Claudin 6 antibody and method for treating cancer

Abstract

The present disclosure provides antigen binding proteins that bind to tight junction protein-6 (CLDN6). In various aspects, the antigen binding protein binds to extracellular loop 2 (EL2) of the extracellular domain of CLDN6. Related polypeptides, nucleic acids, vectors, host cells and conjugates are also provided herein. In addition, kits and pharmaceutical compositions comprising such entities are provided. Methods for preparing antigen binding proteins and methods for treating subjects with cancer are also provided.

Description

[0001] Incorporation by Reference of Electronically Submitted Materials

[0002] Incorporated by reference in its entirety is the computer-readable nucleotide / amino acid sequence listing simultaneously submitted herewith and identified as follows: a 315,776-byte ASCII (text) file named "51836_SeqListing.txt" created on September 18, 2018. Background Art

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

[0004] The U.S. Food and Drug Administration currently has approved more than a dozen monoclonal antibodies for the treatment of cancer. Among these agents is alemtuzumab, which is indicated for chronic lymphocytic leukemia (CLL). and trastuzumab for breast cancer Some antibodies are labeled with chemotherapy drugs, including, for example, brentuximab vedotin and Ado-trastuzumab emtansine Other antibody products, such as blinatumomab (Blincyto), are designed to recognize and bind to two different antigens. Although such antibody products are commercially available, the current incidence of cancer and the number of cancer deaths remain high. It is reported that the incidence of cancer is greater than 450 per 100,000 men and women per year, and the cancer mortality rate is only over 170 per 100,000 men and women per year. Summary of the Invention

[0005] Provided herein are antigen-binding proteins that bind to Claudin-6 (CLDN6). In various aspects, the antigen-binding proteins of the present disclosure bind to human CLDN6 and optionally to mouse CLDN6. In various aspects, the antigen-binding proteins bind to the extracellular domain (ECD) of CLDN6. In various cases, the antigen-binding proteins bind to extracellular loop 2 (EL2) of the ECD of CLDN6. In various aspects, the antigen-binding proteins bind to EL2 and do not bind to extracellular loop 1 (EL1) of the ECD of CLDN6. In various cases, the antigen-binding proteins bind to other members of the human claudin family, including, for example, claudin-3 (CLDN3), claudin-4 (CLDN4), and claudin-9 (CLDN9). In various cases, the antigen-binding proteins bind to CLDN6 and at least one of CLDN4 and CLDN9. In various cases, the antigen-binding proteins bind to CLDN6 and do not bind to any other member of the claudin family. In various aspects, the antigen binding protein binds to CLDN6 endogenously expressed by human ovarian cancer cells (e.g., OVCA429 cells) and exhibits an IC50 of less than about 1200 nM in a FACS affinity assay using OVCA429 cells. In various cases, the antigen binding proteins of the present disclosure inhibit tumor growth in a subject (e.g., a human) in the absence of any other moiety attached to the antigen binding protein. In various cases, the antigen binding protein that is not conjugated to a heterologous moiety (e.g., not conjugated to any chemotherapeutic agent, drug, or toxic moiety) inhibits tumor growth in a subject (e.g., a human).

[0006] In various aspects, the antigen binding protein binds to CLDN6 expressed by human cancer cells. In various aspects, the antigen binding protein inhibits the binding interaction between human CLDN6 and a reference anti-CLDN6 antibody. Without being bound by a particular theory, the inhibitory effects of the antigen binding proteins provided herein allow such entities to be used in methods for reducing tumor growth and treating subjects with tumors or cancer. As further discussed herein, in various aspects, the antigen binding protein is an antibody, an antigen-binding antibody fragment thereof, or an antibody protein product.

[0007] The present disclosure also provides antigen binding proteins comprising at least 3, 4, 5, or all of the amino acid sequences in a specific group. In various aspects, the antigen binding protein comprises at least 3, 4, 5, or 6 complementary determining region (CDR) amino acid sequences of the CLDN6 antibodies disclosed herein.

[0008] The present disclosure also provides antigen-binding proteins comprising the amino acid sequence as described in detail herein. In various aspects, as further described herein, the antigen-binding proteins comprise the amino acid sequence of SEQ ID NO: listed in Table A, Table A1, Table B, Table B1, Table C or Table D or a combination thereof.

[0009] Related polypeptides, nucleic acids, vectors, host cells, and conjugates are also provided herein. Kits and pharmaceutical compositions comprising such entities are also contemplated.

[0010] Also provided are methods of preparing an antigen binding protein. In various embodiments, the method comprises culturing a host cell comprising a nucleic acid encoding an antigen binding protein or polypeptide as described herein so as to express the antigen binding protein or polypeptide.

[0011] Additionally provided herein are methods of treating a subject having cancer. In various embodiments, the method comprises administering to the subject a pharmaceutical composition of the present disclosure in an amount effective to treat the cancer in the subject.

[0012] Also provided are methods of treating a subject having a cancer that expresses CLDN6, the methods comprising administering to the subject a pharmaceutical composition described herein. Also contemplated are methods of inhibiting tumor growth in a subject, the methods comprising administering to the subject a pharmaceutical composition described herein.

[0013] A method of reducing tumor size in a subject or preventing recurrence of cancer in a subject, the method comprising administering to the subject a pharmaceutical composition described herein.

[0014] Also provided herein is a method of treating cancer in a subject diagnosed as a CLDN6 under-overexpresser, the method comprising administering to the subject the pharmaceutical composition described herein.

[0015] In various embodiments, administration induces apoptosis of tumor cells (e.g., cells expressing CLDN6). In various embodiments, administration induces antibody-dependent cell-mediated cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC), tumor necrosis and cell death or exhaustion, and / or disruption of tumor cell adhesion, each of which results in tumor regression or reduced tumor growth. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 Graph showing CLDN6 expression in normal (non-cancerous) tissues.

[0017] Figure 2 Graph representing CLDN6 expression in cancer cell lines as determined by the Agilent 44K method.

[0018] Figure 3Graph representing CLDN6 expression in cancer cell lines as determined by RNASeq.

[0019] Figure 4 Represents a set of fluorescence images depicting the localization of CLDN6-GFP in different cell models.

[0020] Figure 5 The sequence alignment of human CLDN6, human CLDN3, human CLDN4, human CLDN9, and mouse CLDN6 is shown. The sequences of EL1 and EL2 are shown.

[0021] Figure 6A Figure 5 represents the tumor volume (mm) of tumors in mice bearing endometrial tumors after treatment with control IgG2 antibody, AB3, reference Ab1, reference Ab2, reference Ab3, AB2, and AB3. 3 ) over time (days). Figure 6B Figure 5 represents the tumor volume (mm) at day 14 in endometrial tumor-bearing mice treated with control IgG2 antibody, AB3, Reference Ab1, Reference Ab2, Reference Ab3, AB2, or AB3. 3 )Average change graph.

[0022] Figure 7A Figure 5 represents the tumor volume (mm) of tumors in mice bearing bladder tumors after treatment with control IgG2 antibody, AB3, reference Ab1, reference Ab2, and AB3. 3 ) over time (days). Figure 7B Figure 5 represents the tumor volume (mm) at day 35 in bladder tumor-bearing mice treated with control IgG2 antibody, AB3, reference Ab1, reference Ab2, or AB3. 3 )Average change graph.

[0023] Figure 8A represents the tumor volume (mm) of tumors in mice bearing ovarian tumors after treatment with control IgG2 antibody, AB3, reference Ab1, AB2, and AB3. 3 ) over time (days). Figure 8B Figure 5 represents the tumor volume (mm) at day 20 in ovarian tumor-bearing mice treated with control IgG2 antibody, AB3, reference Ab1, AB2, or AB3. 3 )Average change graph.

[0024] Figure 9A Figure 5 represents the tumor volume (mm) of tumors in mice bearing melanoma tumors after treatment with control IgG2 antibody, AB3, reference Ab1, reference Ab2, reference Ab3, and AB3. 3 ) over time (days). Figure 9BFigure 5 represents the tumor volume (mm) at day 21 in melanoma tumor-bearing mice treated with control IgG2 antibody, AB3, reference Ab1, reference Ab2, reference Ab3, or AB3. 3 )Average change graph.

[0025] Figure 10A Graph representing the % inhibition of tumor growth achieved in tumor-bearing mice treated with AB3 relative to mice treated with a control antibody. Figure 10B Images of Western blots showing varying levels of CLDN6 in endometrial cancer cell lines (ARK2), bladder cancer cell lines (UMUC4), ovarian cancer cell lines (OV90), and melanoma cell lines (M202), as well as control cells. α-Tubulin levels were roughly the same, indicating equal protein loading.

[0026] Figure 11 Graph showing % body weight change over time (days) for tumor-bearing mice treated with vehicle control, control antibody, reference Ab1, reference Ab2, reference Ab3, and AB3.

[0027] Figure 12A Figure 5 represents the tumor volume (mm) of tumors in ovarian tumor-bearing mice after treatment with vehicle control, control IgG2 antibody, AB3, reference Ab1, or one of the indicated anti-CLDN6 antibodies. 3 ) over time (days). Figure 12B Figure 5 represents the tumor volume (mm) at day 28 in ovarian tumor-bearing mice treated with vehicle control, control IgG2 antibody, AB3, reference antibody Abl, or one of the indicated anti-CLDN6 antibodies. 3 )Average change graph.

[0028] Figure 13 Graph showing % body weight change over time (days) for tumor-bearing mice treated with vehicle control, control antibody, reference Abl and the indicated anti-CLDN6 antibodies.

[0029] Figure 14 Shown are a series of dose response curves for several anti-CLDN6 antibodies of the invention as well as reference Ab1 and reference 2. Mouse IgG was used as a control.

[0030] Figure 15A Figure 5 represents the tumor volume (mm) at day 35 in bladder tumor-bearing mice treated with vehicle control, control IgG antibody, mouse form of AB3, first humanized form of AB3, and second humanized form of AB3. 3 )Average change graph. Figure 15B express Figure 15A Tumor volume (mm) of each group 3 ) changes in the graph.

[0031] Figure 16A Figure 5 represents the tumor volume (mm) at day 35 in bladder tumor-bearing mice treated with vehicle control, control IgG antibody, mouse form of AB3, first humanized form of AB3, and second humanized form of AB3. 3 ) Average change plot. Two control antibodies (a mouse antibody and a chimeric antibody) were also tested in this experiment. Figure 16B express Figure 16A Tumor volume (mm) of each group 3 ) changes in the graph.

[0032] Figure 17A Figure 35 shows the tumor volume (mm) of bladder tumor-bearing mice treated with vehicle control, control IgG antibody, mouse form of AB1, and humanized form of AB1. 3 )Average change graph. Figure 17B express Figure 17A Tumor volume (mm) of each group 3 ) changes in the graph.

[0033] Figure 18A Figure 35 shows the tumor volume (mm) of bladder tumor-bearing mice treated with vehicle control, control IgG antibody, mouse form of AB4, and humanized form of AB4. 3 )Average change graph. Figure 18B express Figure 18A Tumor volume (mm) of each group 3 ) changes in the graph.

[0034] Figure 19A Figure 2 shows the tumor volume (mm) at day 35 in bladder tumor-bearing mice treated with vehicle control, control IgG antibody, mouse form of AB3, chimeric form of AB3, first humanized form of AB3, second humanized form of AB3, mouse form of Abl, humanized form of AB1, mouse form of AB4, humanized form of AB4. 3 ) average change graph, and four control antibodies (one antibody has a mouse form or a chimeric form, and one antibody has a mouse or human form) were also tested in this experiment. Figure 19B express Figure 19A Tumor volume (mm) of each group 3 ) changes in the graph.

[0035] Figure 20 Indicates that Figure 19A Tumor volume (mm) of the treated bladder tumor-bearing mice on day 55 3 )Average change graph.

[0036] Figure 21Indicates Figure 19A Graph showing the % change in body weight of treated tumor-bearing mice after 32 days of treatment. DETAILED DESCRIPTION

[0037] tight junction protein family

[0038] Tight junctions, also known as occluded junctions or zonular closures, are vertebrate structures located between two adjacent cells that regulate paracellular permeability and maintain cell polarity in epithelial and endothelial cell sheets. The claudin (CLDN) gene family encodes membrane proteins that are important components of tight junctions. CLDN proteins contain four transmembrane (TM) helices (TM1, TM2, TM3, and TM4) and two extracellular loops (EL1 and EL2). The extracellular loops of CLDN proteins from adjacent cells interact with each other to seal the cell sheet and regulate paracellular transport between the lumen and the basolateral space.

[0039] CLDN proteins play a role in various human diseases and pathologies. For example, mutations in the CLDN1 gene have been shown to cause gradual peeling of the skin and bile duct obstruction. Mutants in the CLDN16 gene cause magnesium depletion disorders. CLDN19 mutations can lead to eye conditions such as macular degeneration and myopia, while CLDN14 mutations can cause non-syndromic recessive deafness. CLDN3 and CLDN4 are known to be surface receptors for Clostridium perfringens enterotoxin in the intestine, and CLDN1, CLDN6, and CLDN9 are co-receptors for hepatitis C virus (HCV) entry. Several CLDN proteins have been shown to be abnormally expressed in cancer. For example, CLDN1 is downregulated in breast and colon cancers, while CLDN3 and CLDN4 are highly upregulated in a variety of cancers.

[0040] Claudin-6 (CLDN6) 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 16p13.3 and is conserved in chimpanzees, rhesus monkeys, dogs, cows, mice, rats, zebrafish, and frogs. CLDN6 is typically expressed in humans as a 220-amino acid precursor protein; its first 21 amino acids constitute a signal peptide. The amino acid sequence of the CLDN6 precursor protein is publicly available on the National Center for Biotechnology Information (NCBI) website as NCBI reference sequence NP_067018.2 and is provided herein as SEQ ID NO:1. The amino acid at position 143 of SEQ ID NO:1 is Ile. In some cases, due to a single nucleotide polymorphism (SNP) in the DNA sequence encoding CLDN6, the amino acid at position 143 is Val. The amino acid sequence of human CLDN6 with Val at position 143 is provided herein as SEQ ID NO:178.

[0041] Antigen binding proteins

[0042] Provided herein are antigen-binding proteins that bind to claudin-6 (CLDN6). The antigen-binding proteins of the present disclosure may take any of a variety of forms of antigen-binding proteins known in the art. In various embodiments, the antigen-binding proteins of the present disclosure take the form of antibodies, antigen-binding antibody fragments, or antibody protein products.

[0043] In various embodiments of the present disclosure, the antigen-binding proteins comprise, consist essentially of, or consist of antibodies. As used herein, the term "antibody" refers to a protein having a conventional immunoglobulin pattern comprising a heavy chain and a light chain and comprising a variable region and a constant region. For example, an antibody can be an IgG, which is a "Y-shaped" structure of two pairs of identical polypeptide chains, each pair having a "light" chain (typically having a molecular weight of about 25 kDa) and a "heavy" chain (typically having a molecular weight of about 50-70 kDa). The antibody has a variable region and a constant region. In the IgG format, the variable region is typically about 100 to 110 or more amino acids, comprises three complementary determining regions (CDRs), is primarily responsible for antigen recognition, and is substantially different between 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 composed of the N-terminal region of each light chain and heavy chain, while the constant region is composed of the C-terminal portion of each heavy chain and light chain. (Janeway et al., "Structure of the Antibody Molecule and the Immunoglobulin Genes", Immunobiology: The Immune System in Health and Disease, 4th Edition, Elsevier Science Ltd. / Garland Publishing, (1999)).

[0044] The general structure and properties of antibody CDRs have been described in the art. In short, in an antibody scaffold, CDRs are embedded in the framework of the heavy and light chain variable regions, where they constitute the region primarily responsible for antigen binding and recognition. The variable region is typically in a framework region (by Kabat et al., 1991 named as framework region 1-4, FR1, FR2, FR3 and FR4; See also Chothia and Lesk, 1987, J. Mol. Biol. 196: 901-917) comprising at least three heavy or light chain CDRs (Kabat et al., 1991, Sequences of Proteins of Immunological Interest, Public Health Service NIH, Bethesda, Md.; See also Chothia and Lesk, 1987, supra; Chothia et al., 1989, Nature 342: 877-883).

[0045] Antibodies may comprise any constant region known in the art. Human light chains are divided into kappa and lambda light chains. Heavy chains are divided into μ, δ, γ, α, or ε, and define the isotype of the antibody as IgM, IgD, IgG, IgA, and IgE, respectively. IgG has several subclasses, including but not limited to IgG1, IgG2, IgG3, and IgG4. IgM has subclasses, including but not limited to IgM1 and IgM2. Embodiments of the present disclosure include antibodies of all such classes or isotypes. The light chain constant region may be, for example, a kappa or lambda type light chain constant region, such as a human kappa or lambda type light chain constant region. The heavy chain constant region may be, for example, an α-, δ-, ε-, γ-, or μ-type heavy chain constant region, such as a human α-, δ-, ε-, γ-, or μ-type heavy chain constant region. Therefore, in various embodiments, the antibody is an antibody of isotype IgA, IgD, IgE, IgG, or IgM, including any one of IgG1, IgG2, IgG3, or IgG4. In various aspects, the antibodies comprise a constant region comprising one or more amino acid modifications relative to their naturally occurring counterparts to improve half-life / stability or to make the antibody more suitable for expression / manufacturing. In various cases, the antibodies comprise a constant region in which the C-terminal Lys residue present in the naturally occurring counterpart is removed or cleaved.

[0046] Antibody can be a monoclonal antibody. In some embodiments, antibody comprises a sequence substantially similar to the naturally occurring antibody produced by mammals (e.g., mouse, rabbit, goat, horse, chicken, hamster, people, etc.). In this respect, antibody can be considered to be a mammalian antibody, e.g., mouse antibody, rabbit antibody, goat antibody, horse antibody, chicken antibody, hamster antibody, human antibody, etc. In some aspects, antigen-binding proteins are antibodies, e.g., human antibodies. In some aspects, antigen-binding proteins are chimeric antibodies or humanized antibodies. The term "chimeric antibody" refers to an antibody containing a domain from two or more different antibodies. Chimeric antibodies can, for example, contain a constant domain from a species and a variable domain from a second species, or more generally, can contain a fragment of an amino acid sequence from at least two species. Chimeric antibodies can also contain a domain from two or more different antibodies in the same species. When used relative to an antibody, the term "humanized" refers to an antibody having at least one CDR region from a non-human source, which is engineered to have a structure and immune function more similar to a true human antibody than the original source antibody. In some embodiments, humanization can be directed to a human antibody comprising a CDR from a non-human antibody (e.g., mouse antibody). Humanization can also be directed to selecting amino acid substitutions to make the non-human sequence more similar to the human sequence. The information comprising the sequence information of the constant region of human antibody heavy chain and light chain can be publicly available through other well-known databases in the Uniprot database and antibody engineering and production fields. For example, the IgG2 constant region can be obtained from the Uniprot database with Uniprot numbering P01859, which is incorporated herein by reference.

[0047] Antibodies can be cleaved into fragments by enzymes such as papain and pepsin. Papain cleaves antibodies to produce two Fab fragments and a single Fc fragment. Pepsin cleaves antibodies to produce F(ab')2 fragments and pFc' fragments. In various aspects of the present disclosure, the antigen-binding proteins of the present disclosure are antigen-binding fragments of antibodies (also known as antigen-binding antibody fragments, antigen-binding fragments, antigen-binding portions). In various cases, the antigen-binding antibody fragment is a Fab fragment or a F(ab')2 fragment.

[0048] The architecture of antibodies has been exploited to create a growing number of alternative antibody formats that span a molecular weight range of at least about 12 to 150 kDa and have a valency (n) range from monomer (n=1) to dimer (n=2), trimer (n=3), tetramer (n=4) and potentially higher; such alternative antibody formats are referred to herein as "antibody protein products". Antibody protein products include those based on the structure of intact antibodies and those that mimic antibody fragments that retain full antigen binding ability, such as scFv, 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 the variable (V) region. A soluble, flexible amino acid peptide linker is used to connect the V region to the scFv (single-chain fragment variable) fragment to stabilize the molecule, or a constant (C) domain is added to the V region to generate a Fab fragment [fragment, antigen binding]. Both scFv and Fab fragments can be readily produced in host cells (e.g., prokaryotic host cells). Other antibody protein products include disulfide-stabilized scFv (ds-scFv), single-chain Fab (scFab), and dimeric and multimeric antibody formats (such as di-, tri-, and tetra-antibodies), or miniantibodies (miniAbs) comprising different formats consisting of scFvs connected to oligomerization domains. The smallest fragments are the VHH / VH of camelid heavy chain antibodies and single domain antibodies (sdAbs). The building blocks most commonly used to create new antibody formats are single-chain variable (V) domain antibody fragments (scFvs), which contain the V domains (VH and VL domains) from the heavy and light chains connected by a peptide linker of approximately 15 amino acid residues. Peptibodies or peptide-Fc fusions are another type of 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, for example, Shimamoto et al., mAbs 4(5):586-591 (2012).

[0049] Other antibody protein products include single-chain antibodies (SCAs), diabodies, triabodies, tetrabodies, bispecific or trispecific antibodies, etc. Bispecific antibodies can be divided into five main categories: BsIgG, attached IgG, bispecific antibody (BsAb) fragments, bispecific fusion proteins and BsAb conjugates. See, for example, Spiess et al., Molecular Immunology 67 (2) Part A: 97-106 (2015).

[0050] In various aspects, the antigen-binding proteins of the present disclosure comprise, consist essentially of, or consist of any of these antibody protein products. In various aspects, the antigen-binding proteins of the present disclosure comprise, consist essentially of, or consist of any of scFv, Fab VHH / VH, Fv fragments, ds-scFv, scFab, dimeric antibodies, multimeric antibodies (e.g., diabodies, triabodies, tetrabodies), miniAbs, camelid heavy chain antibody peptibody VHH / VH, sdAbs, diabodies, triabodies, tetrabodies, bispecific or trispecific antibodies, BsIgGs, additional IgGs, BsAb fragments, bispecific fusion proteins, and BsAb conjugates.

[0051] In various cases, the antigen-binding proteins of the present disclosure are antibody protein products in monomeric or polymeric, oligomeric or multimeric form. In certain embodiments in which the antibody comprises two or more different antigen-binding region fragments, the antibody is considered to be bispecific, trispecific or multispecific, or bivalent, trivalent or multivalent, depending on the number of different epitopes recognized and bound by the antibody.

[0052] In various embodiments, the anti-CLDN6 antibody or antibody variant thereof is selected from the group consisting of a human antibody, a humanized antibody, a chimeric antibody, a monoclonal antibody, a recombinant antibody, an antigen-binding antibody fragment, a single-chain antibody, a monomeric antibody, a diabody, a triabody, a tetrabody, a Fab fragment, an IgG1 antibody, an IgG2 antibody, an IgG3 antibody, and an IgG4 antibody.

[0053] In various aspects, the antigen binding proteins of the present disclosure are linked to therapeutic agents. As described below, the therapeutic agent can be any therapeutic agent known in the art, including but not limited to chemotherapeutic agents, cytokines and growth factors, cytotoxic agents, etc. See "Conjugates" below.

[0054] CLDN6 and epitopes

[0055] The antigen binding proteins of the present disclosure bind to CLDN6. In various aspects, CLDN6 is human CLDN6 having the following amino acid sequence:

[0056] wherein X is Ile or Val.

[0057] In various aspects, human CLDN6 comprises the amino acid sequence of any one of SEQ ID NOs: 1, 178, and 200-202.

[0058] In various aspects, the antigen-binding proteins of the present disclosure bind to an epitope within the amino acid sequence of CLDN6. In various aspects, CLDN6 is human CLDN6, and the antigen-binding proteins of the present disclosure bind to an epitope within the amino acid sequence of human CLDN6 (e.g., SEQ ID NOs: 1, 178, and 200-202). An "epitope" refers to a region of CLDN6 or a region within CLDN6 that is bound by an antigen-binding protein. In some embodiments, the epitope is a linear epitope. A "linear epitope" refers to a region of CLDN6 or a region within CLDN6 that is bound by an antigen-binding protein and that is composed of consecutive amino acids in the amino acid sequence of CLDN6. The amino acids of a linear epitope are adjacent to each other in the primary structure of CLDN6. Therefore, a linear epitope is a fragment or portion of the amino acid sequence of an antigen (i.e., CLDN6). In various other embodiments, the epitope is a conformational or structural epitope. A "conformational epitope" or "structural epitope" refers to an epitope composed of amino acids that are adjacent to each other only when CLDN6 is in its properly folded state. Unlike linear epitopes, the amino acids of a conformational or structural epitope are not adjacent to each other in the primary structure (i.e., amino acid sequence) of CLDN6. A conformational or structural epitope does not consist of consecutive amino acids in the amino acid sequence of the antigen (CLDN6).

[0059] In various aspects, the epitope is located within the extracellular domain (ECD) of CLDN6 (e.g., human CLDN6). In various aspects, the antigen-binding protein binds to extracellular loop 2 (EL2) of the ECD of CLDN6 having the amino acid sequence WTAHAIIRDFYNPLVAEAQKREL (SEQ ID NO: 2). In various aspects, the epitope to which the antigen-binding protein binds is within SEQ ID NO: 2. In various aspects, the antigen-binding proteins of the present disclosure bind to the N-terminal portion of SEQ ID NO: 2 (e.g., THAIIRDFYNPL (SEQ ID NO: 3)). In various aspects, the antigen-binding proteins of the present disclosure bind to the C-terminal portion of SEQ ID NO: 2 (e.g., LVAEAQKREL (SEQ ID NO: 4)). In various cases, the antigen-binding proteins of the present disclosure bind to EL2 of CLDN6 but do not bind to extracellular loop 1 (EL1). In various aspects, the antigen-binding proteins of the present disclosure bind to one or more epitopes that are different from the epitope bound by an anti-CLDN6 antibody comprising a light chain variable region comprising the sequence of SEQ ID NO: 185 and a heavy chain variable region comprising the sequence of SEQ ID NO: 186. In various aspects, the antigen-binding proteins of the present disclosure bind to one or more epitopes that are different from the epitope bound by an anti-CLDN6 antibody comprising a light chain variable region comprising the sequence of SEQ ID NO: 181 and a heavy chain variable region comprising the sequence of SEQ ID NO: 182.

[0060] In various aspects, the antigen binding protein binds to human CLDN6 and non-human CLDN6. In various cases, the non-human CLDN6 is CLDN6 from chimpanzee, rhesus monkey, dog, cow, mouse, rat, zebrafish, or frog. In various cases, the antigen binding protein binds to human CLDN6 and mouse CLDN6.

[0061] Affinity and avidity

[0062] The antigen binding proteins provided herein bind to CLDN6 in a non-covalent and reversible manner. In various embodiments, the binding strength of the antigen binding protein to CLDN6 can be described in terms of its affinity (a measure of the strength of the interaction between the binding site of the antigen binding protein and the epitope). In various aspects, the antigen binding proteins provided herein have a high affinity for CLDN6 and thus will bind to a greater amount of CLDN6 in a shorter period of time than low affinity antigen binding proteins. In various aspects, the antigen binding proteins have a binding affinity of at least 10 5 mol -1 , at least 10 6 mol -1 , at least 10 7 mol -1 , at least 10 8 mol -1 , at least 10 9 mol -1 , or at least 10 10 mol -1 , or at least 10 10 mol -1 , at least 10 10 mol -1 The equilibrium association constant K A As will be understood by those skilled in the art, K A Can be affected by factors including pH, temperature and buffer composition.

[0063] In various embodiments, the binding strength of an antigen binding protein to CLDN6 can be described in terms of its sensitivity. D is the equilibrium dissociation constant between the antigen binding protein and CLDN6, i.e., k off / k on The ratio of K D and K A Inversely proportional. K D The value is related to the concentration of the antigen binding protein (the amount of antigen binding protein required for a specific experiment), so K D The lower the value (the lower the concentration), the higher the affinity of the antigen binding protein. In various aspects, the binding strength of the antigen binding protein to CLDN6 can be measured based on K D In various aspects, the K of the antigen binding proteins provided hereinD About 10 -1 , about 10 -2 , about 10 -3 , about 10 -4 , about 10 -5 , about 10 -6 In various aspects, the K of the antigen-binding proteins provided herein is D In various aspects, the K of the antigen-binding proteins provided herein is micromolar, nanomolar, picomolar or femtomolar. D In about 10 -4 to 10 -6 , or 10 -7 to 10 -9 , or 10 -10 to 10 -12 , or 10 -13 to 10 -15 In various aspects, the K of the antigen binding proteins provided herein is D At about 1.0x 10 -12 M to about 1.0x 10 -8 In various aspects, the K of the antigen binding protein D At about 1.0x 10 -11 M to about 1.0x 10 -9 Within the range of M.

[0064] In various aspects, the affinity of antigen-binding proteins is measured or graded using flow cytometry or fluorescence activated cell sorting (FACS)-based assays. Flow cytometry-based binding assays are known in the art. See, for example, Cedeno-Arias et al., Sci Pharm 79 (3): 569-581 (2011); Rathanaswami et al., Analytical Biochem 373: 52-60 (2008); and Geuijen et al., J Immunol Methods 302 (1-2): 68-77 (2005). In various aspects, the affinity of antigen-binding proteins is measured or graded using competitive assays such as Trikha et al., Int J Cancer 110: 326-335 (2004) and Tam et al., Circulation 98 (11): 1085-1091 (1998) and as described below. See the section entitled "Competition Assay" below. In Trikh et al., radioactivity was measured using cells expressing antigen. 125Binding of an I-labeled antigen-binding protein (e.g., an antibody) to a cell surface antigen. In various aspects, the relative affinity of CLDN6 antibodies is determined via a FACS-based assay, in which varying concentrations of CLDN6 antibodies conjugated to a fluorophore are incubated with cells expressing CLDN6 and the emitted fluorescence (which is a direct measure of antibody-antigen binding) is measured. A fluorescence curve is drawn for each dose or concentration. The maximum value is the lowest concentration at which fluorescence stabilizes or reaches a maximum value (i.e., when binding saturation occurs). Half of the maximum value is considered the EC50 or IC50, and the antibody with the lowest EC50 / IC50 is considered to have the highest affinity relative to other antibodies tested in the same manner. Such an assay is described in Example 5 herein.

[0065] In various aspects, as determined in competitive binding inhibition assays, the IC 50 The value is similar to the K of the antigen binding protein D In various cases, as described below, the competition assay is a FACS-based assay performed with a reference antibody, a fluorophore-conjugated secondary antibody, and cells expressing CLDN6. In various aspects, the cells are genetically engineered to overexpress CLDN6. In some aspects, the cells are HEK293T cells transduced with a viral vector to express CLDN6. In alternative aspects, the cells endogenously express CLDN6. Prior to performing the FACS-based assay, in some aspects, cells endogenously expressing CLDN6 are pre-determined as low CLDN6-expressing cells or high CLDN6-expressing cells. In some aspects, the cells are cancer cells or tumor cells. In various aspects, the cells are cells from a cell line, such as an ovarian cell line, an endometrial cell line, a bladder cell line, a lung cell line, a gastrointestinal (GI) cell line, a liver cell line, a lung cell line, and the like. In various aspects, the cells endogenously expressing CLDN6 are selected from the group consisting of: OVCA429 ovarian cells, ARK2 endometrial cells, OAW28 ovarian cells, UMUC-4 bladder cells, PEO14 ovarian cells, OV177 ovarian cells, H1693 lung cells, MKN7 upper GI cells, OV-90 ovarian cells, HUH-7 liver cells, JHOS-4 ovarian cells, H1435 lung cells, and NUGC3 upper GI cells. In various aspects, the antigen binding protein inhibits the binding interaction between human CLDN6 expressed by the cell and a reference antibody that is known to bind CLDN6 but is not an antigen binding protein of the present disclosure. In various cases, the antigen binding protein of the present disclosure competes with the reference antibody for binding to human CLDN6 and thereby reduces the amount of human CLDN6 bound to the reference antibody as determined by an in vitro competitive binding assay. In various aspects, the antigen binding protein of the present disclosure inhibits the binding interaction between human CLDN6 and the reference antibody, and the inhibition is determined by an IC 50Characterization. In various aspects, the antigen binding protein exhibits an IC of less than about 2500 nM for inhibiting the binding interaction between human CLDN6 and a reference antibody 50 In various aspects, the antigen binding protein exhibits an IC of less than about 2000 nM, less than about 1500 nM, less than about 1000 nM, less than about 900 nM, less than about 800 nM, less than about 700 nM, less than about 600 nM, less than about 500 nM, less than about 400 nM, less than about 300 nM, less than about 200 nM, or less than about 100 nM. 50 In various aspects, the antigen binding protein exhibits an IC of less than about 90 nM, less than about 80 nM, less than about 70 nM, less than about 60 nM, less than about 50 nM, less than about 40 nM, less than about 30 nM, less than about 20 nM, or less than about 10 nM. 50 In various cases, the antigen binding proteins of the present disclosure compete for binding to CLDN6 with a reference antibody known to bind CLDN6 (the reference antibody being different from any antigen binding protein of the present disclosure). See further description under competition assays.

[0066] Avidity provides a measure of the overall strength of the antibody-antigen complex. It depends on three main parameters: the affinity of the antigen-binding protein for the epitope, the valency of both the antigen-binding protein and CLDN6, and the structural arrangement of the interacting parts. The greater the valency (number of antigen-binding sites) of the antigen-binding protein, the greater the amount of antigen (CLDN6) it can bind. In various aspects, the antigen-binding protein has a strong affinity for CLDN6. In various aspects, the antigen-binding protein is multivalent. In various aspects, the antigen-binding protein is bivalent. In various cases, the antigen-binding protein is monovalent.

