDN3, CLDN4, and CLDN9.
  • Selectivity may be based on the KD exhibited by the antigen binding protein for CLDN6, or a CLDN family member, wherein the KD may be determined by techniques known in the art, e.g., surface plasmon resonance, FACS-based affinity assays.
  • the antigen-binding protein inhibits a binding interaction between human CLDN6 and a reference antibody, which reference antibody is known to bind to CLDN6 but is not an antigen-binding protein of the present disclosure.
  • the antigen-binding proteins of the present disclosure compete with the reference antibody for binding to human CLDN6 and thereby reduce the amount of human CLDN6 bound to the reference antibody as determined by an in vitro competitive binding assay.
  • the reference antibody binds to an epitope within the amino acid sequence of the cytoplasmic domain of human CLDN6.
  • the antigenbinding proteins of the present disclosure inhibit the binding interaction between human CLDN6 and the reference antibody and the inhibition is characterized by an IC50.
  • the antigen-binding proteins exhibit an IC50 of less than about 2500 nM for inhibiting the binding interaction between human CLDN6 and the reference antibody. In various aspects, the antigen-binding proteins exhibit an IC50 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.
  • the antigen-binding proteins exhibit an IC50 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.
  • the antigen-binding proteins of the present disclosure compete with the reference antibody for binding to human CLDN6 and thereby reduce the amount of human CLDN6 bound to the reference antibody as determined by an in vitro competitive binding assay.
  • the in vitro competitive binding assay is a FACS-based assay in which the fluorescence of a fluorophore-conjugated secondary antibody which binds to the Fc of the reference antibody is measured in the absence or presence of a particular amount of the antigen-binding protein of the present disclosure.
  • the FACS-based assay is carried out with the reference antibody, fluorophore-conjugated secondary antibody and cells which express CLDN6.
  • the cells are genetically engineered to overexpress CLDN6.
  • the cells are HEK293T cells transduced with a viral vector to express CLDN6.
  • the cells endogenously express CLDN6.
  • the cells which endogenously express CLDN6 are pre-determined as low CLDN6-expressing cells or high CLDN6-expressing cells.
  • the cells are cancer or tumor cells.
  • the cells are cells from a cell line, e.g., an ovarian cell line, endometrial cell line, bladder cell line, lung cell line, gastrointestinal (GI) cell line, liver cell line, lung cell line, and the like.
  • a cell line e.g., an ovarian cell line, endometrial cell line, bladder cell line, lung cell line, gastrointestinal (GI) cell line, liver cell line, lung cell line, and the like.
  • the cells which endogenously express CLDN6 as 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.
  • the antigen binding proteins of the present disclosure bind to CLDN6 endogenously expressed by one or more of ARK2 cells, OVCA429 cells, LS513 cells, or MCF7 cells with high affinity.
  • the antigen binding proteins exhibit an IC50 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.
  • the antigen binding proteins exhibit an IC50 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.
  • the antigen binding proteins exhibit an IC50 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.
  • binding assays e.g., competitive binding assays or competition assays, which test the ability of an antibody to compete with a second antibody for binding to an antigen, or to an epitope thereof, are known in the art. See, e.g., Trikha et al., Int J Cancer 110: 326-335 (2004); Tam et al., Circulation 98(11): 1085-1091 (1998).
  • SPR surface plasmon resonance
  • antigen-binding proteins e.g., antibodies, antigenbinding antibody fragments, and antibody protein products
  • standard hybridoma methods for producing antibodies are described in, e.g., 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)).
  • CLDN6 monoclonal antibodies or the present disclosure is provided herein in EXAMPLES.
  • adjuvants can be used to increase the immunological response leading to greater antibody production by the host.
  • adjuvants include but are not limited to Freund's, mineral gels such as aluminum hydroxide, and surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, and dinitrophenol.
  • BCG Bacilli Calmette-Guerin
  • Corynebacterium parvum are potentially useful human adjuvants.
  • Methods of testing antibodies for the ability to bind to the epitope of CLDN6 regardless of how the antibodies are produced are known in the art and include any antibody-antigen binding assay, such as, for example, radioimmunoassay (RIA), ELISA, Western blot, immunoprecipitation, SPR, and competitive inhibition assays (see, e.g., Janeway et al., infra, and U.S. Patent Application Publication No. 2002/0197266, and the above section relating to competition assays).
  • RIA radioimmunoassay
  • ELISA ELISA
  • Western blot Western blot
  • immunoprecipitation e.g., SPR
  • competitive inhibition assays see, e.g., Janeway et al., infra, and U.S. Patent Application Publication No. 2002/0197266, and the above section relating to competition assays.
  • the binding assay was a bead-based flow assay with indirect flow cytometry using beads conjugated with BSA- Peptide conjugates or formaldehyde fixed GST fusion proteins (Press et al., Steroids, 67 (2002) 799-813).
  • antigen-binding proteins comprising (a) a heavy chain (HC) complementarity-determining region (CDR) 1 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%) sequence identity; (b) an HC 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%) sequence identity; (c) an HC 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%) sequence identity; (d) a light chain (LC)
  • LC light chain
  • the antigen-binding protein comprises a LC CDR1 amino acid sequence, a LC CDR2 amino acid sequence, and a LC CDR3 amino acid sequence set forth in Table 4 and at least 1 or 2 of the HC CDR amino acid sequences set forth in Table 4.
  • the antigen-binding protein 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 at least 1 or 2 of the LC CDR amino acid sequences set forth in Table 4.
  • the antigen-binding protein comprises at least 3, 4, or 5 of the amino acid sequences designated by the SEQ ID NOs: in a single row of Table 4. In various embodiments, the antigen-binding protein comprises each of the LC CDR amino acid sequences designated by the SEQ ID NOs: of a single row of Table 4 and at least 1 or 2 of the HC CDR amino acid sequences designated by the SEQ ID NOs: in of a single row of Table 4.
  • the antigen-binding protein comprises each of the HC CDR amino acid sequences designated by the SEQ ID NOs: of a single row of Table 4 and at least 1 or 2 of the LC CDR amino acid sequences designated by the SEQ ID NOs: of a single row of Table 4. In various embodiments, the antigen-binding protein comprises all 6 of the CDR amino acid sequences designated by the SEQ ID NOs: of a single row of 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.
  • the amino acid sequences of 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 acid(s).
  • the antigen-binding protein comprises (a) a heavy chain variable region amino acid sequence set forth in in Table 5 or a sequence selected from the group consisting of: SEQ ID NOs: 16, 32, 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%) 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 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%) sequence identity; or (c) both (a) and (b).
  • 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.
  • the antigen-binding protein comprises an amino acid sequence which is similar to an above-referenced amino acid sequence, yet the antigen-binding protein substantially retains its biological function, e.g., its ability to bind to human CLDN6, detect or quantify its presence in samples.
  • the antigen-binding protein comprises an amino acid sequence which differs by only 1, 2, 3, 4, 5, 6, or more amino acids, relative to the above-referenced amino acid sequence(s).
  • the antigen-binding protein comprises a variant sequence of the referenced sequence, which variant sequence differs by only one or two amino acids, relative to the referenced sequence.
  • the antigen-binding protein comprising one or more amino acid substitutions that occur outside of the CDRs, e.g, the one or more amino acid substitutions occur within the framework region(s) of the heavy or light chain.
  • the antigen-binding protein comprising one or more amino acid substitutions yet the antigen-binding protein retains the amino acid sequences of the six CDRs.
  • the antigen-binding protein comprises an amino acid sequence having only 1, 2, 3, 4, 5, 6, or more conservative amino acid substitutions, relative to the above-referenced amino acid sequence(s).
  • conservative amino acid substitution refers to the substitution of one amino acid with another amino acid having similar properties, e.g., size, charge, hydrophobicity, hydrophilicity, and/or aromaticity, and includes exchanges within one of the following five groups:
  • the conservative amino acid substitution is an exchange within one of the following groups of amino acids:
  • non-aromatic amino acids comprising a side chain hydroxyl: Ser, Thr
  • V acidic amino acid: Glu; Asp
  • amino acid comprising a side chain amide Gin, Asn
  • IX imino acid: Pro, 4-hydroxy-Pro, 4-amino-Pro
  • the antigen-binding protein comprises an amino acid sequence which has greater than or about 30%, greater than or about 50%, or greater than or about 70% sequence identity to the above-referenced amino acid sequence. In various aspects, the antigen-binding protein comprises an amino acid sequence which has 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 has greater than 90% sequence identity to the above-referenced amino acid sequence. In various aspects, the antigen-binding protein comprises an amino acid sequence that has at least 70%, at least 80%, at least 85%, at least 90% or has greater than 90% sequence identity along the full-length of the above-referenced amino acid sequence. In various aspects, the antigen-binding protein comprises an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity along the full-length of the above-referenced amino acid sequence.
  • the antigen-binding protein comprises a variant sequence of the referenced sequence, which variant sequence has at least or about 70% sequence identity, relative to the above-referenced sequence. In various aspects, the antigen-binding protein comprises a variant sequence of the referenced sequence, which variant sequence has at least or about 80% sequence identity, relative to the above-referenced sequence. In various aspects, the antigen-binding protein comprises a variant sequence of the referenced sequence, which variant sequence has at least or about 90% sequence identity, relative to the above-referenced sequence. In various aspects, the antigen-binding protein comprises a variant sequence of the referenced sequence, which variant sequence has at least or about 95% sequence identity, relative to the above-referenced sequence.
  • the antigen-binding protein comprises one, two, three, four, or five sequences of the SEQ ID NOs: in a single row of 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.
  • 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 of the set.
  • the antigen-binding protein comprises a pair of variant sequences having at least or about 70% (e.g., at least about 80%, at least about 85%, at least about 90%, at least about 95%) sequence identity to any of SEQ ID NOs: 16-17, 32-33.
  • the antigen binding protein comprises a pair of variant sequences which have at least or about 70% (e.g., at least about 80%, at least about 85%, at least about 90%, at least about 95%) sequence identity to (a) SEQ ID NOs: 16-17; and (b) SEQ ID NOs: 32- 33.
  • the antigen-binding protein comprises a pair of sequences: one sequence of Table 5 and another sequence which is a variant sequence having at least or about 70% (e.g., at least about 80%, at least about 85%, at least about 90%, at least about 95%) sequence identity to any of SEQ ID NOs: 16-17, 32-33.
  • the antigen-binding protein comprises a pair of sequences: one sequence selected from (a) SEQ ID NOs: 16-17; and (b) SEQ ID NOs: 32-33, and another sequence which is a variant sequence having at least or about 70% (e.g., at least about 80%, at least about 85%, at least about 90%, at least about 95%) sequence identity to a sequence of (a) - (b).
  • the antigen-binding protein comprises an amino acid sequence of an above-referenced amino acid sequence with one or more amino acid substitutions to reduce or eliminate reactive amino acids to decrease or prevent unwanted side chain reactions.
  • the antigen-binding protein comprises an amino acid sequence of an above- referenced amino acid sequence with one or more (i) Trp residues substituted with His, Tyr, or Phe; (ii) Asn residues substituted with Gin, Ser, Ala, or Asp; (iii) Asp residues occurring immediately before a Pro residue substituted with Ala, Ser, or Glu, (iv) Asn residues substituted with Gin, Ser, or Ala; and/or (v) Cys residues substituted with Tyr, Ser, or Ala.
  • the antigen-binding protein comprises an amino acid sequence of an above-referenced amino acid sequence with an amino acid substitution predicted to have greater binding affinity, greater stability, or other positive attribute, based on SHM events or based on statistical analyses of a multitude of other similar antibody sequences.
  • the amino acid sequences of 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 acid(s).
  • nucleic acids comprising a nucleotide sequence encoding an antigen-binding protein of the present disclosure.
  • nucleic acid includes “polynucleotide,” “oligonucleotide,” and “nucleic acid molecule,” and generally means a polymer of DNA or RNA, or modified forms thereof, which can be single-stranded or double- stranded, synthesized or obtained (e.g., isolated and/or purified) from natural sources, which can contain natural, non-natural or altered nucleotides, and which can contain a natural, non-natural or altered inter-nucleotide linkage, such as a phosphoroamidate linkage or a phosphorothioate linkage, instead of the phosphodiester found between the nucleotides of an unmodified oligonucleotide.
  • the nucleic acid can comprise any nucleotide sequence which encodes any of the antigen-binding proteins of the present disclosure.
  • the nucleic acid comprises a nucleotide sequence which encodes an antigen-binding protein comprising (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 which differs by only one or two amino acids or which 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) an 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 which differs by only one or two amino acids or which has at least or about 70% (e.g., at least or about 80%, at least or about 85%, at least or or
  • the nucleic acid comprises a nucleotide sequence encoding an antigenbinding protein comprising a LC CDR1 amino acid sequence, a LC CDR2 amino acid sequence, and a LC CDR3 amino acid sequence set forth in Table 4 and at least 1 or 2 of the HC CDR amino acid sequences set forth in Table 4.
  • the nucleic acid comprises a nucleotide sequence encoding an antigen-binding protein comprising 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 at least 1 or 2 of the LC CDR amino acid sequences set forth in Table 4.
  • the nucleic acid comprises a nucleotide sequence encoding an antigen-binding protein comprising (a) at least 3, 4, or 5 of the amino acid sequences designated by the SEQ ID NOs: in a single row of Table 4, (b) each of the LC CDR amino acid sequences designated by the SEQ ID NOs: of a single row of Table 4 and at least 1 or 2 of the HC CDR amino acid sequences designated by the SEQ ID NOs: in of a single row of Table 4, (c) each of the HC CDR amino acid sequences designated by the SEQ ID NOs: of a single row of Table 4 and at least 1 or 2 of the LC CDR amino acid sequences designated by the SEQ ID NOs: of a single row of Table 4, (d) all 6 of the CDR amino acid sequences designated by the SEQ ID NOs: of a single row of Table 4, and/or (e) six CDR amino acid sequences selected from the group consisting of: (a) SEQ ID NOs: 18- 20,
  • the nucleic acid comprises a nucleotide sequence encoding an antigen-binding protein comprising (a) a heavy chain variable region amino acid sequence set forth in in Table 5 or a sequence selected from the group consisting of: SEQ ID NOs: 16, 32, 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 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, 33, 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 or about 80%, at least or about 85%, at least or about 90%, at least or about 95%) sequence identity; or (c) both (a) and (b).
  • 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.
  • the nucleic acid comprises a nucleotide sequence encoding an antigen-binding protein comprising the amino acid sequences selected from the group listed in Tables 4-5.
  • the nucleic acid does not comprise any insertions, deletions, inversions, and/or substitutions.
  • the nucleic acid comprises one or more insertions, deletions, inversions, and/or substitutions.
  • the nucleic acid comprises a nucleotide sequence which encodes the antigen-binding protein as set forth in Tables 4-5 with 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 in the light chain (LC) variable region, or in both.
  • the nucleic acid comprises a nucleotide sequence which encodes the antigen-binding protein comprising a HC of SEQ ID NO: 16 with 1, 2, 3, 4, or 5 amino acid substitutions.
  • the nucleic acid comprises a nucleotide sequence which encodes the antigen-binding protein comprising a HC CDR1 of SEQ ID NO: 18, aHC CDR2 of SEQ ID NO: 19, a HC CDR3 of SEQ ID NO: 20, or a combination thereof.
  • the nucleic acid comprises a nucleotide sequence which encodes the antigen-binding protein comprising a HC of SEQ ID NO: 16.
  • the nucleic acid comprises a nucleotide sequence which encodes the antigen-binding protein comprising a HC of any one of SEQ ID NOs: 18-20.
  • the nucleic acid comprises a nucleotide sequence which encodes the antigen-binding protein comprising a LC of SEQ ID NO: 17 with 1, 2, 3, 4, or 5 amino acid substitutions.
  • the nucleic acid comprises a nucleotide sequence which encodes the light chain variable region comprising a LC CDR1 of SEQ ID NO: 25, a LC CDR2 of SEQ ID NO: 26, a LC CDR3 of SEQ ID NO: 27, or a combination thereof.
  • the nucleic acid comprises a nucleotide sequence which encodes the antigen-binding protein comprising a LC of any one of SEQ ID NOs: 25-27.