[0067] Cross-reactivity

[0068] In various embodiments, the antigen binding proteins of the present disclosure bind to CLDN6 and do not bind to, e.g., do not cross-react with, any other member of the CLDN family. In various cases, the antigen binding proteins of the present disclosure are specific for CLDN-6. In various embodiments, the selectivity of the antigen binding proteins of the present disclosure for CLDN6 is at least 10-fold, 5-fold, 4-fold, 3-fold, or 2-fold greater than the selectivity of the antigen binding proteins for CLDN3, CLDN4, CLDN9, or a combination thereof. In various embodiments, the selectivity of the antigen binding proteins of the present disclosure for CLDN6 is at least 10-fold, 5-fold, 4-fold, 3-fold, or 2-fold greater than the selectivity of the antigen binding proteins for each of CLDN3, CLDN4, and CLDN9. Selectivity can be based on the K values ​​exhibited by the antigen binding proteins for CLDN6 or a member of the CLDN family. D , where K DIt can be determined by techniques known in the art, such as surface plasmon resonance, FACS-based affinity assays.

[0069] In various aspects, the antigen binding proteins of the present disclosure bind to CLDN6 and do not bind to any of claudin 3 (CLDN3), claudin 4 (CLDN4), and claudin 9 (CLDN9). In various aspects, the antigen binding proteins do not bind to any of CLDN3, CLDN4, and CLDN9 and exhibit an IC of less than about 1200 nM (e.g., less than about 1000 nM, less than about 750 nM, less than about 500 nM, less than about 250 nM) in a FACS-based assay using OVCA429 cells that endogenously express CLDN6. 50 In various aspects, the antigen binding protein does not bind to any of CLDN3, CLDN4, and CLDN9, and the concentration at which 50% binding saturation is achieved with OVCA429 cells that endogenously express CLDN6 is less than about 1200 nM (e.g., less than about 1000 nM, less than about 750 nM, less than about 500 nM, less than about 250 nM). In various aspects, the antigen binding protein is at least 5-fold more selective for CLDN6 than for CLDN3, CLDN4, and CLDN9, and the concentration at which 50% binding saturation is achieved with OVCA429 cells that endogenously express CLDN6 is less than about 1200 nM (e.g., less than about 1000 nM, less than about 750 nM, less than about 500 nM, less than about 250 nM). In various aspects, the antigen binding protein exhibits an IC50 of less than about 1200 nM (e.g., less than about 1000 nM, less than about 750 nM, less than about 500 nM, less than about 250 nM) for artificial and endogenous models of CLDN6 and exhibits a ratio that separates the CLDN6 IC50 from CLDN3, CLDN4, and / or CLDN9 by greater than about 5-fold. In various cases, the antigen binding protein exhibits an IC50 of less than about 1200 nM (e.g., less than about 1000 nM, less than about 750 nM, less than about 500 nM, less than about 250 nM) for CLDN6 and an IC50 of at least 5-fold greater than the IC50 for any of CLDN3, CLDN4, and CLDN9.

[0070] In various embodiments, the antigen-binding proteins of the present disclosure bind to CLDN6 and cross-react (e.g., bind) with at least one other member of the CLDN family. In various aspects, the antigen-binding proteins of the present disclosure bind to CLDN6 and one or more of CLDN3, CLDN4, and CLDN9. In various aspects, the antigen-binding proteins of the present disclosure bind to CLDN6 and CLDN4 or CLDN9, but do not bind to CLDN3. In various cases, the antigen-binding proteins of the present disclosure bind to CLDN6 and CLDN4, but do not bind to CLDN3 or CLDN9. In various cases, the antigen-binding proteins of the present disclosure bind to CLDN6 and CLDN9, but do not bind to CLDN3 or CLDN4.

[0071] Competition assay

[0072] In various embodiments, the antigen binding protein inhibits the binding interaction between human CLDN6 and a reference antibody that is known to bind to CLDN6 but is not an antigen binding protein of the present disclosure. In various cases, the antigen binding protein of the present disclosure competes with the reference antibody for binding to human CLDN6 and thereby reduces the amount of human CLDN6 bound to the reference antibody as determined by an in vitro competitive binding assay. In various embodiments, the reference antibody binds to an epitope within the amino acid sequence of the extracellular domain of human CLDN6 (optionally within EL2 or EL1). In various aspects, the reference antibody comprises a light chain variable sequence encoded by SEQ ID NO: 179 and a heavy chain variable sequence encoded by SEQ ID NO: 180. In various aspects, the reference antibody comprises a light chain variable sequence of SEQ ID NO: 181 and a heavy chain variable sequence of SEQ ID NO: 182. In various aspects, the antigen binding protein of the present disclosure inhibits the binding interaction between human CLDN6 and the reference antibody, and the inhibition is determined by IC 50 Characterization. In various aspects, the antigen binding protein exhibits an IC of less than about 2500 nM for inhibiting the binding interaction between human CLDN6 and a reference antibody 50 In various aspects, the antigen binding protein exhibits an IC of less than about 2000 nM, less than about 1500 nM, less than about 1000 nM, less than about 900 nM, less than about 800 nM, less than about 700 nM, less than about 600 nM, less than about 500 nM, less than about 400 nM, less than about 300 nM, less than about 200 nM, or less than about 100 nM. 50 In various aspects, the antigen binding protein exhibits an IC of less than about 90 nM, less than about 80 nM, less than about 70 nM, less than about 60 nM, less than about 50 nM, less than about 40 nM, less than about 30 nM, less than about 20 nM, or less than about 10 nM. 50 .

[0073] In various cases, the antigen-binding proteins of the present disclosure compete with a reference antibody for binding to human CLDN6, thereby reducing the amount of human CLDN6 bound to the reference antibody, as determined by an in vitro competitive binding assay. In various aspects, the in vitro competitive binding assay is a FACS-based assay in which the fluorescence of a fluorophore-conjugated secondary antibody that binds to the Fc of the reference antibody is measured in the absence or presence of a specified amount of an antigen-binding protein of the present disclosure. Such FACS-based assays are described in the Examples herein. In various aspects, a FACS-based assay is performed using a reference antibody, a fluorophore-conjugated secondary antibody, and cells expressing CLDN6. In various aspects, the cells are genetically engineered to overexpress CLDN6. In some aspects, the cells are HEK293T cells transduced with a viral vector to express CLDN6. In alternative aspects, the cells endogenously express CLDN6. Prior to performing the FACS-based assay, in some aspects, cells endogenously expressing CLDN6 are pre-determined as low CLDN6-expressing cells or high CLDN6-expressing cells. In some aspects, the cells are cancer cells or tumor cells. In various aspects, the cell is a cell from a cell line, such as an ovarian cell line, an endometrial cell line, a bladder cell line, a lung cell line, a gastrointestinal (GI) cell line, a liver cell line, a lung cell line, etc. In various aspects, the cell endogenously expressing CLDN6 is selected from the group consisting of: OVCA429 ovarian cells, ARK2 endometrial cells, OAW28 ovarian cells, UMUC-4 bladder cells, PEO14 ovarian cells, OV177 ovarian cells, H1693 lung cells, MKN7 upper GI cells, OV-90 ovarian cells, HUH-7 liver cells, JHOS-4 ovarian cells, H1435 lung cells, and NUGC3 upper GI cells. In various cases, the antigen binding proteins of the present disclosure bind with high affinity to CLDN6 endogenously expressed by one or more of ARK2 cells, OVCA429 cells, LS513 cells, or MCF7 cells. In various aspects, the antigen binding proteins exhibit an IC of less than about 3000 nM. 50 , as determined in a FACS-based competitive binding inhibition assay using one or more of ARK2 cells, OVCA429 cells, LS513 cells, or MCF7 cells. In various aspects, the antigen binding protein exhibits an IC of less than about 2500 nM, less than about 2000 nM, less than about 1750 nM, less than about 1500 nM, less than about 1250 nM, less than about 1000 nM, less than about 750 nM, or less than about 500 nM. 50, as determined in a FACS-based competitive binding inhibition assay using one or more of ARK2 cells, OVCA429 cells, LS513 cells, or MCF7 cells. In various aspects, the antigen binding protein exhibits an IC of less than about 400 nM, less than about 300 nM, less than about 200 nM, less than about 100 nM, less than about 75 nM, less than about 50 nM, less than about 25 nM, or less than about 10 nM. 50 , as determined in a FACS-based competitive binding inhibition assay using one or more of ARK2 cells, OVCA429 cells, LS513 cells, or MCF7 cells.

[0074] Other binding assays (e.g., competitive binding assays or competitive assays) for the ability of a test antibody to compete with a second antibody for binding to an antigen or its epitope are known in the art. See, for example, Trikha et al., Int J Cancer 110:326-335 (2004); Tam et al., Circulation 98(11):1085-1091(1998); U.S. Patent Application Publication No. US20140178905; Chand et al., Biologicals 46:168-171(2017); Liu et al., Anal Biochem 525:89-91(2017); and Goolia et al., J Vet Diagn Invest 29(2):250-253(2017). Similarly, other methods for comparing two antibodies are known in the art and include, for example, surface plasmon resonance (SPR). SPR can be used to determine the binding constant of an antibody and a second antibody, and the two binding constants can be compared.

[0075] Antibody production methods and related methods

[0076] Suitable methods for preparing antigen-binding proteins (e.g., antibodies, antigen-binding antibody fragments, and antibody protein products) are known in the art. For example, standard hybridoma methods for producing antibodies are described in, for example, Harlow and Lane (eds.), Antibodies: A Laboratory Manual, CSH Press (1988) and CA. Janeway et al. (eds.), Immunobiology, 5th edition, Garland Publishing, New York, NY (2001). Various methods for preparing the CLDN6 monoclonal antibodies disclosed herein are provided in the Examples.

[0077] Depending on the host species, various adjuvants can be used to increase the immune response, thereby causing the host to produce more antibodies. Such adjuvants include, but are not limited to, Freund's adjuvant, mineral gels (such as aluminum hydroxide), and surfactants (such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, and dinitrophenol). BCG (bacillus Calmette-Guérin) and Corynebacterium parvum are potentially useful human adjuvants.

[0078] Other methods of antibody production are summarized in Table 1.

[0079] Table 1

[0080]

[0081] Regardless of how the antibody is produced, methods for testing the ability of an antibody to bind to a CLDN6 epitope are known in the art and include any antibody-antigen binding assay, such as radioimmunoassay (RIA), ELISA, Western blot, immunoprecipitation, SPR, and competitive inhibition assays (see, e.g., Janeway et al., infra, and U.S. Patent Application Publication No. 2002 / 0197266 and the above section on competition assays).

[0082] Sequence / Structure

[0083] Provided herein are antigen binding proteins comprising: (a) a heavy chain (HC) complementarity determining region (CDR) 1 amino acid sequence listed in Table A, or a sequence selected from the group consisting of SEQ ID NO: 11, 17, 23, 29, 35, 41, 47, 53, 59, 65, 71, 77, 83, 89, 95, 101, 107, 113, 119, 125, and 131, or variant sequences thereof that differ by only one or two amino acids or 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; (b) a HC CDR2 amino acid sequence listed in Table A, or a sequence selected from the group consisting of SEQ ID NO: 11, 17, 23, 29, 35, 41, 47, 53, 59, 65, 71, 77, 83, 89, 95, 101, 107, 113, 119, 125, and 131. NO: 12, 18, 24, 30, 36, 42, 48, 54, 60, 66, 72, 78, 84, 90, 86, 102, 108, 114, 120, 126 and 132, or variant sequences thereof that differ by only one or two amino acids or 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; (c) an HC CDR3 amino acid sequence listed in Table A or a sequence selected from the group consisting of: SEQ ID NO: 12, 18, 24, 30, 36, 42, 48, 54, 60, 66, 72, 78, 84, 90, 86, 102, 108, 114, 120, 126 and 132, or variant sequences thereof that differ by only one or two amino acids or 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; NO: 13, 19, 25, 31, 37, 43, 49, 55, 61, 67, 73, 79, 85, 91, 97, 103, 109, 115, 121, 127 and 133, or variant sequences thereof that differ by only one or two amino acids or 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; (d) a light chain (LC) CDR1 amino acid sequence listed in Table A or a sequence selected from the group consisting of: SEQ ID NO: 13, 19, 25, 31, 37, 43, 49, 55, 61, 67, 73, 79, 85, 91, 97, 103, 109, 115, 121, 127 and 133, or variant sequences thereof that differ by only one or two amino acids or 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; NO: 8, 14, 20, 32, 38, 44, 50, 56, 62, 68, 74, 80, 86, 92, 98, 104, 110, 116, 122 and 128, or variant sequences thereof that differ by only one or two amino acids or 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; (e) an LC CDR2 amino acid sequence listed in Table A or a sequence selected from the group consisting of: SEQ ID NO: 8, 14, 20, 32, 38, 44, 50, 56, 62, 68, 74, 80, 86, 92, 98, 104, 110, 116, 122 and 128, or variant sequences thereof that differ by only one or two amino acids or 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; ID NO: 9, 15, 21, 27, 33, 39, 45, 51, 57, 63, 69, 75, 81, 87, 93, 99, 105, 111, 117, 123 and 129, or variant sequences thereof that differ by only one or two amino acids or have at least or about 70% (e.g., at least about 80%, at least about 85%, at least about 90%, at least about 95%) sequence identity;(f) an LC CDR3 amino acid sequence listed in Table A or a sequence selected from the group consisting of SEQ ID NO: 10, 16, 22, 28, 34, 40, 46, 52, 58, 64, 70, 76, 82, 88, 94, 100, 106, 112, 118, 124 and 130, or a variant thereof that differs by only one or two amino acids or has at least or about 70% (e.g., at least about 80%, at least about 85%, at least about 90%, at least about 95%) sequence identity; or a combination of any two or more of (g) (a) to (f).

[0084] Table A

[0085]

[0086] In various aspects, the antigen binding protein comprises at least one or two of the LC CDR1 amino acid sequence, LC CDR2 amino acid sequence, and LC CDR3 amino acid sequence listed in Table A, and the HC CDR amino acid sequences listed in Table A. In various aspects, the antigen binding protein comprises at least one or two of the HC CDR1 amino acid sequence, HC CDR2 amino acid sequence, and HC CDR3 amino acid sequence listed in Table A, and the LC CDR amino acid sequences listed in Table A.

[0087] In various embodiments, the antigen binding protein comprises at least 3, 4, or 5 of the amino acid sequences specified by SEQ ID NO: in a single row of Table A. In various embodiments, the antigen binding protein comprises each of the LC CDR amino acid sequences specified by SEQ ID NO: in a single row of Table A and at least 1 or 2 of the HC CDR amino acid sequences specified by SEQ ID NO: in a single row of Table A. In various embodiments, the antigen binding protein comprises each of the HC CDR amino acid sequences specified by SEQ ID NO: in a single row of Table A and at least 1 or 2 of the LC CDR amino acid sequences specified by SEQ ID NO: in a single row of Table A. In various embodiments, the antigen binding protein comprises all 6 of the CDR amino acid sequences specified by SEQ ID NO: in a single row of Table A. In various embodiments, the antigen binding protein comprises six CDR amino acid sequences selected from the group consisting of: (a) SEQ ID NOs: 74-79; (b) SEQ ID NOs: 50-55; (c) SEQ ID NOs: 122-127; (d) SEQ ID NOs: 26-31; (e) SEQ ID NOs: 128-133; (f) SEQ ID NOs: 38-43; (g) SEQ ID NOs: 62-67; (h) SEQ ID NOs: 80-85; (i) SEQ ID NOs: 44-49; (j) SEQ ID NOs: 86-91; (k) SEQ ID NOs: 104-109; (l) SEQ ID NOs: 56-61; (m) SEQ ID NOs: 32-37; (n) SEQ ID NOs: 110-115; (o) SEQ ID NOs: 98-103; (p) SEQ ID NOs: 92-97; (q) SEQ ID NOs: NO:116-121; (r) SEQ ID NO:8-13; (s) SEQ ID NO:68-73; (t) SEQ ID NO:14-19; and (u) SEQ ID NO:20-25.

[0088] In each case, the amino acid sequences of Table A are separated by at least one or more (e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) intervening amino acids. In each case, there are about 10 to about 20 amino acids between the LC CDR1 and LC CDR2 sequences, and about 25 to about 40 amino acids between the LC CDR2 and LC CDR3 sequences. In each case, there are about 14 to about 16 amino acids between the LC CDR1 and LC CDR2 sequences, and about 30 to about 35 amino acids between the LC CDR2 and LC CDR3 sequences. In each case, there are about 10 to about 20 amino acids between the HC CDR1 and HC CDR2 sequences, and about 25 to about 40 amino acids between the HC CDR2 and HC CDR3 sequences. In each case, there are about 14 to about 16 amino acids between the HC CDR1 and HC CDR2 sequences, and about 30 to about 35 amino acids between the HC CDR2 and HC CDR3 sequences.

[0089] In various embodiments, the antigen binding protein comprises (a) a heavy chain variable region amino acid sequence listed in Table B, or a sequence selected from the group consisting of SEQ ID NO: 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, and 175, or variant sequences thereof that differ by only one or two amino acids or have at least or about 70% (e.g., at least about 80%, at least about 85%, at least about 90%, at least about 95%) sequence identity; or (b) a light chain variable region amino acid sequence listed in Table B, or a sequence selected from the group consisting of SEQ ID NO: 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, and 175. ID NO: 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174 and 176, or variant sequences thereof that differ only by one or two amino acids or have at least or about 70% (e.g., at least about 80%, at least about 85%, at least about 90%, at least about 95%) sequence identity; or (c) both of (a) and (b).

[0090] Table B

[0091] Light chain variable region Heavy chain variable region AB1 134 135 AB2 136 137 AB3 138 139 AB4 140 141 AB5 142 143 AB6 144 145 AB7 146 147 AB8 148 149 AB9 150 151 AB10 152 153 AB11 154 155 AB12 156 157 AB13 158 159 AB14 160 161 AB15 162 163 AB16 164 165 AB17 166 167 AB18 168 169 AB19 170 171 AB20 172 173 AB21 174 175

[0092] In various embodiments, the antigen binding protein comprises a pair of amino acid sequences selected from the group consisting of: (a) SEQ ID NOs: 156 and 157; (b) SEQ ID NOs: 148 and 149; (c) SEQ ID NOs: 172 and 173; (d) SEQ ID NOs: 140 and 141; (e) SEQ ID NOs: 174 and 175; (f) SEQ ID NOs: 144 and 145; (g) SEQ ID NOs: 152 and 153; (h) SEQ ID NOs: 158 and 159; (i) SEQ ID NOs: 146 and 147; (j) SEQ ID NOs: 160 and 161; (k) SEQ ID NOs: 166 and 167; (l) SEQ ID NOs: 150 and 151; (m) SEQ ID NOs: 142 and 143; (n) SEQ ID NOs: 168 and 169; (o) SEQ ID NOs: 164 and 165; (p) SEQ ID NOs: 162 and 163; (q) SEQ ID NOs: 170 and 171; (r) SEQ ID NOs: 134 and 135; (s) SEQ ID NOs: 154 and 155; (t) SEQ ID NOs: 136 and 137; and (u) SEQ ID NOs: 138 and 139.

[0093] In various aspects, the antigen binding protein does not comprise the pair of amino acid sequences encoded by the sequences of SEQ ID NOs: 179 and 180. In various aspects, the antigen binding protein does not comprise the pair of amino acid sequences of SEQ ID NOs: 181 and 182. In various aspects, the antigen binding protein does not comprise the pair of amino acid sequences encoded by the sequences of SEQ ID NOs: 183 and 184. In various aspects, the antigen binding protein does not comprise the pair of amino acid sequences of SEQ ID NOs: 185 and 186.

[0094] In various aspects, the antigen binding protein comprises an amino acid sequence similar to the above amino acid sequence, but the antigen binding protein substantially retains its biological function, such as its ability to bind to human CLDN6, reduce tumor growth, and treat cancer.

[0095] In various aspects, antigen-binding proteins include amino acid sequences that only differ by 1, 2, 3, 4, 5, 6 or more amino acids relative to the above-mentioned one or more amino acid sequences. In various aspects, antigen-binding proteins include variant sequences of the sequence, which only differ by one or two amino acids relative to the sequence. In various aspects, antigen-binding proteins include one or more amino acid replacements that occur outside CDR, for example, one or more amino acid replacements that occur in one or more framework regions of heavy or light chain. In various aspects, antigen-binding proteins include one or more amino acid replacements, but the antigen-binding proteins retain the amino acid sequence of six CDRs. In various aspects, antigen-binding proteins include amino acid sequences that only have 1, 2, 3, 4, 5, 6 or more conservative amino acid replacements relative to the above-mentioned amino acid sequences. As used herein, the term "conservative amino acid replacement" refers to that a kind of amino acid is replaced by another amino acid with similar properties (for example, size, charge, hydrophobicity, hydrophilicity and / or aromaticity), and is included in the exchange within one of the following five groups:

[0096] I. Small aliphatic, non-polar or weakly polar residues:

[0097] Ala, Ser, Thr, Pro, Gly;

[0098] II. Polar, negatively charged residues and their amides and esters:

[0099] Asp, Asn, Glu, Gln, cysteic acid, and homocysteic acid;

[0100] III. Polar, positively charged residues:

[0101] His, Arg, Lys; Ornithine (Orn)

[0102] IV. Large aliphatic, nonpolar residues:

[0103] Met, Leu, Ile, Val, Cys, norleucine (Nle), homocysteine

[0104] V. Large aromatic residues:

[0105] Phe, Tyr, Trp, acetylphenylalanine

[0106] In various aspects, conservative amino acid substitutions are exchanges within one of the following amino acid groups:

[0107] I. Aliphatic amino acids: Gly, Ala, Val, Leu, Ile

[0108] II. Non-aromatic amino acids containing side chain hydroxyl groups: Serc, Thr

[0109] III. Amino acids containing sulfur side chains: Cys, Met

[0110] IV: Amino acids containing aromatic side chains: Phe, Tyr, Trp

[0111] V: acidic amino acids: Glu; Asp

[0112] VI: basic amino acids: Arg; Lys

[0113] VII: Amino acids containing side chain amides: Gln, Asn

[0114] VIII: Amino acids containing side chain imidazole: His, α-dimethylimidazoleacetic acid (DMIA)

[0115] IX: Imino acids: Pro, 4-hydroxy-Pro, 4-amino-Pro

[0116] In various aspects, the antigen binding protein comprises an amino acid sequence having greater than or about 30%, greater than or about 50%, or greater than or about 70% sequence identity to the above amino acid sequence. In various aspects, the antigen binding protein comprises an amino acid sequence having at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, or greater than 90% sequence identity to the above amino acid sequence. In various aspects, the antigen binding protein comprises an amino acid sequence having at least 70%, at least 80%, at least 85%, at least 90%, or greater than 90% sequence identity along the full length of the above 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 amino acid sequence.

[0117] In various aspects, the antigen binding protein comprises a variant sequence of the sequence, the variant sequence having at least or about 70% sequence identity relative to the above sequence. In various aspects, the antigen binding protein comprises a variant sequence of the sequence, the variant sequence having at least or about 80% sequence identity relative to the above sequence. In various aspects, the antigen binding protein comprises a variant sequence of the sequence, the variant sequence having at least or about 90% sequence identity relative to the above sequence. In various aspects, the antigen binding protein comprises a variant sequence of the sequence, the variant sequence having at least or about 95% sequence identity relative to the above sequence.

[0118] In various embodiments, the antigen binding protein comprises one, two, three, four or five sequences of SEQ ID NOs in a single row of Table A 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 one of SEQ ID NOs: 8-133. In various embodiments, the antigen binding protein comprises one, two, three, four or five sequences selected from the group consisting of: (a) SEQ ID NOs: 74-79; (b) SEQ ID NOs: 50-55; (c) SEQ ID NOs: 122-127; (d) SEQ ID NOs: 26-31; (e) SEQ ID NOs: 128-133; (f) SEQ ID NOs: 38-43; (g) SEQ ID NOs: 62-67; (h) SEQ ID NOs: 80-85; (i) SEQ ID NOs: 44-49; (j) SEQ ID NOs: 86-91; (k) SEQ ID NOs: 104-109; (l) SEQ ID NOs: 56-61; (m) SEQ ID NOs: 32-37; (n) SEQ ID NOs: 110-115; (o) SEQ ID NOs: 98-103; (p) SEQ ID NOs: 92-97; (q) SEQ ID NO:116-121; (r) SEQ ID NO:8-13; (s) SEQ ID NO:68-73; (t) SEQ ID NO:14-19; and (u) SEQ ID NO:20-25, wherein the antigen binding protein further comprises a variant sequence having at least or about 70% (e.g., at least about 80%, at least about 85%, at least about 90%, at least about 95%) sequence identity with at least one of the group of sequences. For example, in various aspects, the antigen binding protein comprises four sequences of SEQ ID NOs:74-79, i.e., SEQ ID NOs:74-77, wherein the antigen binding protein comprises two variant sequences: one variant sequence 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 to SEQ ID NO:78 and the other variant sequence 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 to SEQ ID NO:79.

[0119] In various embodiments, the antigen binding protein comprises a pair of variant sequences having at least or about 70% (e.g., at least about 80%, at least about 85%, at least about 90%, at least about 95%) sequence identity to any one of SEQ ID NOs: 134-175. In various cases, 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: (a) SEQ ID NOs: 156 and 157; (b) SEQ ID NOs: 148 and 149; (c) SEQ ID NOs: 172 and 173; (d) SEQ ID NOs: 140 and 141; (e) SEQ ID NOs: 174 and 175; (f) SEQ ID NOs: 144 and 145; (g) SEQ ID NOs: 152 and 153; (h) SEQ ID NOs: 158 and 159; (i) SEQ ID NOs: 146 and 147; (j) SEQ ID NOs: 160 and 161; (k) SEQ ID NOs: 166 and 167; (l) SEQ ID NOs: 150 and 151; (m) SEQ ID NOs: 151 and 152; NO: 142 and 143; (n) SEQ ID NO: 168 and 169; (o) SEQ ID NO: 164 and 165; (p) SEQ ID NO: 162 and 163; (q) SEQ ID NO: 170 and 171; (r) SEQ ID NO: 134 and 135; (s) SEQ ID NO: 154 and 155; (t) SEQ ID NO: 136 and 137; and (u) SEQ ID NO: 138 and 139. In various embodiments, the antigen binding protein comprises a pair of sequences: a sequence of Table B and another sequence that is a variant sequence having at least or about 70% (e.g., at least about 80%, at least about 85%, at least about 90%, at least about 95%) sequence identity to any one of SEQ ID NO: 134-175.In various embodiments, the antigen binding protein comprises a pair of sequences: a sequence selected from the group consisting of: (a) SEQ ID NOs: 156 and 157; (b) SEQ ID NOs: 148 and 149; (c) SEQ ID NOs: 172 and 173; (d) SEQ ID NOs: 140 and 141; (e) SEQ ID NOs: 174 and 175; (f) SEQ ID NOs: 144 and 145; (g) SEQ ID NOs: 152 and 153; (h) SEQ ID NOs: 158 and 159; (i) SEQ ID NOs: 146 and 147; (j) SEQ ID NOs: 160 and 161; (k) SEQ ID NOs: 166 and 167; (l) SEQ ID NOs: 150 and 151; (m) SEQ ID NOs: 142 and 143; (n) SEQ ID NOs: 168 and 169; (o) SEQ ID NOs: 164 and 165; (p) SEQ 135; (s) SEQ ID NOs: 154 and 155; (t) SEQ ID NOs: 136 and 137; and (u) SEQ ID NOs: 138 and 139, and another sequence that is a variant sequence having at least or about 70% (e.g., at least about 80%, at least about 85%, at least about 90%, at least about 95%) sequence identity to the sequence of (a)-(u). For example, in various aspects, the antigen binding protein comprises the sequence of SEQ ID NO: 134, and the antigen binding protein further comprises a variant sequence having at least or about 70% (e.g., at least about 80%, at least about 85%, at least about 90%, at least about 95%) sequence identity to SEQ ID NO 135.

[0120] In various cases, the antigen binding protein comprises an amino acid sequence of the above amino acid sequence with one or more amino acid substitutions to reduce or eliminate reactive amino acids, thereby reducing or preventing undesirable side chain reactions. For example, the antigen binding protein comprises an amino acid sequence of the above amino acid sequence with one or more of the following: (i) Trp residues are substituted with His, Tyr, or Phe; (ii) Asn residues are substituted with Gln, Ser, Ala, or Asp; (iii) Asp residues immediately preceding Pro residues are substituted with Ala, Ser, or Glu; (iv) Asn residues are substituted with Gln, Ser, or Ala; and / or (v) Cys residues are substituted with Tyr, Ser, or Ala. In various aspects, the antigen binding protein comprises an amino acid sequence of the above amino acid sequence with such amino acid substitutions that are predicted to have higher binding affinity, higher stability, or other positive attributes based on SHM events or based on statistical analysis of a large number of other similar antibody sequences.In some aspects, the antigen binding protein comprises (a) an HC CDR1 amino acid sequence listed in Table A1, or a sequence selected from the group consisting of SEQ ID NOs: 452, 455, 461, 465, 71, and 472, or variant sequences thereof that differ by only one or two amino acids or 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; (b) an HC CDR2 amino acid sequence listed in Table A1, or a sequence selected from the group consisting of SEQ ID NOs: 475, 456, 462, 466, 468, and 473, or variant sequences thereof that differ by only one or two amino acids or 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; (c) an HC CDR3 amino acid sequence listed in Table A1, or a sequence selected from the group consisting of SEQ ID NOs: NO: 453, 457, 463, 467, 469 and 474, or variant sequences thereof that differ by only one or two amino acids or 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; (d) the LC CDR1 amino acid sequences listed in Table A1, or a sequence selected from the group consisting of SEQ ID NO: 449, 476, 458, 464, 68 and 470, or variant sequences thereof that differ by only one or two amino acids or 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; (e) the LC CDR2 amino acid sequences listed in Table A1, or a sequence selected from the group consisting of SEQ ID NO: 449, 476, 458, 464, 68 and 470, or variant sequences thereof that differ by only one or two amino acids or 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. NO: 450, 477, 459, 57, 69 and 471, or variant sequences thereof that differ by only one or two amino acids or 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; (f) an LC CDR3 amino acid sequence listed in Table A1 or a sequence selected from the group consisting of SEQ ID NO: 451, 454, 460, 58, 70 and 112, or variant sequences thereof that differ by only one or two amino acids or have at least or about 70% (e.g., at least about 80%, at least about 85%, at least about 90%, at least about 95%) sequence identity, or a combination of any two or more of (g) (a) to (f).

[0121] Table A1

[0122]

[0123] In some aspects, the HC CDR1 comprises a Gly immediately N-terminus of SEQ ID NO: 452, and optionally, in some aspects, the HC CDR1 comprises MX immediately C-terminus of SEQ ID NO: 452, wherein X is H, N, or S. In various aspects, the HC CDR3 comprises an Ala immediately N-terminus of SEQ ID NO: 453. In various aspects, the LC CDR1 further comprises a TAS immediately N-terminus of SEQ ID NO: 449, and optionally, an XH immediately C-terminus of SEQ ID NO: 449, wherein X is H, S, Y, or Q. In some aspects, as described below, the first amino acid of SEQ ID NO: 449 is S or Q. In some aspects, as described below, the first amino acid of SEQ ID NO: 451 is S or Q.

[0124] In various aspects, the HC CDR1 comprises a Gly immediately N-terminus of SEQ ID NO: 455, and optionally, in various aspects, the HC CDR1 comprises an MX immediately C-terminus of SEQ ID NO: 455, wherein X is N, S, or H. In some aspects, the HC CDR2 comprises a Gln immediately N-terminus of SEQ ID NO: 456, and optionally, an H immediately C-terminus of SEQ ID NO: 456. In various aspects, the LC CDR1 comprises an RIS immediately N-terminus of SEQ ID NO: 476, and optionally, an LA immediately C-terminus of SEQ ID NO: 476. In various aspects, the LC CDR2 comprises an XLVE immediately C-terminus of SEQ ID NO: 477, wherein X is I or S.

[0125] In various aspects, the HC CDR1 comprises an MH immediately adjacent to the C-terminus of SEQ ID NO: 461. In various aspects, the HC CDR2 comprises a Tyr immediately adjacent to the N-terminus of SEQ ID NO: 462 and optionally comprises a TH immediately adjacent to the C-terminus of SEQ ID NO: 462. In exemplary aspects, the HC CDR3 does not include the first two amino acids of SEQ ID NO: 463. In various aspects, the LC CDR1 comprises an RSS immediately adjacent to the N-terminus of SEQ ID NO: 458 and optionally an LN immediately adjacent to the C-terminus of SEQ ID NO: 458. In various aspects, the LC CDR2 comprises an XRFS immediately adjacent to the C-terminus of SEQ ID NO: 459, wherein X is Q, S, A, or D.

[0126] In various aspects, the HC CDR1 comprises MH immediately adjacent to the C-terminus of SEQ ID NO: 465. In various aspects, the HC CDR2 comprises YI immediately adjacent to the N-terminus of SEQ ID NO: 466 and optionally comprises Xaa immediately adjacent to the C-terminus of SEQ ID NO: 466, wherein Xaa is N, S, Q, or A. In various aspects, the LC CDR1 comprises LAS immediately adjacent to the N-terminus of SEQ ID NO: 464 and optionally LA immediately adjacent to the C-terminus of SEQ ID NO: 464. In various aspects, the LC CDR2 comprises SLAD immediately adjacent to the C-terminus of SEQ ID NO: 57.

[0127] In various aspects, the HC CDR1 comprises MH immediately adjacent to the C-terminus of SEQ ID NO: 71. In various aspects, the HC CDR2 comprises Tyr immediately adjacent to the N-terminus of SEQ ID NO: 468, and optionally comprises IY immediately adjacent to the C-terminus of SEQ ID NO: 468. In various aspects, the LC CDR1 comprises RAS immediately adjacent to the N-terminus of SEQ ID NO: 68, and optionally comprises SYIH immediately adjacent to the C-terminus of SEQ ID NO: 68. In various aspects, the LC CDR2 comprises XLES immediately adjacent to the C-terminus of SEQ ID NO: 69, wherein X is N, Q, S, A, or D.

[0128] In various aspects, the LC CDR1 comprises KSS of SEQ ID NO: 470 immediately N-terminus and YLA of SEQ 470 immediately C-terminus. In various aspects, the LC CDR2 comprises TRES of SEQ ID NO: 471 immediately C-terminus. In various aspects, the HC CDR1 comprises MN of SEQ ID NO: 472 immediately C-terminus. In various aspects, the HC CDR2 comprises Xaa of SEQ 473 immediately N-terminus, wherein Xaa is N, Q, S, or A; and optionally Thr of SEQ 473 immediately C-terminus.

[0129] In various aspects, the antigen binding protein comprises at least one or two of the LC CDR1 amino acid sequence, LC CDR2 amino acid sequence, and LC CDR3 amino acid sequence listed in Table A1, and the HC CDR amino acid sequences listed in Table A1. In various aspects, the antigen binding protein comprises at least one or two of the HC CDR1 amino acid sequence, HC CDR2 amino acid sequence, and HC CDR3 amino acid sequence listed in Table A1, and the LC CDR amino acid sequences listed in Table A1.

[0130] In various embodiments, the antigen binding protein comprises at least 3, 4, or 5 of the amino acid sequences specified by SEQ ID NO: in a single row of Table A1. In various embodiments, the antigen binding protein comprises each of the LC CDR amino acid sequences specified by SEQ ID NO: in a single row of Table A1 and at least 1 or 2 of the HC CDR amino acid sequences specified by SEQ ID NO: in a single row of Table A1. In various embodiments, the antigen binding protein comprises each of the HC CDR amino acid sequences specified by SEQ ID NO: in a single row of Table A1 and at least 1 or 2 of the LC CDR amino acid sequences specified by SEQ ID NO: in a single row of Table A1. In various embodiments, the antigen binding protein comprises all 6 of the CDR amino acid sequences specified by SEQ ID NO: in a single row of Table A1. In various embodiments, the antigen binding protein comprises six CDR amino acid sequences selected from the group consisting of: (a) SEQ ID NOs: 449-453 and 475; (b) SEQ ID NOs: 476-477, 454-457; (c) SEQ ID NOs: 458-463; (d) SEQ ID NOs: 57, 58, 464-467; (e) SEQ ID NOs: 68-71 and 468-469; and (f) SEQ ID NOs: 112 and 470-474.

[0131] In each case, the amino acid sequences of Table A1 are separated by at least one or more (e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) intervening amino acids. In each case, there are about 10 to about 20 amino acids between the LC CDR1 and LC CDR2 sequences, and about 25 to about 40 amino acids between the LC CDR2 and LC CDR3 sequences. In each case, there are about 14 to about 16 amino acids between the LC CDR1 and LC CDR2 sequences, and about 30 to about 35 amino acids between the LC CDR2 and LC CDR3 sequences. In each case, there are about 10 to about 20 amino acids between the HC CDR1 and HC CDR2 sequences, and about 25 to about 40 amino acids between the HC CDR2 and HC CDR3 sequences. In each case, there are about 14 to about 16 amino acids between the HC CDR1 and HC CDR2 sequences, and about 30 to about 35 amino acids between the HC CDR2 and HC CDR3 sequences.

[0132] In various embodiments, the antigen binding protein comprises (a) a heavy chain variable region amino acid sequence listed in Table B1, or a sequence selected from the group consisting of SEQ ID NOs: 478, 480, 482, 484, 486, and 488, or variant sequences thereof that differ by only one or two amino acids, or have at least or about 70% (e.g., at least about 80%, at least about 85%, at least about 90%, at least about 95%) sequence identity; or (b) a light chain variable region amino acid sequence listed in Table B1, or a sequence selected from the group consisting of SEQ ID NOs: 479, 481, 483, 485, 487, and 489, or variant sequences thereof that differ by only one or two amino acids, or have at least or about 70% (e.g., at least about 80%, at least about 85%, at least about 90%, at least about 95%) sequence identity; or (c) both (a) and (b).

[0133] Table B1

[0134] HC variable region LC variable region AB1* 478 479 AB3* 480 481 AB4* 482 483 AB9* 488 489 AB11* 486 487 AB18* 484 485

[0135] In various embodiments, the antigen binding protein comprises a pair of amino acid sequences selected from the group consisting of: (a) SEQ ID NOs: 478 and 479; (b) SEQ ID NOs: 480 and 481; (c) SEQ ID NOs: 482 and 483; (d) SEQ ID NOs: 484 and 485; (e) SEQ ID NOs: 486 and 487; and (f) SEQ ID NOs: 488 and 489. In various aspects, the antigen binding protein comprises a variant sequence having a sequence of SEQ ID NO: as listed in Table B1, which differs by only one or two amino acids or has at least or about 70% (e.g., at least about 80%, at least about 85%, at least about 90%, at least about 95%) sequence identity, wherein the one or more different amino acids occur at positions in the "humanized antibody" described below.

[0136] Humanized antibodies

[0137] In various aspects, the antigen binding protein is a humanized form of an antigen binding protein described in Table A, Table Al, Table B, or Table Bl.

[0138] Humanized AB1

[0139] , 48, 50, 55, 57, 59, 61, 65, 66, 67, 68, 70, 72, 74, 76, 79, 80, 82, 87, 90, 91, 98, 101, and 116 amino acid substitutions at one or more of the following positions in the heavy chain variable region: In various cases, the antigen binding protein comprises the amino acid sequence of SEQ ID NO: 428. In various aspects, the antigen binding protein is a humanized form of AB1 listed in Table B or Table Bl having one or more amino acid substitutions at one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12) of the following positions in the heavy chain variable region: 20, 31, 35, 48, 50, 59, 67, 70, 74, 79, 98, 101. In various cases, the antigen binding protein comprises the amino acid sequence of SEQ ID NO: 429. In various aspects, the amino acids at the above positions are selected from the amino acids according to the following table:

[0140]

[0141] In various aspects, the antigen binding protein is a humanized form of ABl listed in Table B or Table Bl, which has 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, 35, 36, 37, 94, 95, 96, 101, 107. In each case, the antigen binding protein comprises the amino acid sequence of SEQ ID NO: 430. In various aspects, the antigen binding protein is a humanized form of AB1 listed in Table B or Table B1, which has one or more amino acid substitutions at one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13) of the following positions in the light chain variable region: 4, 21, 32, 34, 48, 51, 53, 61, 67, 79, 84, 91, and 93. In various cases, the antigen binding protein comprises the amino acid sequence of SEQ ID NO: 431. In various aspects, the amino acids at the above positions are selected from the amino acids according to the following table:

[0142]

[0143] Humanized AB3

[0144] 54, 55, 56, 57, 58, 59, 61, 64, 76, 79, 80, 81, 87, 94, 95, 99, 106, 112, 114, 108, 116, 117, 118, 119, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 of the following positions: 3, 5, 18, 19, 23, 31, 33, 35, 40, 42, 49, 50, 52, 53, 54, 55, 56, 57, 58, 59, 61, 64, 76, 79, 80, 81, 87, 94, 95, 99, 106, 112, 114 in the heavy chain variable region. In various cases, the antigen binding protein comprises the amino acid sequence of SEQ ID NO: 432. In various aspects, the antigen binding protein is a humanized form of AB3 listed in Table B or Table B1, which has one or more amino acid substitutions at one or more (e.g., 1, 2, 3, 4, 5, 6, or 7) of the following positions in the heavy chain variable region: 31, 35, 50, 55, 79, 99, 106. In various cases, the antigen binding protein comprises the amino acid sequence of SEQ ID NO: 433. In various aspects, the amino acids at the above positions are selected from the amino acids according to the following table:

[0145]

[0146] In various aspects, the antigen binding protein is a humanized form of AB3 listed in Table B or Table Bl having one or more amino acid substitutions at one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 30, 34, 40, 43, 45, 48, 50, 52, 53, 55, 56, 70, 72, 74, 76, 84, 85, 90, 91, 93, 94, 97, and 100) of the following positions in the light chain variable region. In each case, the antigen binding protein comprises the amino acid sequence of SEQ ID NO: 434. In various aspects, the antigen binding protein is a humanized form of AB3 listed in Table B or Table B1, which has one or more amino acid substitutions at one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, or 9) of the following positions in the light chain variable region: 25, 34, 48, 53, 55, 84, 85, 90, and 93. In various cases, the antigen binding protein comprises the amino acid sequence of SEQ ID NO: 435. In various aspects, the amino acids at the above positions are selected from the amino acids according to the following table:

[0147]

[0148] Humanized AB4

[0149] In various aspects, the antigen binding protein is a humanized form of AB4 listed in Table B or Table B1, which has one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33) of the heavy chain variable region. , 34, 35 or 36) has one or more amino acid substitutions at the following positions: 5, 11, 12, 13, 20, 29, 31, 33, 37, 38, 40, 45, 48, 50, 55, 56, 57, 59, 61, 62, 65, 66, 67, 68, 70, 72, 74, 76, 79, 82, 84, 87, 91, 97, 101, 117. In each case, the antigen binding protein comprises the amino acid sequence of SEQ ID NO: 436. In various aspects, the antigen binding protein is a humanized form of AB4 listed in Table B or Table Bl having one or more amino acid substitutions at one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19) of the following positions in the heavy chain variable region: 20, 29, 31, 37, 45, 48, 56, 59, 61, 62, 65, 66, 68, 70, 74, 79, 84, 97, and 101. In various cases, the antigen binding protein comprises the amino acid sequence of SEQ ID NO: 437. In various aspects, the amino acids at the above positions are selected from the amino acids according to the following table:

[0150]

[0151] In various aspects, the antigen binding protein is a humanized form of AB4 listed in Table B or Table Bl having one or more amino acid substitutions at one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24) of the following positions in the light chain variable region: 7, 14, 17, 18, 31, 33, 39, 41, 42, 44, 50, 51, 55, 57, 60, 81, 88, 92, 94, 95, 96, 99, 100, 105. In each case, the antigen binding protein comprises the amino acid sequence of SEQ ID NO: 438. In various aspects, the antigen binding protein is a humanized form of AB4 listed in Table B or Table B1, having one or more amino acid substitutions at one or more (e.g., 1, 2, 3, 4, 5, 6, or 7) of the following positions in the light chain variable region: 33, 39, 55, 57, 81, 95, and 96. In various cases, the antigen binding protein comprises the amino acid sequence of SEQ ID NO: 439. In various aspects, the amino acids at the above positions are selected from the amino acids according to the following table:

[0152]

[0153] Humanized AB18

[0154] In various aspects, the antigen binding protein is a humanized form of AB18 listed in Table B or Table Bl having one or more amino acid substitutions in the heavy chain variable region at one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25) of the following positions: 5, 9, 11, 12, 20, 38, 40, 41, 43, 44, 48, 61, 65, 67, 68, 70, 72, 74, 76, 79, 82, 84, 87, 91, and 116 (optionally, one or more (e.g., 1, 2, 3, 4, or 5) of the following positions: 20, 48, 68, 70, 79). In each case, the antigen binding protein comprises the amino acid sequence of SEQ ID NO: 440 or 441. In various aspects, the amino acids at the above positions are selected from the amino acids according to the following table:

[0155]

[0156]

[0157] In various aspects, the antigen binding protein is a humanized form of AB18 listed in Table B or Table B1, which has one or more amino acid substitutions in one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16) of the following positions: 1, 3, 9, 15, 18, 19, 21, 22, 49, 51, 69, 93, 84, 78, 105, and 111 (optionally, one or more (e.g., 1, 2, or 3) of the following positions: 19, 21, or 84). In various cases, the antigen binding protein comprises the amino acid sequence of SEQ ID NO: 442 or 443. In various aspects, the amino acids at the above positions are selected from the amino acids according to the following table:

[0158]

[0159] Humanized AB9

[0160] In various aspects, the antigen binding protein is a humanized form of AB9 listed in Table B or Table Bl having one or more amino acid substitutions at one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29) of the following positions in the heavy chain variable region: 1, 5, 9, 11, 12, 20, 38, 40, 41, 43, 44, 48, 61, 63, 65, 67, 69, 70, 72, 73, 74, 76, 79, 84, 87, 91, 93, 112, and 113. In various cases, the antigen binding protein comprises the amino acid sequence of SEQ ID NO: 444. In various aspects, the amino acids at the above positions are selected from the amino acids according to the following table:

[0161]

[0162]

[0163] In various aspects, the antigen binding protein is a humanized form of AB9 listed in Table B or Table B1, which has one or more amino acid substitutions at one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15) of the following positions in the light chain variable region: 9, 11, 15, 17, 18, 43, 45, 70, 72, 73, 74, 80, 84, 85, and 100. In various cases, the antigen binding protein comprises the amino acid sequence of SEQ ID NO: 445. In various aspects, the amino acids at the above positions are selected from the amino acids according to the following table:

[0164]

[0165] Humanized AB11

[0166] In various aspects, the antigen binding protein is a humanized form of AB11 listed in Table B or Table B1, which has one or more amino acid substitutions at one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15) of the following positions in the heavy chain variable region: 1, 15, 18, 19, 42, 49, 63, 75, 76, 78, 80, 84, 88, and 93. In various cases, the antigen binding protein comprises the amino acid sequence of SEQ ID NO: 446. In various aspects, the amino acids at the above positions are selected from the amino acids according to the following table:

[0167]

[0168]

[0169] In various aspects, the antigen binding protein is a humanized form of AB11 listed in Table B or Table B1, which has one or more amino acid substitutions in one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15) of the following positions: 4, 9, 17, 22, 64, 78, 80, 81, 82, 83, 84, 87, 89, 104, and 110 (optionally, one or more of the following positions: 4, 82, 110). In various cases, the antigen binding protein comprises the amino acid sequence of SEQ ID NO: 447 or 448. In various aspects, the amino acids at the above positions are selected from the amino acids according to the following table:

[0170]

[0171] In various embodiments, the antigen binding protein comprises (a) a heavy chain variable region amino acid sequence listed in Table C, or a sequence selected from the group consisting of: 376-379, 384-387, 391-396, 403-408, 412, 413, 416-419, and 422-427, or a sequence thereof that differs by only one or two amino acids or has at least or about 70%, or about 80%, or about 85%, or about 90%, or about 95% sequence identity. variant sequences; or (b) a light chain variable region amino acid sequence listed in Table C or a sequence selected from the group consisting of: 380-383, 388-390, 397-402, 409-411, 414, 415, 420 and 421, or variant sequences thereof that differ only by one or two amino acids or have at least or about 70%, or about 80%, or about 85%, or about 90% or about 95% sequence identity; or (c) both of (a) and (b).

[0172] Table C

[0173]

[0174]

[0175] In various embodiments, the humanized antigen binding protein comprises a pair of amino acid sequences shown in Table D.

[0176] Table D

[0177]

[0178]

[0179] In various embodiments, the antigen binding protein comprises a pair of variant sequences, each 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 SEQ ID NO listed in Table C. In various embodiments, the antigen binding protein comprises a pair of sequences: one sequence selected from SEQ ID NO: listed in Table C and the other sequence being 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 having SEQ ID NO: listed in Table D or SEQ ID NO: listed in Table C.

[0180] In various embodiments, the antigen binding protein comprises a pair of sequences: one sequence selected from SEQ ID NO: 419 and another sequence that is a variant sequence having at least or about 70% (e.g., at least about 80%, at least about 85%, at least about 90%, at least about 95%) sequence identity to the sequence having SEQ ID NO: 419. For example, in various aspects, the antigen binding protein comprises the sequence of SEQ ID NO: 419, and the antigen binding protein further comprises a variant sequence having at least or about 70% (e.g., at least about 80%, at least about 85%, at least about 90%, at least about 95%) sequence identity to SEQ ID NO: 421.

[0181] Nucleic Acids

[0182] The present disclosure also provides nucleic acids comprising the nucleotide sequence encoding the antigen-binding proteins of the present disclosure. "Nucleic acid" as used herein includes "polynucleotides," "oligonucleotides," and "nucleic acid molecules," and generally refers to polymers of DNA or RNA that can be single-stranded or double-stranded, synthesized, or obtained (e.g., isolated and / or purified) from natural sources, or a modified form thereof, which may include natural, non-natural, or altered nucleotides, and which may include natural, non-natural, or altered internucleotide bonds (such as phosphoramidate bonds or phosphorothioate bonds) rather than the phosphodiester present between the nucleotides of unmodified oligonucleotides. Nucleic acids may include any nucleotide sequence encoding any antigen-binding proteins of the present disclosure. In various aspects, the nucleic acid comprises a nucleotide sequence encoding an antigen binding protein comprising (a) a heavy chain (HC) complementarity determining region (CDR) 1 amino acid sequence listed in Table A or Table A1, or a sequence selected from the group consisting of SEQ ID NO: 11, 17, 23, 29, 35, 41, 47, 53, 59, 65, 71, 77, 83, 89, 95, 101, 107, 113, 119, 125, 131, 452, 455, 461, 465, and 472, or variant sequences thereof that differ by only one or two amino acids or have at least or about 70% (e.g., at least or about 80%, at least or about 85%, at least or about 90%, at least or about 95%) sequence identity; (b) a HC CDR2 amino acid sequence listed in Table A or Table A1, or a sequence selected from the group consisting of SEQ ID NO: NO: 12, 18, 24, 30, 36, 42, 48, 54, 60, 66, 72, 78, 84, 90, 86, 102, 108, 114, 120, 126, 132, 475, 456, 462, 466, 468 and 473; or variant sequences thereof that differ by only one or two amino acids or have at least or about 70% (e.g., at least or about 80%, at least or about 85%, at least or about 90%, at least or about 95%) sequence identity; (c) an HC CDR3 amino acid sequence listed in Table A or Table A1, or a sequence selected from the group consisting of: SEQ ID NO: 12, 18, 24, 30, 36, 42, 48, 54, 60, 66, 72, 78, 84, 90, 86, 102, 108, 114, 120, 126, 132, 475, 456, 462, 466, 468 and 473; or variant sequences thereof that differ by only one or two amino acids or have at least or about 70% (e.g., at least or about 80%, at least or about 85%, at least or about 90%, at least or about 95%) sequence identity. NOs: 13, 19, 25, 31, 37, 43, 49, 55, 61, 67, 73, 79, 85, 91, 97, 103, 109, 115, 121, 127, 133, 453, 457, 463, 467, 469, and 474, or variant sequences thereof that differ by only one or two amino acids or have at least or about 70% (e.g., at least or about 80%, at least or about 85%, at least or about 90%, at least or about 95%) sequence identity;(d) a light chain (LC) CDR1 amino acid sequence listed in Table A or Table A1, or a sequence selected from the group consisting of SEQ ID NO: 8, 14, 20, 32, 38, 44, 50, 56, 62, 68, 74, 80, 86, 92, 98, 104, 110, 116, 122, 128, 449, 476, 458, 464, and 470, or a variant thereof that differs by only one or two amino acids or has at least or about 70% (e.g., at least or about 80%, at least or about 85%, at least or about 90%, at least or about 95%) sequence identity; (e) a LC CDR2 amino acid sequence listed in Table A or Table A1, or a sequence selected from the group consisting of SEQ ID NO: 8, 14, 20, 32, 38, 44, 50, 56, 62, 68, 74, 80, 86, 92, 98, 104, 110, 116, 122, 128, 449, 476, 458, 464, and 470. NO:9, 15, 21, 27, 33, 39, 45, 51, 57, 63, 69, 75, 81, 87, 93, 99, 105, 111, 117, 123, 129, 450, 477, 459 and 471; or variant sequences thereof that differ by only one or two amino acids or have at least or about 70% (e.g., at least or about 80%, at least or about 85%, at least or about 90%, at least or about 95%) sequence identity; (f) an LC CDR3 amino acid sequence listed in Table A or a sequence selected from the group consisting of: SEQ ID NO:9, 15, 21, 27, 33, 39, 45, 51, 57, 63, 69, 75, 81, 87, 93, 99, 105, 111, 117, 123, 129, 450, 477, 459 and 471; or variant sequences thereof that differ by only one or two amino acids or have at least 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. NO: 10, 16, 22, 28, 34, 40, 46, 52, 58, 64, 70, 76, 82, 88, 94, 100, 106, 112, 118, 124, 130, 451, 454, and 460, or variant sequences thereof that differ by only one or two amino acids or have at least or about 70% (e.g., at least or about 80%, at least or about 85%, at least or about 90%, at least or about 95%) sequence identity;Or a combination of any two or more of (g)(a)-(f). In various aspects, the nucleic acid comprises a nucleotide sequence encoding an antigen-binding protein comprising at least one or two of the LC CDR1 amino acid sequence, LC CDR2 amino acid sequence, and LC CDR3 amino acid sequence listed in Table A or Table A1, and the HC CDR amino acid sequence listed in Table A or Table A1. In various aspects, the nucleic acid comprises a nucleotide sequence encoding an antigen-binding protein comprising at least one or two of the HC CDR1 amino acid sequence, HC CDR2 amino acid sequence, and HC CDR3 amino acid sequence listed in Table A or Table A1, and the LC CDR amino acid sequence listed in Table A or Table A1. In various embodiments, 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 specified by SEQ ID NO: in a single row of Table A or Table A1, (b) each of the LC CDR amino acid sequences specified by SEQ ID NO: in a single row of Table A or Table A1 and at least 1 or 2 of the HC CDR amino acid sequences specified by SEQ ID NO: in a single row of Table A or Table A1, (c) each of the HC CDR amino acid sequences specified by SEQ ID NO: in a single row of Table A or Table A1 and at least 1 or 2 of the LC CDR amino acid sequences specified by SEQ ID NO: in a single row of Table A or Table A1, (d) all 6 of the CDR amino acid sequences specified by SEQ ID NO: in a single row of Table A, and / or (e) six CDR amino acid sequences selected from the group consisting of: (a) SEQ ID NO: 74-79; (b) SEQ ID NO: 50-55; (c) SEQ ID NO: 122-127; (d) SEQ ID NO: 130-131. NO: 26-31; (e) SEQ ID NO: 128-133; (f) SEQ ID NO: 38-43; (g) SEQ ID NO: 62-67; (h) SEQ ID NO: 80-85; (i) SEQ ID NO: 44-49; (j) SEQ ID NO: 86-91; (k) SEQ ID NO: 104-109; (l) SEQ ID NO: 56-61; (m) SEQ ID NO: 32-37; (n) SEQ ID NO: 110-115; (o) SEQ ID NO: 98-103; (p) SEQ ID NO: 92-97; (q) SEQ ID NO: 116-121; (r) SEQ ID NO: 8-13; (s) SEQ ID NO: 68-73; (t) SEQ ID NO:14-19;(u) SEQ ID NO: 20-25, (v) SEQ ID NO: 449-453 and 475; (w) SEQ ID NO: 476-477, 454-457; (x) SEQ ID NO: 458-463; (y) SEQ ID NO: 57, 58, 464-467; (z) SEQ ID NO: 57, 58, 464-467; NO:68-71 and 468-469; and (aa) SEQ ID NO:112 and 470-474. In various embodiments, the nucleic acid comprises a nucleotide sequence encoding an antigen binding protein comprising (a) a heavy chain variable region amino acid sequence listed in Table B or Table Bl, or a sequence selected from the group consisting of: 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 478, 480, 482, 484, 486, and 488, or a sequence that differs by only one or two amino acids or has at least or about 70% (e.g., at least or about 80%, at least or about 85%, at least or about 90%, at least or about 95%) of the sequence or (b) a light chain variable region amino acid sequence listed in Table B or Table Bl, or a sequence selected from the group consisting of: 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 479, 481, 483, 485, 487, and 489, or variant sequences thereof that differ by only one or two amino acids or have at least or about 70% (e.g., at least or about 80%, at least or about 85%, at least or about 90%, at least or about 95%) sequence identity; or (c) both of (a) and (b). In various embodiments, the nucleic acid comprises a nucleotide sequence encoding an antigen binding protein comprising a pair of amino acid sequences selected from the group consisting of: (a) SEQ ID NOs: 156 and 157; (b) SEQ ID NOs: 148 and 149; (c) SEQ ID NOs: 172 and 173; (d) SEQ ID NOs: 140 and 141; (e) SEQ ID NOs: 174 and 175; (f) SEQ ID NOs: 144 and 145; (g) SEQ ID NOs: 152 and 153; (h) SEQ ID NOs: 158 and 159; (i) SEQ ID NOs: 146 and 147; (j) SEQ ID NOs: 160 and 161; (k) SEQ ID NOs: 166 and 167; (l) SEQ ID NOs: 150 and 151; (m) SEQ ID NOs: 142 and 143;(n) SEQ ID NOs: 168 and 169; (o) SEQ ID NOs: 164 and 165; (p) SEQ ID NOs: 162 and 163; (q) SEQ ID NOs: 170 and 171; (r) SEQ ID NOs: 134 and 135; (s) SEQ ID NOs: 154 and 155; (t) SEQ ID NOs: 136 and 137; and (u) SEQ ID NOs: 138 and 139. In various embodiments, the nucleic acid comprises a nucleotide sequence encoding an antigen binding protein comprising a pair of amino acid sequences selected from the group consisting of the pairs listed in Table D. In various aspects, the nucleic acid comprises a nucleotide sequence comprising the sequence of any one or more of SEQ ID NOs: 208-375. In some embodiments, the nucleic acid does not comprise any insertions, deletions, inversions, and / or substitutions. In other embodiments, the nucleic acid comprises one or more insertions, deletions, inversions, and / or substitutions.

[0183] In some aspects, the nucleic acids of the present disclosure are recombinant. As used herein, the term "recombinant" refers to (i) molecules constructed outside of living cells by linking natural or synthetic nucleic acid fragments to nucleic acid molecules that can replicate in living cells, or (ii) molecules replicated from those described in (i) above. For purposes herein, replication can be in vitro replication or in vivo replication.

[0184] In some aspects, nucleic acids are constructed based on chemical synthesis and / or enzymatic ligation reactions using methods known in the art. See, for example, Sambrook et al., supra; and Ausubel et al., supra. For example, nucleic acids can be chemically synthesized using naturally occurring nucleotides or various modified nucleotides designed to increase the biological stability of the molecule or to increase the physical stability of the duplex formed upon hybridization (e.g., phosphorothioate derivatives and acridine substituted nucleotides). Examples of modified nucleotides that can be used to generate nucleic acids include, but are not limited to, 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxymethyl)uracil, 5-carboxymethylaminomethyl-2-thiouracil, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N 6-Isopentenyl adenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N-substituted adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, β-D-mannosylqueosine, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-substituted-N 6 -isopentenyl adenine, uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, Q nucleoside (queosine), 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methyl ester, 3-(3-amino-3-N-2-carboxypropyl) uracil and 2,6-diaminopurine. Alternatively, one or more nucleic acids of the present disclosure can be purchased from companies such as Macromolecular Resources (Fort Collins, CO) and Synthegen (Houston, TX).

[0185] carrier

[0186] In some aspects, the nucleic acid of the present disclosure is incorporated into a vector. In this regard, the present disclosure provides a vector comprising any currently disclosed nucleic acid. In various aspects, the vector is a recombinant expression vector. For the purposes of this article, the term "recombinant expression vector" refers to a genetically modified oligonucleotide or polynucleotide construct that allows the expression of mRNA, protein, polypeptide or peptide by a host cell when the construct comprises a nucleotide sequence encoding an mRNA, protein, polypeptide or peptide and the vector is in contact with a cell under conditions sufficient to allow the mRNA, protein, polypeptide or peptide to be expressed in the cell. The vector of the present disclosure is not naturally occurring as a whole. However, parts of the vector may be naturally occurring. The currently disclosed vector may comprise any type of nucleotide, including but not limited to DNA and RNA, which may be single-stranded or double-stranded, synthetic or partially obtained from a natural source, and may comprise natural, non-natural or altered nucleotides. The vector may comprise naturally occurring or non-natural internucleotide bonds, or both types of bonds. In some aspects, the altered nucleotides or non-natural internucleotide bonds do not hinder transcription or replication of the vector.

[0187] The carrier of the present disclosure can be any suitable carrier, and can be used for transduction, transformation or transfection any suitable host.Suitable carriers include those designed for breeding and amplification or for expression or both, such as plasmids and viruses.Carrier can be an expression vector based on a plasmid.In various aspects, carrier is selected from the group consisting of: pUC series (Fermentas Life Sciences), pBluescript series (Stratagene, LaJoIIa, CA), pET series (Novagen, Madison, WI), pGEX series (Pharmacia Biotech, Uppsala, Sweden) and pEX series (Clontech, Palo Alto, CA).Phage vectors such as λGT10, λGT11, λZapII (Stratagene), λEMBL4 and λNM1149 can also be used.The example of plant expression vector includes pBIO1, pBI101.2, pBI101.3, pBI121 and pBIN19 (Clontech). In some aspects, the vector is a viral vector, such as a retroviral vector. In various aspects, the vector is an adenoviral vector, an adeno-associated virus (AAV) vector, a herpes simplex virus (HSV) vector, a vesicular stomatitis virus (VSV) vector, a vaccinia virus vector or a lentiviral vector. See, for example, Howarth et al., Cell Biol. Toxicol. 26 (1): 1-20 (2010). In various aspects, the vector is a baculovirus vector that infects arthropods (such as insects). In various aspects, the baculovirus vector is Autographa californica polynuclear virus (AcMNPV) or Bombyx mori nuclear polyhedrosis virus (BmNPV). See, e.g., Khan, Adv Pharm Bull 3(2):257-263 (2013); Miller, Bioessays 11(4):91-96 (1989); Atkinson et al., Pestic Sci 28:215-224 (1990).

[0188] The vectors of the present disclosure can be prepared using standard recombinant DNA techniques, such as those described in Sambrook et al. (supra) and Ausubel et al. (supra). Circular or linear expression vector constructs can be prepared to contain replication systems that function in prokaryotic or eukaryotic host cells. Replication systems can be derived from, for example, CoIE1, 2μ plasmids, lambda, SV40, bovine papilloma virus, and the like.

[0189] In some aspects, the vector contains regulatory sequences, such as transcriptional and translational start and stop codons, which are specific for the type of host into which the vector is to be introduced (e.g., bacteria, fungi, plants, or animals), as appropriate and taking into account whether the vector is DNA-based or RNA-based.

[0190] The vector may include one or more marker genes that allow selection of the transformed or transfected host. Marker genes include biocide resistance (e.g., resistance to antibiotics, heavy metals, etc.), complementation in auxotrophic hosts to provide prototrophy, etc. Suitable marker genes for the currently disclosed expression vectors include, for example, neomycin / G418 resistance genes, hygromycin resistance genes, histidinol resistance genes, tetracycline resistance genes, and ampicillin resistance genes.

[0191] The carrier can comprise a natural or standard promoter operably connected to the nucleotide sequence of the nucleotide sequence of the coded polypeptide (including its functional part and functional variant) or to the nucleotide sequence complementary or hybridized nucleotide sequence of the coded polypeptide. The selection of promoter (such as strong, weak, inducible, tissue-specific and development-specific) is within the ordinary skill of those skilled in the art. Similarly, the combination of nucleotide sequence and promoter is also within the technical scope of those skilled in the art. Promoter can be a non-viral promoter or a viral promoter, such as cytomegalovirus (CMV) promoter, SV40 promoter, RSV promoter and the promoter found in the long terminal repeat sequence of mouse stem cell virus.

[0192] host cells

[0193] Provided herein are host cells comprising nucleic acids or vectors disclosed herein. As used herein, the term "host cell" refers to any type of cell that can comprise a currently disclosed vector and can produce an expression product (e.g., mRNA, protein) encoded by nucleic acid. In some aspects, the host cell is an adherent cell or a suspension cell, i.e., a cell grown in suspension. In various aspects, the host cell is a cultured cell or a primary cell, i.e., a cell directly isolated from an organism (e.g., a human). The host cell can be any cell type, can be derived from any type of tissue, and can have any developmental stage.

[0194] In various aspects, the antigen binding protein is a glycosylated protein and the host cell is a glycosylation competent cell. In various aspects, the glycosylation competent cell is a eukaryotic cell, including but not limited to yeast cells, filamentous fungal cells, protozoan cells, algae cells, insect cells, or mammalian cells. Such host cells are described in the art. See, for example, Frenzel et al., Front Immunol 4:217 (2013). In various aspects, the eukaryotic cell is a mammalian cell. In various aspects, the mammalian cell is a non-human mammalian cell. In some aspects, the cells are Chinese hamster ovary (CHO) cells and their derivatives (e.g., CHO-K1, CHO pro-3), mouse myeloma cells (e.g., NS0, GS-NS0, Sp2 / 0), cells engineered to be deficient in dihydrofolate reductase (DHFR) activity (e.g., DUKX-X11, DG44), human embryonic kidney 293 (HEK293) cells or their derivatives (e.g., HEK293T, HEK293-EBNA), green African monkey kidney cells (e.g., COS cells, VERO cells), human cervical cancer cells (e.g., HeLa), human osteosarcoma epithelial cells U2-OS, adenocarcinoma human alveolar basal epithelial cells A549, human fibrosarcoma cells HT1080, mouse brain tumor cells CAD, embryonic carcinoma cells P19, mouse embryonic fibroblasts NIH 3T3, mouse fibroblast L929, mouse neuroblastoma N2a, human breast cancer MCF-7, retinoblastoma Y79, human retinoblastoma SO-Rb50, human hepatoma Hep G2, mouse B myeloma J558L or baby hamster kidney (BHK) cells (Gaillet et al. 2007; Khan, Adv Pharm Bull 3(2):257-263 (2013)).

[0195] For amplification or replication of the vector, in some aspects, the host cell is a prokaryotic cell, eg, a bacterial cell.