  • the nucleic acid comprises a nucleotide sequence which encodes the antigen-binding protein comprising a HC of SEQ ID NO: 32 with 1, 2, 3, 4, 5, or 6 amino acid substitutions.
  • the nucleic acid comprises a nucleotide sequence which encodes the antigen-binding protein comprising a HC CDR1 of SEQ ID NO: 34, a HC CDR2 of SEQ ID NO: 35, a HC CDR3 of SEQ ID NO: 36, or a combination thereof.
  • the nucleic acid comprises a nucleotide sequence which encodes the antigen-binding protein comprising a HC of SEQ ID NO: 32.
  • the nucleic acid comprises a nucleotide sequence which encodes the antigen-binding protein comprising a HC of any one of SEQ ID NOs: 34-26.
  • the nucleic acid comprises a nucleotide sequence which encodes the antigenbinding protein comprising a LC of SEQ ID NO: 33 with 1, 2, 3, 4, or 5 amino acid substitutions.
  • the nucleic acid comprises a nucleotide sequence which encodes the light chain variable region comprising a LC CDR1 of SEQ ID NO: 41, a LC CDR2 of SEQ ID NO: 42, a LC CDR3 of SEQ ID NO: 43, or a combination thereof.
  • the nucleic acid comprises a nucleotide sequence which encodes the antigenbinding protein comprising a LC of any one of SEQ ID NOs: 41-43.
  • the nucleic acids of the present disclosure are recombinant.
  • the term “recombinant” refers to (i) molecules that are constructed outside living cells by joining natural or synthetic nucleic acid segments to nucleic acid molecules that can replicate in a living cell, or (ii) molecules that result from the replication of those described in (i) above.
  • the replication can be in vitro replication or in vivo replication.
  • nucleic acids in some aspects are constructed based on chemical synthesis and/or enzymatic ligation reactions using procedures known in the art. See, for example, Sambrook et al., supra, and Ausubel et al., supra.
  • a nucleic acid can be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed upon hybridization (e.g., phosphorothioate derivatives and acridine substituted nucleotides).
  • modified nucleotides that can be used to generate the nucleic acids include, but are not limited to, 5 -fluorouracil, 5- bromouracil, 5-chIorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5- (carboxyhydroxymethyl) uracil, 5- carboxymethylaminomethyl-2-thiouridme, 5- carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N 6 - isopentenyladenine, 1-methylguanine, 1 -methylinosine, 2,2-dimethylguanine, 2- methyladenine, 2-methylguanine, 3-methylcytosine, 5 -methylcytosine, N -substituted adenine, 7-methylguanine, 5-methylammomethyluracil, 5- methoxyaminomethyl-2- thiouracil, beta
  • nucleic acids of the present disclosure in some aspects are incorporated into a vector.
  • the present disclosure provides vectors comprising any of the presently disclosed nucleic acids.
  • the vector is a recombinant expression vector.
  • the term "recombinant expression vector” means a genetically- modified oligonucleotide or polynucleotide construct that permits the expression of an mRNA, protein, polypeptide, or peptide by a host cell, when the construct comprises a nucleotide sequence encoding the mRNA, protein, polypeptide, or peptide, and the vector is contacted with the cell under conditions sufficient to have the mRNA, protein, polypeptide, or peptide expressed within the cell.
  • the vectors of the present disclosure are not naturally- occurring as a whole. However, parts of the vectors can be naturally-occurring.
  • the presently disclosed vectors can comprise any type of nucleotides, including, but not limited to DNA and RNA, which can be single- stranded or double-stranded, synthesized or obtained in part from natural sources, and which can contain natural, non-natural or altered nucleotides.
  • the vectors can comprise naturally-occurring or non-naturally-occurring intemucleotide linkages, or both types of linkages.
  • the altered nucleotides or non-naturally occurring intemucleotide linkages do not hinder the transcription or replication of the vector.
  • the vector 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 expansion or for expression or both, such as plasmids and viruses.
  • the vector can be a plasmid based expression vector.
  • the vector is selected from the group consisting of the pUC series (Fermentas Life Sciences), the pBluescript series (Stratagene, LaJoIIa, CA), the pET series (Novagen, Madison, WI), the pGEX series (Pharmacia Biotech, Uppsala, Sweden), and the pEX series (Clontech, Palo Alto, CA).
  • Bacteriophage vectors such as ZGTIO. ZGTI 1, ZZapII (Stratagene), ZEMBL4. and ZNMI 149, also can be used.
  • plant expression vectors include pBIOl, pBI101.2, pBI101.3, pBI121 and pBIN19 (Clontech).
  • animal expression vectors include pEUK-Cl, pMAM and pMAMneo (Clontech).
  • the vector is a viral vector, e.g., a retroviral vector.
  • the vector is an adenovirus vector, an adeno- associated virus (AAV) vector, a Herpes Simplex Virus (HSV) vector, a Vesicular stomatitis virus (VSV) vector, vaccinia virus vector, or lentivirus vector. See, e.g., Howarth et al., Cell Biol. Toxicol. 26(1): 1-20 (2010).
  • the vector is a baculovirus vector which infects arthropods, e.g., insects.
  • the baculovirus vector is an Autographacalifomica multiple nuclear virus (AcMNPV) or a Bombyxmorinuclear polyhedrosis (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).
  • AcMNPV Autographacalifomica multiple nuclear virus
  • BmNPV Bombyxmorinuclear polyhedrosis
  • Table 6 Expression vectors used to produce anti-CLDN6 antibodies
  • the vectors of the present disclosure can be prepared using standard recombinant DNA techniques described in, for example, Sambrook et al., supra, and Ausubel et al., supra.
  • Constructs of expression vectors which are circular or linear, can be prepared to contain a replication system functional in a prokaryotic or eukaryotic host cell.
  • Replication systems can be derived, e.g., from CoIEl, 2 p plasmid, X, SV40, bovine papilloma virus, and the like.
  • the vector comprises regulatory sequences, such as transcription and translation initiation and termination codons, which are specific to the type of host (e.g., bacterium, fungus, plant, or animal) into which the vector is to be introduced, as appropriate and taking into consideration whether the vector is DNA- or RNA- based.
  • regulatory sequences such as transcription and translation initiation and termination codons, which are specific to the type of host (e.g., bacterium, fungus, plant, or animal) into which the vector is to be introduced, as appropriate and taking into consideration whether the vector is DNA- or RNA- based.
  • the vector can include one or more marker genes, which allow for selection of transformed or transfected hosts.
  • Marker genes include biocide resistance, e.g., resistance to antibiotics, heavy metals, etc., complementation in an auxotrophic host to provide prototrophy, and the like.
  • Suitable marker genes for the presently disclosed expression vectors include, for instance, neomycin/G418 resistance genes, hygromycin resistance genes, histidinol resistance genes, tetracycline resistance genes, and ampicillin resistance genes.
  • the vector can comprise a native or normative promoter operably linked to the nucleotide sequence encoding the polypeptide (including functional portions and functional variants thereof), or to the nucleotide sequence which is complementary to or which hybridizes to the nucleotide sequence encoding the polypeptide.
  • a native or normative promoter operably linked to the nucleotide sequence encoding the polypeptide (including functional portions and functional variants thereof), or to the nucleotide sequence which is complementary to or which hybridizes to the nucleotide sequence encoding the polypeptide.
  • the promoter can be a non-viral promoter or a viral promoter, e.g., a cytomegalovirus (CMV) promoter, an SV40 promoter, an RSV promoter, and a promoter found in the long-terminal repeat of the murine stem cell virus.
  • CMV cytomegalovirus
  • host cells comprising a nucleic acid or vector of the present disclosure.
  • the term "host cell” refers to any type of cell that can contain the presently disclosed vector and is capable of producing an expression product encoded by the nucleic acid (e.g., mRNA, protein).
  • the host cell in some aspects is an adherent cell or a suspended cell, i.e., a cell that grows in suspension.
  • the host cell in various aspects is a cultured cell or a primary cell, i.e., isolated directly from an organism, e.g., a human.
  • the host cell can be of any cell type, can originate from any type of tissue, and can be of any developmental stage.
  • the antigen-binding protein is a glycosylated protein and the host cell is a glycosylation-competent cell.
  • the glycosylation-competent cell is an eukaryotic cell, including, but not limited to, a yeast cell, filamentous fungi cell, protozoa cell, algae cell, insect cell, or mammalian cell. Such host cells are described in the art. See, e.g., Frenzel, et al., Front Immunol 4: 217 (2013).
  • the eukaryotic cells are mammalian cells.
  • the mammalian cells are nonhuman mammalian cells.
  • the cells are 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 be deficient in dihydrofolatereductase (DHFR) activity (e.g., DUKX-X11, DG44), human embryonic kidney 293 (HEK293) cells or derivatives thereof (e.g., HEK293T, HEK293-EBNA), green African monkey kidney cells (e.g., COS cells, VERO cells), human cervical cancer cells (e.g., HeLa), human bone osteosarcoma epithelial cells U2-OS, adenocarcinomic human alveolar basal epithelial cells A549, human fibrosarcoma cells HT1080, mouse brain tumor cells CAD, embryonic carcinoma cells Pl 9, mouse embryo fibroblast cells NIH 3T3, mouse fibro
  • a population of cells comprising at least one host cell described herein.
  • the population of cells in some aspects is a heterogeneous population comprising the host cell comprising vectors described, in addition to at least one other cell, which does not comprise any of the vectors.
  • the population of cells is a substantially homogeneous population, in which the population comprises mainly host cells (e.g., consisting essentially of) comprising the vector.
  • the population in some aspects is a clonal population of cells, in which all cells of the population are clones of a single host cell comprising a vector, such that all cells of the population comprise the vector.
  • the population of cells is a clonal population comprising host cells comprising a vector as described herein.
  • the method comprises culturing a host cell comprising a nucleic acid comprising a nucleotide sequence encoding the antigenbinding protein as described herein in a cell culture medium and harvesting the antigenbinding protein from the cell culture medium.
  • the host cell can be any of the host cells described herein.
  • the host cell is selected from the group consisting of: CHO cells, NS0 cells, COS cells, VERO cells, and BHK cells.
  • the step of culturing a host cell comprises culturing the host cell in a growth medium to support the growth and expansion of the host cell.
  • the growth medium increases cell density, culture viability and productivity in a timely manner.
  • the growth medium comprises amino acids, vitamins, inorganic salts, glucose, and serum as a source of growth factors, hormones, and attachment factors.
  • the growth medium is a fully chemically defined media consisting of amino acids, vitamins, trace elements, inorganic salts, lipids and insulin or insulin-like growth factors. In addition to nutrients, the growth medium also helps maintain pH and osmolality.
  • growth media are commercially available and are described in the art. See, e.g., Arora, “Cell Culture Media: A Review” MATER METHODS 3:175 (2013).
  • the method comprises culturing the host cell in a feed medium.
  • the method comprises culturing in a feed medium in a fed- batch mode.
  • Methods of 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).
  • the method making an antigen-binding protein can comprise one or more steps for purifying the protein from a cell culture or the supernatant thereof and preferably recovering the purified protein.
  • the method comprises one or more chromatography steps, e.g., affinity chromatography (e.g., protein A affinity chromatography), ion exchange chromatography, hydrophobic interaction chromatography.
  • the method comprises purifying the protein using a Protein A affinity chromatography resin.
  • the method further comprises steps for formulating the purified protein, etc., thereby obtaining a formulation comprising the purified protein.
  • steps for formulating the purified protein, etc. thereby obtaining a formulation comprising the purified protein.
  • the antigen-binding protein linked to a polypeptide and the antigen-binding protein is part of a fusion protein.
  • the present disclosure further provides methods of producing a fusion protein comprising an antigen-binding protein which binds to CLDN6.
  • the method comprises culturing a host cell comprising a nucleic acid comprising a nucleotide sequence encoding the fusion protein as described herein in a cell culture medium and harvesting the fusion protein from the cell culture medium.
  • the present disclosure also provides antigen-binding proteins attached, linked or conjugated to a second moiety (e.g., a heterologous moiety, a conjugate moiety).
  • a second moiety e.g., a heterologous moiety, a conjugate moiety
  • the present disclosure provides a conjugate comprising an antigen-binding protein and a heterologous moiety.
  • heterologous moiety is synonymous with “conjugate moiety” and refers to any molecule (chemical or biochemical, naturally-occurring or non-coded) which is different from the antigen-binding proteins of the present disclosure.
  • heterologous moi eties include, but are not limited to, a polymer, a carbohydrate, a lipid, a nucleic acid, an oligonucleotide, a DNA or RNA, an amino acid, peptide, polypeptide, protein, a detectable label, therapeutic agent, (e.g., a cytotoxic agent, cytokine), or a diagnostic agent.
  • the heterologous moiety is a detectable label (as used herein, a "detectable label” is a synonym for a "detectable marker”).
  • detectable labels include, but are not limited to, a fluorophore, radioactive label, colorometric label, or wherein the detectable label is an enzyme.
  • the detectable label is horse radish peroxidase (HRP).
  • HRP horse radish peroxidase
  • a product of horse radish peroxidase (HRP) enzymatic reaction is a chromogen.
  • HRP horse radish peroxidase
  • the oxidation reaction catalyzed by HRP comprises HRP, an HRP chromogenic substrate and an oxidant.
  • the oxidation reaction catalyzed by HRP additionally comprises a metal and/or imidazole.
  • the oxidant is hydrogen peroxide (H2O2).
  • the metal is selected from the group consisting of nickel (Ni 2+ ), copper (Cu 2+ ), silver (Ag 2+ ) and cobalt (Co 2+ ).
  • the metal is selected from the group consisting of nickel (Ni 2+ ) and cobalt (Co 2+ ).
  • the HRP chromogenic substrate is selected from the group consisting of 2,2’-azino-bis(3- ethylbenzthiazoline-6-sulfonic acid) (ABTS), aminoethyl carbazole (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-trihydroxy benzene (Pyrogallol), hydroxybenzene (Phenol), 4-methylphenol (p-Cresol), 3,3 ’-dimethoxybenzidine (0- Dianisidine), 1 -amino-4-methylbenzene (p-Toluidine), 3,3 ’-dimethylbenzidine (Tolidine), 1 ,4-dihydroxybenzene (ABTS),
  • the chromogenic HRP substrate is selected from the group consisting of of 2,2’-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS), aminoethyl carbazole (AEC), 3,3 ’,5,5 ’-tetramethylbenzidine (TMB), 3,3’-diaminobenzidine (DAB), 0- phenylenediamine (OPD), and 4-chl oro-1 -naphthol (4-CN).
  • ABTS 2,2’-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid)
  • AEC aminoethyl carbazole
  • TMB 3,3 ’,5,5 ’-tetramethylbenzidine
  • DAB 3,3’-diaminobenzidine
  • OPD 0- phenylenediamine
  • a brown chromogen following oxidation of DAB (3,3'- diaminobenzidine) by HRP is detected.
  • a grayish blue chromogen following oxidation of DAB (3,3'-diaminobenzidine) in the presence of copper (Cu 2+ ) by HRP is detected.
  • a gray-black, dark blue, or purplish blue chromogen following oxidation of DAB (3,3'-diaminobenzidine) in the presence of nickel (Ni 2+ ) by HRP is detected.
  • a dark blue or bluish black chromogen following oxidation of DAB (3, 3 '-diaminobenzidine) in the presence of cobalt (Co 2+ ) by HRP is detected.
  • a blue/dark blue chromogen following oxidation of TMB (3,3’,5,5’-tetramethylbenzidine) by HRP is detected.
  • a red chromogen following oxidation of AEC (aminoethyl carbazole) by HRP is detected.
  • a dark blue/purple chromogen following oxidation of 4-chloro-l -naphthol (4-CN) by HRP is detected.
  • the product of horse radish peroxidase (HRP) enzymatic reaction is a fluorogenic label.
  • the detectable label is a fluorogenic label or fluorophore following an oxidation reaction catalyzed by HRP.
  • the fluorogenic label or fluorophore is an oxidized compound following an oxidation reaction catalyzed by HRP.
  • the oxidized compound is fluorescent.