[0196] The present disclosure also provides a cell mass comprising at least one host cell as described herein. In some aspects, a cell mass is a heterogeneous colony comprising a host cell and at least one other cell, and this host cell comprises the vector, and this at least one other cell does not comprise any vector. Alternatively, in some aspects, a cell mass is a substantially homogeneous colony, wherein this colony mainly comprises a host cell, and this host cell comprises a vector (for example, being substantially composed thereof). In some aspects, a colony is a clonal colony of a cell, wherein all cells of the colony are clones of a single host cell comprising a vector, so that all cells of the colony comprise a vector. In various embodiments of the present disclosure, a cell mass is a clonal colony comprising a host cell, and this host cell comprises a vector as described herein.

[0197] Manufacturing method

[0198] Also provided herein are methods for producing antigen-binding proteins that bind to CLDN6. In various embodiments, the method comprises culturing host cells comprising a nucleic acid comprising a nucleotide sequence encoding an antigen-binding protein as described herein in a cell culture medium, and harvesting the antigen-binding protein from the cell culture medium. The host cells can be any of the host cells described herein. In various aspects, the host cells are selected from the group consisting of CHO cells, NS0 cells, COS cells, VERO cells, and BHK cells. In various aspects, the step of culturing the host cells comprises culturing the host cells in a growth medium to support the growth and expansion of the host cells. In various aspects, the growth medium increases cell density, culture viability, and productivity in a timely manner. In various aspects, the growth medium comprises amino acids, vitamins, inorganic salts, glucose, and serum as sources of growth factors, hormones, and attachment factors. In various aspects, the growth medium is a completely chemically defined medium composed of amino acids, vitamins, trace elements, inorganic salts, lipids, and insulin or insulin-like growth factor. In addition to nutrients, the growth medium also helps maintain pH and osmolality. Several growth media are commercially available and described in the art. See, e.g., Arora, “Cell Culture Media: A Review” MATER METHODS 3: 175 (2013).

[0199] In various aspects, the method comprises culturing the host cell in a feed medium. In various aspects, the method comprises culturing the host cell in a feed medium in a fed batch mode. Methods for recombinant protein production are known in the art. See, for example, Li et al., "Cell culture processes for monoclonal antibody production" MAbs 2(5):466-477 (2010).

[0200] The method for preparing the antigen binding protein may comprise one or more steps for purifying the protein from the cell culture or its supernatant and preferably recovering the purified protein. In various aspects, the method comprises one or more chromatography steps, such as affinity chromatography (e.g., Protein A affinity chromatography), ion exchange chromatography, hydrophobic interaction chromatography. In various aspects, the method comprises purifying the protein using a Protein A affinity chromatography resin.

[0201] In various embodiments, the method further comprises steps for formulating the purified protein, etc., thereby obtaining a formulation comprising the purified protein. These steps are described in Formulation and Process Development Strategies for Manufacturing, edited by Jameel and Hershenson, John Wiley & Sons, Inc. (Hoboken, NJ), 2010.

[0202] In various aspects, the antigen binding protein linked to the polypeptide and the antigen binding protein are part of a fusion protein. Therefore, the present disclosure also provides methods for producing a fusion protein comprising an antigen binding protein that binds to CLDN6. In various embodiments, the method comprises culturing a host cell comprising a nucleic acid comprising a nucleotide sequence encoding a fusion protein as described herein in a cell culture medium, and harvesting the fusion protein from the cell culture medium.

[0203] Conjugate

[0204] The present disclosure also provides antigen-binding proteins that are attached, connected or conjugated to a second portion (e.g., a heterologous portion, a conjugated portion). Thus, the present disclosure provides conjugates comprising an antigen-binding protein and a heterologous portion. As used herein, the term "heterologous portion" is synonymous with "conjugated portion" and refers to any molecule (chemical or biochemical, naturally occurring or non-encoded) that is different from the antigen-binding protein of the present disclosure. Various heterologous portions include, but are not limited to, polymers, carbohydrates, lipids, nucleic acids, oligonucleotides, DNA or RNA, amino acids, peptides, polypeptides, proteins, therapeutic agents (e.g., cytotoxic agents, cytokines) or diagnostic agents.

[0205] In some embodiments, the heterologous moiety is a polymer. The polymer can be branched or unbranched. The polymer can have any molecular weight. In some embodiments, the polymer has an average molecular weight between about 2kDa and about 100kDa (the term "about" means that in the preparation of water-soluble polymers, some molecules will be larger than the molecular weight, and some molecules will be smaller than the molecular weight). In some aspects, the average molecular weight of the polymer is between about 5kDa and about 50kDa, between about 12kDa and about 40kDa, or between about 20kDa and about 35kDa.

[0206] In some embodiments, the polymer is modified to have a single reactive group, such as an active ester for acylation or an aldehyde for alkylation, so that the degree of polymerization can be controlled. In some embodiments, the polymer is water-soluble so that the protein to which it is attached does not precipitate in an aqueous environment (such as a physiological environment). In some embodiments, when, for example, the composition is used for therapeutic purposes, the polymer is pharmaceutically acceptable. In addition, in some aspects, the polymer is a mixture of polymers, such as a copolymer, a block copolymer.

[0207] In some embodiments, the polymer is selected from the group consisting of: polyamides; polycarbonates; polyalkylenes and their derivatives, including polyalkylene glycols, polyalkylene oxides, polyalkylene terephthalates; polymers of acrylic and methacrylates, including poly(methyl methacrylate), poly(ethyl methacrylate), poly(butyl methacrylate), poly(isobutyl methacrylate), poly(hexyl methacrylate), poly(isodecyl methacrylate), poly(lauryl methacrylate), poly(phenyl methacrylate), poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutyl acrylate), and poly(octadecyl acrylate); polyethylene polymers, including Polyvinyl alcohol, polyvinyl ether, polyvinyl ester, polyvinyl halide, poly(vinyl acetate) and polyvinyl pyrrolidone; polyglycolide, polysiloxane, polyurethane and copolymers thereof; cellulose, including alkyl cellulose, hydroxyalkyl cellulose, cellulose ether, cellulose ester, nitrocellulose, methyl cellulose, ethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, hydroxybutyl methyl cellulose, cellulose acetate, cellulose propionate, cellulose acetate butyrate, cellulose acetate phthalate, carboxyethyl cellulose, cellulose triacetate, cellulose sulfate sodium salt; polypropylene; polyethylene, including poly(ethylene glycol), poly(ethylene oxide) and poly(ethylene terephthalate); and polystyrene.

[0208] A particularly preferred water-soluble polymer for use herein is polyethylene glycol (PEG). As used herein, polyethylene glycol is intended to encompass any form of PEG that can be used to derivatize other proteins, such as mono-(C1-C10) alkoxy- or aryloxy-polyethylene glycol. PEG is a linear or branched neutral polyether available in a wide range of molecular weights and soluble in water and most organic solvents.

[0209] In some embodiments, the heterologous moiety is a carbohydrate. In some embodiments, the carbohydrate is a monosaccharide (e.g., glucose, galactose, fructose), a disaccharide (e.g., sucrose, lactose, maltose), an oligosaccharide (e.g., raffinose, stachyose), a polysaccharide (starch, amylase, amylopectin, cellulose, chitin, callose, laminarin, xylan, mannan, fucoidan, galactomannan.

[0210] In some embodiments, the heterologous moiety is a lipid. In some embodiments, the lipid is a fatty acid, an eicosanoid, a prostaglandin, a leukotriene, a thromboxane, an N-acylethanolamine), a glycerolipid (e.g., a mono-, di-, or tri-substituted glycerol), a glycerophospholipid (e.g., phosphatidylcholine, phosphatidylinositol, phosphatidylethanolamine, phosphatidylserine), a sphingolipid (e.g., sphingosine, ceramide), a sterol lipid (e.g., a steroid, cholesterol), a prenol lipid, a glycolipid or polyketide, an oil, a wax, cholesterol, a sterol, a fat-soluble vitamin, a monoglyceride, a diglyceride, a triglyceride, or a phospholipid.

[0211] In some embodiments, the heterologous moiety is a therapeutic agent. The therapeutic agent can be any therapeutic agent known in the art. Examples of therapeutic agents contemplated herein include, but are not limited to, natural enzymes, proteins derived from natural sources, recombinant proteins, natural peptides, synthetic peptides, cyclic peptides, antibodies, receptor agonists, cytotoxic agents, immunoglobulins, beta-adrenergic blockers, calcium channel blockers, coronary vasodilators, cardiac glycosides, antiarrhythmics, antiarrhythmic agents, angiotensin converting enzyme (ACE) inhibitors, diuretics, inotropes, cholesterol and triglyceride lowering agents, bile acid sequestrants, fibrates, 3-hydroxy-3-methylglutaric acid (HMG)-coenzyme A reductase inhibitors, nicotinic acid derivatives, antiadrenergic agents, alpha-adrenergic blockers, centrally acting antiadrenergic agents, vasodilators, potassium sparing agents, thiothiazolinone, ... Azine and related drugs, angiotensin II receptor antagonists, peripheral vasodilators, antiandrogens, estrogens, antibiotics, retinoids, insulin and analogs, α-glucosidase inhibitors, biguanides, meglitinides, sulfonylureas, thiazolidinediones, androgens, progestogens, bone metabolism regulators, anterior pituitary hormones, hypothalamic hormones, posterior pituitary hormones, gonadotropins, gonadotropin-releasing hormone antagonists, ovulation stimulants, selective estrogen receptor modulators, antithyroid agents, thyroid hormones, volume formers, laxatives, anthelmintics, flora improvers, intestinal adsorbents, intestinal anti-infectives, anorexia drugs, anti-cachexia drugs, anti-bulimia drugs, appetite suppressants, anti-obesity drugs, antacids, upper gastrointestinal drugs, anticholinergics, aminosalicylic acid derivatives, biological response modifiers, corticosteroids, antispasmodics, 5-HT4 partial agonists, antihistamines, cannabinoids, dopamine antagonists, serotonin antagonists, cytoprotective agents, histamine H2-receptor antagonists, mucosal protective agents, proton pump inhibitors, Helicobacter pylori eradication therapy, erythropoietin stimulating agents, hematopoietic agents, anemic agents, heparin, antifibrinolytics, hemostatics, coagulation factors, adenosine diphosphate inhibitors, glycoprotein receptor inhibitors, fibrinogen-platelet binding inhibitors, thromboxane-A2 inhibitors, plasminogen activators, antithrombotic agents, glucocorticoids, mineralocorticoids, corticosteroids, selective immunosuppressants, antifungal agents, preventive treatment Drugs, AIDS-related infections, cytomegalovirus, non-nucleoside reverse transcriptase inhibitors, nucleoside analog reverse transcriptase inhibitors, protease inhibitors, anemia, Kaposi's sarcoma, aminoglycosides, carbapenems, cephalosporins, glycopeptides, lincosamides, macrolides, oxazolidinones, penicillins, streptogramins, sulfonamides, trimethoprim and derivatives, tetracyclines, anthelmintics, amebamycin, biguanides, cinchona alkaloids, folic acid antagonists, quinoline derivatives, Pneumocystis carinii therapy, hydrazides, imidazoles, triazoles, nitroimidazoles, cycloamines, neuraminidase inhibitors, nucleosides, phosphate binders, cholinesterase inhibitors, adjunctive therapy, barbiturates and derivatives, benzodiazepines, gamma-aminobutyric acid derivatives,Hydantoin derivatives, iminodiphenylethylene derivatives, succinimide derivatives, anticonvulsants, ergot alkaloids, antimigrane preparations), biological response modifiers, carbamates, tricyclic derivatives, depolarizing agents, non-depolarizing agents, neuromuscular paralytics, CNS stimulants, dopaminergic agents, monoamine oxidase inhibitors, COMT inhibitors, alkyl sulfonates, ethyleneimines, imidazotetrazines, nitrogen mustard analogs, nitrosoureas, platinum-containing compounds, antimetabolites, purine analogs, pyrimidine analogs, urea derivatives, anthracyclines, actinomycins, camptothecin derivatives, epipodophyllotoxins, taxanes, vinca alkaloids and analogs, antiandrogens, antiestrogens, nonsteroidal aromatase inhibitors, protein kinase inhibitors, antineoplastic agents, azaspirodecanedione derivatives, anxiolytics, stimulants, monoamine reuptake inhibitors, selective serotonin reuptake inhibitors, antidepressants, benzisoxazole derivatives, butylphenone derivatives, dibenzodiazepine derivatives, dibenzothiazepine derivatives, diphenylbutylpiperidine derivatives, phenothiazines, thienobenzodiazepine derivatives, Thioxanthene derivatives, allergenic extracts, nonsteroidal drugs, leukotriene receptor antagonists, xanthines, endothelin receptor antagonists, prostaglandins, pulmonary surfactants, mucolytics, antimitotics, uricosuric agents, xanthine oxidase inhibitors, phosphodiesterase inhibitors, methylamine salts, nitrofuran derivatives, quinolones, smooth muscle relaxants, parasympathomimetics, halogenated hydrocarbons, aminobenzoates, amides (e.g., lidocaine, articaine hydrochloride, bupivacaine hydrochloride), antipyretics , hypnotics and sedatives, cyclopyrrolidones, pyrazolopyrimidines, nonsteroidal anti-inflammatory drugs, opioids, para-aminophenol derivatives, alcohol dehydrogenase inhibitors, heparin antagonists, adsorbents, emetics, opioid antagonists, cholinesterase reactivators, nicotine replacement therapy, vitamin A analogues and antagonists, vitamin B analogues and antagonists, vitamin C analogues and antagonists, vitamin D analogues and antagonists, vitamin E analogues and antagonists, vitamin K analogues and antagonists.

[0212] The antigen binding proteins of the present invention can be conjugated to one or more cytokines and growth factors that are effective in inhibiting tumor metastasis, and wherein the cytokine or growth factor has been shown to have an anti-proliferative effect on at least one cell population. Such cytokines, lymphokines, growth factors or other hematopoietic factors include, but are not limited to, M-CSF, GM-CSF, TNF, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IFN, TNFα, TNF1, TNF2, G-CSF, Meg-CSF, GM-CSF, thrombopoietin, stem cell factor and erythropoietin.Additional growth factors for use herein include angiogenin, bone morphogenetic protein-1, bone morphogenetic protein-2, bone morphogenetic protein-3, bone morphogenetic protein-4, bone morphogenetic protein-5, bone morphogenetic protein-6, bone morphogenetic protein-7, bone morphogenetic protein-8, bone morphogenetic protein-9, bone morphogenetic protein-10, bone morphogenetic protein-11, bone morphogenetic protein-12, bone morphogenetic protein-13, bone morphogenetic protein-14, bone morphogenetic protein-15, bone morphogenetic protein-16, bone morphogenetic protein-17, bone morphogenetic protein-18, bone morphogenetic protein-19, bone morphogenetic protein-20, bone morphogenetic protein-21, bone morphogenetic protein-22, bone morphogenetic protein-23, bone morphogenetic protein-24, bone morphogenetic protein-25, bone morphogenetic protein-26, bone morphogenetic protein-27, bone morphogenetic protein-28, bone morphogenetic protein-29, bone morphogenetic protein-30, bone morphogenetic protein-31, bone morphogenetic protein-32, bone morphogenetic protein-33, bone morphogenetic protein-34, bone morphogenetic protein-35, bone morphogenetic protein-36, bone morphogenetic protein-37, bone morphogenetic protein-38, bone morphogenetic protein-39, bone morphogenetic protein-40, bone morphogenetic protein-41, bone morphogenetic protein-42, bone morphogenetic protein-43, bone morphogenetic protein-44, bone morphogenetic protein-45, bone morphogenetic protein-46, bone morphogenetic protein-47, bone morphogenetic protein-48, bone morphogenetic protein-49, bone morphogenetic protein-50, bone morphogenetic protein-5 Leukocyte receptor IA, bone morphogenetic protein receptor IB, brain-derived neurotrophic factor, ciliary neurotrophic factor, ciliary neurotrophic factor receptor α, cytokine-induced neutrophil chemoattractant 1, cytokine-induced neutrophil chemoattractant 2α, cytokine-induced neutrophil chemoattractant 2β, endothelial cell growth factor β, endothelin 1, epithelial-derived neutrophil attractant, glial cell line-derived neutrophil factor receptor α1, glial cell line-derived neutrophil factor receptor α2, growth factor Related proteins, growth-related protein alpha, growth-related protein beta, growth-related protein gamma, heparin-binding epidermal growth factor, hepatocyte growth factor, hepatocyte growth factor receptor, insulin-like growth factor I, insulin-like growth factor receptor, insulin-like growth factor II, insulin-like growth factor binding protein, keratinocyte growth factor, leukemia inhibitory factor, leukemia inhibitory factor receptor alpha, nerve growth factor, nerve growth factor receptor, neurotrophin-3, neurotrophin-4, pre-B cell growth stimulating factor, stem cell factor, stem cell factor receptor, transforming growth factor alpha, transforming growth factor beta, transforming growth factor beta1, transforming growth factor beta1.2, transforming growth factor beta2, transforming growth factor beta3, transforming growth factor beta5, latent transforming growth factor beta1, transforming growth factor beta binding protein I, transforming growth factor beta binding protein II, transforming growth factor beta binding protein III, tumor necrosis factor receptor type I, tumor necrosis factor receptor type II, urokinase-type plasminogen activator receptor and their chimeric proteins and biologically active or immunologically active fragments.

[0213] In some embodiments, the conjugate comprises an antigen-binding protein as described herein and a cytotoxic agent. Cytotoxic agents are any (chemical or biochemical) molecules that are toxic to cells. In some aspects, when cytotoxic agents are put together with antigen-binding proteins of the present disclosure, the results obtained are synergistic. In other words, the effect of antigen-binding proteins and cytotoxic agent combination therapy is synergistic, that is, the effect is greater than the desired effect of the respective summed individual effects. Therefore, the dosage of the cytotoxic agent can be reduced, and therefore the risk of toxicity problems and other side effects can be reduced. In some embodiments, the cytotoxic agent is a chemotherapeutic agent. Chemotherapeutic agents are known in the art, including but not limited to platinum coordination compounds, topoisomerase inhibitors, antibiotics, antimitotic alkaloids and difluoronucleosides, as described in U.S. Patent number 6,630,124.

[0214] In some embodiments, the chemotherapeutic agent is a platinum coordination compound. The term "platinum coordination compound" refers to any platinum coordination compound that provides platinum in ionic form and inhibits tumor cell growth. In some embodiments, cisplatin is the platinum coordination compound used in the compositions and methods of the present disclosure. In some embodiments, the chemotherapeutic agent is a topoisomerase inhibitor. In some aspects, the topoisomerase inhibitor is camptothecin or a camptothecin analog. In other embodiments of the present disclosure, the chemotherapeutic agent is an antibiotic compound. Suitable antibiotics include, but are not limited to, doxorubicin, mitomycin, bleomycin, daunorubicin, and streptozotocin. In some embodiments, the chemotherapeutic agent is an antimitotic alkaloid. Antimitotic alkaloids can generally be extracted from Catharanthus roseus and have been shown to be effective as anticancer chemotherapeutic agents. In other embodiments of the present disclosure, the chemotherapeutic agent is a difluoronucleoside. 2'-deoxy-2',2'-difluoronucleosides are known in the art to have antiviral activity. Such compounds are disclosed and taught in U.S. Patent Nos. 4,526,988 and 4,808,614. European Patent Application Publication 184,365 discloses that these same difluoronucleosides have oncolytic activity.

[0215] The present disclosure also provides a conjugate comprising an antigen-binding protein of the present disclosure connected to a polypeptide, such that the conjugate is a fusion protein. Therefore, the present disclosure provides a fusion protein comprising an antigen-binding protein of the present disclosure connected to a polypeptide. In various embodiments, the polypeptide is a diagnostic marker, such as a fluorescent protein (such as green fluorescent protein), or other labels, such as a Myc tag. In various aspects, the polypeptide is a cytokine, a lymphokine, a growth factor, or one of the other hematopoietic factors listed above.

[0216] connector

[0217] In some embodiments, the conjugate is directly connected to the heterologous part. In alternative embodiments, the conjugate comprises a joint that connects the compound of the present disclosure to the heterologous part. In some aspects, the joint comprises an atomic chain of 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. In some embodiments, the chain atoms are all carbon atoms. In some embodiments, the chain atoms in the main chain of the joint are selected from the group consisting of C, O, N and S. Chain atoms and joints can be selected according to their expected solubility (hydrophilicity) to provide a more soluble conjugate. In some embodiments, the joint provides a functional group that is easily cracked by an enzyme or other catalyst or hydrolysis condition found in the target tissue or organ or cell. In some embodiments, the length of the joint is long enough to reduce the possibility of steric hindrance. In some embodiments, the joint is an amino acid or peptidyl joint. Such peptidyl joints can be of any length. Various linkers are between about 1 and 50 amino acids in length, between 5 and 50, 3 and 5, 5 and 10, 5 and 15, or 10 and 30 amino acids in length.

[0218] Compositions, pharmaceutical compositions and preparations

[0219] Provided herein are compositions comprising antigen-binding proteins, nucleic acids, carriers, host cells or conjugates as currently disclosed. In some respects, compositions comprise antigen-binding proteins in separation and / or purification form. In some respects, compositions comprise antigen-binding proteins of a single type (e.g., structure) of the present disclosure or a combination of two or more antigen-binding proteins of the present disclosure, wherein the combination comprises antigen-binding proteins of two or more different types (e.g., structure).

[0220] In some aspects, the composition comprises an agent that enhances the chemo-physical characteristics of the antigen binding protein, e.g., by stabilizing the antigen binding protein at certain temperatures (e.g., room temperature), increasing shelf life, reducing degradation (e.g., oxidative protease-mediated degradation), increasing the half-life of the antigen binding protein, etc. In some aspects, the composition comprises any agent disclosed herein as a heterologous moiety or conjugate moiety, optionally admixed with or conjugated to an antigen binding protein of the disclosure.

[0221] In various aspects of the present disclosure, the composition further comprises a pharmaceutically acceptable carrier, diluent or excipient. In some embodiments, the antigen-binding proteins, nucleic acids, carriers, host cells or conjugates (hereinafter referred to as "active agents") disclosed herein are formulated into pharmaceutical compositions comprising active agents and pharmaceutically acceptable carriers, diluents or excipients. In this regard, the present disclosure also provides pharmaceutical compositions comprising active agents intended to be administered to subjects (e.g., mammals).

[0222] In some embodiments, the active agent is present in the pharmaceutical composition at a purity level suitable for administration to a patient. In some embodiments, the active agent has a purity level of at least about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%, and a pharmaceutically acceptable diluent, carrier, or excipient. In some embodiments, the composition comprises the active agent at a concentration of about 0.001 to about 30.0 mg / ml.

[0223] In various aspects, the pharmaceutical composition comprises a pharmaceutically acceptable carrier. As used herein, the term "pharmaceutically acceptable carrier" includes any standard pharmaceutical carrier, such as phosphate buffered saline, water, emulsions (e.g., oil / water or water / oil emulsions), and various types of wetting agents. The term also encompasses any agent approved by a U.S. federal government regulatory agency or listed in the U.S. Pharmacopoeia for use in animals (including humans).

[0224] The pharmaceutical composition may contain any pharmaceutically acceptable ingredient, including, for example, acidifiers, additives, adsorbents, aerosol propellants, air displacers, alkalizers, anti-caking agents, anticoagulants, antimicrobial preservatives, antioxidants, preservatives, bases, binders, buffers, chelating agents, coating agents, colorants, desiccants, detergents, diluents, disinfectants, disintegrants, dispersants, dissolution enhancers, dyes, softeners, emulsifiers, emulsion stabilizers, fillers, film formers, flavor enhancers, flavorings, flow enhancers, gelling agents, granulating agents, wetting agents, lubricants, mucoadhesives, ointment bases, ointments, oily vehicles, organic bases, lozenge bases, pigments, plasticizers, polishing agents, preservatives, chelating agents, skin penetrants, solubilizers, solvents, stabilizers, suppository bases, surface active agents,

[0013] Examples of the present invention include, but are not limited to, surfactants, surfactants, suspending agents, sweeteners, therapeutic agents, thickeners, tonicity agents, toxicants, viscosity enhancers, water absorbents, water miscible cosolvents, water softeners, or wetting agents. See, for example, Handbook of Pharmaceutical Excipients, 3rd ed., A.H. Kibbe (Pharmaceutical Press, London, UK, 2000), which is incorporated by reference in its entirety. Remington's Pharmaceutical Sciences, 16th ed., E.W. Martin (Mack Publishing Co., Easton, Pa., 1980), which is incorporated by reference in its entirety.

[0225] In various aspects, the pharmaceutical composition is included in a formulation material that is nontoxic to the recipient at the dosage and concentration used. In a specific embodiment, the pharmaceutical composition comprises an active agent and one or more pharmaceutically acceptable salts, polyols, surfactants, osmotic balance agents, tension agents, antioxidants, antibiotics, antifungals, fillers, lyoprotectants, defoamers, chelating agents, preservatives, coloring agents, analgesics or other medicaments. In various aspects, optionally in addition to one or more excipients, the pharmaceutical composition comprises one or more polyols and / or one or more surfactants, including but not limited to pharmaceutically acceptable salts, osmotic balance agents (tonicity agents), antioxidants, antibiotics, antifungals, fillers, lyoprotectants, defoamers, chelating agents, preservatives, coloring agents and analgesics.

[0226] In certain embodiments, pharmaceutical compositions may contain formulation materials for altering, maintaining, or preserving, for example, the pH, osmolarity, viscosity, clarity, color, isotonicity, odor, sterility, stability, dissolution or release rate, absorption, or permeation of the composition. In such embodiments, suitable formulation materials include, but are not limited to, amino acids (such as glycine, glutamine, asparagine, arginine, or lysine); antimicrobial agents; antioxidants (such as ascorbic acid, sodium sulfite, or sodium bisulfite); buffers (such as borates, bicarbonates, Tris-HCl, citrates, phosphates, or other organic acids); bulking agents (such as mannitol or glycine); chelating agents (such as ethylenediaminetetraacetic acid (EDTA)); complexing agents (such as caffeine, polyvinylpyrrolidone, β-cyclodextrin, or hydroxypropyl-β-cyclodextrin); fillers; monosaccharides; disaccharides; and other carbohydrates (such as glucose, mannose, or dextrin); proteins (such as serum albumin, gelatin, or immunoglobulins); colorants, flavorings, and diluents; emulsifiers; hydrophilic polymers (such as polyvinylpyrrolidone ); low molecular weight polypeptides; salt-forming counterions (such as sodium); preservatives (such as benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenylethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid or hydrogen peroxide); solvents (such as glycerol, propylene glycol or polyethylene glycol); sugar alcohols (such as mannitol or sorbitol); suspending agents; surfactants or wetting agents (such as pluronics, PEG, sorbitan esters, polysorbates (such as polysorbate 20, polysorbate 80), triton, tromethamine, lecithin, cholesterol, tyloxapol); stability enhancers (such as sucrose or sorbitol); tonicity enhancers (such as alkali metal halides, preferably sodium chloride or potassium chloride, mannitol sorbitol); delivery vehicles; diluents; excipients and / or pharmaceutical adjuvants. See, REMINGTON'S PHARMACEUTICAL SCIENCES, 18th ed., (AR Genrmo ed.), 1990, Mack Publishing Company.

[0227] The pharmaceutical composition can be formulated to achieve a physiologically compatible pH. In some embodiments, the pH of the pharmaceutical composition can be, for example, between about 4 or about 5 and about 8.0, or between about 4.5 and about 7.5, or between about 5.0 and about 7.5. In various embodiments, the pH of the pharmaceutical composition is between 5.5 and 7.5.

[0228] The present disclosure provides methods for producing pharmaceutical compositions. In various aspects, the method comprises combining an antigen binding protein, a conjugate, a fusion protein, a nucleic acid, a vector, a host cell, or a combination thereof with a pharmaceutically acceptable carrier, diluent, or excipient.

[0229] Route of administration

[0230] With respect to the present disclosure, the active agent or pharmaceutical composition comprising the active agent can be administered to a subject via any suitable route of administration. For example, the active agent can be administered to a subject via parenteral, nasal, oral, pulmonary, topical, vaginal, or rectal administration. The following discussion of routes of administration is intended only to illustrate various embodiments and should not be construed as limiting the scope in any way.

[0231] Preparations suitable for parenteral administration include aqueous and non-aqueous isotonic sterile injection solutions, which may contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions, which may include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives. The term "parenteral" means not through the digestive tract, but through some other route, such as subcutaneous, intramuscular, intraspinal, or intravenous. The active agents of the present disclosure can be administered with a physiologically acceptable diluent in a pharmaceutical carrier such as a sterile liquid or liquid mixture including water, saline, aqueous dextrose and related sugar solutions, alcohols such as ethanol or cetyl alcohol, glycols such as propylene glycol or polyethylene glycol, dimethyl sulfoxide, glycerol, ketals such as 2,2-dimethyl-153-dioxolane-4-methanol, ethers, poly(ethylene glycol) 400, oils, fatty acids, fatty acid esters, or glycerides, or acetylated fatty acid glycerides, with or without the addition of pharmaceutically acceptable surfactants such as soaps or detergents, suspending agents such as pectin, carbomer, methylcellulose, hydroxypropylmethylcellulose or carboxymethylcellulose, or emulsifiers and other pharmaceutical adjuvants.

[0232] The oil that can be used for parenteral preparation comprises petroleum, animal oil, vegetable oil or synthetic oil.The specific example of oil comprises peanut oil, soybean oil, sesame oil, cottonseed oil, corn oil, olive oil, petrolatum and mineral oil.The suitable fatty acid that is used for parenteral preparation comprises oleic acid, stearic acid and isostearic acid.Ethyl oleate and isopropyl myristate are examples of suitable fatty acid esters.

[0233] Suitable soaps for parenteral formulations include fatty alkali metal salts, ammonium salts and triethanolamine salts, and suitable detergents include (a) cationic detergents such as dimethyldialkylammonium halides and alkylpyridinium halides, (b) anionic detergents such as alkyl, aryl and olefin sulfonates, alkyl, olefin, ether and monoglyceride sulfates, and sulfosuccinates, (c) nonionic detergents such as fatty amine oxides, fatty acid alkanolamides and polyoxyethylene polypropylene copolymers, (d) amphoteric detergents such as alkyl-β-aminopropionates and 2-alkyl-imidazoline quaternary ammonium salts, and (e) mixtures thereof.

[0234] In some embodiments, the parenteral formulation comprises an active agent of the present disclosure of about 0.5% to about 25% by weight in the solution. Preservatives and buffers can be used. In order to minimize or eliminate the irritation of the injection site, such compositions may include one or more nonionic surfactants having a hydrophilic-lipophilic balance (HLB) of about 12 to about 17. The amount of surfactant in such formulations is generally about 5% to about 15% by weight. Suitable surfactants include polyethylene glycol sorbitan fatty acid esters (such as sorbitan monooleate) and high molecular weight adducts of ethylene oxide and hydrophobic bases formed by the condensation of propylene oxide and propylene glycol. In some aspects, the parenteral formulation is present in unit dose or multi-dose sealed containers (such as ampoules and vials) and can be stored under freeze-dried (lyophilized) conditions, requiring only the addition of sterile liquid excipients (such as water) for injection immediately before use. In some aspects, extemporaneous injection solutions and suspensions are prepared by sterile powders, granules, and tablets of the aforementioned types.

[0235] Injectable formulations are in accordance with the present disclosure. The requirements for effective pharmaceutical carriers for injectable compositions are well known to those of ordinary skill in the art (see, for example, Pharmaceutics and Pharmacy Practice, JB Lippincott Company, Philadelphia, PA, Banker and Chalmers, eds., pp. 238-250 (1982), and ASHP Handbook on Injectable Drugs, Toissel, 4th edition, pp. 622-630 (1986)).

[0236] dose

[0237] It is believed that the activating agent of the present invention can be used for the method for suppressing tumor growth and other methods as further described herein, including the method for treating or preventing cancer. For the purposes of the present disclosure, the amount or dosage of the activating agent administered should be sufficient to achieve, for example, treatment or prevention response in a subject or animal within a reasonable time frame. For example, the dosage of the activating agent of the present invention should be sufficient to treat cancer as described herein within a time period of about 1 to 4 minutes, 1 to 4 hours, or 1 to 4 weeks or longer (e.g., 5 to 20 weeks or more weeks) from the time of administration. In certain embodiments, the time period can be even longer. The dosage will be determined by the efficacy of the specific activating agent and the condition of the animal (e.g., people) and the weight of the animal (e.g., people) to be treated.

[0238] Many assays for determining the dosage to be administered are known in the art. For the purposes of this article, a starting dose to be administered to a mammal can be determined using an assay that includes comparing the extent to which cancer is treated after a given dose of an active agent of the present disclosure is administered to a group of mammals, each group of which is given a different dose of the active agent. The extent to which cancer is treated after administration of a certain dose can be represented by the degree of tumor regression achieved, for example, with the active agent in a mouse xenograft model. Methods for determining tumor regression are known in the art and are described herein in the Examples.

[0239] The dosage of the active agent of the present invention will also be determined by the presence, nature and extent of any adverse side effects that may accompany the administration of the specific active agent of the present invention. Typically, the attending physician will consider a variety of factors (such as age, body weight, general health, diet, sex, the active agent of the present invention to be administered, the route of administration and the severity of the disease being treated) to determine the dosage of the active agent of the present invention used for treating each individual patient. By way of example and not intended to limit the present disclosure, the dosage of the active agent of the present invention can be about 0.0001 to about 1g / kg of the body weight / day of the treated subject, about 0.0001 to about 0.001g / kg body weight / day or about 0.01mg to about 1g / kg body weight / day.

[0240] controlled-release preparations

[0241] In some embodiments, the active agent described herein can be modified into a depot form so that the mode of release of the active agent of the present disclosure into the body to which it is applied is controlled in terms of time and position in the body (see, for example, U.S. Patent No. 4,450,150). The depot form of the active agent of the present disclosure can be, for example, an implantable composition comprising an active agent and a porous or non-porous material (such as a polymer), wherein the active agent is encapsulated by the material or diffused throughout the degradation of the material and / or non-porous material. The depot is then implanted in the desired position in the subject's body and the active agent is released from the implant at a predetermined rate.

[0242] In some aspects, the pharmaceutical composition comprising an active agent is modified to have any type of in vivo release profile. In some aspects, the pharmaceutical composition is an immediate release, controlled release, sustained release, extended release, delayed release or biphasic release formulation. Methods for preparing peptides for controlled release are known in the art. See, for example, Qian et al., J Pharm 374: 46-52 (2009), and International Patent Application Publication Nos. WO 2008 / 130158, WO 2004 / 033036, WO 2000 / 032218 and WO 1999 / 040942.

[0243] The compositions of the present invention may also comprise, for example, micelles or liposomes or some other encapsulated form, or may be administered in an extended release form to provide prolonged storage and / or delivery.

[0244] use

[0245] The antigen binding proteins of the present disclosure can be used to inhibit tumor growth. Without being bound by a particular theory, the inhibitory effects of the antigen binding proteins provided herein allow such entities to be used in methods for treating cancer.

[0246] Thus, provided herein are methods for inhibiting tumor growth in a subject and methods for reducing tumor size in a subject. In various embodiments, these methods comprise administering to a subject a pharmaceutical composition of the present disclosure in an amount effective to inhibit tumor growth in the subject or reduce tumor size in the subject. In various aspects, the growth of an ovarian tumor, a melanoma tumor, a bladder tumor, or an endometrial tumor is inhibited. In various aspects, the size of an ovarian tumor, a melanoma tumor, a bladder tumor, or an endometrial tumor is reduced.

[0247] As used herein, the terms "inhibit" or "reduce" and words derived therefrom may not be 100% or complete inhibition or reduction. On the contrary, one of ordinary skill in the art recognizes that there are varying degrees of inhibition or reduction with potential benefits or therapeutic effects. In this regard, the antigen-binding proteins of the present disclosure can inhibit tumor growth or reduce tumor size to any amount or level. In various embodiments, the inhibition provided by the methods of the present disclosure is at least or about 10% inhibition (e.g., at least or about 20% inhibition, at least or about 30% inhibition, at least or about 40% inhibition, at least or about 50% inhibition, at least or about 60% inhibition, at least or about 70% inhibition, at least or about 80% inhibition, at least or about 90% inhibition, at least or about 95% inhibition, at least or about 98% inhibition). In various embodiments, the reduction provided by the methods of the present disclosure is at least or about a 10% reduction (e.g., at least or about a 20% reduction, at least or about a 30% reduction, at least or about a 40% reduction, at least or about a 50% reduction, at least or about a 60% reduction, at least or about a 70% reduction, at least or about an 80% reduction, at least or about a 90% reduction, at least or about a 95% reduction, at least or about a 98% reduction).

[0248] Further provided herein are methods of treating a subject having cancer (e.g., a cancer expressing CLDN6). In various embodiments, the method comprises administering to the subject a pharmaceutical composition of the present disclosure in an amount effective to treat the cancer in the subject.

[0249] For purposes herein, the cancer of the methods disclosed herein can be any cancer, eg, any malignant growth or tumor caused by abnormal and uncontrolled cell division that can spread to other parts of the body through the lymphatic system or bloodstream. In some aspects, the cancer is a cancer selected from the group consisting of acute lymphocytic carcinoma, acute myeloid leukemia, alveolar rhabdomyosarcoma, bone cancer, brain cancer, breast cancer, cancer of the anus, anal canal or anorectum, eye cancer, intrahepatic bile duct cancer, joint cancer, cancer of the neck, gallbladder or pleura, cancer of the nose, nasal cavity or middle ear, oral cancer, vulvar cancer, chronic lymphocytic leukemia, chronic myeloid carcinoma, colon cancer, esophageal cancer, cervical cancer, gastrointestinal carcinoid, Hodgkin lymphoma, hypopharyngeal cancer, kidney cancer, laryngeal cancer, liver cancer, lung cancer, malignant mesothelioma, melanoma, multiple myeloma, nasopharyngeal cancer, non-Hodgkin lymphoma, ovarian cancer, pancreatic cancer, cancer of the peritoneum, omentum and mesentery, pharyngeal cancer, prostate cancer, rectal cancer, kidney cancer (e.g., renal cell carcinoma (RCC)), small intestine cancer, soft tissue cancer, stomach cancer, testicular cancer, thyroid cancer, ureteral cancer and bladder cancer. In particular aspects, the cancer is selected from the group consisting of head and neck cancer, ovarian cancer, cervical cancer, bladder cancer and esophageal cancer, pancreatic cancer, gastrointestinal cancer, gastric cancer, breast cancer, endometrial cancer and colorectal cancer, hepatocellular carcinoma, glioblastoma, bladder cancer, lung cancer (e.g., non-small cell lung cancer (NSCLC), bronchoalveolar carcinoma). In various aspects, the cancer is ovarian cancer, melanoma, bladder cancer, lung cancer, liver cancer, endometrial cancer. In various aspects, the cancer is any cancer characterized by moderate to high expression of CLDN6. See, e.g., Figures 1 to 3 In various aspects, the cancer is acute myeloid leukemia, large B-cell lymphoma, gastric cancer, prostate cancer, melanoma, colon cancer, rectal cancer, bladder cancer, cervical cancer, liver cancer, breast cancer, renal clear cell carcinoma, head and neck cancer, sarcoma, renal chromophobe cell carcinoma, low-grade glioma, adrenocortical carcinoma, glioblastoma, renal papillary cell carcinoma, lung squamous cell carcinoma, thyroid cancer, lung adenocarcinoma, pancreatic cancer, endometrioid carcinoma, uterine carcinosarcoma, or ovarian cancer. In various aspects, the cancer is selected from ovarian cancer, endometrioid carcinoma, uterine cancer, lung cancer, gastric cancer, breast cancer, head and neck squamous cell carcinoma (HNSCC), cervical cancer, and bladder cancer.

[0250] As used herein, the term "treatment" and words related thereto do not necessarily mean 100% or complete treatment. On the contrary, one of ordinary skill in the art recognizes that there are different degrees of treatment with potential benefits or therapeutic effects. In this regard, the methods of treating cancer disclosed herein can provide any amount or any level of treatment. In addition, the treatment provided by the methods of the present disclosure can include treating one or more conditions or symptoms or signs of the cancer being treated. In addition, the treatment provided by the methods of the present disclosure can cover slowing the progression of cancer. For example, these methods can treat cancer by enhancing T cell activity or an immune response against cancer, reducing tumor or cancer growth, reducing tumor cell metastasis, increasing cell death of tumor or cancer cells, etc. In various aspects, these methods treat by delaying the onset or recurrence of 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 longer. In various aspects, these methods treat by increasing the survival of the subject.

[0251] The antigen-binding proteins of the present disclosure can also be used to detect CLDN6 in a sample or diagnose CLDN6-positive cancers. Therefore, the present disclosure provides a method for detecting claudin 6 (CLDN6) in a sample. In various embodiments, the method comprises contacting the sample with an antigen-binding protein, conjugate, or fusion protein as described herein, and measuring an immune complex comprising the antigen-binding protein, conjugate, or fusion protein that binds to CLDN6. The present disclosure also provides a method for diagnosing claudin 6 (CLDN6)-positive cancer in a subject. In various embodiments, the method comprises contacting a biological sample comprising cells or tissues obtained from a subject with an antigen-binding protein, conjugate, or fusion protein as described herein, and measuring an immune complex comprising the antigen-binding protein, conjugate, or fusion protein that binds to CLDN6.

[0252] Subjects

[0253] In some embodiments of the present disclosure, the subject is a mammal, including but not limited to: rodents, such as mice and hamsters; and lagomorphs, such as rabbits; carnivores, including felines (cats) and canines (dogs); artiodactyls, including bovines (cows) and suids (pigs), or odd-toed ungulates, including equines (horses). In some aspects, the mammal is a primate, ceboids, or simoids (monkeys), or anthropoids (humans and apes). In some aspects, the mammal is a human.

[0254] Reagent test kit

[0255] In some embodiments, the antigen-binding proteins of the present disclosure are provided in a kit. In various aspects, the kit comprises one or more antigen-binding proteins as a unit dose. For the purposes of this article, a "unit dose" refers to a discrete amount dispersed in a suitable carrier. In various aspects, a unit dose is an amount sufficient to provide a subject with a desired effect, such as inhibiting tumor growth, reducing tumor size, or treating cancer. Therefore, provided herein are kits comprising the antigen-binding proteins of the present disclosure optionally provided in unit doses. In various aspects, the kit comprises several unit doses, such as a weekly or monthly supply of unit doses, optionally each of which is individually packaged or otherwise separated from other unit doses. In some embodiments, the components of the kit / unit dose are packaged together with instructions for administering to a patient. In some embodiments, the kit comprises one or more devices for administering to a patient, such as a needle and a syringe. In some aspects, the antigen-binding proteins of the present disclosure, their pharmaceutically acceptable salts, conjugates comprising the antigen-binding proteins, or multimers or dimers comprising the antigen-binding proteins are pre-packaged in a ready-to-use form, such as a syringe, an intravenous bag, etc. In some aspects, the kit further comprises other therapeutic or diagnostic agents or pharmaceutically acceptable carriers (e.g., solvents, buffers, diluents, etc.), including any of those described herein. In specific aspects, the kit comprises an antigen binding protein of the present disclosure and an agent (e.g., a therapeutic agent) for chemotherapy or radiotherapy.

[0256] Various implementation plans

[0257] In various embodiments of the present disclosure, the antigen binding protein binds to human Claudin 6 (CLDN6) protein (SEQ ID NO: 200), wherein (a) the antigen binding protein binds to extracellular loop 2 (EL2) of the extracellular domain (ECD) of CLDN6 but not to extracellular loop 1 (EL1) of the ECD of CLDN6; or (b) does not bind to any of Claudin 3 (CLDN3), Claudin 4 (CLDN4), and Claudin 9 (CLDN9), and inhibits binding of a reference antibody to CLDN6 endogenously expressed in OVCA429 cells by less than about 1200 nM; or (c) a combination thereof. In each case, the antigen binding protein binds to an epitope within the amino acid sequence WTAHAIIRDFYNPLVAEAQKREL (SEQ ID NO: 2), or binds to the amino acid sequence THAIIRDFYNPL (SEQ ID NO: 3) or LVAEAQKREL (SEQ ID NO: 4) of CLDN 6. In various aspects, the antigen binding protein does not bind to any one or more of claudin 3 (CLDN3), claudin 4 (CLDN4), and claudin 9 (CLDN9). In various cases, the antigen binding protein does not bind to CLDN3. In various cases, the antigen binding protein binds to CLDN6, CLDN4, and CLDN9, but not to CLDN3. In various cases, the antigen binding protein binds to CLDN6 and CLDN4, but not to CLDN3 or CLDN9. In various aspects, the antigen binding protein binds to CLDN6 and CLDN9, but not to CLDN3 or CLDN4.

[0258] In various cases, the antigen binding proteins of the present disclosure inhibit binding of a reference antibody to CLDN6 endogenously expressed in OVCA429 cells at less than about 1200 nM, and the reference antibody comprises a light chain variable sequence of SEQ ID NO: 181 and a heavy chain variable sequence of SEQ ID NO: 182, or a light chain variable sequence of SEQ ID NO: 185 and a heavy chain variable sequence of SEQ ID NO: 186. In various aspects, the antigen binding proteins of the present disclosure inhibit binding of a reference antibody to CLDN6 endogenously expressed in OVCA429 cells at less than about 1000 nM or less than 750 nM (e.g., less than about 500 nM, less than about 250 nM, less than about 100 nM), and the reference antibody comprises a light chain variable sequence of SEQ ID NO: 181 and a heavy chain variable sequence of SEQ ID NO: 182, or a light chain variable sequence of SEQ ID NO: 185 and a heavy chain variable sequence of SEQ ID NO: 186.

[0259] In various embodiments, the antigen binding protein comprises (a) a heavy chain CDR1 amino acid sequence listed in Table A or Table A1, or a sequence selected from the group consisting of SEQ ID NO: 11, 17, 23, 29, 35, 41, 47, 53, 59, 65, 71, 77, 83, 89, 95, 101, 107, 113, 119, 125, 131, 452, 455, 461, 465, and 472, or variant sequences thereof that differ by only one or two amino acids or have at least or about 70% (e.g., at least or about 85%, at least or about 90%) sequence identity; (b) a heavy chain CDR2 amino acid sequence listed in Table A or Table A1, or a sequence selected from the group consisting of SEQ ID NO: NO: 12, 18, 24, 30, 36, 42, 48, 54, 60, 66, 72, 78, 84, 90, 86, 102, 108, 114, 120, 126, 132, 475, 456, 462, 466, 468 and 473, or variant sequences thereof that differ by only one or two amino acids or have at least or about 70% (e.g., at least or about 85%, at least or about 90%) sequence identity; (c) a heavy chain CDR3 amino acid sequence listed in Table A or Table A1, or a sequence selected from the group consisting of: SEQ ID NO: 12, 18, 24, 30, 36, 42, 48, 54, 60, 66, 72, 78, 84, 90, 86, 102, 108, 114, 120, 126, 132, 475, 456, 462, 466, 468 and 473, or variant sequences thereof that differ by only one or two amino acids or have at least or about 70% (e.g., at least or about 85%, at least or about 90%) sequence identity. NO: 13, 19, 25, 31, 37, 43, 49, 55, 61, 67, 73, 79, 85, 91, 97, 103, 109, 115, 121, 127, 133, 453, 457, 463, 467, 469 and 474, or variant sequences thereof that differ by only one or two amino acids or have at least or about 70% (e.g., at least or about 85%, at least or about 90%) sequence identity; (d) a light chain CDR1 amino acid sequence listed in Table A or Table A1, or a sequence selected from the group consisting of: SEQ ID NO: 13, 19, 25, 31, 37, 43, 49, 55, 61, 67, 73, 79, 85, 91, 97, 103, 109, 115, 121, 127, 133, 453, 457, 463, 467, 469 and 474, or variant sequences thereof that differ by only one or two amino acids or have at least or about 70% (e.g., at least or about 85%, at least or about 90%) sequence identity. NO: 8, 14, 20, 32, 38, 44, 50, 56, 62, 68, 74, 80, 86, 92, 98, 104, 110, 116, 122, 128, 449, 476, 458, 464 and 470, or variant sequences thereof that differ by only one or two amino acids or have at least or about 70% (e.g., at least or about 85%, at least or about 90%) sequence identity; (e) a light chain CDR2 amino acid sequence listed in Table A or Table A1, or a sequence selected from the group consisting of: SEQ ID NO: 8, 14, 20, 32, 38, 44, 50, 56, 62, 68, 74, 80, 86, 92, 98, 104, 110, 116, 122, 128, 449, 476, 458, 464 and 470, or variant sequences thereof that differ by only one or two amino acids or have at least or about 70% (e.g., at least or about 85%, at least or about 90%) sequence identity; NOs: 9, 15, 21, 27, 33, 39, 45, 51, 57, 63, 69, 75, 81, 87, 93, 99, 105, 111, 117, 123, 129, 450, 477, 459, and 471, or variant sequences thereof that differ by only one or two amino acids, or have at least or about 70% (e.g., at least or about 85%, at least or about 90%) sequence identity;(f) a light chain CDR3 amino acid sequence listed in Table A or Table A1, or a sequence selected from the group consisting of SEQ ID NO: 10, 16, 22, 28, 34, 40, 46, 52, 58, 64, 70, 76, 82, 88, 94, 100, 106, 112, 118, 124, 130, 451, 454, and 460, or a variant thereof that differs by only one or two amino acids or has at least or about 70% (e.g., at least or about 85%, at least or about 90%) sequence identity; (g) a combination of any two or more of (a) to (f).

[0260] In various aspects, the antigen binding protein comprises a light chain CDR1 amino acid sequence, a light chain CDR2 amino acid sequence, and a light chain CDR3 amino acid sequence listed in Table A or Table A1, and one or two of the heavy chain CDR amino acid sequences listed in Table A or Table A1. In some cases, the antigen binding protein comprises a heavy chain CDR1 amino acid sequence, a heavy chain CDR2 amino acid sequence, and a heavy chain CDR3 amino acid sequence listed in Table A or Table A1, and one or two of the light chain CDR amino acid sequences listed in Table A or Table A1. In various aspects, the antigen binding protein comprises six CDR amino acid sequences selected from the group consisting of: (a) SEQ ID NOs: 74-79; (b) SEQ ID NOs: 50-55; (c) SEQ ID NOs: 122-127; (d) SEQ ID NOs: 26-31; (e) SEQ ID NOs: 128-133; (f) SEQ ID NOs: 38-43; (g) SEQ ID NOs: 62-67; (h) SEQ ID NOs: 80-85; (i) SEQ ID NOs: 44-49; (j) SEQ ID NOs: 86-91; (k) SEQ ID NOs: 104-109; (l) SEQ ID NOs: 56-61; (m) SEQ ID NOs: 32-37; (n) SEQ ID NOs: 110-115; (o) SEQ ID NOs: 98-103; (p) SEQ ID NOs: 92-97; (q) SEQ ID NOs: NO: 116-121; (r) SEQ ID NO: 8-13; (t) SEQ ID NO: 68-73; (u) SEQ ID NO: 14-19; (v) SEQ ID NO: 20-25, (v) SEQ ID NO: 449-453 and 475; (w) SEQ ID NO: 476-477, 454-457, (x) SEQ ID NO:458-463; (y) SEQ ID NO:57, 58, 464-467; (z) SEQ ID NO:68-71 and 468-469; and (aa) SEQ ID NO:112 and 470-474.In various aspects, the antigen binding protein comprises (a) a heavy chain variable region amino acid sequence listed in Table B, or a sequence selected from the group consisting of SEQ ID NO: 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173 and 175, or variant sequences thereof that differ by only one or two amino acids or have at least or about 70% (e.g., at least or about 85%, at least or about 90%) sequence identity; (b) a light chain variable region amino acid sequence listed in Table B, or a sequence selected from the group consisting of SEQ ID NO: NO:134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174 and 176, or variant sequences thereof that differ only by one or two amino acids, or having at least or about 70% (e.g., at least or about 85%, at least or about 90%) sequence identity; or both (a) and (b). In various aspects, the antigen binding protein comprises a pair of amino acid sequences selected from the group consisting of: (a) SEQ ID NOs: 156 and 157; (b) SEQ ID NOs: 148 and 149; (c) SEQ ID NOs: 172 and 173; (d) SEQ ID NOs: 140 and 141; (e) SEQ ID NOs: 174 and 175; (f) SEQ ID NOs: 144 and 145; (g) SEQ ID NOs: 152 and 153; (h) SEQ ID NOs: 158 and 159; (i) SEQ ID NOs: 146 and 147; (j) SEQ ID NOs: 160 and 161; (k) SEQ ID NOs: 166 and 167; (l) SEQ ID NOs: 150 and 151; (m) SEQ ID NOs: 142 and 143; (n) SEQ ID NOs: 168 and 169; (o) SEQ ID NOs: 164 and 165; (p) SEQ ID NOs: 166 and 167; NOs: 162 and 163; (q) SEQ ID NOs: 170 and 171; (r) SEQ ID NOs: 134 and 135; (s) SEQ ID NOs: 154 and 155; (t) SEQ ID NOs: 136 and 137; and (u) SEQ ID NOs: 138 and 139.

[0261] In various embodiments, the antigen binding protein comprises (a) a heavy chain variable region amino acid sequence listed in Table Bl or Table C, or a sequence selected from the group consisting of SEQ ID NOs: 376-379, 384-387, 391-396, 403-408, 412, 413, 416-419, 422-427, 478, 480, 482, 484, 486, and 488, or variant sequences thereof that differ by only one or two amino acids or have at least or about 70%, or about 80%, or about 90%, or about 95% sequence identity; (b) a light chain variable region amino acid sequence listed in Table Bl or Table C, or a sequence selected from the group consisting of SEQ ID NOs: NO:380-383, 388-390, 397-402, 409-411, 414, 415, 420, 421 and 479, 481, 483, 485, 487 and 489, or variant sequences thereof that differ by only one or two amino acids or have at least or about 70%, or about 80%, or about 90% or about 95% sequence identity; or (c) (a) and (b) both. In various aspects, the antigen binding protein comprises a pair of amino acid sequences listed in Table D.

[0262] The present disclosure provides an antigen binding protein comprising: (A) a HC CDR1 comprising the amino acid sequence YTFTXYT (SEQ ID NO: 452), wherein X is T, V, D or S, optionally comprising the amino acid sequence YTFTTYT (SEQ ID NO: 11); (B) a HC CDR2 comprising the amino acid sequence IXPSSGYT (SEQ ID NO: 475), wherein X is Q, S, A or N, optionally comprising the amino acid sequence INPSSGYT (SEQ ID NO: 12); (C) a HC CDR3 comprising the amino acid sequence AXGDYYVAY (SEQ ID NO: 453), wherein X is N, Q, H or D, optionally comprising the amino acid sequence ANGDYYVAY (SEQ ID NO: 13); (D) a LC CDR1 comprising the amino acid sequence SSVSSXY (SEQ ID NO: 449), wherein X is T, V, F or D, optionally comprising the amino acid sequence SSVSSTY (SEQ ID NO: 8); (E) a LC CDR2 comprising the amino acid sequence of XTX (SEQ ID NO: 450), wherein X at position 1 is S, T, Q or A and X at position 3 is S, T, D or Q, optionally comprising the amino acid sequence of STS (SEQ ID NO: 9); and (F) LC CDR3 comprising the amino acid sequence of HXYXRSPLT (SEQ ID NO: 451), wherein X at position 2 is Q, H or S and X at position 4 is H, Y, Q or S, optionally comprising the amino acid sequence of HQYHRSPLT (SEQ ID NO: 10).

[0263] An antigen-binding protein comprising: (A) a HC CDR1 comprising the amino acid sequence of FTFSXYX (SEQ ID NO: 455), wherein X at position 5 is N, S, R, Q or A and X at position 7 is W, H, Y or F, optionally comprising the amino acid sequence of FTFSNYW (SEQ ID NO: 23); (B) a HC CDR2 comprising the amino acid sequence of IRLKXDXYAT (SEQ ID NO: 456), wherein X at position 5 is S, N, A or T and X at position 7 is Q, S, A or N, optionally comprising the amino acid sequence of IRLKSDNYAT (SEQ ID NO: 24); (C) a HC CDR3 comprising the amino acid sequence of XDGPPSGX (SEQ ID NO: 457), wherein X at position 1 is N, D or T and X at position 8 is S, T, A, C or Y, optionally comprising the amino acid sequence of NDGPPSGC (SEQ ID NO: 25); NO: 25); (D) LC CDR1 comprising the amino acid sequence EXIYSY (SEQ ID NO: 476), wherein X is Q, S, A, D or N, optionally comprising the amino acid sequence ENIYSY (SEQ ID NO: 20); (E) LC CDR2 comprising the amino acid sequence XAK (SEQ ID NO: 477), wherein X at position 1 is Q, S, A, D or N, optionally comprising the amino acid sequence NAK (SEQ ID NO: 21); and (F) LC CDR3 comprising the amino acid sequence QXHYXVPWT (SEQ ID NO: 454), wherein X at position 2 is H, Q, S or T and X at position 5 is T, S, N or G, optionally comprising the amino acid sequence QHHYTVPWT (SEQ ID NO: 22).

[0264] An antigen binding protein comprising: (A) a HC CDR1 comprising the amino acid sequence YTXTXYT (SEQ ID NO: 461), wherein X at position 3 is F, Y, S or T and X at position 5 is S, T, Y or D, optionally comprising the amino acid sequence YTFTSYT (SEQ ID NO: 29); (B) a HC CDR2 comprising the amino acid sequence IXPSSXYT (SEQ ID NO: 462), wherein X at position 2 is Q, S, A or N and X at position 6 is T, S, V, D or G, optionally comprising the amino acid sequence INPSSTYT (SEQ ID NO: 30); (C) a HC CDR3 comprising the amino acid sequence XRGEXGGFAY (SEQ ID NO: 463), wherein X at position 1 is S, A, T or V and X at position 5 is L, V or F, optionally comprising the amino acid sequence SRGELGGFAY (SEQ ID NO: 464). NO: 31); (D) LC CDR1 comprising the amino acid sequence of QSLVHSXGXTY (SEQ ID NO: 458), wherein X at position 7 is D, N, E, Q, S or A and X at position 9 is Q, S, A, D or N, optionally comprising the amino acid sequence of QSLVHSDGNTY (SEQ ID NO: 26); (E) LC CDR2 comprising the amino acid sequence of XVX (SEQ ID NO: 459), wherein X at position 1 is K, Q or R and X at position 3 is S, T or V, optionally comprising the amino acid sequence of KVS (SEQ ID NO: 27); and (F) LC CDR3 comprising the amino acid sequence of SXXTHVPYT (SEQ ID NO: 460), wherein X at position 2 is Q, H or T and X at position 3 is S, G, T or D, optionally comprising the amino acid sequence of SQSTHVPYT (SEQ ID NO: 28).

[0265] In various aspects, the antigen binding proteins of the present disclosure are antibodies, such as monoclonal antibodies. In various cases, the antigen binding proteins are IgG. In various aspects, the antigen binding proteins inhibit colony growth by at least about 50% in a soft agar 3D proliferation assay or inhibit tumor growth in xenograft mice injected with human cancer cells. In various aspects, the antigen binding proteins inhibit tumor growth in xenograft mice injected with ovarian cancer cells, melanoma cancer cells, bladder cancer cells, or endometrial cancer cells. In various cases, the antigen binding proteins inhibit tumor growth by at least 50% in xenograft mice injected with ovarian cancer cells, bladder cancer cells, or endometrial cancer cells.

[0266] The present disclosure provides conjugates comprising an antigen-binding protein as described herein and a heterologous moiety. The present disclosure also provides fusion proteins comprising the antigen-binding protein as described herein. The present disclosure also provides nucleic acids comprising a nucleotide sequence encoding the antigen-binding protein, conjugate, or fusion protein of the present disclosure. The present disclosure provides vectors comprising a nucleic acid comprising a nucleotide sequence encoding the antigen-binding protein, conjugate, or fusion protein of the present disclosure. The present disclosure further provides host cells comprising a nucleic acid or vector of the present disclosure.

[0267] The present disclosure provides a method for producing an antigen-binding protein that binds to a Claudin 6 (CLDN6) protein, the method comprising (i) culturing a host cell of the present disclosure in a cell culture medium, wherein the host cell comprises a nucleic acid comprising a nucleotide sequence encoding the antigen-binding protein of any one of the preceding claims, and (ii) harvesting the antigen-binding protein from the cell culture medium. Furthermore, a method for producing a fusion protein comprising an antigen-binding protein that binds to a Claudin 6 (CLDN6) protein is provided, the method comprising (i) culturing a host cell of the present disclosure in a cell culture medium, wherein the host cell comprises a nucleic acid comprising a nucleotide sequence encoding the fusion protein of the present disclosure, and (ii) harvesting the fusion protein from the cell culture medium.

[0268] The present disclosure also provides a method for producing a pharmaceutical composition, comprising combining an antigen-binding protein, conjugate, fusion protein, nucleic acid, vector, host cell, or combination thereof of the present disclosure and a pharmaceutically acceptable carrier, diluent, or excipient. Also provided is a pharmaceutical composition comprising an antigen-binding protein, conjugate, fusion protein, nucleic acid, vector, host cell, or combination thereof of the present disclosure and a pharmaceutically acceptable carrier, diluent, or excipient.

[0269] Provided herein are methods for treating a subject having a cancer that expresses CLDN6, comprising administering to the subject a pharmaceutical composition described herein in an amount effective to treat the cancer. Also provided are methods for inhibiting tumor growth in a subject, comprising administering to the subject a pharmaceutical composition described herein in an amount effective to inhibit tumor growth. The present disclosure provides methods for reducing tumor size in a subject, comprising administering to the subject a pharmaceutical composition described herein in an amount effective to reduce tumor size. Also provided are methods for preventing cancer recurrence in a subject, comprising administering to the subject a pharmaceutical composition described herein in an amount effective to prevent cancer recurrence.

[0270] The present disclosure provides a method for detecting claudin 6 (CLDN6) in a sample, the method comprising contacting the sample with an antigen-binding protein, conjugate, or fusion protein of the present disclosure, and measuring immune complexes comprising the antigen-binding protein, conjugate, or fusion protein that binds to CLDN6. Also provided herein is a method for diagnosing a claudin 6 (CLDN6)-positive cancer in a subject, the method comprising contacting a biological sample comprising cells or tissue obtained from the subject with an antigen-binding protein, conjugate, or fusion protein of the present disclosure, and measuring immune complexes comprising the antigen-binding protein, conjugate, or fusion protein that binds to CLDN6.

[0271] The present disclosure also provides methods for treating cancer in subjects diagnosed with low overexpression of CLDN6. In various embodiments, the method comprises administering a currently disclosed pharmaceutical composition to a subject in an amount effective to prevent cancer recurrence. In some aspects, administration induces apoptosis in tumor cells, and optionally, administration induces apoptosis in cells expressing CLDN6. In various aspects, the subject has a tumor, and the tumor is semi-quantitatively classified into one of four groups: high expressers, moderate expressers, low expressers, and non-expressers. In various cases, high expressers are defined as having CLDN6 RNA greater than 12 log fragments per kilobase pair (FPKM), where CLDN6 RNA is measured by RNASeq, or CLDN6 protein levels greater than 3+ as measured by immunohistochemistry (IHC). In various cases, moderate expressers are defined as having CLDN6 RNA greater than 10 log FPKM, where CLDN6 RNA is measured by RNASeq, or CLDN6 protein levels greater than 2+ as measured by IHC. In various cases, low expressers are defined as CLDN6 RNA greater than 6 log FPKM, where CLDN6 RNA is measured by RNASeq, or CLDN6 protein levels greater than 1+ as measured by IHC. In various cases, non-expressers are defined as CLDN6 RNA less than 6 log FPKM, where CLDN6 RNA is measured by RNASeq, or CLDN6 protein levels below the limit of detection of IHC. In various aspects, subjects with the tumors are also described as high expressers, intermediate expressers, low expressers, or non-expressers of CLDN6.

[0272] The following examples are given merely to illustrate the present disclosure and are not intended to limit its scope in any way.

[0273] Example

[0274] Example 1

[0275] This example illustrates the analysis of CLDN6 RNA levels in different cell and tissue sources.

[0276] To establish a baseline for CLDN6 expression in materials from different sources, the expression levels of CLDN6 were determined in patient samples, normal tissues, and cell lines created by the Translational Oncology Research Laboratory (TORL).

[0277] Levels of CLDN6 RNA in patient samples were measured using information contained in the Cancer Genome Atlas (TCGA) database, managed by the National Cancer Institute (NCI). CLDN6 levels in normal tissues were measured using information from the Genotype-Tissue Expression (GTEx) database, maintained by the Common Fund. Analysis of tissues from the GTEx database showed that CLDN6 was detectable in a variety of sites, including the brain, pituitary gland, pancreas, kidney, lung, thyroid gland, and cervix, among other tissues. Figure 1 ).

[0278] CLDN6 expression levels in TORL cancer cell lines were measured using Agilent 44K microarrays (4x44K array chips, Agilent Technologies, Santa Clara, CA) and RNA sequencing (RNA-seq) assays. RNA-seq was performed by BGI Americas (Cambridge, MA) using their "RNASeq for Quantification" service. Figure 2 and Figure 3 As shown in Figure 3, although CLDN6 expression levels can be detected in breast cancer, kidney cancer, colon cancer, sarcoma and liver cancer cells, ovarian cancer, head and neck cancer, lung cancer and bladder cancer cells have the highest CLDN6 expression levels.

[0279] Example 2

[0280] This example demonstrates the generation of cells engineered to overexpress CLDN6.

[0281] Models engineered to overexpress CLDN6 were generated. These models were used to determine the efficacy of the CLDN6 antibodies described in Example 5. Briefly, the nucleotide sequence encoding CLDN6 was engineered into a bicistronic vector containing a CMV promoter and an attenuated internal ribosome entry site (IRES) of encephalomyocarditis virus (EMCV). The IRES was located between the gene of interest (GOI) cDNA (CLDN6) and the puromycin cDNA. A woodchuck posttranscriptional regulatory element (WPRE) was located downstream of the puromycin cDNA. This vector also expressed a GFP marker sequence or a MycDDK tag. The sequence of the expression vector containing GFP is provided herein as SEQ ID NO: 189.

[0282] The expression vector was virally transduced into HEK293T cells (for screening purposes) and NIH3T3 cells (for immunization). Positively transduced cells were selected based on their survival in medium containing puromycin (1 μg / ml). Positive cells were subcloned to obtain a stable, homogeneous clonal population of CLDN6-overexpressing cells.

[0283] A reference CLDN6 monoclonal antibody (mAb) was used in the BD Biosciences Accuri TM The expression of subcloned CLDN6 was confirmed by flow cytometry on a flow cytometer (San Jose, CA). Secondary antibodies and conjugates: Alexa 647 goat anti-mouse IgG (minimum x reactivity) antibody (Biolegend, San Diego, CA; catalog number 405322) was used to detect the binding activity between the reference CLDN6 mAb and the subcloned expressed CLDN6.

[0284] By using The cellular localization of CLDN6 was determined by fluorescence microscopy using a CLDN6 imaging system (Synentec (Mountain View, CA)) and cells expressing a CLDN6-green fluorescent protein (GFP) fusion protein. Figure 4 As shown, GFP fluorescence was detected in the cell membrane, demonstrating that CLDN6 was located on the cell membrane.