  • the oxidized compound comprises fluorescence emission and/or absorption substantially different from its unoxidized compound from which the oxidized compound is derived.
  • the oxidation reaction catalyzed by HRP comprises HRP, an HRP fluorogenic substrate and an oxidant.
  • the oxidation reaction catalyzed by HRP additionally comprises a metal and/or imidazole.
  • the oxidant is hydrogen peroxide (H2O2).
  • the metal is selected from the group consisting of nickel (Ni 2+ ), copper, silver and cobalt (Co 2+ ).
  • the HRP fluorogenic substrate is selected from the group comprising 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).
  • the detectable label is 7-hydroxy-3H-phenoxazine-3-one (resorufin) following oxidation of 10-acetyl-3,7-dihydroxyphenoxazine (ADHP or AmplexRed) by HRP.
  • the detectable label is 2,2'-(6,6'-dihydroxy-5-methoxy-[l,l'-biphenyl]-3,3'- diyl)diacetic acid (HVA dimer) following oxidation of 2-(4-hydroxy-3- methoxyphenyl)acetic acid (homovanillic acid or HVA monomer) by HRP.
  • 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- xanthene-3,6-diol (DCFH-1) by HRP.
  • HRP chromogenic kits include, but are not limited to, DAB Peroxidase (HRP) Substrate Kit with Nickel (Vector Labs, catalog #: SK-4100), ImmPACT® DAB Peroxidase (HRP) Substrate Kit (Vector Labs, catalog #: SK-4105), ImmPACT® DAB EqV Peroxidase (HRP) Substrate Kit (Vector Labs, catalog #: SK- 4103), Vector® VIP Peroxidase (HRP) Substrate Kit (Vector Labs, catalog #: SK-4600), ImmPACT® VIP Peroxidase (HRP) Substrate Kit (Vector Labs, catalog #: SK-4605), Vector® SG Peroxidase (HRP) Substrate Kit (Vector Labs, catalog #: SK-4700), I
  • the detectable label is a product of alkaline phosphatase (AP) enzymatic reaction.
  • the product of alkaline phosphatase (AP) enzymatic reaction is a chromogen.
  • the detectable label is a chromogen following a dephosphorylation reaction catalyzed by AP.
  • the dephosphorylation reaction catalyzed by AP comprises AP and an AP substrate.
  • the dephosphylation reaction catalyzed by AP additionally comprises a diazonium salt.
  • the dephosphylation reaction catalyzed by AP comprises AP, an AP substrate, and a diazonium salt.
  • the product of alkaline phosphatase (AP) enzymatic reaction reacts with diazonium salts to yield a chromogen.
  • the dephosphylation reaction catalyzed by alkaline phosphatase (AP) is a hydrolysis reaction resulting in release of a phosphate group.
  • the detectable label is a chromogen following a hydrolysis reaction catalyzed by AP.
  • the hydrolysis reaction catalyzed by AP comprises AP, a substrate and a diazonium salt.
  • 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-chl oro-3 -indolyl- phosphate-toluidine salt (salmon phosphate) and 5-bromo-6-chloro-3-indolyl phosphate p- toluidine salt (magenta phosphate).
  • BCIP 5-bromo-4-chloro-3-indolyl phosphate
  • Naphthol AS-MX phosphate 3-hydroxy-2-naphthoic acid 2,4-dimethylanilide phosphate
  • 6-chl oro-3 -indolyl- phosphate-toluidine salt sodium phosphate
  • the detectable label is a blue chromogen following hydrolysis of BCIP (5-bromo-4-chloro-3-indolyl phosphate) by AP (alkaline phosphatase) in the presence of NBT (p-nitroblue tetrazolium chloride).
  • the detectable label is a purple chromogen following hydrolysis of BCIP (5- bromo-4-chloro-3-indolyl phosphate) by AP in the presence of TNBT (tetranitroblue tetrazolium).
  • the detectable label is a blue chromogen following hydrolysis of Naphthol AS-MX phosphate (3-Hydroxy-2-naphthoic acid 2,4- dimethylanilide phosphate) by AP in the presence of Fast Blue BB.
  • the detectable label is a red chromogen following hydrolysis of Naphthol AS-MX phosphate by AP in the presence of Red TR.
  • the detectable label is a red chromogen following hydrolysis of Naphthol AS-MX phosphate by AP in the presence of new fuchsin.
  • the detectable label is a green chromogen using a green AP chromogen/substrate system, such as HIGHDEF Green AP chromogen/substrate (Enzo, catalog #: ENZ-ACC130) or STAYGREEN/AP Plus (Abeam, catalog #: abl56428).
  • a green AP chromogen/substrate system such as HIGHDEF Green AP chromogen/substrate (Enzo, catalog #: ENZ-ACC130) or STAYGREEN/AP Plus (Abeam, catalog #: abl56428).
  • the product of alkaline phosphatase (AP) enzymatic reaction is a fluorogenic label.
  • the detectable label is a fluorogenic label or fluorophore following a hydrolysis reaction catalyzed by AP.
  • the hydrolyzed compound is fluorescent.
  • the AP fluorogenic substrate is selected from a group consisting of 2-(Benzothiazol-2-yl)-4-bromophenol phosphate (BT3- Phos), Vector red (Vector Labs, catalog #: SK-5100), ImmPACT Vector red (Vector Labs, catalog #: SK5105), and Vector blue (Vector Labs, catalog #: SK-5300).
  • kits for detection of AP enzymatic activity are known in the art and readily available.
  • AP chromogenic kits include, but are not limited to ImmPRESS® Duet Double Staining Polymer Kit (Vector Labs, catalog #: MP-7714 ), ImmPRESS® Duet Double Staining Polymer Kit (Vector Labs, catalog #: MP-7724), Human on human IHC kit (AP/Permanent Red; Abeam, catalog #: ab214753), DoubleStain IHC Kit: M&R on human tissue (DAB & AP/Red; Abeam, catalog #: ab210059), TripleStain IHC Kit: M&R&G on human tissue (DAB, AP/Red & HRP/Green: Abeam, catalog #: abl 83290), TripleStain IHC Kit: M&R&Rt on rodent tissue (DAB, DAB/Ni & AP/Red; Abeam, catalog
  • the heterologous moiety is a polymer.
  • the polymer can be branched or unbranched.
  • the polymer can be of any molecular weight.
  • the polymer in some embodiments has an average molecular weight of between about 2 kDa to about 100 kDa (the term "about” indicating that in preparations of a water soluble polymer, some molecules will weigh more, some less, than the stated molecular weight).
  • the average molecular weight of the polymer is in some aspect between about 5 kDa and about 50 kDa, between about 12 kDa to about 40 kDa or between about 20 kDa to about 35 kDa.
  • 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.
  • the polymer in some embodiments is water soluble so that the protein to which it is attached does not precipitate in an aqueous environment, such as a physiological environment.
  • the polymer when, for example, the composition is used for therapeutic use, the polymer is pharmaceutically acceptable.
  • the polymer is a mixture of polymers, e.g., a co-polymer, a block co-polymer.
  • the polymer is selected from the group consisting of: polyamides, polycarbonates, polyalkylenes and derivatives thereof including, polyalkylene glycols, polyalkylene oxides, polyalkylene terepthalates, polymers of acrylic and methacrylic esters, including poly(methyl methacrylate), poly(ethyl methacrylate), poly(butylmethacrylate), poly(isobutyl methacrylate), poly(hexylmethacrylate), poly(isodecyl methacrylate), poly(lauryl methacrylate), poly(phenyl methacrylate), poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutyl acrylate), and poly(octadecyl acrylate), polyvinyl polymers including polyvinyl alcohols, polyvinyl ethers, polyvinyl esters, polyvinyl halides, poly(vinyl acetate), and poly(octadecy
  • a particularly preferred water-soluble polymer for use herein is polyethylene glycol (PEG).
  • PEG polyethylene glycol
  • polyethylene glycol is meant to encompass any of the forms of PEG that can be used to derivatize other proteins, such as mono-(Cl-ClO) alkoxy- or aryloxy -polyethylene glycol.
  • PEG is a linear or branched neutral polyether, available in a broad range of molecular weights, and is soluble in water and most organic solvents.
  • the heterologous moiety is a carbohydrate.
  • the carbohydrate is a monosaccharide (e.g., glucose, galactose, fructose), a disaccharide (e.g., sucrose, lactose, maltose), an oligosaccharide (e.g., raffinose, stachyose), a polysaccharide (a starch, amylase, amylopectin, cellulose, chitin, callose, laminarin, xylan, mannan, fucoidan, galactomannan.
  • a monosaccharide e.g., glucose, galactose, fructose
  • a disaccharide e.g., sucrose, lactose, maltose
  • an oligosaccharide e.g., raffinose, stachyose
  • a polysaccharide a starch,
  • the heterologous moiety is a lipid.
  • the lipid in some embodiments, is a fatty acid, eicosanoid, prostaglandin, leukotriene, thromboxane, N-acyl ethanolamine), glycerolipid (e.g., mono-, di-, tri-substituted glycerols), glycerophospholipid (e.g., phosphatidylcholine, phosphatidylinositol, phosphatidylethanolamine, phosphatidylserine), sphingolipid (e.g., sphingosine, ceramide), sterol lipid (e.g., steroid, cholesterol), prenol lipid, saccharolipid, or a polyketide, oil, wax, cholesterol, sterol, fatsoluble vitamin, monoglyceride, diglyceride, triglyceride, a phospholipid.
  • glycerolipid e
  • 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) to detect CLDN6 positive tumors in a subject, e.g., a human.
  • the conjugated antigen-binding protein is a monoclonal antibody conjugated to a heterologous moiety.
  • the heterologous moiety may be a polypeptide. While the polypeptidic heterologous moiety can be chemically conjugated to e.g., the monoclonal antibody, the polypeptidic heterologous moiety may be coexpressed as a fusion protein of the monoclonal antibody.
  • the present disclosure also provides conjugates comprising an antigen-binding protein of the present disclosure linked to a polypeptide, such that the conjugate is a fusion protein. Therefore, the present disclosure provides fusion proteins comprising an antigen-binding protein of the present disclosure linked to a polypeptide.
  • the polypeptide may be a reporter protein.
  • 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, -lactamase, or blue fluorescent protein (BFP).
  • the polypeptide is a diagnostic label, e.g., a fluorescent protein, such as green fluorescent protein, red fluorescent protein, and blue fluorescent protein.
  • the polypeptide is an epitope tag, e.g., myc tag, hemagglutinin (HA) tag, hexa-histidine tag, FLAGTM tag, VSV-G tag and V5 tag.
  • the present disclosure also provides conjugates comprising an antigen-binding protein of the present disclosure linked to a small molecule.
  • the small molecules is, but not limited to, biotin, digoxigenin (DIG), fluorescein isothiocyanate (FITC) or allophycocyanin (APC).
  • the antigen-binding protein or its conjugate may be detected by a secondary antibody linked to a label or detection moiety, wherein the secondary antibody binds the antigen-binding protein or its conjugate.
  • the label or detection moiety may be a reporter protein.
  • 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, P-lactamase, or blue fluorescent protein (BFP).
  • the label or detection moiety may be an epitope tag or a small molecule.
  • the epitope tag includes, but is not limited to, myc tag, hemagglutinin (HA) tag, hexa-histidine tag, FLAGTM tag, VSV-G tag and V5 tag.
  • the small molecule includes, but is not limited to, biotin DIG, FITC and APC.
  • the antigen-binding protein of the invention is bound by a secondary antibody conjugated to an enzyme and the detectable label is a product of an enzymatic reaction.
  • the antigen-binding protein of the invention may be a monoclonal antibody or its fragment.
  • the antigen-binding protein may be free of or not conjugated to a heterologous moiety.
  • the enzyme may be horseradish peroxidase (HRP), alkaline phosphatase (AP), chloramphenicol acetyltransferase (CAT), beta-galactosidase, glucose oxidase (GO), beta-glucuronidase (GUS), luciferase, or -lactamase.
  • the enzyme is HRP or AP.
  • the product of an enzymatic reaction is a chromogen, a fluorogenic label, or a fluorophore.
  • the antigen-binding protein of the invention is bound by a secondary antibody conjugated to HRP and the detectable label is a product of an HRP enzymatic reaction.
  • the product of an HRP enzymatic reaction is a chromogen, a fluorogenic label, or a fluorophore following oxidation of an HRP substrate by HRP.
  • the product of an HRP enzymatic reaction is a chromogen.
  • the HRP substrate may be an HRP chromogenic substrate or an HRP fluorogenic substrate.
  • the HRP substrate is an HRP chromogenic substrate.
  • the antigen-binding protein of the invention is bound by a secondary antibody conjugated to AP and the detectable label is a product of an AP enzymatic reaction.
  • the product of an AP enzymatic reaction is a chromogen, a fluorogenic label, or a fluorophore.
  • the product of an AP enzymatic reaction is a chromogen.
  • the AP substrate may be an AP chromogenic substrate or an AP fluorogenic substrate.
  • the AP substrate is an AP chromogenic substrate.
  • the antigen-binding protein of the invention is bound by a secondary antibody conjugated to biotin (i.e., biotinylated secondary antibody).
  • the label or detection moiety is conjugated to biotin (i.e., biotinylated label or detection moiety).
  • the label or detection moiety is a reporter protein.
  • the reporter protein may be an enzyme.
  • the enzyme is conjugate to biotin.
  • the enzyme conjugated to biotin may be horseradish peroxidase (HRP), alkaline phosphatase (AP), chloramphenicol acetyltransferase (CAT), beta-galactosidase, glucose oxidase (GO), betaglucuronidase (GUS), luciferase, or P-lactamase.
  • HRP horseradish peroxidase
  • AP alkaline phosphatase
  • CAT chloramphenicol acetyltransferase
  • beta-galactosidase glucose oxidase
  • GUS betaglucuronidase
  • luciferase or P-lactamase.
  • the enzyme conjugated to biotin i.e., biotinylated enzyme
  • HRP horseradish peroxidase
  • AP alkaline phosphatase
  • CAT chloramphenicol acetyltransferase
  • immunodetection of CLDN6 comprises an immunocomplex comprising the antigen-binding protein of the invention, biotinylated secondary antibody, biotinylated enzyme, and biotin-binding protein selected from the group comprising avidin, streptavidin and NeutrAvidin.
  • the immunodetection further comprises an enzymatic substrate to produce an enzymatic product, which is a detectable label.
  • immunodetection of CLDN6 comprises an immunocomplex comprising the antigen-binding protein of the invention, biotinylated secondary antibody, biotinylated HRP or AP, and biotin-binding protein selected from the group comprising avidin, streptavidin and NeutrAvidin (de-glycosylated avidin).
  • the immunodetection further comprises an HRP or AP enzymatic substrate to produce an enzymatic product, which is a chromogen, fluorogenic label or fluorophore.
  • the enzymatic product is a chromogen.
  • the antigen-binding protein of the invention is bound by a secondary antibody conjugated to biotin (i.e., biotinylated secondary antibody).
  • the label or detection moiety is conjugated to biotin (i.e., biotinylated label or detection moiety).
  • the label or detection moiety is a reporter protein.
  • the reporter protein may be an enzyme.
  • the enzyme is conjugate to avidin, streptavidin or NeutrAvidin.
  • the enzyme conjugated to avidin, streptavidin or NeutrAvidin may be horseradish peroxidase (HRP), alkaline phosphatase (AP), chloramphenicol acetyltransferase (CAT), beta-galactosidase, glucose oxidase (GO), betaglucuronidase (GUS), luciferase, or P-lactamase.
  • HRP horseradish peroxidase
  • AP alkaline phosphatase
  • CAT chloramphenicol acetyltransferase
  • beta-galactosidase beta-galactosidase
  • glucose oxidase GO
  • betaglucuronidase GUS
  • luciferase or P-lactamase.
  • the enzyme conjugated to avidin, streptavidin or NeutrAvidin is horse radish peroxidase (HRP) or alkaline phosphatase (AP).
  • immunodetection of CLDN6 comprises an immunocomplex comprising the antigen-binding protein of the invention, biotinylated secondary antibody, and avidin-, streptavidin- or NeutrAvidin-conjugated enzyme.