[0285] Example 3

[0286] This example illustrates the generation of reference and control antibodies.

[0287] By cloning the antibody heavy chain variable region and light chain variable region into ExpiCHO TM Benchmark (reference) CLDN6-specific antibodies and control antibodies were prepared by producing recombinant mouse IgG2A chimeric antibodies in a CLDN6-expression system (ThermoFisher Scientific, Waltham, MA). These antibodies were tested together with the newly generated CLDN6-specific antibodies described in Example 5.

[0288] Briefly, ExpiCHO was used according to the manufacturer's protocol. TM Plasmids containing control and reference antibody sequences were transfected using the ExpiCHO expression system (Cat. No. A29133, ThermoFisher Scientific, USA). On day 1 after transfection, cells were cultured at 37°C and 8% CO2, and then cultured in the medium provided in the kit at 32°C and 5% CO2. The cells were centrifuged at 1,000 g for 10 minutes and then at 5,000 g for 30 minutes. TMThe culture medium is clarified to purify the antibody. The supernatant is then filtered using a 0.45 μm filter and subsequently using a 0.22 μm filter. Subsequently, according to the manufacturer's protocol, protein A / G resin (Life Technologies, Carlsbad, CA; catalog number 20424) is used to affinity purify the supernatant. Before ELISA purification, the antibody titer in the culture medium is roughly determined to ensure that the amount of the loaded culture medium occupies less than 80% resin binding capacity. After incubation, the resin is washed with PBS and eluted with elution buffer (Life Technologies, catalog number 21004). By adding the Tris buffer of pH 8.0, the eluted fraction is immediately adjusted to physiological pH. Subsequently, Amicon Ultra-15 centrifugal filter devices (Life Technologies, catalog number UFC900324) are used to carry out buffer exchange and protein concentration in PBS buffer to the purified antibody. Antibody concentration is determined by BCA protein assay. SDS-PAGE and Coomassie staining are carried out to test antibody purity. The purified protein was aliquoted and stored at -80°C for long term storage or at 4°C for immediate use.

[0289] The integrity of the antibody was verified by SDS-PAGE followed by Coomassie staining under non-reducing and reducing conditions; under non-reducing conditions, a major band was observed around 150 kDa, while under reducing conditions, two bands were observed, 50 kDa and 25 kDa.

[0290] In substantially the same manner, pairs of sequences with similarity ( Figure 5 ) are antibodies specific for other CLDN family members (i.e., CLDN3, CLDN4, and CLDN9), except that the antibody sequence contained in the plasmid is an antibody sequence specific for CLDN3, CLDN4, or CLDN9.

[0291] Example 4

[0292] This example illustrates the characterization of cell lines with high endogenous CLDN6 expression.

[0293] A panel of cancer cell lines was analyzed for endogenous expression of CLDN6 by FACS and Western blotting. Briefly, binding of the antibodies to the target was validated by FACS using cells overexpressing CLDN6 (e.g., HEK293T cells overexpressing CLDN6 as described in Example 2) and cell lines endogenously expressing CLDN6 at high or low levels as determined in Example 1. Cells expressing CLDN6 were incubated on ice for 30 minutes with reference or control antibodies (described in Example 3), washed, and incubated with Alexa Fluor 500 antibody. 647 conjugated goat anti-mouse IgG (minimum x reactivity) antibody (Biolegne catalog number 405322) was incubated on ice for 30 minutes. TM Fluorescence was read on a flow cytometer (San Jose, CA).

[0294] Western blotting was performed on nitrocellulose using reference and control antibodies. Briefly, samples from cell lysates were boiled to denature the protein content. SDS-PAGE (SDS-polyacrylamide gel electrophoresis) was used to separate the denatured proteins according to the length of the polypeptides. The separated proteins were then transferred from the acrylamide gel to a nitrocellulose membrane. The membrane was blocked with a 2% bovine serum albumin (BSA) solution to minimize nonspecific antibody binding. The membrane was incubated with a reference or control antibody. The membrane was stained with a horseradish peroxidase (HRP)-conjugated secondary antibody that recognized the reference or control antibody, and the secondary antibody was detected via chemiluminescence.

[0295] Overexpressing lines are used to validate control and reference antibodies, and once validated, control and reference antibodies are used to characterize the endogenous cell line. Cells overexpressing CLDN6 are included in these assays as positive controls.

[0296] FACS analysis showed that four ovarian cancer cell lines, in addition to endometrial, bladder, lung, and upper gastrointestinal cancer cell lines, expressed high levels of CLDN6 on their surfaces. High levels of CLDN6 expression were also detected by Western blotting. Two other ovarian cancer cell lines, another liver cancer cell line, another lung cancer cell line, and another upper gastrointestinal cancer cell line showed moderate surface expression of CLDN6 as detected by Western blotting. Endometrial and bladder tumor cells also expressed high levels of CLDN6 as in vivo xenografts. Table 2 summarizes the endogenous expression levels of CLDN6 in the tested cancer cell lines.

[0297] Table 2

[0298]

[0299]

[0300]

[0301] Example 5

[0302] This example demonstrates the immunization of mice to generate CLDN6-specific antibodies.

[0303] CLDN6-specific antibodies were generated by immunizing Balb / c and CD1 mice with a mixture of three different peptide immunogens according to Fred Hutchinson Cancer Research Center technology. These three peptides span the second loop of the CLDN6 extracellular domain (i.e., EL2). These peptides include full-length EL2, a peptide spanning the front (N-terminal) half of EL2, and a peptide spanning the back (C-terminal) half of EL2. The sequences of the three peptides are provided in Table 3.

[0304] Table 3

[0305]

[0306] Mice were also immunized with 3T3 cells overexpressing full-length CLDN6 using a plasmid containing the human CLDN6-myc-DDK expression vector.

[0307] Splenocytes were harvested from immunized mice and fused with myeloma cell lines by BTX electrofusion (BTX, Holliston, MA) to generate hybrid cells. 7,680 primary hybrid cell cultures were generated and cultured in 384-well plates. The ability of the antibodies to bind to the peptides was assessed by bead arrays using beads expressing three different peptide targets. 1,920 potential positive antibodies were rearranged into 96-well plates and screened by flow cytometry against endogenous and artificial cell line models.

[0308] Positively hybridizing cell supernatants were then counterscreened by flow cytometry against endogenous and artificial models of proteins with sequence similarity to the target region (e.g., other CLDN proteins). From the secondary screening and counterscreening, approximately 20 CLDN6-specific antibodies were selected for further studies. These antibodies were subcloned, and the variable heavy and light chain sequences were determined. See Table B and the Sequence Listing.

[0309] Using ExpiCHO TM The CLDN6 antibody was formatted as a full-length IgG antibody. The heavy and light chain variable regions of the antibody were cloned into an antibody expression vector based on pcDNA3. TM 3.4- The vector (catalog number: A14697, ThermoFisher Scientific, USA) was engineered and cloned by (according to the kit (ExpiCHO TM The antibodies were purified and cell surface binding of the antibodies to CLDN6 and the IC50 of the antibodies were determined by FACS. The CLDN6 antibodies were stained with Alexa Fluor 500 according to the manufacturer's protocol. CLDN6 antibodies were directly conjugated with 647 NHS ester (succinimidyl ester) (Cat. No. A20106, ThermoFisher Scientific). CLDN6 antibodies were tested in a 150,000 cell system in a volume of 50 μl from 0.32 nM to 1000 nM (serial dilution 1:5, 6 points).

[0310] Cells expressing CLDN6 were used in FACS assays to determine the ability of CLDN6 antibodies to bind to CLDN6 on the cell surface and to cross-react with other CLDN family members. HEK293 T cells engineered to express human CLDN6 fused to GFP, mouse CLDN6 fused to GFP, CLDN9-GFP, CLDN4-GFP, or CLDN3-GFP, or GFP alone (without CLDN6) were used as artificial models of CLDN6 expression. ARK2, OVCA429, LS513, and MCF7 cells were used as endogenous models of CLDN6 expression and as models for CLDN3 / 4 expression.

[0311] For each cell type and each mAb tested, cells were detached from the culture flask surface using EDTA (rather than trypsin) to protect cell surface proteins. The detached cells were then incubated with Alexa Fluor -labeled CLDN6 mAb at a predetermined concentration was incubated on ice in the dark for 30 minutes. 647NHS ester (succinimidyl ester) labeling. After washing, the BD Accuri TM Cells were read on a flow cytometer C6 to detect antibody-antigen protein binding in channel FL4H. Each antibody was tested at different concentrations to establish a dose-fluorescence curve. The EC50 / IC50 (antibody concentration at half the maximum) of the antibody was calculated based on the value of FL4H (gated on live singlet cells) using the Very Simple IC50 toolkit available online. The toolkit allows for plotting biological dose-response data and fitting them to a curve type to obtain EC50 / IC50. The maximum value is the lowest antibody concentration that maximizes fluorescence. The antibodies were also screened for their ability to cross-react with other CLDN proteins (such as CLDN9, CLDN3, and CLDN4). These values ​​were used to determine the relative affinity of each antibody in the panel of antibodies tested. Cross-reactivity data were obtained using a similar method, but the cells had different expression profiles for CLDN6, CLDN3, CLDN4, and CLDN9.

[0312] The relative affinity data and cross-reactivity data determined in this manner are listed in Tables 4 and 5.

[0313] Table 4

[0314]

[0315]

[0316] AB# corresponds to AB# listed in Table A and Table B.

[0317] Table 5

[0318]

[0319]

[0320] AB# corresponds to AB# listed in Table A and Table B.

[0321] Example 6

[0322] This example illustrates the characterization of a chimeric mouse IgG mAb.

[0323] Soft agar 3D proliferation assays and xenograft binding assays were performed to further characterize the mAbs described in Example 5. Briefly, 250 μL of a top layer mixture containing 10,000 cells in 0.6% SeaPlaque agarose in 1x RPMI medium was layered on top of a 250 μL solidified bottom layer of 0.6% SeaPlaque agarose in 1x RPMI medium in each well of a 48-well plate. A 250 μL liquid feed layer containing 1x RPMI medium was placed on top of the solidified top layer. All three layers of the soft agar assay were prepared with or without trastuzumab, Cldn6 mAb, or mouse IgG2a control, starting at a concentration of 150 ng / mL (1 uM) and ending at a concentration of 1.5 ng / mL, diluted 1:10. Each test condition was performed in duplicate. Cells were allowed to form colonies for 3 weeks and then stained with 0.05% neutral red and imaged on an EVOS XL inverted light microscope. Treated cell lines showing a ≥20% reduction in colony number compared to controls were considered sensitive.

[0324] As shown in Table 6, many cell lines exhibited a decrease in colony number when treated with the indicated antibodies.

[0325] Table 6

[0326] In vitro 3D activity ReferenceAb1** 70％ Reference Ab2** 50％ AB1 50％ AB2 50％ AB3 50％ AB4 70％ AB5 70％ AB6 70％ AB7 50％ AB8 0％ AB9 0％ AB10 50％ AB11 50％ AB12 0％ AB13 50％ AB14 0％ AB19 50％ AB16 25％ AB17 0％ AB18 0％ AB19 0％

[0327] In vivo binding studies were performed in mice injected with xenografts of human cancer cell lines. Briefly, xenograft models of human cancer cell lines were established in six-week-old CD-1 athymic nude mice (Charles River Laboratories). The following conditions were followed for subcutaneous injection of each cell line: ARK2 0.75 × 107 , UMUC4 1.0×10 7 ,OV90 1.0×10 7 and M202 0.5×10 7 cells, all containing 50% Matrigel (BD Biosciences). A sufficient number of mice were injected to achieve 8 mice per treatment group. When tumors reached 150 to 300 mm 3 Mice were randomized to treatment groups at an average size of 400 mg / kg. For treatment, each therapeutic antibody (AB3, AB2, Reference Ab1, Reference Ab2, Reference Ab3 (trastuzumab) and non-targeted IgG2 control) was diluted in sterile saline to a working concentration of 1 mg / ml for intravenous tail vein (IV) injection. For the M202 study, trametinib (DMSO solvate, MedChem Express) was administered PO at 1.0 mg / kg (10% Cremaphor, 10% PEG400) on a 5-day dosing, 2-day resting schedule for the first weekly cycle, then reduced to 0.5 mg / kg for the remaining two weeks of dosing. Tumor xenografts were measured three times per week with a caliper and the height in mm was determined by multiplying the height × width × length. 3 The tumor volume was measured in units of . The mice were treated for 2-7 weeks. At the end of the study, the animals were euthanized, and the tumor tissue was excised and separated to be stored as quick-frozen or formalin-fixed paraffin-embedded (FFPE) tissue for biomarker analysis. All animal work was performed according to protocols approved by the IACUC and the University of California, Los Angeles Animal Research Committee. Data were analyzed using StudyLog software from StudyDirector (San Francisco, CA). The results are expressed as the mean volume of each group. Error bars represent the standard error of the mean (SE).

[0328] The results of the xenograft assay are shown in Figures 6 to 10. Figure 6A and Figure 6B As shown, in mice bearing endometrial tumors, AB2 and AB3 each caused substantial mean changes in tumor volume at day 14 relative to a control IgG2 antibody. Figure 7A and Figure 7B As shown, in bladder tumor-bearing mice, AB3 caused a substantial mean change in tumor volume at day 35 relative to a control IgG2 antibody. Figure 8A and Figure 8B It was shown that in ovarian tumor-bearing mice, each of AB2 and AB3 caused a substantial mean change in tumor volume at day 20 relative to a control IgG2 antibody. Figure 9A and Figure 9BThis suggests that AB3 acts in a CLDN-specific manner because Figure 9A and Figure 9B The model used in does not express any of CLDN6, CLDN3, CLDN4, and CLDN9 and was therefore used as a negative control. Figure 9A and Figure 9B The data also showed that AB3 had lower off-target activity than reference Ab1 and reference Ab2. Figure 10A The results of Figures 6 to 9 are summarized. Figure 10A As shown, AB3 significantly reduced tumor growth in mice bearing endometrial, bladder, and ovarian tumors, all of which expressed CLDN6, but did not reduce tumor growth in mice bearing melanoma tumors that did not express CLDN6 ( Figure 10B ).like Figure 11 As shown, there was no significant change in the mean body weight of mice during the treatment period, indicating the safety of the treatment.

[0329] A second set of experiments was conducted in a xenograft model of the human ovarian cancer cell line OV90. Mice were injected with one of the 10 mAbs described in Example 5, a control antibody (mouse IgG2a antibody, reference CLDN6 ab), or a PBS vehicle control. Each group consisted of 8 mice, and each animal was injected intravenously with 10 mg / kg of antibody every 4 days. Figure 12A and Figure 12B As shown, several antibodies described in Example 5 reduced tumor volume in mice bearing ovarian tumors. Although all antibodies tested reduced tumor volume relative to vehicle control, the best performing antibodies were AB3, AB4, AB7, and AB10. Figure 13 As shown, there was no significant change in body weight of animals treated with AB3, AB4, AB7 or AB10, indicating their safety.

[0330] Example 7

[0331] This example illustrates further characterization of the chimeric mouse IgG mAb.

[0332] Internalization Quantification Assay Briefly, studies of CLDN6 protein internalization triggered by binding of reference antibodies Abl, AB3, or AB4 were performed with a positive control being the ubiquitously expressed transferrin receptor (TfR), a cell surface receptor known to be internalized upon antibody binding.

[0333] TfR and CLDN6 antibodies were detected with Texas Red TM-X succinimidyl ester (mixed isomers, catalog number T6134, ThermoFisher Scientific) labeling. One day before antibody treatment, cells were seeded in μ-slide 8-well chambers (catalog number 80826, ibidi Cells In Focus Inc.) to allow cells to attach and grow. The cells were incubated with the labeled antibodies on ice in the dark for 30 minutes. Then, before internalization, the chamber containing CLDN6 or TfR labeled cells was read by an Echo laboratory fluorescence microscope to collect images. The chamber was then incubated at 37 ° C for 40 minutes to allow the internalization process, and images were collected again by an Echo laboratory fluorescence microscope. Compared to the results caused by reference Ab1, AB3 and AB4 caused a greater degree of CLDN6 internalization (data not shown).

[0334] Example 8

[0335] This example illustrates further characterization of the chimeric mouse IgG mAb.

[0336] Two-dimensional (2D) proliferation assays were performed as follows using the selected antibodies described in Example 5: cells were seeded in duplicate in 24-well plates at 5,000 to 20,000 cells per well. The next day, cells were treated with six 1-5 diluted mAbs (starting with 100 nM trastuzumab, Cldn6 mAb, or mouse IgG2A controls) and a fixed concentration of 1 ng / μL monomethyl auristatin E (MMAE)-conjugated anti-mouse secondary antibody (Moradec, LLC) to generate a dose-response curve. Untreated cell wells were quantified on the 1st day, on the antibody-treated day, and on the 6th day thereafter to determine the extent of cell growth. The wells treated with mAbs were quantified on the 6th day, and the growth under each treatment condition was determined as a standardized percentage relative to untreated cell growth. Quantification was performed on a Z1 particle counter (Beckman Coulter, Inc).

[0337] The results are shown in Figure 14 AB2, AB3, AB4, and AB5 showed the greatest efficacy in inhibiting proliferation. The IC50 values ​​for each of these antibodies ranged from 0.1 nM to 1 nM. AB7, AB10, AB11, and AB15 also demonstrated the ability to inhibit proliferation in this assay, albeit to a lesser extent than AB2, AB3, AB4, and AB5.

[0338] Example 9

[0339] This example illustrates the humanization of the antibodies of the present disclosure.

[0340] The antibody subsets listed in Table A were selected for humanization analysis. The heavy chain variable (VH) and light chain variable (VL) sequences of AB1, AB3, AB4, AB9, AB11, and AB18 antibodies were compared to a library of known human germline sequences from human VH genes and human VLκ genes ( the international ImMunoGeneTics information www.imgt.org; Founder and Director: Marie-Paule Lefranc, Montpellier, France); The databases used are the IMGT human VH gene (F+ORF, 273 germline sequences) and the IMGT human VLκ gene (F+ORF, 74 germline sequences). The acceptor human germline is selected from those that are closest to the parent antibody sequence.

[0341] Table 7 provides information on the human germline sequences selected as acceptor sequences and the selected human heavy chain joining regions (J genes) for each VH and VL of each antibody. the internationalImMunoGeneTics information www.imgt.org (Founder and Director: Marie-Paule Lefranc, Montpellier, France) compiled human joiner region sequences.

[0342] Table 7

[0343]

[0344] CDRs are defined according to the AbM definition (for a table comparing CDR definitions, see Dr. Andrew CR Martin's website www.bioinf.org.uk / abs / ).

[0345] It may be necessary to change the human germline framework (ie, non-CDR residues in VH and VL) positions to the corresponding parental murine sequences to optimize binding of the humanized antibody. The sequences of humanized antibody versions are provided as SEQ ID NOs: 376-421.

[0346] For AB1, based on sequence and conformation, each of Asn52 (sequential numbering) of CDR2 in the HC and Asn54 of CDR2 in the LC was determined to have low deamidation potential.

[0347] For AB3, based on sequence and conformation, each of Asn31 (sequential numbering) of HC's CDR1, Asn57 of HC's CDR2, Asn28 of LC's CDR1, and Asn50 of LC's CDR2 were determined to have low deamidation potential. Trp33 of HC's CDR1 was determined to be potentially solvent-exposed and to have oxidation potential, particularly under stress conditions. In HC's CDR3, it was determined that there was a free Cys106 in the CDR, which could be problematic when manufacturing antibodies because it is likely to be solvent-exposed. It was suggested that this Cys residue be changed to Tyr, Ser, or Ala. These altered antibodies were tested for maintenance of binding. Ile53 of LC's CDR2 was determined to be solvent-exposed and could result in nonspecific binding. It was suggested that this Ile residue be changed to Ser. These altered antibodies were tested for maintenance of binding.

[0348] For AB4, based on sequence and conformation, Asn52 (sequential numbering) of CDR2 of HC and Asn58 of CDR2 of LC were determined to have low deamidation potential. The DGNT sequence in CDR1 of LC was identified as problematic due to its high potential for isoaspartate formation (at the DG sequence) and deamidation (at the NT sequence). Changes to this sequence are recommended.

[0349] For AB9, Asn33 (sequential numbering) in the CDR1 of the HC and Asn52 and Asn59 in the CDR2 of the HC were determined to have low deamidation potential based on sequence and conformation. Asn54 was determined to have moderate deamidation potential based on sequence and conformation. The NGG sequence in the CDR2 of the HC was determined to have high / moderate potential for deamidation followed by isoaspartic acid formation. Therefore, alterations to this amino acid sequence were recommended. When manufacturing antibodies, the free Cys106 in the CDR3 of the HC could be problematic as it was determined to be potentially solvent-exposed. It was proposed to alter this Cys residue to Tyr, Ser, or Ala. These altered antibodies were tested for binding maintenance. Arg28 in the CDR1 of the HC is not common in human antibodies. This residue was altered to Thr and tested for binding maintenance. For AB 9, Trp32 (sequential numbering) in the CDR1 of the LC was determined to be potentially solvent-exposed and potentially subject to oxidation, particularly under stress conditions. Leu24 in the same CDR is not common in human antibodies. This residue was changed to Arg and tested for maintenance of binding.

[0350] For AB11, Asp54-Ser55 (sequential numbering) in CDR2 of HC were determined to have low isoaspartate formation potential. Based on sequence and conformation, Asn57 in CDR2 of LC was determined to have low deamidation potential.

[0351] For AB18, based on sequence and conformation, Asn33 in CDR1 of the HC and Asn50 in CDR-H2 (numbered sequentially) were determined to have low deamidation potential. In CDR2 of the HC, the Asp-Pro (DP) sequence was determined to have the potential to undergo cleavage under acidic conditions. In the VL domain, based on sequence and conformation, Asn34 and Asn37 in CDR1 of the LC were determined to have low deamidation potential. In CDR3 of the LC, Trp56 was determined to be likely exposed to solvents and could undergo oxidation, especially under stress conditions.

[0352] Table 8 shows the scheme for combining humanized VH and VL. If no humanized form is equivalent to the chimeric mAb, the preferred pair is shown in bold underlined text.

[0353] Table 8

[0354]

[0355]

[0356] The humanized antibodies described in Table 8 were constructed and expressed essentially as described in Example 5. FACS assays were performed essentially as described in Example 5 to determine the relative antigen binding strength of the humanized antibodies. Two doses (1.5 μg or 0.3 μg) of humanized antibodies were tested for binding to human CLDN6 or murine CLDN6 proteins expressed from engineered 293T clones. The results of the assays are provided in Table 9.

[0357] Table 9

[0358]

[0359]

[0360] FACS assays were also performed to determine the relative antigen binding strength of humanized antibodies (at 1.5 μg or 0.3 μg) for binding to CLDN6 expressed by the indicated cancer cell lines. The results of the assays are provided in Table 10. Only the second Ab was used as a negative control. 64A-chim, h64A, and SC27-108-chim were used as reference antibodies. The corresponding parental antibodies (antibodies before humanization) were used as controls and designated "chim."

[0361] Table 10

[0362]

[0363]

[0364] A "chim" is a pre-humanized form of an antibody.

[0365] Based on in vitro antigen binding data, three humanized antibodies were selected for further testing and development. The antibodies were derived from AB1, AB3, and AB4.

[0366] In vivo binding studies of humanized forms of AB1, AB3, and AB4 were performed in mice injected with xenografts of the bladder cancer cell line UMUC4, essentially as described in Example 6. Briefly, a xenograft model of UMUC4 was established in six-week-old CD-1 athymic nude mice (Charles River Laboratories). 3 The mice were randomized into treatment groups after the average size of the humanized antibodies was measured. The humanized antibodies were diluted in sterile saline to a working concentration of 1 mg / ml for intravenous tail vein (IV) injection. Tumor xenografts were measured three times a week with calipers and the size in mm was determined by multiplying the height × width × length. 3 The tumor volume was measured in units of 100 μg / mL. The mice were treated for 2-7 weeks. At the end of the study, the animals were euthanized and the tumor tissue was excised and separated and stored as snap-frozen or formalin-fixed paraffin-embedded (FFPE) tissue for biomarker analysis.

[0367] The results of the xenograft assay are shown in Figures 15 to Figure 21 Figure 15 shows the results of xenograft assays of two forms of humanized AB3 (AB3-2 and AB3-4), where the treatment involved administering 10 mg / kg Q4D. Controls included vehicle control (PBS), human IgG (10 mg / kg Q4D), and a mouse form of AB3 (10 mg / kg Q4D). As shown in the figure, humanized AB3-4 showed a decrease in tumor volume over the 35-day treatment period. Figure 16 shows the results of xenograft assays of the same treatment as the experiment shown in Figure 15, except that two additional controls (64A MSE and its chimeric form (64A-CHIM)) were performed. As in Figure 15, Figure 16 shows a significant decrease in tumor volume after treatment with humanized AB3-4.

[0368] Figure 17 shows the results of xenograft assays of humanized AB1-5. Controls included vehicle control (PBS), human IgG (10 mg / kg Q4D), and the murine form of AB1 (10 mg / kg Q4D). As shown in Figure 17, mice treated with humanized AB1-5 showed a significant reduction in tumor volume.

[0369] Figure 18 shows the results of xenograft assays of humanized AB4-3. Controls included vehicle control (PBS), human IgG (10 mg / kg Q4D), and the murine form of AB4 (10 mg / kg Q4D). Mice treated with humanized AB4-3 did not show a significant reduction in tumor volume.

[0370] Figure 19 to Figure 21 Results from xenograft assays are shown, in which all four humanized antibodies from Figures 15 to 18 were tested. Mouse and chimeric versions of the reference CLDN6 antibody served as controls. As shown in Figure 19, mice treated with humanized AB3-4 and AB1-5 showed a reduction in tumor volume. Figure 20 The tumor volumes are shown over a 55-day period. The body weights of the mice were measured as follows: Figure 21 shown.

[0371] Example 10

[0372] Computer analysis was performed with different AB1, AB3 and AB4 sequences. In particular, for each antibody, the sequences of (a) the original parent clone, (b) the closest mouse germline sequence, (c) the closest people germline sequence and (d) the humanized sequence were compared. The amino acids that were considered to have undergone affinity maturation were marked with asterisks, while the amino acids that were different from the amino acids at that position according to the antibody database information were marked with #. The CDRs of each sequence were boxed. Based on this analysis, several humanized antibodies with the sequences listed in Table 10 were prepared.

[0373] Table 10

[0374] Parental clone Consensus sequence HC Consensus sequence LC AB1 422 423 AB3 424 425 AB4 426 427

[0375] Multiple antibodies having sequences defined by these consensus sequences were prepared essentially as described in Example 5 and tested in vitro for antigen binding via FACS (essentially as described in Example 5) and in vivo for the ability to reduce tumor volume in mice (essentially as described in Example 6).

[0376] All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein.

[0377] The terms "a," "an," and "the," and similar referents used in the context of describing the present disclosure (especially in the context of the claims below) should be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms "comprising," "having," "including," and "containing" should be construed as open-ended terms (i.e., meaning "including, but not limited to") unless otherwise noted.

[0378] Recitation of ranges of values ​​herein are merely intended to serve as a shorthand method of referring individually to each separate value and endpoint falling within the range, unless otherwise indicated herein, and each separate value and endpoint is incorporated into the specification as if it were individually recited herein.

[0379] Unless otherwise indicated herein or clearly contradicted by the context, all methods described herein can be performed in any suitable order. Unless otherwise required, the use of any and all examples or various languages ​​(e.g., "such as") provided herein is intended only to better illustrate the present disclosure and does not limit the scope of the present disclosure. No language in the specification should be construed as indicating that any unclaimed element is essential for the practice of the present disclosure.