  • the immunodetection further comprises an enzymatic substrate to produce an enzymatic product, which is a detectable label.
  • immunodetection of CLDN6 comprises an immunocomplex comprising the antigen-binding protein of the invention, biotinylated secondary antibody, and avidin-, streptavidin- or NeutrAvidin- conjugated HRP or AP.
  • the immunodetection further comprises an HRP or AP enzymatic substrate to produce an enzymatic product, which is a chromogen, fluorogenic label or fluorophore.
  • the enzymatic product is a chromogen.Z/ «fer5
  • the protein of the present disclosure (e.g., antigenbinding protein, fusion protein, etc.) is directly linked to the heterologous moiety.
  • the conjugate comprises a linker that joins the compound of the present disclosure to the heterologous moiety.
  • the linker comprises a chain of atoms from 1 to about 60, or 1 to 30 atoms or longer, 2 to 5 atoms, 2 to 10 atoms, 5 to 10 atoms, or 10 to 20 atoms long.
  • the chain atoms are all carbon atoms.
  • the chain atoms in the backbone of the linker are selected from the group consisting of C, O, N, and S.
  • Chain atoms and linkers can be selected according to their expected solubility (hydrophilicity) so as to provide a more soluble conjugate.
  • the linker provides a functional group that is subject to cleavage by an enzyme or other catalyst or hydrolytic conditions found in the target tissue or organ or cell.
  • the length of the linker is long enough to reduce the potential for steric hindrance.
  • the linker is an amino acid or a peptidyl linker. Such peptidyl linkers can be any length.
  • Various linkers are from about 1 to 50 amino acids in length, 5 to 50, 3 to 5, 5 to 10, 5 to 15, or 10 to 30 amino acids in length.
  • linker can be cleavable (a cleavable linker).
  • the linker can be bonded to a chemically reactive group on the antibody moiety, e.g., to a free amino, imino, hydroxyl, thiol, or carboxyl group (e.g., to the N- or C- terminus, to the epsilon amino group of one or more lysine residues, to the free carboxylic acid group of one or more glutamic acid or aspartic acid residues, to the sulfhydryl group of one or more cysteinyl residues, or to the hydroxyl group of one or more serine or threonine residues).
  • a chemically reactive group on the antibody moiety e.g., to a free amino, imino, hydroxyl, thiol, or carboxyl group (e.g., to the N- or C- terminus, to the epsilon amino group of one or more lysine residues, to the free carboxylic acid group of one or more glutamic acid or aspartic
  • the linker or the heterologous moiety is attached to an interchain disulfide of an antibody (see e.g., ADCETRIS®; see also Jain et al. (2015) Pharmaceutical Research 32:3526-3540, which is incorporated herein by reference).
  • the site to which the linker is bound can be a natural residue in the amino acid sequence of the antibody moiety, or it can be introduced into the antibody moiety, e.g., by DNA recombinant technology (e.g., by introducing a cysteine or protease cleavage site in the amino acid sequence) or by protein biochemistry (e.g., reduction, pH adjustment, or proteolysis).
  • the site to which the linker is bound can also be a non-natural amino acids.
  • the site to which the linker is bound can also be a glycan on the antibody.
  • the linker is substantially inert under conditions for which the two groups it is connecting are linked.
  • the term “bifunctional crosslinking agent,” “bifunctional linker” or “crosslinking agent” refers to a modifying agent that possess two reactive groups at each end of the linker, such that one reactive group can be first reacted with the cytotoxic compound to provide a compound bearing the linker moiety and a second reactive group, which can then react with the antibody.
  • one end of the bifunctional crosslinking agent can be first reacted with the antibody to provide an antibody bearing a linker moiety and a second reactive group, which can then react with the cytotoxic compound.
  • the linking moiety may contain a chemical bond that allows for the release of the cytotoxic moiety at a particular 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, for example, 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;
  • the bonds are disulfide bonds, thioether, and/or protease/peptidase labile bonds.
  • Other linkers that can be used in the present invention include non-cleavable linkers, such as those described in detail in US 20050169933, charged linkers, or hydrophilic linkers, such as those described in US 2009/0274713, US 2010/0129314, and WO 2009/134976, each of which is expressly incorporated herein by reference.
  • the linker is a hydrophilic linker that confers hydrophilicity to the conjugate.
  • the hydrophilic linker comprises polyethylene glycol (PEG).
  • the hydrophilic linker is CL2A.
  • the CL2A linker has the following structure:
  • the hydrophilic linker is CL2E.
  • the CL2E has the following structure:
  • the linker is cleavable by a cleaving agent that is present in the intracellular environment (e.g., within a lysosome or endosome or caveolea).
  • the linker can be, e.g., 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.
  • the peptide linker comprises at least two, at least three, at least four, or at least five amino acids long.
  • the peptide linker is VC-PAB, comprising 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:
  • MC-VC-PAB has been described in U.S. Patent Nos. 7,659,241; 7,829,531; 6,884,869; 6,214,345; and 6,214,345.
  • the peptide linker is glycine-glycine-phenylalanine- glycine (GGFG). In some such embodiments, the peptide linker is maleimidocaproyl glycine-glycine-phenylalanine-glycine (MC-GGFG). In some embodiments, the MC- GGFG linker has the following structure: MC-GGFG has been described in U.S. Patent Nos. 9,808,537 and 10,195,288.
  • the cleavable linker is pH-sensitive, i.e., sensitive to hydrolysis at certain pH values.
  • the pH-sensitive linker is hydrolyzable under acidic conditions.
  • an acid-labile linker that is hydrolyzable in the lysosome e.g., a hydrazone, semicarbazone, thiosemicarbazone, cisaconitic amide, orthoester, acetal, ketal, or the like
  • an acid-labile linker that is hydrolyzable in the lysosome e.g., a hydrazone, semicarbazone, thiosemicarbazone, cisaconitic amide, orthoester, acetal, ketal, or the like
  • can be used see, e.g., US Patent Nos. 5,122,368; 5,824,805; 5,622,929; Dubowchik and Walker, 1999, Pharm.
  • the hydrolyzable linker is a thioether linker (such as, e.g., a thioether attached to the therapeutic agent via an acylhydrazone bond (see, e.g., US Patent No. 5,622,929).
  • the linker is cleavable under reducing conditions (e.g., a disulfide linker).
  • Bifunctional crosslinking agents that enable the linkage of an antibody with cytotoxic compounds via disulfide bonds 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), N-succinimidyl-4-(2-pyridyldithio)-2- sulfo butanoate (sulfo-SPDB).
  • Sulfo-SPDB is described, e.g., in US Patent 8,236,319, incorporated herein by reference.
  • crosslinking agents that introduce thiol groups such as 2-iminothiolane, homocysteine thiolactone, or S-acetylsuccinic anhydride can be used.
  • the linker may contain a combination of one or more of the peptide, pH-sensitive, or disulfide linkers described previously.
  • Heterobifunctional crosslinking agents are bifunctional crosslinking agents having two different reactive groups. Heterobifunctional crosslinking agents containing both an amine-reactive N-hydroxysuccinimide group (NHS group) and a carbonyl-reactive hydrazine group can also be used to link cytotoxic compounds with an antibody. Examples of such commercially available heterobifunctional crosslinking agents include succinimidyl 6-hydrazinonicotinamide acetone hydrazone (SANH), succinimidyl 4- hydrazidoterephthalate hydrochloride (SHTH) and succinimidyl hydrazinium nicotinate hydrochloride (SHNH).
  • SSH succinimidyl 6-hydrazinonicotinamide acetone hydrazone
  • SHTH succinimidyl 4- hydrazidoterephthalate hydrochloride
  • SHNH succinimidyl hydrazinium nicotinate hydrochloride
  • Conjugates bearing an acid-labile linkage can also be prepared using a hydrazine-bearing benzodiazepine derivative of the present invention.
  • bifunctional crosslinking agents include succinimidyl-p-formyl benzoate (SFB) and succinimidyl-p-formylphenoxyacetate (SFPA).
  • linkers described herein may be used in any combination with the heterologous moiety described herein.
  • the linkers described herein can have any chemical reactive moi eties (e.g., mal eimide, cysteine, sulfhydryl, etc.) that can react with any part (e.g., an amino acid, disulfide bond, carbohydrate (e.g., those from the post- translational modification), etc.) of the antigen-binding protein of the present disclosure.
  • any part e.g., an amino acid, disulfide bond, carbohydrate (e.g., those from the post- translational modification), etc.
  • All of the above-listed linkers and heterologous moiety described herein are available commercially and/or can be prepared by conventional techniques including those described in the above-listed references.
  • the heterologous moiety -to-antigen-binding protein ratio represents the number of a heterologous moiety linked per antigen-binding molecule.
  • the HAR ranges from 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.
  • the HAR ranges from 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.
  • the HAR is about 2, about 2.5, about 3, about 4, about 5, or about 6.
  • the HAR ranges from about 2 to about 4.
  • the HAR may be characterized by conventional means such as mass spectrometry, UV/Vis spectroscopy, ELISA assay, and/or HPLC.
  • the conjugates are heterogeneous conjugates (also referred to as “conventional”), wherein the antigen-binding proteins are conjugated to a different number of the heterologous moiety.
  • the heterogeneous conjugates follow a Gaussian distribution or quasi-Gaussian distribution of the conjugates, wherein the distribution centers on the average heterologous moiety loading value with some antigen-binding proteins conjugated with higher than average and some antigenbinding proteins conjugated with lower than the average.
  • the conjugates are homogeneous conjugates, wherein the substantial percentage of the antigen-binding proteins are conjugated to a defined number of the heterologous moiety.
  • the homogeneous conjugates comprise the HAR of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.
  • the homogeneous conjugates comprise the HAR of 2, 4, 6, or 8.
  • the homogeneous conjugates comprise the HAR of 4.
  • the homogeneous conjugates comprise the HAR of 2.
  • the homogeneous conjugates comprise greater than or equal to 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 conjugates with the defined HAR.
  • the homogeneous conjugates comprise 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 conjugates with the defined HAR.
  • the homogeneous conjugates comprise 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 conjugates with the defined HAR.
  • the homogeneous conjugates comprise the HAR distribution that is not Gaussian or quasi-Gaussian distribution.
  • the homogeneity of the homogeneous conjugates is determined by a chromatogram, e.g, HPLC or any suitable chromatography.
  • the chromatogram is a HIC chromatogram.
  • the homogeneous conjugate may be generated by a site-specific conjugation.
  • the heterologous moiety is conjugated to the antigenbinding protein (e.g., antibody) in a site-specific manner.
  • antigenbinding protein e.g., antibody
  • Various site-specific conjugation methods are known in the art, e.g., thiomab or TDC or conjugation at an unpaired cysteine residue (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 bridge linker (Behrens et al. (2015) Mol. Pharm.
  • compositions comprising an antigen-binding protein, a nucleic acid, a vector, a host cell, or a conjugate as presently disclosed are provided herein.
  • the compositions in some aspects comprise the antigen-binding proteins in isolated and/or purified form.
  • the composition comprises a single type (e.g., structure) of an antigenbinding protein of the present disclosure or comprises 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).
  • the composition comprises agents which enhance the chemico- physico features of the antigen-binding protein, e.g., via stabilizing the antigen-binding protein at certain temperatures, e.g., room temperature, increasing shelflife, reducing degradation, e.g., oxidation protease mediated degradation, increasing half-life of the antigen-binding protein, etc.
  • the composition comprises any of the agents disclosed herein as a heterologous moiety or conjugate moiety, optionally in admixture with the antigen-binding proteins of the present disclosure or conjugated to the antigen-binding proteins.
  • the composition additionally comprises a pharmaceutically acceptable carrier, diluents, or excipient.
  • a pharmaceutically acceptable carrier diluents, or excipient.
  • the antigen-binding protein, a nucleic acid, a vector, a host cell, or a conjugate as presently disclosed (hereinafter referred to as “active agents”) is formulated into a pharmaceutical composition comprising the active agent, along with a pharmaceutically acceptable carrier, diluent, or excipient.
  • the active agent is present in the composition 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%, and a pharmaceutically acceptable diluent, carrier or excipient.
  • the compositions contain an active agent at a concentration of about 0.001 to about 30.0 mg/ml.
  • compositions comprise a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carrier includes any of the standard pharmaceutical carriers, such as a phosphate buffered saline solution, water, emulsions such as an oil/water or water/oil emulsion, and various types of wetting agents. The term also encompasses any of the agents approved by a regulatory agency of the US Federal government or listed in the US Pharmacopeia for use in animals, including humans.
  • the composition can comprise any pharmaceutically acceptable ingredients, including, for example, acidifying agents, additives, adsorbents, aerosol propellants, air displacement agents, alkalizing agents, anticaking agents, anticoagulants, antimicrobial preservatives, antioxidants, antiseptics, bases, binders, buffering agents, chelating agents, coating agents, coloring agents, desiccants, detergents, diluents, disinfectants, disintegrants, dispersing agents, dissolution enhancing agents, dyes, emollients, emulsifying agents, emulsion stabilizers, fillers, film forming agents, flavor enhancers, flavoring agents, flow enhancers, gelling agents, granulating agents, humectants, lubricants, mucoadhesives, ointment bases, ointments, oleaginous vehicles, organic bases, pastille bases, pigments, plasticizers, polishing agents, preservatives, sequestering agents, skin
  • the composition comprises formulation materials that are nontoxic to recipients at the dosages and concentrations employed.
  • compositions comprising an active agent and one or more pharmaceutically acceptable salts; polyols; surfactants; osmotic balancing agents; tonicity agents; antioxidants; antibiotics; antimycotics; bulking agents; lyoprotectants; anti-foaming agents; chelating agents; preservatives; colorants; analgesics; or additional pharmaceutical agents.
  • the composition comprises 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 balancing agents (tonicity agents); anti-oxidants; antibiotics; antimycotics; bulking agents; lyoprotectants; anti-foaming agents; chelating agents; preservatives; colorants; and analgesics.
  • pharmaceutically acceptable salts including but not limited to, pharmaceutically acceptable salts; osmotic balancing agents (tonicity agents); anti-oxidants; antibiotics; antimycotics; bulking agents; lyoprotectants; anti-foaming agents; chelating agents; preservatives; colorants; and analgesics.
  • the composition can contain formulation materials for modifying, maintaining or preserving, for example, the pH, osmolarity, viscosity, clarity, color, isotonicity, odor, sterility, stability, rate of dissolution or release, adsorption or penetration of the composition.
  • formulation materials for modifying, maintaining or preserving for example, the pH, osmolarity, viscosity, clarity, color, isotonicity, odor, sterility, stability, rate of dissolution or release, adsorption or penetration of the composition.
  • suitable formulation materials include, but are not limited to, amino acids (such as glycine, glutamine, asparagine, arginine or lysine); antimicrobials; antioxidants (such as ascorbic acid, sodium sulfite or sodium hydrogen-sulfite); buffers (such as borate, bicarbonate, Tris-HCl, citrates, phosphates or other organic acids); bulking agents (such as mannitol or glycine); chelating agents (such as ethylenediamine tetraacetic acid (EDTA)); complexing agents (such as caffeine, polyvinylpyrrolidone, beta-cyclodextrin or hydroxypropyl-beta-cyclodextrin); fillers; monosaccharides; disaccharides; and other carbohydrates (such as glucose, mannose or dextrins); proteins (such as serum albumin, gelatin or immunoglobulins); coloring, flavoring and diluting agents; emulsifying agents;
  • amino acids
  • the ompositions can be formulated to achieve a physiologically compatible pH.
  • the pH of the composition can be for example between about 4 or about 5 and about 8.0 or about 4.5 and about 7.5 or about 5.0 to about 7.5.
  • the pH of the composition is between 5.5 and 7.5.
  • the present disclosure provides methods of producing a composition.
  • the method comprises combining the antigen-binding protein, conjugate, fusion protein, nucleic acid, vector, host cell, or a combination thereof, with a pharmaceutically acceptable carrier, diluent, or excipient.
  • the antigen-binding proteins of the present disclosure are useful for detecting or monitoring tumors or tumor growth.
  • the antigen-binding proteins are useful in methods or assays for detecting or monitoring tumors or tumor growth.
  • 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), fluoroimmunoassay (FIA), chemiluminescent immunoassay (CLIA), counting immunoassay (CIA), and immunoblotting (e.g., Western blot), wherein the antigen-binding proteins of the present disclosure are used to detect the presence of CLDN6 protein in a sample.
  • IHC immunohistochemistry
  • IF immunofluorescence
  • SPR surface plasmon resonance
  • RIA radioimmuno
  • the method or assay is immunohistochemistry (IHC).
  • the sample may be newly excised or extracted sample from a subject, a fresh sample, a frozen sample, flash-frozen sample, a formalin-fixed sample, a paraffin-embedded sample, or a lysate.
  • the sample may be bodily fluid from a subject, such as, blood, plasma, saliva, secretion or excrement, or a tissue from a subject.
  • the tumors may be benign or cancerous.
  • the antigen-binding proteins of the present disclosure may be used in conjunction with a therapeutic agent to treat benign or cancerous tumors, wherein the antigen-binding proteins of the present disclosure are used to monitor tumors or tumor growth.
  • the method comprises administering to the subject the pharmaceutical composition of the present disclosure or any cancer therapy known in the art in an amount effective for treating the cancer in the subject.
  • the cancer of the methods disclosed herein can be any cancer, e.g., any malignant growth or tumor caused by abnormal and uncontrolled cell division that may spread to other parts of the body through the lymphatic system or the blood stream.
  • the cancer in some aspects is one selected from the group consisting of acute lymphocytic cancer, acute myeloid leukemia, alveolar rhabdomyosarcoma, bone cancer, brain cancer, breast cancer, cancer of the anus, anal canal, or anorectum, cancer of the eye, cancer of the intrahepatic bile duct, cancer of the joints, cancer of the neck, gallbladder, or pleura, cancer of the nose, nasal cavity, or middle ear, cancer of the oral cavity, cancer of the vulva, chronic lymphocytic leukemia, chronic myeloid cancer, colon cancer, esophageal cancer, cervical cancer, gastrointestinal carcinoid tumor, Hodgkin lymphoma, hypopharynx cancer, kidney cancer, larynx
  • the cancer is selected from the group consisting of: head and neck, ovarian, cervical, bladder and oesophageal cancers, pancreatic, gastrointestinal cancer, gastric, breast, endometrial and colorectal cancers, hepatocellular carcinoma, glioblastoma, bladder, lung cancer, e.g., nonsmall cell lung cancer (NSCLC), bronchioloalveolar carcinoma.
  • the cancer is ovarian cancer, melanoma, bladder cancer, lung cancer, liver cancer, endometrial cancer.
  • the cancer is any cancer characterized by moderate to high expression of CLDN6. See, e.g., FIG. 4.
  • the cancer is acute myeloid leukemia, large B-cell lymphoma, stomach cancer, prostate cancer, melanoma, colon cancer, rectal cancer, bladder cancer, cervical cancer, liver cancer, breast cancer, kidney clear cell carcinoma, head and neck cancer, sarcoma, kidney chromophobe cancer, lower grade glioma, adrenocortical cancer, glioblastoma, kidney papillary cell carcinoma, lung squamous cell carcinoma, thyroid cancer, lung adenocarcinoma, pancreatic cancer, endometroid cancer, uterine carcinsarcoma, or ovarian cancer.
  • the cancer is selected from ovarian cancer, endometrioid cancer, uterine cancer, lung cancer, gastric cancer, breast cancer Head and Neck Squamous Cell Carcinoma (HNSCC) cancer, cervical cancer, and bladder.
  • HNSCC Head and Neck Squamous Cell Carcinoma
  • the term “treat,” as well as words related thereto, do not necessarily imply 100% or complete treatment. Rather, there are varying degrees of treatment of which one of ordinary skill in the art recognizes as having a potential benefit or therapeutic effect.
  • the methods of treating cancer of the present disclosure can provide any amount or any level of treatment.
  • the treatment provided by the method of the present disclosure can include treatment of one or more conditions or symptoms or signs of the cancer being treated. Also, the treatment provided by the methods of the present disclosure can encompass slowing the progression of the cancer.
  • the methods can treat cancer by virtue of enhancing the T cell activity or an immune response against the cancer, reducing tumor or cancer growth, reducing metastasis of tumor cells, increasing cell death of tumor or cancer cells, and the like.
  • the methods treat by way of delaying the onset or recurrence of the cancer by at least 1 day, 2 days, 4 days, 6 days, 8 days, 10 days, 15 days, 30 days, two months, 3 months, 4 months, 6 months, 1 year, 2 years, 3 years, 4 years, or more.
  • the methods treat by way increasing the survival of the subject.
  • the antigen binding proteins of the present disclosure also may be used to detect or determine the quantity of Claudin-6 (CLDN6) in a sample, diagnose a Claudin-6 (CLDN6)-positive cancer in a subject, monitor a course of a Claudin-6 (CLDN6)-positive cancer in a subject, type 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 having a Claudin-6 (CLDN6)-positive cancer. Therefore, the present disclosure provides methods of detecting or quantitating Claudin-6 (CLDN6) in a sample.
  • the method comprises contacting the sample with an antigen-binding protein, a conjugate, or a fusion protein, as described herein, and assaying for an immunocomplex comprising the antigen-binding protein, conjugate or fusion protein bound to CLDN6.
  • the present disclosure also provides methods of monitoring, typing or diagnosing a Claudin-6 (CLDN6)-positive cancer in a subject.
  • the method comprises contacting a biological sample comprising cells or tissue obtained from the subject with an antigen-binding protein, a conjugate, or a fusion protein, as described herein, and assaying for an immunocomplex comprising the antigen-binding protein, conjugate or fusion protein bound to CLDN6.
  • the present disclosure also provides methods of determining whether a cancer is treatable by a cancer therapy targeting CLDN6-positive cancer in a subject or determining the prognosis of a subject having a Claudin-6 (CLDN6)-positive cancer in a subject.
  • the method comprises contacting a biological sample comprising cells or tissue obtained from the subject with an antigen-binding protein, a conjugate, or a fusion protein, as described herein, and assaying for an immunocomplex comprising the antigen-binding protein, conjugate or fusion protein bound to CLDN6.
  • the present disclosure provides also assays to detect or determine the quantity of Claudin-6 (CLDN6) in a sample, diagnose a Claudin-6 (CLDN6)-positive cancer in a subject, monitor a course of a Claudin-6 (CLDN6)-positive cancer in a subject, type 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 having a Claudin-6 (CLDN6)-positive cancer
  • the assay comprises contacting a sample with a first antigen-binding protein which is specific for the CLDN6 to form a first-antigen-binding protein-CLDN6 immunocomplex, contacting the immunocomplex so formed with a second antigen-binding protein labeled with a detectable label to form a second immunocomplex comprising first antigen-binding protein-CLDN6- second antigen-binding protein and detecting the second immunocomplex so formed indicative of
  • the present invention provides, in part, methods, systems, and code for accurately classifying whether a biological sample comprises CLDN6 and/or whether the levels of CLDN6 are modulated (e.g., upregulated or downregulated), thereby indicative of the state of a disorder of interest, such as cancer.
  • 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 a statistical algorithm and/or empirical data (e.g., the presence, absence, or level of CLDN6).
  • An exemplary method for detecting the level of CLDN6, and thus useful for classifying whether a sample is associated with a cancer or a clinical subtype thereof or different stages of a 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 is detected in the biological sample.
  • an antigen-binding protein of the present invention capable of detecting CLDN6 such that the level of CLDN6 is detected in the biological sample.
  • at least one antigen-binding protein is used, wherein two, three, four, five, six, seven, eight, nine, ten, or more such antibodies or antibody fragments can be used in combination (e.g., in sandwich ELISAs) or in serial.
  • the statistical algorithm is a single learning statistical classifier system.
  • a single learning statistical classifier system can be used to classify a sample as a cancer sample based upon a prediction or probability value and the presence or level of CLDN6.
  • the use of a single learning statistical classifier system typically classifies the 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%,
  • learning statistical classifier systems include a machine learning algorithmic technique capable of adapting to complex data sets (e.g., panel of markers of interest) and making decisions based upon such data sets.
  • a single learning statistical classifier system such as a classification tree (e.g., random forest) is used.
  • a combination of 2, 3, 4, 5, 6, 7, 8, 9, 10, or more learning statistical classifier systems are used, preferably in tandem.
  • learning statistical classifier systems include, but are not limited to, those using inductive learning (e.g., decision/classification trees such as random forests, classification and regression trees (C&RT), boosted trees, etc.), Probably Approximately Correct (PAC) learning, connect!
  • inductive learning e.g., decision/classification trees such as random forests, classification and regression trees (C&RT), boosted trees, etc.
  • PAC Probably Approximately Correct
  • onist learning e.g., neural networks (NN), artificial neural networks (ANN), neuro fuzzy networks (NFN), 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 such as naive learning, adaptive dynamic learning, and temporal difference learning, passive learning in an unknown environment, active learning in an unknown environment, learning action-value functions, applications of reinforcement learning, etc.
  • genetic algorithms and evolutionary programming e.g., neural networks (NN), artificial neural networks (ANN), neuro fuzzy networks (NFN), 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 such as naive learning, adaptive dynamic learning, and temporal difference learning, passive learning in an unknown environment, active learning in an unknown environment, learning action-value functions, applications of reinforcement
  • the method of the present invention further comprises sending the sample classification results to a clinician (a non-specialist, e.g., primary care physician; and/or a specialist, e.g., a histopathologist or an oncologist).
  • a clinician e.g., a non-specialist, e.g., primary care physician; and/or a specialist, e.g., a histopathologist or an oncologist.
  • the method of the present disclosure further provides a diagnosis in the form of a probability that the individual has a cancer.
  • the individual can have about a 0%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or greater probability of having the cancer.
  • the method of the present invention further provides a prognosis of the cancer in the individual.
  • the method of classifying a sample as a cancer sample may be further based on the symptoms (e.g., clinical factors) of the individual from which the sample is obtained.
  • the symptoms or group of symptoms can be, for example, lymphocyte count, white cell count, erythrocyte sedimentation rate, diarrhea, abdominal pain, bloating, pelvic pain, lower back pain, cramping, fever, anemia, weight loss, anxiety, depression, and combinations thereof.
  • the method of classifying a sample as a cancer sample may be further based on genetic mutations and/or predisposition to cancer, irrespective of the symptoms.
  • the diagnosis of an individual as having a cancer is followed by administering to the individual a therapeutically effective amount of a cancer therapy (e.g., chemotherapeutic agents).
  • a cancer therapy e.g., chemotherapeutic agents
  • An exemplary method for detecting the presence or absence of CLDN6 comprises using an antigen-binding protein of the present disclosure, capable of binding to CLDN6, preferably an antigen-binding protein with a detectable label.
  • An antigen-binding protein can be an antibody.
  • An antibody can be polyclonal, or more preferably, monoclonal. Such agents can be labeled.
  • the term “labeled”, with regard to the antibody, is intended to encompass direct labeling of the probe or antibody by coupling (i.e., physically linking) a detectable substance to the probe or antibody, as well as indirect labeling of the probe or antibody by reactivity with another reagent that is directly labeled.
  • Examples of indirect labeling include detection of a primary antibody using a fluorescently labeled secondary antibody.
  • biological sample is intended to include tissues, cells, and biological fluids isolated from a subject, such as serum, 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 a CLDN6 include enzyme linked immunosorbent assays (ELISAs), immunoprecipitations, immunohistochemistry (IHC), flow cytometry and related techniques, and immunofluorescence.
  • the methods further involve obtaining a control biological sample (e.g., biological sample from a subject who does not have a cancer), a biological sample from the subject during remission or before developing a cancer, or a biological sample from the subject during treatment for developing a cancer.
  • a control biological sample e.g., biological sample from a subject who does not have a cancer
  • the methods comprise contacting the control sample with a compound or agent capable of detecting CLDN6 such that the presence and/or the level of CLDN6 is detected in the biological sample, and comparing the presence or the level of CLDN6 in the control sample with the presence or the level of CLDN6 in the test sample.
  • a biological sample is a serum, blood, saliva, tissue, tumor microenvironment, peritumoral, or intratumoral, isolated by conventional means from a subject.
  • the antigen-binding proteins can be associated with a component or device for the use of the antibodies in an ELISA or RIA.
  • Non-limiting examples include antigen-binding proteins immobilized on solid surfaces for use in these assays (e.g., linked and/or conjugated to a detectable label based on light or radiation emission as described above).
  • the antigen-binding proteins are associated with a device or strip for detection of CLDN6 by use of an immunochromatographic or immunochemical assay, such as in a “sandwich” or competitive assay, immunohistochemistry, immunofluorescence microscopy, and the like. Additional examples of such devices or strips are those designed for home testing or rapid point of care testing.
  • an unlabeled antibody of the invention may be applied to a “capture” CLDN6 in a biological sample and the captured (or immobilized) CLDN6 may be bound to a labeled form of an antigen-binding protein of the present disclosure for detection.
  • Other embodiments of immunoassays are well-known the skilled artisan, including assays based on, for example, immunodiffusion, immunoelectrophoresis, immunohistopathology, immunohistochemistry, and histopathology.
  • the compositions and methods of the present disclosure can be used to determine a grade of a cancer, based on the level of CLDN6 determined as described herein.
  • a cancer’s grade describes how abnormal the cancer cells and tissue look under a microscope when compared to healthy cells. Cancer cells that look and organize most like healthy cells and tissue are low grade tumors. Doctors describe these cancers as being well differentiated. Lower grade cancers are typically less aggressive and have a better prognosis. The more abnormal the cells look and organize themselves, the higher the cancer’s grade. Cancer cells with a high grades tend to be more aggressive. They are called poorly differentiated or undifferentiated. Some cancers have their own system for grading tumors. Many others use a standard 1-4 grading scale. [00180] Grade 1 : Tumor cells and tissue looks most like healthy cells and tissue. These are called well-differentiated tumors and are considered low grade.
  • Grade 2 The cells and tissue are somewhat abnormal and are called moderately differentiated. These are intermediate grade tumors.
  • Grade 3 Cancer cells and tissue look very abnormal. These cancers are considered poorly differentiated, since they no longer have an architectural structure or pattern.
  • Grade 3 tumors are considered high grade.
  • Grade 4 These undifferentiated cancers have the most abnormal looking cells. These are the highest grade and typically grow and spread faster than lower grade tumors.
  • low grade cancer refers to Grade I cancer
  • high grade cancer refers to cancer of Grades 2-4.
  • compositions and methods of the present disclosure can be used to determine a grade of a cancer, based on the level of CLDN6 determined as described herein.
  • a cancer’s stage explains how large the primary tumor is and how far the cancer has spread in the patient’s body.
  • staging systems Many of these have been created for specific kinds of cancers. Others can be used to describe several types of cancer.
  • Stage 0 is for abnormal cells that haven’t spread and are not considered cancer, though they could become cancerous in the future. This stage is also called “in-situ.”
  • Stage I through Stage III are for cancers that haven’t spread beyond the primary tumor site or have only spread to nearby tissue. The higher the stage number, the larger the tumor and the more it has spread.
  • Stage IV cancer has spread to distant areas of the body.
  • cancer at the early /low stage refers to cancer at Stage I; and cancer at the late/high/advanced stage includes cancer at Stage II to Stage IV.
  • the compositions and methods of the present disclosure can be used to determine the tumor burden of a subject, based on the level of CLDN6 determined as described herein.
  • Tumor burden (or tumor load) is defined as the total amount of tumor (cells/mass) distributed in the patients’ body, including bone marrow.
  • RECIST Response Evaluation Criteria in Solid Tumors
  • tumor burden is considered the sum of the longest diameters of all measurable lesions.
  • CT computed tomography
  • MR magnetic resonance
  • RECIST classification describes lesions’ size and distinguishes 4 types of treatment response - stable disease (SD), partial response (PR), complete response (CR) or progressive disease (PD).
  • prognosis includes a prediction of the probable course and outcome of cancer or the likelihood of recovery from the disease.
  • use of statistical algorithms provides a prognosis of cancer in an individual.
  • the prognosis can be surgery, development of a clinical subtype of cancer (e.g., solid tumors, such as lung cancer, melanoma, and renal cell carcinoma), development of one or more clinical factors, development of intestinal cancer, or recovery from the disease.
  • a clinical subtype of cancer e.g., solid tumors, such as lung cancer, melanoma, and renal cell carcinoma
  • the assays described herein can be utilized 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 inhibition therapy, or other drug candidate) to treat a cancer.
  • an agent e.g., an agonist, antagonist, peptidomimetic, polypeptide, peptide, nucleic acid, small molecule, immunotherapy, immune checkpoint inhibition therapy, or other drug candidate
  • agent e.g., an agonist, antagonist, peptidomimetic, polypeptide, peptide, nucleic acid, small molecule, immunotherapy, immune checkpoint inhibition therapy, or other drug candidate
  • agent e.g., an agonist, antagonist, peptidomimetic, polypeptide, peptide, nucleic acid, small molecule, immunotherapy, immune checkpoint inhibition therapy, or other drug candidate
  • the present disclosure provides methods for determining whether a subject can be effectively treated with one
  • the methods described herein may be performed, for example, by utilizing pre-packaged diagnostic kits comprising at least one antibody reagent described herein, which may be conveniently used, e.g., in clinical settings to diagnose patients exhibiting symptoms or family history of a cancer.
  • compositions and methods described herein for association and/or stratification analyses include uses of the compositions and methods described herein for association and/or stratification analyses in which CLDN6 in biological samples from individuals with a cancer, are analyzed and the information is compared to that of controls (e.g., individuals who do not have the cancer; controls may be also referred to as “healthy” or “normal” individuals or at early timepoints in a given time lapse study) who are preferably of similar age and race.
  • controls e.g., individuals who do not have the cancer; controls may be also referred to as “healthy” or “normal” individuals or at early timepoints in a given time lapse study
  • the appropriate selection of patients and controls may be important to the success of association and/or stratification studies. Therefore, a pool of individuals with well-characterized phenotypes is extremely desirable. Criteria for cancer diagnosis, cancer predisposition screening, predicting clinical outcomes, cancer prognosis, determining drug responsiveness (pharmacogenomics), drug toxicity screening, etc. are described
  • Different study designs may be used for genetic association and/or stratification studies (Modem Epidemiology, Lippincott Williams & Wilkins (1998), 609- 622). Observational studies are most frequently carried out in which the response of the patients is not interfered with.
  • the first type of observational study identifies a sample of persons in whom the suspected cause of the disease is present and another sample of persons in whom the suspected cause is absent, and then the frequency of development of disease in the two samples is compared. These sampled populations are called cohorts, and the study is a prospective study.
  • the other type of observational study is case-control or a retrospective study.
  • case-control studies samples are collected from individuals with the phenotype of interest (cases) such as certain manifestations of a disease, and from individuals without the phenotype (controls) in a population (target population) that conclusions are to be drawn from. Then the possible causes of the disease are investigated retrospectively. As the time and costs of collecting samples in case-control studies are considerably less than those for prospective studies, case-control studies are the more commonly used study design in genetic association studies, at least during the exploration and discovery stage.
  • An important decision in the performance of genetic association tests is the determination of the significance level at which significant association can be declared when the p-value of the tests reaches that level.
  • an unadjusted p-value ⁇ 0.2 (a significance level on the lenient side), for example, may be used for generating hypotheses for significant association of a CLDN6 level with certain phenotypic characteristics of a cancer. It is preferred that a p-value ⁇ 0.05 (a significance level traditionally used in the art) is achieved in order for the level to be considered to have an association with a cancer.
  • a classification/prediction scheme can be set up to predict the category (for instance, disease or no-disease) that an individual will be in depending on his phenotype and/or genotype and other non-genetic risk factors.
  • Logistic regression for discrete trait and linear regression for continuous trait are standard techniques for such tasks (Applied Regression Analysis, Draper and Smith, Wiley (1998)).
  • other techniques can also be used for setting up classification.
  • Such techniques include, but are not limited to, MART, CART, neural network, and discriminant analyses that are suitable for use in comparing the performance of different methods (The Elements of Statistical Learning, Hastie, Tibshirani & Friedman, Springer (2002)).
  • the therapeutic agents of the present invention can be used alone or can be administered in combination therapy with, e.g., chemotherapeutic agents, hormones, antiangiogens, radiolabelled, compounds, or with surgery, cryotherapy, immunotherapy, cancer vaccine, immune cell engineering (e.g., CAR-T), and/or radiotherapy.
  • chemotherapeutic agents e.g., hormones, antiangiogens, radiolabelled, compounds, or with surgery, cryotherapy, immunotherapy, cancer vaccine, immune cell engineering (e.g., CAR-T), and/or radiotherapy.
  • the preceding treatment methods can be administered in conjunction with other forms of conventional therapy (e.g., standard-of-care treatments for cancer well-known to the skilled artisan), either consecutively with, pre- or post-conventional therapy.
  • agents of the present invention can be administered with a therapeutically effective dose of chemotherapeutic agent.
  • agents of the present invention are administered in conjunction with chemotherapy to enhance the activity and efficacy of the chemotherapeutic agent.
  • the Physicians’ Desk Reference discloses dosages of chemotherapeutic agents that have been used in the treatment of various cancers. The dosing regimen and dosages of these aforementioned chemotherapeutic drugs that are therapeutically effective will depend on the particular cancer being treated, the extent of the disease and other factors familiar to the physician of skill in the art, and can be determined by the physician.
  • Immunotherapy is a targeted therapy that may comprise, for example, the use of cancer vaccines and/or sensitized antigen presenting cells.
  • an oncolytic virus is a virus that is able to infect and lyse cancer cells, while leaving normal cells unharmed, making them potentially useful in cancer therapy. Replication of oncolytic viruses both facilitates tumor cell destruction and also produces dose amplification at the tumor site. They may also act as vectors for anticancer genes, allowing them to be specifically delivered to the tumor site.
  • the immunotherapy can involve passive immunity for short-term protection of a host, achieved by the administration of pre-formed antibody directed against a cancer antigen or disease antigen (e.g, administration of a monoclonal antibody, optionally linked to a chemotherapeutic agent or toxin, to a tumor antigen).
  • a cancer antigen or disease antigen e.g, administration of a monoclonal antibody, optionally linked to a chemotherapeutic agent or toxin, to a tumor antigen.
  • anti-VEGF is known to be effective in treating renal cell carcinoma.
  • Immunotherapy can also focus on using the cytotoxic lymphocyte-recognized epitopes of cancer cell lines.
  • Immunotherapy also encompasses immune checkpoint modulators.
  • Immune checkpoints are a group of molecules on the cell surface of CD4+ and/or CD8+ T cells that fine-tune immune responses by down-modulating or inhibiting an anti-tumor immune response.
  • Immune checkpoint proteins are well-known in the art and include, without limitation, CTLA-4, PD-1, VISTA, B7-H2, B7-H3, PD-L1, B7-H4, B7-H6, 2B4, ICOS, HVEM, PD-L2, CD 160, gp49B, PIR-B, KIR family receptors, TIM-1, TIM-3, TIM-4, LAG-3, BTLA, SIRPalpha (CD47), CD48, 2B4 (CD244), B7.1, B7.2, ILT-2, ILT-4, TIGIT, HHLA2, TMIDG2, KIR3DL3, and A2aR (see, for example, WO 2012/177624).
  • Inhibition of one or more immune checkpoint inhibitors can block or otherwise neutralize inhibitory signaling to thereby upregulate an immune response in order to more efficaciously treat cancer.
  • the cancer vaccine is administered in combination with one or more inhibitors of immune checkpoints (immune checkpoint inhibition therapy), such as PD1, PD-L1, and/or CD47 inhibitors.
  • Adoptive cell-based immunotherapies can be combined with the therapies of the present invention.
  • Well-known adoptive cell-based immunotherapeutic modalities including, without limitation, 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 CAR T cell therapy, autologous immune enhancement therapy (AIET), cancer vaccines, and/or antigen presenting cells.
  • Such cellbased immunotherapies can be further modified to express one or more gene products to further modulate immune responses, such as expressing cytokines like GM-CSF, and/or to express tumor-associated antigen (TAA) antigens, such as Mage-1, gp-100, and the like.
  • TAA tumor-associated antigen
  • chimeric antigen receptor refers to engineered T cell receptors (TCR) having a desired antigen specificity.
  • T lymphocytes recognize specific antigens through interaction of the T cell receptor (TCR) with short peptides presented by major histocompatibility complex (MHC) class I or II molecules.
  • MHC major histocompatibility complex
  • naive T cells are dependent on professional antigen-presenting cells (APCs) that provide additional co-stimulatory signals. TCR activation in the absence of costimulation can result in unresponsiveness and clonal anergy.
  • APCs professional antigen-presenting cells
  • CARs have been constructed that consist of binding domains derived from natural ligands or antibodies specific for cell-surface components of the TCR-associated CD3 complex. Upon antigen binding, such chimeric antigen receptors link to endogenous signaling pathways in the effector cell and generate activating signals similar to those initiated by the TCR complex. Since the first reports on chimeric antigen receptors, this concept has steadily been refined and the molecular design of chimeric receptors has been optimized and routinely use any number of well-known binding domains, such as scFV and another protein binding fragments described herein.
  • immunotherapy comprises non-cell-based immunotherapies.
  • compositions comprising antigens with or without vaccine-enhancing adjuvants are used.
  • Such compositions exist in many well- known forms, such as peptide compositions, oncolytic viruses, recombinant antigen comprising fusion proteins, and the like.
  • immunomodulatory cytokines such as interferons, G-CSF, imiquimod, TNF alpha, and the like, as well as modulators thereof (e.g, blocking antibodies or more potent or longer lasting forms) are used.
  • immunomodulatory interleukins such as IL-2, IL-6, IL-7, IL- 12, IL-17, IL-23, and the like, as well as modulators thereof (e.g, blocking antibodies or more potent or longer lasting forms) are used.
  • immunomodulatory chemokines such as CCL3, CCL26, and CXCL7, and the like, as well as modulators thereof (e.g, blocking antibodies or more potent or longer lasting forms) are used.
  • immunomodulatory molecules targeting immunosuppression such as STAT3 signaling modulators, NFkappaB signaling modulators, and immune checkpoint modulators, are used.
  • immunomodulatory drugs such as immunocytostatic drugs, glucocorticoids, cytostatics, immunophilins and modulators thereof (e.g, rapamycin, a calcineurin inhibitor, tacrolimus, ciclosporin (cyclosporin), pimecrolimus, abetimus, gusperimus, ridaforolimus, everolimus, temsirolimus, zotarolimus, etc.), hydrocortisone (cortisol), cortisone acetate, prednisone, prednisolone, methylprednisolone, dexamethasone, betamethasone, triamcinolone, beclometasone, fludrocortisone acetate, deoxycorticosterone acetate (doca) aldosterone, a non-glucocorticoid steroid, a pyrimidine synthesis inhibitor, leflunomide, teriflunomide,
  • immunomodulatory antibodies or protein are used.
  • antibodies that bind to CD40, Toll-like receptor (TLR), 0X40, GITR, CD27, or to 4-1BB T-cell bispecific antibodies, an anti-IL-2 receptor antibody, an anti-CD3 antibody, 0KT3 (muromonab), otelixizumab, teplizumab, visilizumab, an anti-CD4 antibody, clenoliximab, keliximab, zanolimumab, an anti-CDl l a antibody, efalizumab, an anti-CD18 antibody, erlizumab, rovelizumab, an anti-CD20 antibody, afutuzumab, ocrelizumab, ofatumumab, pascolizumab, rituximab, an anti-CD23 antibody, lumiliximab, an anti-CD40 antibody, teneliximab,
  • Nutritional supplements that enhance immune responses such as vitamin A, vitamin E, vitamin C, and the like, are well-known in the art (see, for example, U.S. Pat. Nos. 4,981,844 and 5,230,902 and PCT Publ. No. WO 2004/004483) can be used in the methods described herein.
  • various agents or a combination thereof can be used to treat a cancer.
  • Chemotherapy includes the administration of a chemotherapeutic agent.
  • a chemotherapeutic agent may be, but is not limited to, those selected from among the following groups of compounds: platinum compounds, cytotoxic antibiotics, antimetabolites, anti-mitotic agents, alkylating agents, arsenic compounds, DNA topoisomerase inhibitors, taxanes, nucleoside analogues, plant alkaloids, and toxins; and synthetic derivatives thereof.
  • Exemplary compounds include, but are not limited to, alkylating agents: cisplatin, treosulfan, and trofosfamide; plant alkaloids: vinblastine, paclitaxel, docetaxol; DNA topoisomerase inhibitors: teniposide, crisnatol, and mitomycin; anti-folates: 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.
  • alkylating agents cisplatin, treosulfan, and trofosfamide
  • compositions comprising one or more chemotherapeutic agents (e.g, FLAG, CHOP) may also be used.
  • FLAG comprises fludarabine, cytosine arabinoside (Ara-C) and G-CSF.
  • CHOP comprises cyclophosphamide, vincristine, doxorubicin, and prednisone.
  • PARP e.g., PARP-1 and/or PARP-2
  • 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. Pat. Re.
  • the mechanism of action is generally related to the ability of PARP inhibitors to bind PARP and decrease its activity.
  • PARP catalyzes the conversion of .beta. -nicotinamide adenine dinucleotide (NAD+) into nicotinamide and poly-ADP-ribose (PAR).
  • NAD+ nicotinamide adenine dinucleotide
  • PARP poly-ADP-ribose
  • Both poly (ADP-ribose) and PARP have been linked to regulation of transcription, cell proliferation, genomic stability, and carcinogenesis (Bouchard V. J. et.al. Experimental Hematology, Volume 31, Number 6, June 2003, pp. 446-454(9); Herceg Z.; Wang Z.-Q.
  • PARP1 Poly(ADP-ribose) polymerase 1
  • SSBs DNA singlestrand breaks
  • DSBs DNA double-strand breaks
  • chemotherapeutic agents are illustrative, and are not intended to be limiting.
  • radiation therapy is used.
  • the radiation used in radiation therapy can be ionizing radiation.
  • Radiation therapy can also be gamma rays, X-rays, or proton beams.
  • Examples of radiation therapy include, but are not limited to, external-beam radiation therapy, interstitial implantation of radioisotopes (1-125, palladium, iridium), radioisotopes such as strontium-89, thoracic radiation therapy, intraperitoneal P-32 radiation therapy, and/or total abdominal and pelvic radiation therapy.
  • the radiation therapy can be administered as external beam radiation or teletherapy wherein the radiation is directed from a remote source.
  • the radiation treatment can also be administered as internal therapy or brachytherapy wherein a radioactive source is placed inside the body close to cancer cells or a tumor mass.
  • photodynamic therapy comprising the administration of photosensitizers, such as hematoporphyrin and its derivatives, Vertoporfin (BPD-MA), phthalocyanine, photosensitizer Pc4, demethoxy-hypocrellin A; and 2BA-2-DMHA.
  • hormone therapy is used.
  • Hormonal therapeutic treatments can comprise, for example, hormonal agonists, hormonal antagonists (e.g., flutamide, bicalutamide, tamoxifen, raloxifene, leuprolide acetate (LUPRON), LH-RH antagonists), inhibitors of hormone biosynthesis and processing, and steroids (e.g., dexamethasone, retinoids, deltoids, betamethasone, cortisol, cortisone, prednisone, dehydrotestosterone, glucocorticoids, mineralocorticoids, estrogen, 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).
  • hormonal antagonists e.g., flutamide, bicalu
  • photodynamic therapy also called PDT, photoradiation therapy, phototherapy, or photochemotherapy
  • PDT photoradiation therapy
  • phototherapy phototherapy
  • photochemotherapy is used for the treatment of some types of cancer. It is based on the discovery that certain chemicals known as photosensitizing agents can kill one-celled organisms when the organisms are exposed to a particular type of light.
  • laser therapy is used to harness high-intensity light to destroy cancer cells. This technique is often used to relieve symptoms of cancer such as bleeding or obstruction, especially when the cancer cannot be cured by other treatments. It may also be used to treat cancer by shrinking or destroying tumors.
  • 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 from the order Carnivora, including Felines (cats) and Canines (dogs), mammals from the order Artiodactyla, including Bovines (cows) and Swines (pigs) or of the order Perssodactyla, including Equines (horses).
  • the mammals are of the order Primates, Ceboids, or Simoids (monkeys) or of the order Anthropoids (humans and apes).
  • the mammal is a human.
  • the antigen-binding proteins of the present disclosure are provided in a kit.
  • the kit comprises the antigen-binding protein(s) as a unit assay.
  • unit assay refers to a discrete amount for a single assay use.
  • each unit assay may comprise multiple reagents, each separated from each as required in the assay.
  • kits comprising an antigen-binding protein of the present disclosure optionally provided in unit assays.
  • the kit comprises multiple unit assays, e.g., a supply of 5, 10, 25, 50, 100, 500 or 1,000 unit assays, optionally, each of which is individually packaged or otherwise separated from other unit assays .
  • the kit comprises multiple unit assays, wherein each reagent is packaged in a single vessel or container for multiple assays.
  • the components of the kit/unit dose are packaged with instructions for use.
  • the kit comprises one or more devices for use, e.g., a needle and syringe, and the like.
  • the antigen-binding protein of the present disclosure, a pharmaceutically acceptable salt thereof, a conjugate comprising the antigenbinding protein, or a multimer or dimer comprising the antigen-binding protein is prepackaged in a ready to use form, e.g., all reagents in solution avoiding a need to resuspend or add additional components.
  • the kit further comprises therapeutic or additional diagnostic agents or pharmaceutically acceptable carriers (e.g., solvents, buffers, diluents, etc.), including any of those described herein.
  • the kit comprises an antigen-binding protein of the present disclosure, along with an agent, e.g., a therapeutic agent, used in chemotherapy or radiation therapy.
  • the kit comprises an antigen-binding protein of the present disclosure, along with a second antigen-binding protein which is labeled with a detectable marker.
  • the kit comprises an antigen-binding protein of the present disclosure, along with a second antigen-binding protein which is labeled with an epitope or small molecule.
  • the kit comprises an antigen-binding protein of the present disclosure, along with a second antigen-binding protein which is labeled with an epitope or small molecule, and a label or detection moiety that binds to the epitope or small molecule.
  • the kit comprises an antigen-binding protein of the present disclosure, along with a second antigen-binding protein which is labeled with an epitope or small molecule, and a label or detection moiety linked to a binding partner for the epitope or small molecule.
  • the secondary antigen-binding protein binds the antigen-binding protein of the present disclosure.
  • the antigen-binding protein binds to a human Claudin-6 (CLDN6) protein (SEQ ID NOs: 1-2) which does not bind to any of Claudin3 (CLDN3), Claudin4 (CLDN4), and Claudin9 (CLDN9).
  • CLDN6 human Claudin-6
  • CLDN9 Claudin9
  • the antigen-binding protein binds to an epitope within the amino acid sequence of CCTCPSGGSQGPSHYMARYSTSAPAISRGPSEYPTKNYV (SEQ ID NO: 3), or binds to the amino acid sequence of MASAGMQILGVVLTLLGWVNGLVSCALPMWKVTAFIGNSIVVAQVVWEGLWMS CVVQSTGQMQCKVYDSLLALPQDLQAARALCVIALLVALFGLLVYLAGAKCTTC VEEKDSKARLVLTSGIVFVISGVLTLIPVCWTAHAIIRDFYNPLVAEAQKRELGASL YLGWAASGLLLLGGGLLCCTCPSGGSQGPSHYMARYSTSAPAISRGPSEYPTKNYV (SEQ ID NO: 1) or MASAGMQILGVVLTLLGWVNGLVSCALPMWKVTAFIGNSIVVAQVVWEGLWMS CVVQSTGQMQCKVYDSLLALPQDLQAARALCVI
  • an antigen-binding protein does not bind to any one or more of Claudin3 (CLDN3), Claudin4 (CLDN4), and Claudin9 (CLDN9).
  • an antigen-binding protein comprises: (a) CDRs 1-3 derived from a heavy chain variable region comprising the amino acid sequence:
  • EIQLQQSGAELVRPGALVRLSCKPSGFNIKDYYIHWVKERPEQGLEWIGWIDPDNG DTLYDSKFQGKASLTADTSSNTAYLQLTSLTSEDTAVYYCATYRYSFAYWGQGTL VTVSA (SEQ ID NO: 16) or a variant sequence thereof which differs by only one or two amino acids or which 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: DIQMNQSPSSLSASLGDTITITCHASQNINVWLNWYQQKPGNIPKLLIFKASNLYTG VPSRFSGGGSGTGFTLTISSLQPEDIATYYCQQGQTYPLTFGGGTKLEIK (SEQ ID NO: 17), or a variant sequence thereof which differs by only one or two amino acids or which has at least or about 80% sequence identity.
  • the antigenbinding protein binds to: (a) human Claudin-6 (CLDN6), wherein said 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) human Claudin-6 (CLDN6), wherein said 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) human Claudin-6 (CLDN6), wherein said antigen-binding protein binds to CLDN6 by binding at least to an epitope within CLDN6 having the amino acid sequence RYSTSAPAISRGPSE (SEQ ID NO: 100); and/or (d) human Claudin-6 (CLDN6), wherein said antigen-binding protein binds to:
  • 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).
  • an antigen-binding protein which specifically binds the cytoplasmic domain of CLDN6 comprises: (a) a heavy chain CDR1 comprising the amino acid sequence of: GFNIKDYY (SEQ ID NO: 18) or a variant sequence thereof which differs by only one or two amino acids or which has at least or about 70% sequence identity; (b) a heavy chain CDR2 comprising the amino acid sequence of: IDPDNGDT (SEQ ID NO: 19) or a variant sequence thereof which differs by only one or two amino acids or which has at least or about 70% sequence identity; (c) a heavy chain CDR3 comprising the amino acid sequence of: ATYRYSFAY (SEQ ID NO: 20) or a variant sequence thereof which differs by only one or two amino acids or which has at least or about 70% sequence identity; (d) a light chain CDR1 comprising the amino acid sequence of: QNINVW (SEQ ID NO: 25) or a variant sequence thereof which differs by only one or
  • the antigen-binding protein binds to: (a) human Claudin-6 (CLDN6), wherein said 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) human Claudin-6 (CLDN6), wherein said 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) human Claudin-6 (CLDN6), wherein said antigen-binding protein binds to CLDN6 by binding at least to an epitope within CLDN6 having the amino acid sequence RYSTSAPAISRGPSE (SEQ ID NO: 100); and/or (d) human Claudin-6 (CLDN6), wherein said antigen-binding protein binds to: (a
  • the antigenbinding 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).
  • an antigen-binding protein which specifically binds the cytoplasmic of CLDN6 comprises: (a) a heavy chain CDR1 comprising the amino acid sequence of: GFNIKDYY (SEQ ID NO: 18); (b) a heavy chain CDR2 comprising the amino acid sequence of: IDPDNGDT (SEQ ID NO: 19); (c) a heavy chain CDR3 comprising the amino acid sequence of: ATYRYSFAY (SEQ ID NO: 20); (d) a light chain CDR1 comprising the amino acid sequence of: QNINVW (SEQ ID NO: 25); (e) alight chain CDR2 comprising the amino acid sequence of: KAS (SEQ ID NO: 26); and (f) alight chain CDR3 comprising the amino acid sequence of: QQGQTYPLT (SEQ ID NO: 27); and binds to: (a) human Claudin-6 (CLDN6), wherein said antigen-binding protein binds to CLDN
  • the antigenbinding protein binds to one or more of the following peptides having the amino acid sequence of any of: CCTCPSGGSQGPSHYMARYSTSAPAISRGPSEYPTKNYV (SEQ ID NO: 3), RYSTSAPAISRGPSEYPTKN (SEQ ID NO: 10), RYSTSAPAISRGPSE (SEQ ID NO: 100), or APAISRGPSEYPTKN (SEQ ID NO: 101).
  • an antigen-binding protein comprisies: (a) CDRs 1-3 derived from a heavy chain variable region comprising the amino acid sequence: EVQLQQFGAELVKPGASVKISCRTSGYTFTDYNIDWVRQSHGKSLEWIGDINPNSE NTDYNQKFKGKATLTVDKSSSTAYLELRSLTSEDTAVYYCARSPYGNYVGYLMD YWGQGTSVTVSS (SEQ ID NO: 32) or a variant sequence thereof which differs by only one or two amino acids or which 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: DVVLTQTPLSLPVNIGDQASISCRSTKSLLNSDGFTYLDWYLQKPGQSPQVLIYLISN RFSGVPDRFSGNGSGTDFTLKISRVEAEDLGVYYCFQSNYIPLTFGAGTKLELK (SEQ ID NO: 32) or a variant sequence thereof which
  • the antigen-binding protein binds to: (a) human Claudin-6 (CLDN6), wherein said antigenbinding protein binds to CLDN6 by binding at least to an epitope within CLDN6 having the amino acid sequence CCTCPSGGSQGPSHYMARYSTSAPAISRGPSEYPTKNYV (SEQ ID NO: 3); (b) human Claudin-6 (CLDN6), wherein said 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) human Claudin-6 (CLDN6), wherein said antigen-binding protein binds to CLDN6 by binding at least to an epitope within CLDN6 having the amino acid sequence CPSGGSQGPSHYMARYSTS (SEQ ID NO: 96); (d) human Claudin-6 (CLDN6), wherein said antigen-binding protein binds to CLDN
  • the antigen-binding protein binds to one or more of the following peptides having the amino acid sequence of any of: 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), and/or SHYMARYSTSAPAIS (SEQ ID NO: 99).
  • CCTCPSGGSQGPSHYMARYSTSAPAISRGPSEYPTKNYV SEQ ID NO: 3
  • CPSGGSQGPSHYMARYSTSAPAIS SEQ ID NO: 95
  • CPSGGSQGPSHYMARYSTS SEQ ID NO: 96
  • SQGPSHYMARYSTSAPAIS SEQ ID NO: 97
  • SHYMAR
  • an antigen-binding protein which specifically binds the cytoplasmic domain of CLDN6 comprises: (a) a heavy chain CDR1 comprising the amino acid sequence of: GYTFTDYN (SEQ ID NO: 34) or a variant sequence thereof which differs by only one or two amino acids or which has at least or about 70% sequence identity; (b) a heavy chain CDR2 comprising the amino acid sequence of: INPNSENT (SEQ ID NO: 35) or a variant sequence thereof which differs by only one or two amino acids or which has at least or about 70% sequence identity; (c) a heavy chain CDR3 comprising the amino acid sequence of: ARSPYGNYVGYLMDY (SEQ ID NO: 36) or a variant sequence thereof which differs by only one or two amino acids or which has at least or about 70% sequence identity;
  • a light chain CDR1 comprising the amino acid sequence of: KSLLNSDGFTY (SEQ ID NO: 41) or a variant sequence thereof which differs by only one or two amino acids or which has at least or about 70% sequence identity
  • alight chain CDR2 comprising the amino acid sequence of: LIS (SEQ ID NO: 42) or a variant sequence thereof which differs by only one or two amino acids or which has at least or about 70% sequence identity
  • alight chain CDR3 comprising the amino acid sequence of: FQSNYIPLT (SEQ ID NO: 43) or a variant sequence thereof which differs by only one or two amino acids or which has at least or about 70% sequence identity
  • the antigen-binding protein binds to: (a) human Claudin-6 (CLDN6), wherein said 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);
  • human Claudin-6 (CLDN6), wherein said 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) human Claudin-6 (CLDN6), wherein said antigen-binding protein binds to CLDN6 by binding at least to an epitope within CLDN6 having the amino acid sequence CPSGGSQGPSHYMARYSTS (SEQ ID NO: 96); (d) human Claudin-6 (CLDN6), wherein said 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) human Claudin-6 (CLDN6), wherein said antigen-binding protein binds to CLDN6 by binding at least to an epitope within CLDN6 having the amino acid sequence SHYMAR (SEQ
  • the antigen-binding protein binds to one or more of the following peptides having the amino acid sequence of any of: 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), and/or SHYMARYSTSAPAIS (SEQ ID NO: 99).
  • CCTCPSGGSQGPSHYMARYSTSAPAISRGPSEYPTKNYV SEQ ID NO: 3
  • CPSGGSQGPSHYMARYSTSAPAIS SEQ ID NO: 95
  • CPSGGSQGPSHYMARYSTS SEQ ID NO: 96
  • SQGPSHYMARYSTSAPAIS SEQ ID NO: 97
  • SHYMAR
  • an antigen-binding protein which specifically binds the cytoplasmic domain of CLDN6 comprises: (a) a heavy chain CDR1 comprising the amino acid sequence of: GYTFTDYN (SEQ ID NO: 34); (b) a heavy chain CDR2 comprising the amino acid sequence of: INPNSENT (SEQ ID NO: 35);
  • a heavy chain CDR3 comprising the amino acid sequence of: ARSPYGNYVGYLMDY (SEQ ID NO: 36);
  • a light chain CDR1 comprising the amino acid sequence of: KSLLNSDGFTY (SEQ ID NO: 41);
  • a light chain CDR2 comprising the amino acid sequence of: LIS (SEQ ID NO: 42);
  • a light chain CDR3 comprising the amino acid sequence of: FQSNYIPLT (SEQ ID NO: 43).
  • the antigen-binding protein binds to: (a) human Claudin-6 (CLDN6), wherein said 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) human Claudin-6 (CLDN6), wherein said 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) human Claudin-6 (CLDN6), wherein said antigen-binding protein binds to CLDN6 by binding at least to an epitope within CLDN6 having the amino acid sequence CPSGGSQGPSHYMARYSTS (SEQ ID NO: 96); (d) human Claudin-6 (CLDN6), wherein said antigen-binding protein binds to CL
  • the antigen-binding protein binds to one or more of the following peptides having the amino acid sequence of any of: 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), and/or SHYMARYSTSAPAIS (SEQ ID NO: 99).
  • CCTCPSGGSQGPSHYMARYSTSAPAISRGPSEYPTKNYV SEQ ID NO: 3
  • CPSGGSQGPSHYMARYSTSAPAIS SEQ ID NO: 95
  • CPSGGSQGPSHYMARYSTS SEQ ID NO: 96
  • SQGPSHYMARYSTSAPAIS SEQ ID NO: 97
  • SHYMAR
  • 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.
  • the antigen-binding protein is a fusion protein, an antibody or antigen-binding antibody fragment.
  • the antibody is a monoclonal antibody, a fragment thereof, a human antibody, a humanized antibody, or a chimeric antibody.
  • the antigen-binding protein is an IgG.
  • the IgG can be selected from the group consisting of IgGl, IgG2, IgG3 and IgG4.
  • the antigen-binding protein is an antibody fragment selected from the group consisting of scFv, F(ab')2, Fab, Fab' and Fv.
  • an antigen-binding protein disclosed herein inhibits tumor growth in xenograft mice injected with human cancer cells.
  • an antigen-binding protein disclosed herein comprises aFc polypeptide comprising an afucosylated glycan.
  • an antigen-binding protein disclosed herein is conjugated with a detectable label.
  • the detectable label is at least one detectable label coupled to an antigen-binding protein or coexpressed with an antigen-binding protein.
  • the detectable label comprises a fluorophore, radioactive label, colorometric label, or the detectable label is the product of an enzymatic reaction.
  • the label or agent is conjugated to the antigen-binding protein via a cleavable linker such as, for example, VC-PAB.
  • the label or agent is conjugated at a specific site of the antigen-binding protein, for example, the specific site is an unpaired cysteine residue.
  • an antigen-binding protein disclosed herein is conjugated with a detectable label, wherein the antigen-binding protein is an antibody.
  • the conjugated antigen-binding protein is a conjugated fusion protein.
  • the conjugated antigen-binding protein is a conjugated antibody.
  • 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.
  • the conjugated antigen-binding protein is an IgG.
  • the conjugated IgG can be selected from the group consisting of IgGl, IgG2, IgG3 and IgG4.
  • the conjugated antigen-binding protein is an antibody fragment selected from the group consisting of scFv, F(ab')2, Fab, Fab' and Fv.
  • the conjugated antigen-binding protein is conjugated with an average number of units of the label or agent per antigen-binding protein in a range of 1 to 8, preferably wherein the average number of units of the label or agent conjugated per antigen-binding protein is in a range of 3-8.
  • the conjugate is a heterogeneous or homogeneous conjugate.
  • the conjugated antigen-binding protein comprises a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 16 and SEQ ID NO: 17 conjugated to horseradish peroxidase (HRP).
  • the conjugated antigen-binding protein comprises a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 32 and SEQ ID NO: 33 conjugated to horseradish peroxidase (HRP).
  • HRP horseradish peroxidase
  • a nucleic acid molecule comprising a nucleotide sequence encodes the antigen binding protein or the conjugated antigen binding protein.
  • the nucleic acid is a cDNA.
  • the nucleic acid molecule is inserted into a vector (e.g., expression vector). Additionally, the vector can comprise an internal ribosome entry site (IRES).
  • the vector comprising the nucleic acid encoding the antigen binding protein, or the conjugated thereof is transfected or transformed into a host cell.
  • the host cell is a bacterial or eukaryotic cell.
  • the eukaryotic cell is a mammalian cell e.g., Chinese hamster ovary (CHO) cell.
  • Certain embodiments of the invention provide a method of producing an antigen-binding protein, or a conjugate thereof, that binds to a Claudin-6 (CLDN6) protein, comprising (i) culturing the host cell in a cell culture medium, and (ii) harvesting the antigen-binding protein, or conjugate thereof, from the cell culture medium.
  • CLDN6 Claudin-6
  • Certain embodiments of the invention provide a method of producing a fusion protein comprising an antigen-binding protein that binds to a Claudin-6 (CLDN6) protein, comprising (i) culturing the host cell in a cell culture medium, and (ii) harvesting the fusion protein from the cell culture medium.
  • CLDN6 Claudin-6
  • Certain embodiments of the invention provide a method of producing a composition, the method comprising combining (a) an antigen-binding protein, a conjugate of thereof, a fusion protein, a nucleic acid, a vector and/or a host cell, each described herein, or any combination thereof; and (b) a pharmaceutically acceptable carrier, diluent and/or excipient.
  • composition comprising (a) an antigen-binding protein, a conjugate of thereof, a fusion protein, a nucleic acid, a vector and/or a host cell, each described herein, or any combination thereof; and (b) a pharmaceutically acceptable carrier, diluent and/or excipient.
  • Certain embodiments of the invention provide a method of treating a subject with a CLDN6-expressing cancer comprising administering to the subject a composition described herein in a therapeutic amount effective to treat the cancer.
  • Certain embodiments of the invention provide a method of 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 an immunocomplex or quantifying an amount of the immunocomplexes so assayed comprising the antigen-binding protein bound to CLDN6, the immunocomplex indicative of the presence and quantity of CLDN6; (c) thereby detecting or determining the quantity of CLDN6 in the sample.
  • the method further comprises contacting the immunocomplex so formed with an antibody labeled with a detectable marker to form a second immunocomplex comprising the antigen-binding protein-CLDN6- labeled antibody and detecting the second immunocomplex so formed.
  • Certain embodiments of the invention provide a method of diagnosing a Claudin-6 (CLDN6)-positive cancer in a subject, comprising: (a) contacting a biological sample comprising cells or tissue obtained from the subject with an antigenbinding protein described herein; (a) assaying for an immunocomplex comprising the antigen-binding protein or quantifying the immunocomplexes so assayed, the presence of the immunocomplex indicative of the presence of CLDN6-positive cancer; (b) thereby diagnosing the CLDN6-positive cancer in the subject.
  • the method further comprises contacting the immunocomplex so formed with an antibody labeled with a detectable marker to form a second immunocomplex comprising the antigenbinding protein-CLDN6-labeled antibody and detecting the second immunocomplex so formed.
  • a method for monitoring a course of a Claudin-6 (CLDN6)-positive cancer in a subject comprises: (a) quantitatively determining in a first sample from the subject the presence of CLDN6 in the sample by the formation of immunocomplexes as described herein; and (b) comparing the amount so determined with the amount present in a second sample from the subject, such samples being taken at different points in time, and (c) a difference in the amounts determined being indicative of the course of the cancer.
  • the method further comprises contacting the immunocomplex so formed with an antibody labeled with a detectable marker to form a second immunocomplex comprising the antigen-binding protein-CLDN6-labeled antibody and detecting the second immunocomplex so formed.
  • a method for typing tumors in a subject comprises: (a) detecting in a sample from the subject the presence of Claudin-6 (CLDN6) in the sample by the formation of immunocomplexes as described herein; and (b) the presence thereof being indicative of a specific Claudin-6 (CLDN6) tumor type.
  • the method further comprises contacting the immunocomplex so formed with an antibody labeled with a detectable marker to form a second immunocomplex comprising the antigen-binding protein-CLDN6-labeled antibody and detecting the second immunocomplex so formed.
  • a method of determining whether a cancer is treatable by a cancer therapy targeting Claudin-6 (CLDN6)-positive cancer in a subject comprises: (a) quantitatively determining in a first sample from the subject the presence of CLDN6 in the sample by the formation of immunocomplexes as described herein; and (b) comparing the amount so determined with the amount present in a second sample from the subject, such samples being taken at different points in time. A decrease over time of the quantity of CLDN6 determined indicative that the CLDN6- positive cancer is treatable in the subject.
  • the method further comprises contacting the immunocomplex so formed with an antibody labeled with a detectable marker to form a second immunocomplex comprising the antigen-binding protein-CLDN6-labeled antibody and detecting the second immunocomplex so formed.
  • a method of determining the prognosis of a subject having a Claudin-6 (CLDN6)-positive cancer comprises: (a) quantitatively determining in a first sample from the subject the presence of CLDN6 in the sample by the formation of immunocomplexes as described herein; and (b) comparing the amount so determined with the amount present in a second sample from the subject, such samples being taken at different points in time. The quantity of CLDN6 determined over time indicative of the prognosis of the CLDN6-positive cancer in the subject.
  • the method further comprises contacting the immunocomplex so formed with an antibody labeled with a detectable marker to form a second immunocomplex comprising the antigen-binding protein-CLDN6-labeled antibody and detecting the second immunocomplex so formed.
  • Certain embodiments of the invention provide a method of detecting or quantifying the presence of CLDN6 in a sample comprising: (a) contacting the sample under suitable conditions with a first antigen-binding protein which is specific for the Claudin-6 (CLDN6) to form a first-antigen-binding protein-CLDN6 immunocomplex; (b) contacting the immunocomplex so formed with a second antigen-binding protein labeled with a detectable marker to form a second immunocomplex comprising first antigenbinding protein-CLDN6-second antigen-binding protein and detecting the second immunocomplex so formed; (c) thereby detecting or quantifying the presence of CLDN6 in the sample.
  • a first antigen-binding protein which is specific for the Claudin-6 (CLDN6) to form a first-antigen-binding protein-CLDN6 immunocomplex
  • a second antigen-binding protein labeled with a detectable marker to form a second immunocomplex comprising first antigenbinding protein-CLDN6
  • Certain embodiments of the invention provide a method of producing a conjugate comprising an antigen-binding protein, or a fusion protein thereof, that binds to a Claudin-6 (CLDN6) protein, comprising: (a) culturing a host cell in a cell culture medium, wherein the host cell comprises a nucleotide sequence 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; and (c) attaching the antigen binding protein or a fusion protein to a second moiety so as to produce the conjugate, wherein the second moiety is a detectable label.
  • CLDN6 Claudin-6
  • Certain embodiments of the invention provide a method of using the antigenbinding proteins, or conjugates thereof, described herein for the assays and methods of assaying described herein.
  • an antigen-binding protein which is an antibody or antigen-binding antibody fragment.
  • the antibody is a monoclonal antibody.
  • the antibody is a human or humanized antibody, or a chimeric antibody.
  • the antigen-binding protein is an IgG.
  • the IgG is selected from the group consisting of IgGl, IgG2, IgG3 and IgG4.
  • the antigen-binding antibody fragment is selected from the group consisting of scFv, F(ab')2, Fab, Fab' and Fv.
  • the antibody is one of a selected combination of antibodies described herein which are specific for the Claudin-6 (CLDN6).
  • the antigen-binding protein is labeled with a detectable label.
  • the detectable label comprises a fluorophore, radioactive label, colorometric label, or wherein the detectable label is the product of an enzymatic reaction.
  • the detectable label comprises horse radish peroxidase (HRP).
  • Certain embodiments of the invention provide a diagnostic kit comprising: (a) a 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) above.
  • the label is selected from the group consisting of enzymes, radiolabels, chromophores and fluorophores.
  • the method comprises culturing a host cell comprising a nucleic acid encoding an antigen-binding protein, a fusion protein or a polypeptide as described herein so as to express the antigen-binding protein, fusion protein or polypeptide, harvesting the antigen-binding protein, fusion protein or the fusion protein from the cell culture medium then attaching the antigen binding protein, fusion protein or polypeptide to a second moiety so as to produce the conjugate.
  • the second moiety is a detectable label.
  • the detectable label is horseradish peroxidase (HRP).
  • mice were immunized with a mixture of peptides and fusion proteins (Table 7) using standard methods infra.
  • Splenocytes were harvested from the immunized mice and fused with myeloma lines by Electrofusion to generate hydridomas. Approximately 2000 primary hybridoma cultures were generated and cultured in 96-well plates. BSA conjugated peptides 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 and map antibody binding epitopes to the CLDN6 cytoplasmic domain using bead-based indirect flow cytometry and ELISA (Fig 1 and Fig 2).
  • CLDN6-specfic antibodies 3D07 and 3H11 were chosen for additional 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, adjoin, France). See Tables 8-9 below and sequence listing.
  • CLDN6 antibodies were formated as full-length IgG antibodies using ExpiCHOTM expression.
  • the heavy and light chain variable regions of the antibodies were cloned into an antibody expression vector which was engineered in the lab based on a pcDNATM3.4-TOPO® vector (Catalog Number: A14697, ThermoFisher Scientific, USA) and transfected into CHO cells by (According to protocol provided in the kit (ExpiCHOTM Expression System, Catalog Number: A29133, ThermoFisher Scientific, USA)).
  • Antibodies were purified and binding of the antibodies to CLDN6 was assessed as described in EXAMPLES 3-4 below.
  • Peptides were designed spanning the cytoplasmic domain of CLDN6 (SEQ ID NO: 3). The peptides were offset by several amino acids i.e., each peptide was overlapping another peptide preceding and/or following it by several amino acids as shown in FIGS 1-2 and Table 7 above. [00269] A binding assay was performed using a bead-based flow assay with indirect flow cytometry.
  • BSA conjugated peptides and GST fusion proteins were formalin fixed using published methods (Press et al., Steroids, 67 (2002) 799-813) were conjugated to beads and used to identify and map antibody binding epitopes to the CLDN6 cytoplasmic domain using bead-based indirect flow cytometry.
  • the binding assay identified overlapping peptides 4 and 5 with a 10 amino acid (APAISRGPSE, SEQ ID NO: 15) core region for binding in the cytoplasmic domain of CLDN6 (FIG 1).
  • the binding assay identified overlapping peptides 1R, 2 and 3 with a 6 amino acid (SHYMAR, SEQ ID NO: 98) core region for binding in the cytoplasmic domain of CLDN6 (FIG 1).
  • SHYMAR SEQ ID NO: 98
  • FIG 2 shows that 3D07 does not bind to SHYMA thus the R in SHYMAR is required.
  • Example 1 the two antibodies described in Example 1, 3H11 and 3D07, were characterized for their binding to CLDN6 in HEK293 T cells.
  • CLDN6-expressing cells were used in an assay to determine each CLDN6 antibody’s ability to bind to CLDN6 on the surface of cells and to cross-react with other CLDN family members (CLDN3, CLDN4 or CLDN9).
  • CLDN3, CLDN4 or CLDN9 CLDN family members
  • Samples of CLDN6, 3, 4, and 9 engineered HEK293 cells were prepared as follows. Formalin-fixed paraffin-embedded cells was cut into 4 mm thin sections and mounted on slides. The cell mounted slides were dried overnight and baked at 60°C for an hour, ran through xylene 3 times for 5 minutes, treated with 100%, 95%, 70%, and 50% ethanol 2 times for 2 minutes, washed in dH2O 2 times for 2 minutes, soaked in 0.3% H2O2 for 15 minutes, rinsed with dH2O, and finally washed 2 times for 2 minutes in lx TBS.
  • the slides were washed in lx TBST (IxTBS + 0.1% Tween 20) for 15 minutes, 2 times for 2 minutes in TBS, then incubated with the Goat polyclonal antibody (pAb) to mouse IgG HRP polymer (Abeam, #ab214879) for 30 minutes at room temperature, washed in lx TBST for 20 minutes, rinsed 2 times for 2 minutes with IxTBS, stained with ImmPACT® DAB EqV Peroxidase (HRP) Substrate (Vectorlab, # SK-4103-400) for 6 minutes, transferred to a large container of dH2O, finally, the slides were taken out and stained with Hematoxylin (Abeam, ab220365) for 15 seconds, rinsed under continuous sink water for 3 minutes, washed 2 times for 2 minutes with dH2O, dehydrated through treatment with 95% and 100% ethanol 2 times for 2 minutes, xylene 3 times for 5 minutes, and mounted with per
  • Tissue samples of human ovarian cancer were prepared as follows. Formalin- fixed paraffin-embedded tissue was cut into 4 pm thin sections and mounted on slides. The tissue mounted slides were dried overnight and baked at 60°C for an hour, ran through xylene 3 times for 5 minutes, treated with 100%, 95%, 70%, and 50% ethanol 2 times for 2 minutes, washed in dH2O 2 times for 2 minutes, soaked in 0.3% H2O2 for 15 minutes, rinsed with dH2O, and finally washed 2 times for 2 minutes in lx TBS.
  • the slides were washed in lx TBST (IxTBS + 0.1% Tween 20) for 15 minutes, 2 times for 2 minutes in TBS, then incubated with the Goat polyclonal antibody (pAb) to mouse IgG HRP polymer (Abeam, #ab214879) for 30 minutes at room temperature, washed in lx TBST for 20 minutes, rinsed 2 times for 2 minutes with IxTBS, stained with ImmPACT® DAB EqV Peroxidase (HRP) Substrate (Vectorlab, # SK-4103-400) for 6 minutes, transferred to a large container of dH2O, finally, the slides were taken out and stained with Hematoxylin (Abeam, ab220365) for 15 seconds, rinsed under continuous sink water for 3 minutes, washed 2 times for 2 minutes with dH2O, dehydrated through treatment with 95% and 100% ethanol 2 times for 2 minutes, xylene 3 times for 5 minutes, and mounted with Per
  • FIG 4 shows the results of the binding of antibodies 3H11 and 3D07 to tissues sections of human ovarian cancer.
  • the results show that the antibodies stained human CLDN6-positive ovarian cancer tissue but did not stain CLDN6-negative ovarian cancer tissue.
  • the antibodies 3H11 and 3D07 identified the sections of ovarian cancer tissue having CLDN6-positive cells.
  • amino acid sequences for antibody 3H11 heavy and light chain (SEQ ID NOs: 16-17) and 3D07 heavy chain and light chain (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.

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Abstract

La présente divulgation concerne des protéines de liaison à l'antigène qui se lient à la claudine-6 (CLDN6). Dans divers aspects, les protéines de liaison à l'antigène se lient au domaine cytoplasmique de CLDN6. L'invention concerne en outre des polypeptides, des acides nucléiques, des vecteurs, des cellules hôtes et des conjugués associés. L'invention concerne en outre des kits et des compositions pharmaceutiques comprenant de telles entités. L'invention concerne également des procédés de fabrication d'une protéine de liaison à l'antigène et des procédés de détection et de quantification de claudine-6 (CLDN6) dans des échantillons provenant d'un sujet et de traitement d'un sujet atteint d'un cancer.
PCT/US2023/029856 2022-08-09 2023-08-09 Anticorps de claudine-6 et leurs conjugués et utilisations WO2024035787A2 (fr)

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