[0380] Preferred embodiments of the present disclosure are described herein, including the best mode known to the inventor for carrying out the present disclosure. After reading the foregoing description, variations of those preferred embodiments will become apparent to those of ordinary skill in the art. The inventor expects that the skilled person will appropriately adopt these variations, and the inventor intends that the present disclosure be implemented in a manner different from that specifically described herein. Therefore, the present disclosure includes all modifications and equivalents of the subject matter recited in the appended claims as permitted by applicable law. In addition, unless otherwise indicated herein or clearly contradicted by the context, the present disclosure encompasses any combination of the above-mentioned elements in all their possible variations. Sequence Listing <110> Regents of the University of California <120> Claudin 6 antibodies and methods for treating cancer <130> 32424 / 51836 <150> US 62 / 560,143 <151> 2017-09-18 <160> 489 <170> PatentIn version 3.5 <210> 1 <211> 220 <212> PRT <213> Homo sapiens <300> <308> NCBI / NP_067018.2 <309> 2017-06-04 <313> (1)..(220) <400> 1 Met Ala Ser Ala Gly Met Gln Ile Leu Gly Val Val Leu Thr Leu Leu 1 5 10 15 Gly Trp Val Asn Gly Leu Val Ser Cys Ala Leu Pro Met Trp Lys Val 20 25 30 Thr Ala Phe Ile Gly Asn Ser Ile Val Val Ala Gln Val Val Trp Glu 35 40 45 Gly Leu Trp Met Ser Cys Val Val Gln Ser Thr Gly Gln Met Gln Cys 50 55 60 Lys Val Tyr Asp Ser Leu Leu Ala Leu Pro Gln Asp Leu Gln Ala Ala 65 70 75 80 Arg Ala Leu Cys Val Ile Ala Leu Leu Val Ala Leu Phe Gly Leu Leu 85 90 95 Val Tyr Leu Ala Gly Ala Lys Cys Thr Thr Cys Val Glu Glu Lys Asp 100 105 110 Ser Lys Ala Arg Leu Val Leu Thr Ser Gly Ile Val Phe Val Ile Ser 115 120 125 Gly Val Leu Thr Leu Ile Pro Val Cys Trp Thr Ala His Ala Ile Ile 130 135 140 Arg Asp Phe Tyr Asn Pro Leu Val Ala Glu Ala Gln Lys Arg Glu Leu 145 150 155 160 Gly Ala Ser Leu Tyr Leu Gly Trp Ala Ala Ser Gly Leu Leu Leu Leu 165 170 175 Gly Gly Gly Leu Leu Cys Cys Thr Cys Pro Ser Gly Gly Ser Gln Gly 180 185 190 Pro Ser His Tyr Met Ala Arg Tyr Ser Thr Ser Ala Pro Ala Ile Ser 195 200 205 Arg Gly Pro Ser Glu Tyr Pro Thr Lys Asn Tyr Val 210 215 220 <210> 2 <211> 23 <212> PRT <213> Homo sapiens <400> 2 Trp Thr Ala His Ala Ile Ile Arg Asp Phe Tyr Asn Pro Leu Val Ala 1 5 10 15 Glu Ala Gln Lys Arg Glu Leu 20 <210> 3 <211> 13 <212> PRT <213> Homo sapiens <400> 3 Thr Ala His Ala Ile Ile Arg Asp Phe Tyr Asn Pro Leu 1 5 10 <210> 4 <211> 10 <212> PRT <213> Homo sapiens <400> 4 Leu Val Ala Glu Ala Gln Lys Arg Glu Leu 1 5 10 <210> 5 <211> 220 <212> PRT <213> Homo sapiens <300> <308> NCBI / NP_001297.1 <309> 2017-07-10 <313> (1)..(220) <400> 5 Met Ser Met Gly Leu Glu Ile Thr Gly Thr Ala Leu Ala Val Leu Gly 1 5 10 15 Trp Leu Gly Thr Ile Val Cys Cys Ala Leu Pro Met Trp Arg Val Ser 20 25 30 Ala Phe Ile Gly Ser Asn Ile Ile Thr Ser Gln Asn Ile Trp Glu Gly 35 40 45 Leu Trp Met Asn Cys Val Val Gln Ser Thr Gly Gln Met Gln Cys Lys 50 55 60 Val Tyr Asp Ser Leu Leu Ala Leu Pro Gln Asp Leu Gln Ala Ala Arg 65 70 75 80 Ala Leu Ile Val Val Ala Ile Leu Leu Ala Ala Phe Gly Leu Leu Val 85 90 95 Ala Leu Val Gly Ala Gln Cys Thr Asn Cys Val Gln Asp Asp Thr Ala 100 105 110 Lys Ala Lys Ile Thr Ile Val Ala Gly Val Leu Phe Leu Leu Ala Ala 115 120 125 Leu Leu Thr Leu Val Pro Val Ser Trp Ser Ala Asn Thr Ile Ile Arg 130 135 140 Asp Phe Tyr Asn Pro Val Val Pro Glu Ala Gln Lys Arg Glu Met Gly 145 150 155 160 Ala Gly Leu Tyr Val Gly Trp Ala Ala Ala Ala Leu Gln Leu Leu Gly 165 170 175 Gly Ala Leu Leu Cys Cys Ser Cys Pro Pro Arg Glu Lys Lys Tyr Thr 180 185 190 Ala Thr Lys Val Val Tyr Ser Ala Pro Arg Ser Thr Gly Pro Gly Ala 195 200 205 Ser Leu Gly Thr Gly Tyr Asp Arg Lys Asp Tyr Val 210 215 220 <210> 6 <211> 209 <212> PRT <213> Homo sapiens <300> <308> NCBI / NP_001296.1 <309> 2017-07-10 <313> (1)..(209) <400> 6 Met Ala Ser Met Gly Leu Gln Val Met Gly Ile Ala Leu Ala Val Leu 1 5 10 15 Gly Trp Leu Ala Val Met Leu Cys Cys Ala Leu Pro Met Trp Arg Val 20 25 30 Thr Ala Phe Ile Gly Ser Asn Ile Val Thr Ser Gln Thr Ile Trp Glu 35 40 45 Gly Leu Trp Met Asn Cys Val Val Gln Ser Thr Gly Gln Met Gln Cys 50 55 60 Lys Val Tyr Asp Ser Leu Leu Ala Leu Pro Gln Asp Leu Gln Ala Ala 65 70 75 80 Arg Ala Leu Val Ile Ile Ser Ile Ile Val Ala Ala Leu Gly Val Leu 85 90 95 Leu Ser Val Val Gly Gly Lys Cys Thr Asn Cys Leu Glu Asp Glu Ser 100 105 110 Ala Lys Ala Lys Thr Met Ile Val Ala Gly Val Val Phe Leu Leu Ala 115 120 125 Gly Leu Met Val Ile Val Pro Val Ser Trp Thr Ala His Asn Ile Ile 130 135 140 Gln Asp Phe Tyr Asn Pro Leu Val Ala Ser Gly Gln Lys Arg Glu Met 145 150 155 160 Gly Ala Ser Leu Tyr Val Gly Trp Ala Ala Ser Gly Leu Leu Leu Leu 165 170 175 Gly Gly Gly Leu Leu Cys Cys Asn Cys Pro Pro Arg Thr Asp Lys Pro 180 185 190 Tyr Ser Ala Lys Tyr Ser Ala Ala Arg Ser Ala Ala Ala Ser Asn Tyr 195 200 205 Val <210> 7 <211> 217 <212> PRT <213> Homo sapiens <300> <308> NCBI / NP_066192.1 <309> 2017-04-15 <313> (1)..(217) <400> 7 Met Ala Ser Thr Gly Leu Glu Leu Leu Gly Met Thr Leu Ala Val Leu 1 5 10 15 Gly Trp Leu Gly Thr Leu Val Ser Cys Ala Leu Pro Leu Trp Lys Val 20 25 30 Thr Ala Phe Ile Gly Asn Ser Ile Val Val Ala Gln Val Val Trp Glu 35 40 45 Gly Leu Trp Met Ser Cys Val Val Gln Ser Thr Gly Gln Met Gln Cys 50 55 60 Lys Val Tyr Asp Ser Leu Leu Ala Leu Pro Gln Asp Leu Gln Ala Ala<65 70 75 80 Arg Ala Leu Cys Val Ile Ala Leu Leu Leu Ala Leu Leu Gly Leu Leu 85 90 95 Val Ala Ile Thr Gly Ala Gln Cys Thr Thr Cys Val Glu Asp Glu Gly 100 105 110 Ala Lys Ala Arg Ile Val Leu Thr Ala Gly Val Ile Leu Leu Leu Ala 115 120 125 Gly Ile Leu Val Leu Ile Pro Val Cys Trp Thr Ala His Ala Ile Ile 130 135 140 Gln Asp Phe Tyr Asn Pro Leu Val Ala Glu Ala Leu Lys Arg Glu Leu 145 150 155 160 Gly Ala Ser Leu Tyr Leu Gly Trp Ala Ala Ala Ala Leu Leu Met Leu 165 170 175 Gly Gly Gly Leu Leu Cys Cys Thr Cys Pro Pro Pro Gln Val Glu Arg 180 185 190 Pro Arg Gly Pro Arg Leu Gly Tyr Ser Ile Pro Ser Arg Ser Gly Ala 195 200 205 Ser Gly Leu Asp Lys Arg Asp Tyr Val 210 215 <210> 8 <211> 7 <212> PRT <213> artificial sequence <220> <223> synthetic peptides <400> 8 Ser Ser Val Ser Ser Thr Tyr 1 5 <210> 9 <211> 3 <212> PRT <213> Artificial sequence <220> <223> synthetic peptides <400> 9 Ser Thr Ser 1 <210> 10 <211> 9 <212> PRT <213> Artificial sequence <220> <223> synthetic peptides <400> 10 His Gln Tyr His Arg Ser Pro Leu Thr 1 5 <210> 11 <211> 8 <212> PRT <213> Artificial sequence <220> <223> synthetic peptides <400> 11 Gly Tyr Thr Phe Thr Thr Tyr Thr 1 5 <210> 12 <211> 8 <212> PRT <213> Artificial sequence <220> <223> synthetic peptides <400> 12 Ile Asn Pro Ser Ser Gly Tyr Thr 1 5 <210> 13 <211> 9 <212> PRT <213> Artificial sequence <220> <223> synthetic peptides <400> 13 Ala Asn Gly Asp Tyr Tyr Val Ala Tyr 1 5 <210> 14 <211> 6 <212> PRT <213> Artificial sequence <220> <223> synthetic peptides <400> 14 Glu Asn Ile Tyr Ser Tyr 1 5 <210> 15 <211> 3 <212> PRT <213> Artificial sequence <220> <223> synthetic peptides <400> 15 Asn Ala Lys 1 <210> 16 <211> 9 <212> PRT <213> Artificial sequence <220> <223> synthetic peptides <400> 16 Gln His His Tyr Thr Val Pro Trp Thr 1 5 <210> 17 <211> 8 <212> PRT <213> Artificial sequence <220> <223> synthetic peptides <400> 17 Gly Phe Thr Phe Ser Asp Tyr Trp 1 5 <210> 18 <211> 10 <212> PRT <213> Artificial sequence <220> <223> synthetic peptides <400> 18 Ile Arg Leu Lys Ser Asp Asn Tyr Ala Thr 1 5 10 <210> 19 <211> 8 <212> PRT <213> Artificial sequence <220> <223> synthetic peptides <400> 19 Asn Asp Gly Pro Pro Ser Gly Cys 1 5 <210> 20 <211> 6 <212> PRT <213> Artificial sequence <220> <223> synthetic peptides <400> 20 Glu Asn Ile Tyr Ser Tyr 1 5 <210> twenty one <211> 3 <212> PRT <213> Artificial sequence <220> <223> synthetic peptides <400> twenty one Asn Ala Lys 1 <210> twenty two <211> 9 <212> PRT <213> Artificial sequence <220> <223> synthetic peptides <400> twenty two Gln His His Tyr Thr Val Pro Trp Thr 1 5 <210> twenty three <211> 8 <212> PRT <213> Artificial sequence <220> <223> synthetic peptides <400> twenty three Gly Phe Thr Phe Ser Asn Tyr Trp 1 5 <210> twenty four <211> 10 <212> PRT <213> Artificial sequence <220> <223> synthetic peptides <400> twenty four Ile Arg Leu Lys Ser Asp Asn Tyr Ala Thr 1 5 10 <210> 25 <211> 8 <212> PRT <213> Artificial sequence <220> <223> synthetic peptides <400> 25 Asn Asp Gly Pro Pro Ser Gly Cys 1 5 <210> 26 <211> 11 <212> PRT <213> Artificial sequence <220> <223> synthetic peptides <400> 26 Gln Ser Leu Val His Ser Asp Gly Asn Thr Tyr 1 5 10 <210> 27 <211> 3 <212> PRT <213> Artificial sequence <220> <223> synthetic peptides <400> 27 Lys Val Ser 1 <210> 28 <211> 9 <212> PRT <213> Artificial sequence <220> <223> synthetic peptides <400> 28 Ser Gln Ser Thr His Val Pro Tyr Thr 1 5 <210> 29 <211> 8 <212> PRT <213> Artificial sequence <220> <223> synthetic peptides <400> 29 Gly Tyr Thr Phe Thr Ser Tyr Thr 1 5 <210> 30 <211> 8 <212> PRT <213> Artificial sequence <220> <223> synthetic peptides <400> 30 Ile Asn Pro Ser Ser Thr Tyr Thr 1 5 <210> 31 <211> 10 <212> PRT <213> Artificial sequence <220> <223> synthetic peptides <400> 31 Ser Arg Gly Glu Leu Gly Gly Phe Ala Tyr 1 5 10 <210> 32 <211> 11 <212> PRT <213> Artificial sequence <220> <223> synthetic peptides <400> 32 Gln Ser Ile Val His Ser Asn Gly Asn Thr Tyr 1 5 10 <210> 33 <211> 3 <212> PRT <213> Artificial sequence <220> <223> synthetic peptides <400> 33 Lys Val Ser 1 <210> 34 <211> 9 <212> PRT <213> Artificial sequence <220> <223> synthetic peptides <400> 34 Phe Gln Gly Ser His Val Pro Phe Thr 1 5 <210> 35 <211> 8 <212> PRT <213> Artificial sequence <220> <223> synthetic peptides <400> 35 Gly Tyr Ile Phe Thr His Tyr Ile 1 5 <210> 36 <211> 8 <212> PRT <213> Artificial sequence <220> <223> synthetic peptides <400> 36 Ile Asn Pro Tyr Asn Asp Gly Thr 1 5 <210> 37 <211> 13 <212> PRT <213> Artificial sequence <220> <223> synthetic peptides <400> 37 Ala Arg Tyr Tyr Gly Tyr Pro Tyr Tyr Ser Met Asp Tyr 1 5 10 <210> 38 <211> 12 <212> PRT <213> Artificial sequence <220> <223> synthetic peptides <400> 38 Gln Ser Leu Leu Asn Ser Arg Thr Arg Lys Asn Tyr 1 5 10 <210> 39 <211> 3 <212> PRT <213> Artificial sequence <220> <223> synthetic peptides <400> 39 Trp Ala Ser 1 <210> 40 <211> 8 <212> PRT <213> Artificial sequence <220> <223> synthetic peptides <400> 40 Lys Gln Ser Tyr Tyr Leu Tyr Thr 1 5 <210> 41 <211> 9 <212> PRT <213> Artificial sequence <220> <223> synthetic peptides <400> 41 Gly Tyr Ser Ile Thr Ser Gly Tyr Tyr 1 5 <210> 42 <211> 7 <212> PRT <213> Artificial sequence <220> <223> synthetic peptides <400> 42 Ile Ser Tyr Asp Gly Gly Ile 1 5 <210> 43 <211> 10 <212> PRT <213> Artificial sequence <220> <223> synthetic peptides <400> 43 Ala Arg Phe Gly Lys Gly Ala Met Asp Tyr 1 5 10 <210> 44 <211> 7 <212> PRT <213> Artificial sequence <220> <223> synthetic peptides <400> 44 Ser Ser Val Ser Ser Ser Tyr 1 5 <210> 45 <211> 3 <212> PRT <213> Artificial sequence <220> <223> synthetic peptides <400> 45 Ser Thr Ser 1 <210> 46 <211> 9 <212> PRT <213> Artificial sequence <220> <223> synthetic peptides <400> 46 His Gln Tyr His Arg Ser Pro Pro Thr 1 5 <210> 47 <211> 8 <212> PRT <213> Artificial sequence <220> <223> synthetic peptides <400> 47 Gly Tyr Ser Phe Thr Gly Tyr Thr 1 5 <210> 48 <211> 8 <212> PRT <213> Artificial sequence <220> <223> synthetic peptides <400> 48 Ile Asn Pro Tyr Asn Gly Gly Thr 1 5 <210> 49 <211> 12 <212> PRT <213> Artificial sequence <220> <223> synthetic peptides <400> 49 Ala Arg Gly Val Tyr Asp Tyr Asp Gly Phe Thr Tyr 1 5 10 <210> 50 <211> 11 <212> PRT <213> Artificial sequence <220> <223> synthetic peptides <400> 50 Gln Ser Leu Val His Ser Asp Gly Asn Thr Tyr 1 5 10 <210> 51 <211> 3 <212> PRT <213> Artificial sequence <220> <223> synthetic peptides <400> 51 Lys Val Ser 1 <210> 52 <211> 9 <212> PRT <213> Artificial sequence <220> <223> synthetic peptides <400> 52 Ser Gln Ser Thr His Val Pro Tyr Thr 1 5 <210> 53 <211> 8 <212> PRT <213> Artificial sequence <220> <223> synthetic peptides <400> 53 Gly Tyr Thr Phe Thr Thr Tyr Thr 1 5 <210> 54 <211> 8 <212> PRT <213> Artificial sequence <220> <223> synthetic peptides <400> 54 Ile Asn Pro Arg Ser Gly Tyr Ser 1 5 <210> 55 <211> 10 <212> PRT <213> Artificial sequence <220> <223> synthetic peptides <400> 55 Ser Arg Gly Glu Leu Gly Gly Phe Ala Tyr 1 5 10 <210> 56 <211> 6 <212> PRT <213> Artificial sequence <220> <223> synthetic peptides <400> 56 Gln Thr Ile Gly Thr Trp 1 5 <210> 57 <211> 3 <212> PRT <213> Artificial sequence <220> <223> synthetic peptides <400> 57 Ala Ala Ala 1 <210> 58 <211> 9 <212> PRT <213> Artificial sequence <220> <223> synthetic peptides <400> 58 Gln Gln Leu Tyr Ser Ile Pro Arg Thr 1 5 <210> 59 <211> 8 <212> PRT <213> Artificial sequence <220> <223> synthetic peptides <400> 59 Gly Tyr Arg Phe Thr Asp Tyr Asn 1 5 <210> 60 <211> 8 <212> PRT <213> Artificial sequence <220> <223> synthetic peptides <400> 60 Ile Asn Pro Asn Asn Gly Gly Thr 1 5 <210> 61 <211> 10 <212> PRT <213> Artificial sequence <220> <223> synthetic peptides <400> 61 Ala Arg Asp Tyr Leu Tyr Phe Phe Asp Cys 1 5 10 <210> 62 <211> 11 <212> PRT <213> Artificial sequence <220> <223> synthetic peptides <400> 62 Gln Ser Leu Val His Ser Asn Gly Asn Thr Tyr 1 5 10 <210> 63 <211> 3 <212> PRT <213> Artificial sequence <220> <223> synthetic peptides <400> 63 Lys Val Ser 1 <210> 64 <211> 9 <212> PRT <213> Artificial sequence <220> <223> synthetic peptides <400> 64 Ser Gln Ile Thr His Val Pro Tyr Thr 1 5 <210> 65 <211> 8 <212> PRT <213> Artificial sequence <220> <223> synthetic peptides <400> 65 Gly Tyr Thr Phe Thr Asp Tyr Ser 1 5 <210> 66 <211> 8 <212> PRT <213> Artificial sequence <220> <223> synthetic peptides <400> 66 Ile Ser Thr Glu Thr Gly Glu Pro 1 5 <210> 67 <211> 10 <212> PRT <213> Artificial sequence <220> <223> synthetic peptides <400> 67 Thr Arg Gly Leu Trp Ser Ser Phe Ala Tyr 1 5 10 <210> 68 <211> 10 <212> PRT <213> Artificial sequence <220> <223> synthetic peptides <400> 68 Lys Ser Val Ser Thr Ser Gly Tyr Ser Tyr 1 5 10 <210> 69 <211> 3 <212> PRT <213> Artificial sequence <220> <223> synthetic peptides <400> 69 Leu Ala Ser 1 <210> 70 <211> 9 <212> PRT <213> Artificial sequence <220> <223> synthetic peptides <400> 70 Gln His Ser Arg Glu Leu Pro Leu Thr 1 5 <210> 71 <211> 8 <212> PRT <213> Artificial sequence <220> <223> synthetic peptides <400> 71 Gly Phe Thr Phe Ser Ser Phe Gly 1 5 <210> 72 <211> 8 <212> PRT <213> Artificial sequence <220> <223> synthetic peptides <400> 72 Ile Ser Ser Asp Ser Arg Thr Ile 1 5 <210> 73 <211> 11 <212> PRT <213> Artificial sequence <220> <223> synthetic peptides <400> 73 Ala Arg Asp Tyr Gly Arg Thr Tyr Glu Ala Tyr 1 5 10 <210> 74 <211> 6 <212> PRT <213> Artificial sequence <220> <223> synthetic peptides <400> 74 Gln Asp Ile Gly Gly Asn 1 5 <210> 75 <211> 3 <212> PRT <213> Artificial sequence <220> <223> synthetic peptides <400> 75 Ser Thr Ser 1 <210> 76 <211> 9 <212> PRT <213> Artificial sequence <220> <223> synthetic peptides <400> 76 Leu Gln Arg Asn Ala Tyr Pro Leu Thr 1 5 <210> 77 <211> 8 <212> PRT <213> Artificial sequence <220> <223> synthetic peptides <400> 77 Gly Phe Thr Phe Ser Ser Tyr Ala 1 5 <210> 78 <211> 7 <212> PRT <213> Artificial sequence <220> <223> synthetic peptides <400> 78 Ile Arg Ser Gly Gly Thr Thr 1 5 <210> 79 <211> 12 <212> PRT <213> Artificial sequence <220> <223> synthetic peptides <400> 79 Ala Lys Val Gly Gly Asn Pro Tyr Pro Met Asp Tyr 1 5 10 <210> 80 <211> 7 <212> PRT <213> Artificial sequence <220> <223> synthetic peptides <400> 80 Ser Ser Ile Ser Ser Asn Tyr 1 5 <210> 81 <211> 3 <212> PRT <213> Artificial sequence <220> <223> synthetic peptides <400> 81 Arg Thr Ser 1 <210> 82 <211> 9 <212> PRT <213> Artificial sequence <220> <223> synthetic peptides <400> 82 Gln Gln Gly Ser Ser Ile Pro Leu Thr 1 5 <210> 83 <211> 8 <212> PRT <213> Artificial sequence <220> <223> synthetic peptides <400> 83 Gly Tyr Ala Phe Ser Asn Tyr Leu 1 5 <210> 84 <211> 8 <212> PRT <213> Artificial sequence <220> <223> synthetic peptides <400> 84 Ile Asn Pro Gly Ser Gly Gly Thr 1 5 <210> 85 <211> 15 <212> PRT <213> Artificial sequence <220> <223> synthetic peptides <400> 85 Ala Arg Ser Tyr Phe Gly Arg Ser Tyr Pro Tyr Thr Met Asp Tyr 1 5 10 15 <210> 86 <211> 10 <212> PRT <213> Artificial sequence <220> <223> synthetic peptides <400> 86 Gln Ser Val Asp Tyr Asp Gly Asp Asn Tyr 1 5 10 <210> 87 <211> 3 <212> PRT <213> Artificial sequence <220> <223> synthetic peptides <400> 87 Ala Ala Ser 1 <210> 88 <211> 9 <212> PRT <213> Artificial sequence <220> <223> synthetic peptides <400> 88 Gln Gln Ser Asn Glu Asp Pro Phe Thr 1 5 <210> 89 <211> 8 <212> PRT <213> Artificial sequence <220> <223> synthetic peptides <400> 89 Gly Tyr Thr Phe Thr Asp Tyr Ala 1 5 <210> 90 <211> 8 <212> PRT <213> Artificial sequence <220> <223> synthetic peptides <400> 90 Ile Ser Thr Tyr Ser Gly Asn Thr 1 5 <210> 91 <211> 13 <212> PRT <213> Artificial sequence <220> <223> synthetic peptides <400> 91 Ala Arg Arg Gly Asp Tyr Ser Leu Tyr Ala Met Asp Tyr 1 5 10 <210> 92 <211> 12 <212> PRT <213> Artificial sequence <220> <223> synthetic peptides <400> 92 Gln Ser Val Leu Phe Ser Ser Asn Gln Lys Asn Tyr 1 5 10 <210> 93 <211> 3 <212> PRT <213> Artificial sequence <220> <223> synthetic peptides <400> 93 Trp Ala Ser 1 <210> 94 <211> 8 <212> PRT <213> Artificial sequence <220> <223> synthetic peptides <400> 94 His Gln Tyr Leu Ser Ser Arg Thr 1 5 <210> 95 <211> 8 <212> PRT <213> Artificial sequence <220> <223> synthetic peptides <400> 95 Gly Phe Thr Phe Ser Ser Phe Gly 1 5 <210> 96 <211> 8 <212> PRT <213> Artificial sequence <220> <223> synthetic peptides <400> 96 Ile Ser Ser Asp Ser Arg Thr Ile 1 5 <210> 97 <211> 11 <212> PRT <213> Artificial sequence <220> <223> synthetic peptides <400> 97 Ala Arg Asp Tyr Gly Arg Thr Tyr Glu Ala Tyr 1 5 10 <210> 98 <211> 10 <212> PRT <213> Artificial sequence <220> <223> synthetic peptides <400> 98 Glu Ser Val Asp Asn Tyr Gly Ile Ser Phe 1 5 10 <210> 99 <211> 3 <212> PRT <213> Artificial sequence <220> <223> synthetic peptides <400> 99 Ala Ala Ser 1 <210> 100 <211> 9 <212> PRT <213> Artificial sequence <220> <223> synthetic peptides <400> 100 Gln Gln Ser Lys Glu Val Pro Leu Thr 1 5 <210> 101 <211> 8 <212> PRT <213> Artificial sequence <220> <223> synthetic peptides <400> 101 Gly Phe Pro Phe Ser Ser Ser Ala 1 5 <210> 102 <211> 7 <212> PRT <213> Artificial sequence <220> <223> synthetic peptides <400> 102 Ile Asn Ser Asp Gly Asn Thr 1 5 <210> 103 <211> 13 <212> PRT <213> Artificial sequence <220> <223> synthetic peptides <400> 103 Thr Arg Asn Gly Asp Tyr Arg Tyr Asp Glu Phe Ala Tyr 1 5 10 <210> 104 <211> 7 <212> PRT <213> Artificial sequence <220> <223> synthetic peptides <400> 104 Ser Ser Val Ser Ser Ser Tyr 1 5 <210> 105 <211> 3 <212> PRT <213> Artificial sequence <220> <223> synthetic peptides <400> 105 Ser Thr Ser 1 <210> 106 <211> 9 <212> PRT <213> Artificial sequence <220> <223> synthetic peptides <400> 106 His Gln Tyr His Arg Ser Pro Pro Thr 1 5 <210> 107 <211> 8 <212> PRT <213> Artificial sequence <220> <223> synthetic peptides <400> 107 Gly Tyr Thr Phe Thr Gly Tyr Trp 1 5 <210> 108 <211> 8 <212> PRT <213> Artificial sequence <220> <223> synthetic peptides <400> 108 Ile Asn Pro Ser Thr Gly Tyr Thr 1 5 <210> 109 <211> 12 <212> PRT <213> Artificial sequence <220> <223> synthetic peptides <400> 109 Ala Arg Glu Gly Ile Thr Thr Val Leu Val Asp Tyr 1 5 10 <210> 110 <211> 12 <212> PRT <213> Artificial sequence <220> <223> synthetic peptides <400> 110 Gln Ser Val Leu Phe Ser Ser Asn Gln Lys Asn Tyr 1 5 10 <210> 111 <211> 3 <212> PRT <213> Artificial sequence <220> <223> synthetic peptides <400> 111 Trp Ala Ser 1 <210> 112 <211> 8 <212> PRT <213> Artificial sequence <220> <223> synthetic peptides <400> 112 His Gln Tyr Leu Ser Ser Arg Thr 1 5 <210> 113 <211> 8 <212> PRT <213> Artificial sequence <220> <223> synthetic peptides <400> 113 Gly Tyr Ser Phe Thr Gly Tyr Asn 1 5 <210> 114 <211> 8 <212> PRT <213> Artificial sequence <220> <223> synthetic peptides <400> 114 Ile Asp Pro Tyr Tyr Gly Gly Ser 1 5 <210> 115 <211> 14 <212> PRT <213> Artificial sequence <220> <223> synthetic peptides <400> 115 Ala Arg Glu Arg Ser Gly Tyr Val Phe Ser Ala Met Asp Tyr 1 5 10 <210> 116 <211> 12 <212> PRT <213> Artificial sequence <220> <223> synthetic peptides <400> 116 Gln Ser Val Leu Phe Ser Ser Asn Gln Lys Asn Tyr 1 5 10 <210> 117 <211> 3 <212> PRT <213> Artificial sequence <220> <223> synthetic peptides <400> 117 Trp Ala Ser 1 <210> 118 <211> 8 <212> PRT <213> Artificial sequence <220> <223> synthetic peptides <400> 118 His Gln Tyr Leu Ser Ser Arg Thr 1 5 <210> 119 <211> 8 <212> PRT <213> Artificial sequence <220> <223> synthetic peptides <400> 119 Gly Tyr Ser Phe Thr Gly Tyr Thr 1 5 <210> 120 <211> 8 <212> PRT <213> Artificial sequence <220> <223> synthetic peptides <400> 120 Ile Asn Pro Tyr Asn Gly Val Thr 1 5 <210> 121 <211> 16 <212> PRT <213> Artificial sequence <220> <223> synthetic peptides <400> 121 Thr Arg Asp Pro Leu Tyr Tyr Gly Tyr Arg Asp Ser Thr Met Asp Tyr 1 5 10 15 <210> 122 <211> 11 <212> PRT <213> Artificial sequence <220> <223> synthetic peptides <400> 122 Gln Ser Leu Val His Ser Asp Gly Asn Thr Tyr 1 5 10 <210> 123 <211> 3 <212> PRT <213> Artificial sequence <220> <223> synthetic peptides <400> 123 Lys Val Ser 1 <210> 124 <211> 9 <212> PRT <213> Artificial sequence <220> <223> synthetic peptides <400> 124 Ser Gln Ser Thr His Val Pro Tyr Thr 1 5 <210> 125 <211> 8 <212> PRT <213> Artificial sequence <220> <223> synthetic peptides <400> 125 Gly Tyr Thr Phe Thr Ser Tyr Thr 1 5 <210> 126 <211> 8 <212> PRT <213> Artificial sequence <220> <223> synthetic peptides <400> 126 Ile Asn Pro Ser Ser Thr Tyr Thr 1 5 <210> 127 <211> 10 <212> PRT <213> Artificial sequence <220> <223> synthetic peptides <400> 127 Ser Arg Gly Glu Leu Gly Gly Phe Ala Tyr 1 5 10 <210> 128 <211> 6 <212> PRT <213> Artificial sequence <220> <223> synthetic peptides <400> 128 Gln Gly Ile Arg Gly Asn 1 5 <210> 129 <211> 3 <212> PRT <213> Artificial sequence <220> <223> synthetic peptides <400> 129 Ser Thr Ser 1 <210> 130 <211> 9 <212> PRT <213> Artificial sequence <220> <223> synthetic peptides <400> 130 Leu Gln Arg Asn Ala Tyr Pro Leu Thr 1 5 <210> 131 <211> 8 <212> PRT <213> Artificial sequence <220> <223> synthetic peptides <400> 131 Gly Phe Thr Phe Ser Ser Phe Ala 1 5 <210> 132 <211> 7 <212> PRT <213> Artificial sequence <220> <223> synthetic peptides <400> 132 Ile Arg Ser Gly Gly Ile Thr 1 5 <210> 133 <211> 13 <212> PRT <213> artificial sequence <220> <223> synthetic peptide <400> 133 Ala Arg Val Ser Thr Ala Thr Tyr Tyr Gly Met Asp Tyr 1 5 10 <210> 134 <211> 108 <212> PRT <213> artificial sequence <220> <223> synthetic peptide <400> 134 Gln Ile Val Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Leu Gly 1 5 10 15 Glu Arg Val Thr Met Thr Cys Thr Ala Ser Ser Ser Val Ser Ser Thr 20 25 30 Tyr Phe His Trp Tyr Gln Gln Lys Pro Gly Ser Ser Pro Lys Leu Trp 35 40 45 Ile Tyr Ser Thr Ser Asn Leu Ala Ser Gly Val Pro Arg Arg Phe Ser 50 55 60 Gly Ser Ala Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu 65 70 75 80 Ala Glu Asp Ala Ala Thr Tyr Tyr Cys His Gln Tyr His Arg Ser Pro 85 90 95 Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys 100 105 <210> 135 <211> 116 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Peptide <400> 135 Gln Val Gln Leu Gln Gln Ser Ala Ala Glu Leu Ala Arg Pro Gly Ala 1 5 10 15 Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Thr Tyr 20 25 30 Thr Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Phe Ile Asn Pro Ser Ser Gly Tyr Thr Asp Tyr Asn Gln Lys Phe 50 55 60 Lys Asp Arg Thr Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Val Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Asn Gly Asp Tyr Tyr Val Ala Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ala 115 <210> 136 <211> 107 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <400> 136 Asp Ile Gln Met Thr Gln Ser Pro Ala Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Glu Thr Val Thr Ile Thr Cys Arg Ile Ser Glu Asn Ile Tyr Ser Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Gln Gly Lys Ser Pro Gln Leu Leu Val 35 40 45 Tyr Asn Ala Lys Ile Leu Val Glu Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Gln Phe Ser Leu Lys Ile Asn Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Gly Asn Tyr Tyr Cys Gln His His Tyr Thr Val Pro Trp 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 137 <211> 117 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <400> 137 Glu Val Lys Leu Glu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Asp Ile Gln Met Thr Gln Ser Pro Ala Ser Leu Ser Ala Ser Val Gly 20 25 30 Glu Thr Val Thr Ile Thr Cys Arg Ile Ser Glu Asn Ile Tyr Ser Tyr 35 40 45 Ser Met Lys Leu Ser Cys Val Ala Ser Gly Phe Thr Phe Ser Asp Tyr 50 55 60 Trp Met Asn Trp Val Arg Gln Ser Pro Glu Lys Gly Leu Glu Trp Val 65 70 75 80 Ala Gln Ile Arg Leu Lys Ser Asp Asn Tyr Ala Thr His Tyr Ala Glu 85 90 95 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Arg Ser 100 105 110 Val Tyr Leu Gln Met Asn Asn Leu Arg Ala Glu Asp Thr Gly Thr Tyr 115 <210> 138 <211> 107 <212> PRT <213> Synthetic sequence <220> <223> Synthetic peptide <400> 138 Asp Ile Gln Met Thr Gln Ser Pro Ala Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Glu Thr Val Thr Ile Thr Cys Arg Ile Ser Glu Asn Ile Tyr Ser Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Gln Gly Lys Ser Pro Gln Leu Leu Val 35 40 45 Tyr Asn Ala Lys Ile Leu Val Glu Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Gln Phe Ser Leu Lys Ile Asn Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Gly Asn Tyr Tyr Cys Gln His His Tyr Thr Val Pro Trp 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 139 <211> 117 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Peptide <400> 139 Glu Val Lys Leu Glu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Met Lys Leu Ser Cys Val Ala Ser Gly Phe Thr Phe Ser Asn Tyr 20 25 30 Trp Met Asn Trp Val Arg Gln Ser Pro Glu Lys Gly Leu Glu Trp Val 35 40 45 Ala Gln Ile Arg Leu Lys Ser Asp Asn Tyr Ala Thr His Tyr Ala Glu 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Arg Ser 65 70 75 80 Val Tyr Leu Gln Met Asn Asn Leu Arg Ala Glu Asp Thr Gly Thr Tyr 85 90 95 Tyr Cys Asn Asp Gly Pro Pro Ser Gly Cys Trp Gly Gln Gly Thr Thr 100 105 110 Leu Ile Val Ser Ser 115 <210> 140 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Peptide <400> 140 Asp Val Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly 1 5 10 15 Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser 20 25 30 Asp Gly Asn Thr Tyr Leu Asn Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Arg Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Phe Cys Ser Gln Ser 85 90 95 Thr His Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 141 <211> 117 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <400> 141 Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala 1 5 10 15 Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Thr Met His Trp Ile Lys Gln Arg Pro Gly Gln Gly Gln Glu Trp Ile 35 40 45 Gly Tyr Ile Asn Pro Ser Ser Thr Tyr Thr His Tyr Ile Lys Lys Phe 50 55 60 Lys Asp Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Arg Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ser Arg Gly Glu Leu Gly Gly Phe Ala Tyr Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ala 115 <210> 142 <211> 112 <212> PRT <213> Artificial Sequence <220>​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​ <211> 120 <212> PRT <213> artificial sequence <220> <223> synthetic peptide <400> 143 Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Ile Phe Thr His Tyr 20 25 30 Ile Met His Trp Val Lys Gln Lys Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Cys Ile Asn Pro Tyr Asn Asp Gly Thr Lys Tyr Asn Glu Lys Phe 50 55 60 Lys Gly Lys Ala Thr Leu Thr Ser Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Tyr Tyr Gly Tyr Pro Tyr Tyr Ser Met Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Ser Val Thr Val Ser Ser 115 120 <210> 144 <211> 112 <212> PRT <213> artificial sequence <220> <223> synthetic peptide <400> 144 Ala Ile Val Met Phe Gln Ser Pro Ser Ser Leu Val Val Ser Ala Gly 1 5 10 15 Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser 20 25 30 Arg Thr Arg Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 Ser Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Val Tyr Tyr Cys Lys Gln 85 90 95 Ser Tyr Tyr Leu Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 145 <211> 117 <212> PRT <213> artificial sequence <220> <223> synthetic peptide <400> 145 Asp Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Ser Ser Gln 1 5 10 15 Ser Leu Ser Leu Thr Cys Ser Val Thr Gly Tyr Ser Ile Thr Ser Gly 20 25 30 Tyr Tyr Trp Lys Trp Ile Arg Gln Phe Pro Gly Asn Lys Leu Glu Trp 35 40 45 Met Gly Tyr Ile Ser Tyr Asp Gly Gly Ile Asn Tyr Asn Pro Ser Leu 50 55 60 Lys Asn Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln Phe Phe 65 70 75 80 Leu Lys Leu Asn Ser Val Thr Thr Glu Asp Thr Ala Lys Tyr Tyr Cys 85 90 95 Ala Arg Phe Gly Lys Gly Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser 100 105 110 Val Thr Val Ser Ser 115 <210> 146 <211> 108 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Peptide <400> 146 Gln Ile Val Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Leu Gly 1 5 10 15 Asp Arg Val Thr Met Thr Cys Thr Ala Ser Ser Ser Val Ser Ser Ser 20 25 30 Tyr Leu His Trp Tyr Gln Gln Lys Pro Gly Ser Ser Pro Lys Leu Trp 35 40 45 Ile Tyr Ser Thr Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu 65 70 75 80 Ala Glu Asp Ala Ala Thr Tyr Tyr Cys His Gln Tyr His Arg Ser Pro 85 90 95 Pro Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 147 <211> 119 <212> PRT <213> Synthetic Sequence <220> <223> Synthetic Peptide <400> 147 Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Met Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Gly Tyr 20 25 30 Thr Met Asn Trp Val Lys Gln Ser His Gly Lys Asn Leu Glu Trp Ile 35 40 45 Gly Leu Ile Asn Pro Tyr Asn Gly Gly Thr Asn Tyr Asn Gln Lys Phe 50 55 60 Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Leu Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Val Tyr Asp Tyr Asp Gly Phe Thr Tyr Trp Gly Gln Gly 100 105 110<******>Thr Leu Val Thr Val Ser Ala<******>115<******><210> 148<******><211> 112<******><212> PRT<******><213> Artificial Sequence<******><220><******><223> Synthetic Peptide<******><400> 148<******>Asp Val Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly<******>1 5 10 15<******>Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser<******>20 25 30<******>Asp Gly Asn Thr Tyr Leu Tyr Trp Tyr Leu Gln Lys Pro Gly Gln Ser<******>35 40 45<******>Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro<******>50 55 60<******>Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 It should be noted that the two tags " " and " " in the original text are likely to be misspelled. I assume they should be " " and " " respectively, and I have translated them as such. If this is not what you intended, please correct the original text and I will be happy to provide a more accurate translation. Also, the two tags " " and " " seem to be incomplete or misspelled in the original. I have translated them as best as possible based on the context. If there are specific requirements for these tags, please let me know. Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Phe Cys Ser Gln Ser 85 90 95 Thr His Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 149 <211> 117 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <400> 149 Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala 1 5 10 15 Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Thr Tyr 20 25 30 Thr Met His Trp Leu Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile<tmp 35 40 45 Gly Tyr Ile Asn Pro Arg Ser Gly Tyr Ser Asn Tyr Asn Gln Lys Phe 50 55 60<00027{tmp Lys Asp Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Asn Thr Ala Tyr 65 70 75 80 Met Gln Leu Asn Thr Leu Thr Ser Glu Asp Ser Lys Val Tyr Tyr Cys 85 90 95 It should be noted that there seems to be an error in the original text where "<tmp " and "<00027{tmp " are not in a correct format. This translation is done as accurately as possible based on the provided text.Ser Arg Gly Glu Leu Gly Gly Phe Ala Tyr Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ala 115 <210> 150 <211> 107 <212> PRT <213> artificial sequence <220> <223> synthetic peptide <400> 150 Asp Ile Gln Met Thr Gln Ser Pro Ala Ser Gln Ser Ala Ser Leu Gly 1 5 10 15 Glu Ser Val Thr Ile Thr Cys Leu Ala Ser Gln Thr Ile Gly Thr Trp 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ser Pro Gln Leu Leu Ile 35 40 45 Tyr Ala Ala Ala Ser Leu Ala Asp Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Arg Phe Ser Phe Lys Ile Ser Ser Leu Gln Ala 65 70 75 80 Glu Asp Phe Val Ser Tyr Tyr Cys Gln Gln Leu Tyr Ser Ile Pro Arg 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 151 <211> 117 <212> PRT <213> Artificial sequence <220> <223> synthetic peptides <400> 151 Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Arg Phe Thr Asp Tyr 20 25 30 Asn Met His Trp Val Lys Gln Ser His Gly Lys Ser Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Asn Pro Asn Asn Gly Gly Thr Asn Tyr Asn Gln Asn Phe 50 55 60 Lys Gly Lys Ala Thr Leu Thr Val Asn Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Arg Ser Leu Thr Ser Glu Asp Ser Ala Ala Tyr Tyr Cys 85 90 95 Ala Arg Asp Tyr Leu Tyr Phe Phe Asp Cys Trp Gly Gln Gly Thr Thr 100 105 110 Leu Thr Val Ser Ser 115 <210> 152 <211> 112 <212> PRT <213> Artificial sequence <220> <223> synthetic peptides <400> 152 Asp Val Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly 1 5 10 15 Asp Gln Ala Ser Ile Ser Cys Arg Ala Ser Gln Ser Leu Val His Ser 20 25 30 Asn Gly Asn Thr Tyr Leu His Trp Phe Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Arg Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Phe Cys Ser Gln Ile 85 90 95 Thr His Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 153 <211> 117 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Peptide <400> 153 Gln Ile Gln Leu Val Gln Ser Gly Pro Ala Leu Lys Lys Pro Gly Glu 1 5 10 15 Thr Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Ser Met His Trp Ile Lys Gln Ala Pro Gly Lys Gly Leu Lys Trp Met 35 40 45 Gly Trp Ile Ser Thr Glu Thr Gly Glu Pro Thr Tyr Ala Asp Gly Phe 50 55 60 Lys Gly Arg Phe Asp Phe Ser Leu Glu Thr Ser Ala Asp Thr Ala Tyr 65 70 75 80 Leu Ser Ile Asn Asn Leu Thr Asn Glu Asp Thr Ala Thr Tyr Phe Cys 85 90 95 Thr Arg Gly Leu Trp Ser Ser Phe Ala Tyr Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ala 115 <210> 154 <211> 111 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Peptide <400> 154 Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Lys Ser Val Ser Thr Ser 20 25 30 Gly Tyr Ser Tyr Ile His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro 35 40 45 Lys Leu Leu Ile Tyr Leu Ala Ser Asn Leu Glu Ser Gly Val Pro Ala 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile His 65 70 75 80 Pro Val Glu Glu Glu Asp Ala Ala Thr Tyr Tyr Cys Gln His Ser Arg 85 90 95 Glu Leu Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys 100 105 110 <210> 155 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Peptide <400> 155 Asp Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Arg Gly 1 5 10 15 Ser Arg Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Phe 20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Glu Lys Gly Leu Glu Trp Val 35 40 45 Ala Tyr Ile Ser Ser Asp Ser Arg Thr Ile Tyr Tyr Ala Asp Thr Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Pro Thr Asn Thr Leu Phe 65 70 75 80 Leu Gln Met Thr Ser Leu Arg Ser Glu Asp Thr Ala Met Tyr Tyr Cys 85 90 95 Ala Arg Asp Tyr Gly Arg Thr Tyr Glu Ala Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ala 115 <210> 156 <211> 107 <212> PRT <213> artificial sequence <220> <223> synthetic peptide <400> 156 Asp Ile Gln Met Ile Gln Ser Pro Ser Ser Met Phe Ala Ser Leu Gly 1 5 10 15 Asp Arg Val Ser Leu Ser Cys Arg Ala Ser Gln Asp Ile Gly Gly Asn 20 25 30 Leu Asp Trp Tyr Gln Gln Lys Pro Gly Gly Thr Ile Lys Leu Leu Ile 35 40 45 Tyr Ser Thr Ser Asn Leu Asn Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Ser Asp Tyr Ser Leu Thr Ile Thr Ser Leu Glu Ser 65 70 75 80 Glu Asp Phe Ala Asp Tyr Tyr Cys Leu Gln Arg Asn Ala Tyr Pro Leu 85 90 95 Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys 100 105 <210> 157 <211> 118 <212> PRT <213> artificial sequence <220> <223> synthetic peptide <400> 157 Glu Val Lys Leu Met Glu Ser Gly Gly Asp Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln Thr Pro Glu Lys Arg Leu Glu Trp Val 35 40 45 Ala Ser Ile Arg Ser Gly Gly Thr Thr Tyr Tyr Pro Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Arg Asn Ile Leu Tyr Leu 65 70 75 80 Arg Met Ser Ser Leu Arg Ser Glu Asp Thr Ala Ile Tyr Tyr Cys Ala 85 90 95 Lys Val Gly Gly Asn Pro Tyr Pro Met Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Ser Val Thr Val Ser Ser 115 <210> 158 <211> 108 <212> PRT <213> Artificial sequence <220> <223> synthetic peptides <400> 158 Glu Ile Val Leu Thr Gln Ser Pro Thr Thr Met Ala Ala Ser Pro Gly 1 5 10 15 Glu Lys Ile Thr Ile Thr Cys Ser Ala Ser Ser Ser Ile Ser Ser Asn 20 25 30 Tyr Leu His Trp Tyr Gln Gln Lys Pro Gly Phe Ser Pro Lys Leu Leu 35 40 45 Ile Tyr Arg Thr Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Gly Thr Met Glu 65 70 75 80 Ala Glu Asp Val Ala Thr Tyr Tyr Cys Gln Gln Gly Ser Ser Ile Pro 85 90 95 Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys 100 105 <210> 159 <211> 122 <212> PRT <213> Artificial sequence <220> <223> synthetic peptides <400> 159 Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Arg Pro Gly Thr 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ala Phe Ser Asn Tyr 20 25 30 Leu Ile Glu Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Val Ile Asn Pro Gly Ser Gly Gly Thr Asn Tyr Asn Glu Lys Phe 50 55 60 Lys Gly Lys Ala Thr Met Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met His Leu Ser Asn Leu Thr Ser Glu Asp Ser Val Val Tyr Phe Cys 85 90 95 Ala Arg Ser Tyr Phe Gly Arg Ser Tyr Pro Tyr Thr Met Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Ser Val Thr Val Ser Ser 115 120 <210> 160 <211> 111 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Peptide <400> 160 Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Gln Arg Ala Thr Ile Ser Cys Lys Ala Ser Gln Ser Val Asp Tyr Asp 20 25 30 Gly Asp Asn Tyr Val Asn Trp Tyr Gln Gln Lys Val Gly Gln Pro Pro 35 40 45 Lys Leu Leu Ile Ser Ala Ala Ser Asn Leu Glu Ser Gly Ile Pro Ala 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile His 65 70 75 80 Pro Val Glu Glu Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Ser Asn 85 90 95 Glu Asp Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 161 <211> 120 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Peptide <400> 161 Gln Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Arg Pro Gly Val 1 5 10 15 Ser Val Lys Ile Ser Cys Lys Gly Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Ala Met His Trp Val Lys Gln Ser His Ala Lys Ser Leu Glu Trp Ile 35 40 45 Gly Val Ile Ser Thr Tyr Ser Gly Asn Thr Asn Tyr Asn Gln Lys Phe 50 55 60 Gln Asp Lys Ala Thr Met Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Ala Leu Ala Arg Leu Thr Ser Asp Asp Ser Ala Ile Tyr Tyr Cys 85 90 95 Ala Arg Arg Gly Asp Tyr Ser Leu Tyr Ala Met Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Ser Val Thr Val Ser Ser 115 120 <210> 162 <211> 112 <212> PRT <213> artificial sequence <220> <223> synthetic peptide <400> 162 Asn Ile Met Met Thr Gln Ser Pro Ser Ser Leu Ala Val Ser Ala Gly 1 5 10 15 Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Val Leu Phe Ser 20 25 30 Ser Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 Ser Pro Arg Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Asn Val Gln Ala Glu Asp Leu Ala Val Tyr Tyr Cys His Gln 85 90 95 Tyr Leu Ser Ser Arg Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys 100 105 110 <210> 163 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Peptide <400> 163 Asp Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Arg Gly 1 5 10 15 Ser Arg Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Phe 20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Glu Lys Gly Leu Glu Trp Val 35 40 45 Ala Tyr Ile Ser Ser Asp Ser Arg Thr Ile Tyr Tyr Ala Asp Thr Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Pro Thr Asn Thr Leu Phe 65 70 75 80 Leu Gln Met Thr Ser Leu Arg Ser Glu Asp Thr Ala Met Tyr Tyr Cys 85 90 95 Ala Arg Asp Tyr Gly Arg Thr Tyr Glu Ala Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ala 115 <210> 164 <211> 111 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Peptide <400> 164 Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Leu Ser Leu Gly 1 5 10 15 Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Glu Ser Val Asp Asn Tyr 20 25 30 Gly Ile Ser Phe Met Asn Trp Phe Gln Gln Lys Pro Gly Gln Pro Pro 35 40 45 Lys Leu Leu Ile Tyr Ala Ala Ser Asn Gln Gly Ser Gly Val Pro Ala 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Ser Leu Asn Ile His 65 70 75 80 Pro Met Glu Glu Asp Asp Thr Ala Met Tyr Phe Cys Gln Gln Ser Lys 85 90 95 Glu Val Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys 100 105 110 <210> 165 <211> 119 <212> PRT <213> artificial sequence <220> <223> synthetic peptide <400> 165 Glu Val Arg Leu Val Glu Ser Gly Gly Gly Leu Met Gln Pro Gly Gly 1 5 10 15 Ser Leu Lys Leu Pro Cys Ala Ala Ser Gly Phe Pro Phe Ser Ser Ser 20 25 30 Ala Met Ser Trp Val Arg Gln Thr Pro Glu Lys Arg Leu Glu Trp Val 35 40 45 Ala Ser Ile Asn Ser Asp Gly Asn Thr Tyr Tyr Pro Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Ser Ala Arg Asn Ile Leu Tyr Leu 65 70 75 80 Gln Met Ser Ser Leu Arg Ser Glu Asp Thr Ala Met Tyr Tyr Cys Thr 85 90 95 Arg Asn Gly Asp Tyr Arg Tyr Asp Glu Phe Ala Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ala 115 <210> 166 <211> 108 <212> PRT <213> artificial sequence <220> <223> synthetic peptide <400> 166 Gln Ile Val Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Leu Gly 1 5 10 15 Glu Arg Val Thr Met Thr Cys Thr Ala Ser Ser Ser Val Ser Ser Ser 20 25 30 Tyr Leu His Trp Tyr Gln Gln Lys Pro Gly Ser Ser Pro Lys Leu Trp 35 40 45 Ile Tyr Ser Thr Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu 65 70 75 80 Ala Glu Asp Ala Ala Thr Tyr Tyr Cys His Gln Tyr His Arg Ser Pro 85 90 95 Pro Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys 100 105 <210> 167 <211> 119 <212> PRT <213> artificial sequence <220> <223> Synthetic peptide <400> 167 Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Ala Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Gly Tyr 20 25 30 Trp Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Leu 35 40 45 Gly Tyr Ile Asn Pro Ser Thr Gly Tyr Thr Glu Ser Asn Gln Lys Phe 50 55 60 Lys Asp Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Thr Thr Ala Tyr 65 70 75 80 Met Gln Leu Arg Ser Leu Thr Pro Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Glu Gly Ile Thr Thr Val Leu Val Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Thr Leu Thr Val Ser Ser 115 <210> 168 <211> 112 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <400> 168 Asn Ile Met Met Thr Gln Ser Pro Ser Ser Leu Ala Val Ser Ala Gly 1 5 10 15 Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Val Leu Phe Ser 20 25 30 Ser Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 Ser Pro Arg Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Asn Val Gln Ala Glu Asp Leu Ala Val Tyr Tyr Cys His Gln 85 90 95 Tyr Leu Ser Ser Arg Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys 100 105 110 [[ID=2{28}]<210> 169 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Peptide <400> 169 Gln Val Gln Leu Lys Gln Ser Gly Pro Glu Leu Glu Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Gly Tyr 20 25 30 Asn Met Asn Trp Val Lys Gln Ser Asn Gly Lys Ser Leu Glu Trp Ile 35 40 45 Gly Asn Ile Asp Pro Tyr Tyr Gly Gly Ser Thr Tyr Asn Gln Lys Phe 50 55 60 Thr Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Lys Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Glu Arg Ser Gly Tyr Val Phe Ser Ala Met Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Ser Val Thr Val Ser Ser 115 120 <210> 170 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Peptide <400> 170 Asn Ile Met Met Thr Gln Ser Pro Ser Ser Leu Ala Val Ser Ala Gly 1 5 10 15 Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Val Leu Phe Ser 20 25 30 Ser Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 Ser Pro Arg Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Asn Val Gln Ala Glu Asp Leu Ala Val Tyr Tyr Cys His Gln 85 90 95 Tyr Leu Ser Ser Arg Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys 100 105 110 <210> 171 <211> 123 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Peptide <400> 171 Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Met Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Gly Tyr 20 25 30 Thr Met Asn Trp Val Lys Arg Ser His Gly Lys Asn Leu Glu Trp Ile 35 40 45 Gly Leu Ile Asn Pro Tyr Asn Gly Val Thr Thr Tyr Asn Gln Asn Phe 50 55 60 Lys Gly Lys Ala Thr Leu Ala Val Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Leu Gly Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Thr Arg Asp Pro Leu Tyr Tyr Gly Tyr Arg Asp Ser Thr Met Asp Tyr 100 105 110 Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser 115 120 <210> 172 <211> 112<~ <212> PRT <213> Artificial Sequence <220> <223> Synthetic Peptide <400> 172 Asp Val Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly<~ 1 5 10 15 Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser 20 25 30 Asp Gly Asn Thr Tyr Leu Asn Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 y Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Note: There seems to be a formatting issue with and in the original text which might be a special character encoding problem. I've translated them as best as possible while keeping the tags intact. If this is a specific format that needs to be corrected, more context would be helpful. Also, <210>, <211>, <212>, <213>, <220>, <223>, <400> are likely some sort of sequence or format identifiers in the patent context and are translated literally here. Arg Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Phe Cys Ser Gln Ser 85 90 95 Thr His Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 173 <211> 117 <212> PRT <213> Synthetic Sequence <220> <223> Synthetic Peptide <400> 173 Glu Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala 1 5 10 15 Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Thr Met His Trp Ile Lys Gln Arg Pro Gly Gln Gly Gln Glu Trp Ile 35 40 45 Gly Tyr Ile Asn Pro Ser Ser Thr Tyr Thr His Tyr Ile Lys Lys Phe 50 55 60 Lys Asp Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Arg Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ser Arg Gly Glu Leu Gly Gly Phe Ala Tyr Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ala 115 <210> 174 <211> 107 <212> PRT <213> artificial sequence <220> <223> synthetic peptide <400> 174 Asp Ile Gln Met Ile Gln Ser Pro Ser Ser Met Phe Ala Ser Leu Gly 1 5 10 15 Asp Arg Val Ser Leu Ser Cys Arg Ala Ser Gln Gly Ile Arg Gly Asn 20 25 30 Leu Asp Trp Tyr Gln Gln Lys Pro Gly Gly Thr Ile Lys Leu Leu Ile 35 40 45 Tyr Ser Thr Ser Ile Leu Asn Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Ser Asp Tyr Ser Leu Thr Ile Thr Ser Leu Glu Ser 65 70 75 80 Glu Asp Phe Ala Asp Tyr Tyr Cys Leu Gln Arg Asn Ala Tyr Pro Leu 85 90 95 Thr Phe Gly Ser Gly Thr Lys Leu Glu Leu Lys 100 105 <210> 175 <211> 119 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Peptide <400> 175 Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Met Lys Pro Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Phe 20 25 30 Ala Leu Ser Trp Val Arg Gln Thr Pro Glu Lys Arg Leu Glu Trp Val 35 40 45 Ala Ser Ile Arg Ser Gly Gly Ile Thr Tyr His Ala Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Gly Asn Ile Leu Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Ser Glu Asp Thr Ala Met Tyr Phe Cys Ala 85 90 95<00​​​​​​​​​​​​​​​​​​​​<309> 2017-08-07 <313> (1)..(219) <400> 176 Met Ala Ser Thr Gly Leu Gln Ile Leu Gly Ile Val Leu Thr Leu Leu 1 5 10 15 Gly Trp Val Asn Ala Leu Val Ser Cys Ala Leu Pro Met Trp Lys Val 20 25 30 Thr Ala Phe Ile Gly Asn Ser Ile Val Val Ala Gln Met Val Trp Glu 35 40 45 Gly Leu Trp Met Ser Cys Val Val Gln Ser Thr Gly Gln Met Gln Cys 50 55 60 Lys Val Tyr Asp Ser Leu Leu Ala Leu Pro Gln Asp Leu Gln Ala Ala 65 70 75 80 Arg Ala Leu Cys Val Val Thr Leu Leu Ile Val Leu Leu Gly Leu Leu 85 90 95 Val Tyr Leu Ala Gly Ala Lys Cys Thr Thr Cys Val Glu Asp Arg Asn 100 105 110 Ser Lys Ser Arg Leu Val Leu Ile Ser Gly Ile Ile Phe Val Ile Ser 115 120 125 Gly Val Leu Thr Leu Ile Pro Val Cys Trp Thr Ala His Ser Ile Ile 130 135 140 Gln Asp Phe Tyr Asn Pro Leu Val Ala Asp Ala Gln Lys Arg Glu Leu 145 150 155 160 Gly Ala Ser Leu Tyr Leu Gly Trp Ala Ala Ser Gly Leu Leu Leu Leu 165 170 175 Gly Gly Gly Leu Leu Cys Cys Ala Cys Ser Ser Gly Gly Thr Gln Gly 180 185 190 Pro Arg His Tyr Met Ala Cys Tyr Ser Thr Ser Val Pro His Ser Arg 195 200 205 Gly Pro Ser Glu Tyr Pro Thr Lys Asn Tyr Val 210 215 <210> 177 <211> 80 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Peptide <400> 177 Met Ala Ser Ala Gly Met Gln Ile Leu Gly Val Val Leu Thr Leu Leu 1 5 10 15 Gly Trp Val Asn Gly Leu Val Ser Cys Ala Leu Pro Met Trp Lys Val 20 25 30 Thr Ala Phe Ile Gly Asn Ser Ile Val Val Ala Gln Val Val Trp Glu 35 40 45 Gly Leu Trp Met Ser Cys Val Val Gln Ser Thr Gly Gln Met Gln Cys 50 55 60 Lys Val Tyr Asp Ser Leu Leu Ala Leu Pro Gln Asp Leu Gln Ala Ala 65 70 75 80 <210> 178 <211> 220 <212> PRT <213> artificial sequence <220> <223> synthetic peptide <400> 178 Met Ala Ser Ala Gly Met Gln Ile Leu Gly Val Val Leu Thr Leu Leu 1 5 10 15 Gly Trp Val Asn Gly Leu Val Ser Cys Ala Leu Pro Met Trp Lys Val 20 25 30 Thr Ala Phe Ile Gly Asn Ser Ile Val Val Ala Gln Val Val Trp Glu 35 40 45 Gly Leu Trp Met Ser Cys Val Val Gln Ser Thr Gly Gln Met Gln Cys 50 55 60 Lys Val Tyr Asp Ser Leu Leu Ala Leu Pro Gln Asp Leu Gln Ala Ala 65 70 75 80 Arg Ala Leu Cys Val Ile Ala Leu Leu Val Ala Leu Phe Gly Leu Leu 85 90 95 Val Tyr Leu Ala Gly Ala Lys Cys Thr Thr Cys Val Glu Glu Lys Asp 100 105 110 Ser Lys Ala Arg Leu Val Leu Thr Ser Gly Ile Val Phe Val Ile Ser 115 120 125 Gly Val Leu Thr Leu Ile Pro Val Cys Trp Thr Ala His Ala Val Ile 130 135 140 Arg Asp Phe Tyr Asn Pro Leu Val Ala Glu Ala Gln Lys Arg Glu Leu 145 150 155 160 Gly Ala Ser Leu Tyr Leu Gly Trp Ala Ala Ser Gly Leu Leu Leu Leu 165 170 175 Gly Gly Gly Leu Leu Cys Cys Thr Cys Pro Ser Gly Gly Ser Gln Gly 180 185 190 Pro Ser His Tyr Met Ala Arg Tyr Ser Thr Ser Ala Pro Ala Ile Ser 195 200 205 Arg Gly Pro Ser Glu Tyr Pro Thr Lys Asn Tyr Val 210 215 220 <210> 179 <211> 322 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Peptide <400> 179 Cys Ala Gly Ala Thr Cys Gly Thr Gly Cys Thr Gly Ala Cys Thr Cys 1 5 10 15 Ala Gly Ala Gly Thr Cys Cys Thr Thr Cys Ala Ala Thr Thr Ala Thr 20 25 30 Gly Thr Cys Cys Gly Thr Gly Ala Gly Cys Cys Cys Ala Gly Gly Cys 35 40 45 Gly Ala Gly Ala Ala Gly Gly Thr Cys Ala Cys Cys Ala Thr Cys Ala 50 55 60 Cys Ala Thr Gly Cys Ala Gly Thr Gly Cys Cys Thr Cys Cys Ala Gly 65 70 75 80 Cys Thr Cys Thr Gly Thr Cys Thr Cys Ala Thr Ala Cys Ala Thr Gly 85 90 95 Cys Ala Cys Thr Gly Gly Thr Thr Cys Cys Ala Gly Cys Ala Gly Ala 100 105 110 Ala Gly Cys Cys Ala Gly Gly Gly Ala Cys Cys Ala Gly Thr Cys Cys 115 120 125 Cys Ala Ala Gly Cys Thr Gly Thr Gly Cys Ala Thr Cys Thr Ala Cys 130 135 140 Thr Cys Thr Ala Cys Ala Thr Cys Gly Ala Ala Cys Cys Thr Gly Gly 145 150 155 160 Cys Cys Thr Cys Cys Gly Gly Ala Gly Thr Gly Cys Cys Cys Gly Cys 165 170 175 Ala Ala Gly Gly Thr Thr Thr Ala Gly Cys Gly Gly Thr Cys Gly Gly 180 185 190 Gly Gly Cys Thr Cys Thr Gly Gly Ala Ala Cys Thr Thr Cys Ala Thr 195 200 205 Ala Cys Thr Cys Cys Cys Thr Gly Ala Cys Cys Ala Thr Cys Thr Cys 210 215 220 Gly Cys Gly Gly Gly Thr Gly Gly Cys Cys Gly Cys Thr Gly Ala Gly 225 230 235 240 Gly Ala Thr Gly Cys Ala Gly Cys Ala Ala Cys Ala Thr Ala Cys Thr 245 250 255 Ala Thr Thr Gly Cys Cys Ala Gly Cys Ala Gly Ala Gly Gly Thr Cys 260 265 270 Cys Ala Ala Thr Thr Ala Thr Cys Cys Cys Cys Cys Thr Thr Gly Gly 275 280 285 Ala Cys Ala Thr Thr Cys Gly Gly Cys Gly Gly Ala Gly Gly Thr Ala 290 295 300 Cys Cys Ala Ala Ala Cys Thr Cys Gly Ala Gly Ala Thr Thr Ala Ala 305 310 315 320 Gly Cys <210> 180 <211> 351 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Peptide <400> 180 Gly Ala Ala Gly Thr Cys Cys Ala Gly Cys Thr Gly Cys Ala Gly Cys 1 5 10 15 Ala Gly Thr Cys Thr Gly Gly Cys Cys Cys Thr Gly Ala Ala Cys Thr 20 25 30 Gly Gly Thr Gly Ala Ala Gly Cys Cys Thr Gly Gly Cys Gly Cys Cys 35 40 45 Ala Gly Cys Ala Thr Gly Ala Ala Gly Ala Thr Cys Thr Cys Cys Thr 50 55 60 Gly Cys Ala Ala Gly Gly Cys Cys Ala Gly Cys Gly Gly Cys Thr Ala 65 70 75 80 Cys Thr Cys Cys Thr Thr Cys Ala Cys Cys Gly Gly Cys Thr Ala Thr 85 90 95 Ala Cys Ala Ala Thr Gly Ala Ala Cys Thr Gly Gly Gly Thr Gly Ala 100 105 110 Ala Gly Cys Ala Gly Thr Cys Cys Cys Ala Cys Gly Gly Cys Ala Ala 115 120 125 Gly Ala Ala Thr Cys Thr Gly Gly Ala Gly Thr Gly Gly Ala Thr Cys 130 135 140 Gly Gly Cys Cys Thr Gly Ala Thr Cys Ala Ala Cys Cys Cys Ala Thr 145 150 155 160 Ala Cys Ala Ala Thr Gly Gly Cys Gly Gly Cys Ala Cys Cys Ala Thr 165 170 175 Cys Thr Ala Cys Ala Ala Cys Cys Ala Gly Ala Ala Gly Thr Thr Thr 180 185 190 Ala Ala Gly Gly Gly Cys Ala Ala Gly Gly Cys Cys Ala Cys Cys Cys 195 200 205 Thr Gly Ala Cys Ala Gly Thr Gly Gly Ala Cys Ala Ala Gly Ala Gly 210 215 220 Cys Thr Cys Cys Thr Cys Thr Ala Cys Cys Gly Cys Cys Thr Ala Cys 225 230 235 240 Ala Thr Gly Gly Ala Gly Cys Thr Gly Cys Thr Gly Thr Cys Thr Cys 245 250 255 Thr Gly Ala Cys Ala Ala Gly Cys Gly Ala Gly Gly Ala Cys Thr Cys 260 265 270 Cys Gly Cys Cys Gly Thr Gly Thr Ala Cys Thr Ala Thr Thr Gly Cys 275 280 285 Gly Cys Cys Cys Gly Gly Gly Ala Cys Thr Ala Cys Gly Gly Cys Thr 290 295 300 Thr Cys Gly Thr Gly Cys Thr Gly Gly Ala Cys Thr Ala Thr Thr Gly 305 310 315 320 Gly Gly Gly Cys Cys Ala Gly Gly Gly Cys Ala Cys Cys Ala Cys Ala 325 330 335 Cys Thr Gly Ala Cys Ala Gly Thr Gly Ala Gly Cys Thr Cys Cys 340 345 350 <210> 181 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Peptide <400> 181 Gln Ile Val Leu Thr Gln Ser Pro Ser Ile Met Ser Val Ser Pro Gly 1 5 10 15 Glu Lys Val Thr Ile Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr Met 20 25 30 His Trp Phe Gln Gln Lys Pro Gly Thr Ser Pro Lys Leu Cys Ile Tyr 35 40 45 Ser Thr Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Arg 50 55 60 Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Arg Val Ala Ala Glu 65 70 75 80 Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Arg Ser Asn Tyr Pro Pro Trp 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys[[ID=***]] 100 105 <210> 182 <211> 117 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <400> 182 Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Met Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Gly Tyr 20 25 30 Thr Met Asn Trp Val Lys Gln Ser His Gly Lys Asn Leu Glu Trp Ile 35 40 45 Gly Leu Ile Asn Pro Tyr Asn Gly Gly Thr Ile Tyr Asn Gln Lys Phe 50 55 60 Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Leu Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Tyr Gly Phe Val Leu Asp Tyr Trp Gly Gln Gly Thr Thr 100 105 110 Leu Thr Val Ser Ser 115 <210> 183 <211> 324 <212> DNA <213> Artificial sequence <220> <223> Synthetic peptide <400> 183 gaaattgtgc tcacccagtc tccagcactc atggctgcat ctccagggga gaaggtcacc 60 atcacctgca gtgtcagctc aagtataagt tccagcaact tgcactggta ccagcagaag 120 tcaggaacct cccccaaact ctggatttat ggcacatcca acctggcttc tggagtccct 180 gttcgcttca gtggcagtgg atctgggacc tcttattctc tcacaatcag caacatggag 240 gctgaagatg ctgccactta ttactgtcaa cagtggagta gttacccaca cacgttcgga 300 ggggggacca agctggaaat aaaa 324 <210> 184 <211> 378 <212> DNA <213> Artificial sequence <220> <223> Synthetic peptide <400> 184 caggtccaaa tgcagcagtc tggagctgag ctggtaaggc ctgggacttc agtgaaggtg 60 tcctgcaagg cttctggata cgccttcact aattacttga tagagtgggt aaagcagagg 120 cctggacagg gccttgagtg gattggactg attaatcctg gaagtggtgg tactaattac 180 aatgagaagt tcaagggcaa ggcaacactg actgcagaca aatcctccac cactgcctac 240 atgcagctca gcagcctgac atctgatgac tctgcggttt atttctgtgc aagacggtcc 300 cctctaggga gttggatcta ctatgcttac gacggtgttg cttactgggg ccaagggact 360 ctggtcactg tctctgca 378 <210> 185 <211> 108 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Peptide <400> 185 Glu Ile Val Leu Thr Gln Ser Pro Ala Leu Met Ala Ala Ser Pro Gly 1 5 10 15 Glu Lys Val Thr Ile Thr Cys Ser Val Ser Ser Ser Ile Ser Ser Ser 20 25 30 Asn Leu His Trp Tyr Gln Gln Lys Ser Gly Thr Ser Pro Lys Leu Trp 35 40 45 Ile Tyr Gly Thr Ser Asn Leu Ala Ser Gly Val Pro Val Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Asn Met Glu 65 70 75 80 Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Tyr Pro 85 90 95 His Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 186 <211> 126 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <400> 186 Gln Val Gln Met Gln Gln Ser Gly Ala Glu Leu Val Arg Pro Gly Thr 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ala Phe Thr Asn Tyr 20 25 30 Leu Ile Glu Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Leu Ile Asn Pro Gly Ser Gly Gly Thr Asn Tyr Asn Glu Lys Phe 50 55 60 Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Thr Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Thr Ser Asp Asp Ser Ala Val Tyr Phe Cys 85 90 95 Ala Arg Arg Ser Pro Leu Gly Ser Trp Ile Tyr Tyr Ala Tyr Asp Gly 100 105 110 Val Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ala 115 120 125 <210> 187 <211> 645 <212> DNA <213> Mus musculus <400> 187 gatattgtgc taactcagtc tccagccacc ctgtctgtga ctccaggaaa tagcgtcagt 60 ctttcctgca gggccagcca aagtattggc ggtaacctac actggtatca acaaaaatca 120 catgagtctc caaggcttct catcaagtat gcttcccagt ccatctctgg gatcccctcc 180 aggttcagtg gcagtggatc agggacagat ttcactctca gtatcaacag tgtggagact 240 gaagattttg gaatgtattt ctgtcaacag agtaacagct ggccttacac gttcggaggg 300 gggaccaagc tggaaataaa acgggcagat gctgcaccaa ctgtatccat cttcccacca 360 tccagtgagc agttaacatc tggaggtgcc tcagtcgtgt gcttcttgaa caacttctac 420 cccaaagaca tcaatgtcaa gtggaagatt gatggcagtg aacgacaaaa tggcgtcctg 480 aacagttgga ctgatcagga cagcaaagac agcacctaca gcatgagcag caccctcacg 540 ttgaccaagg acgagtatga acgacataac agctatacct gtgaggccac tcacaagaca 600 tcaacttcac ccattgtcaa gagcttcaac aggaatgagt gttag 645 <210> 188 <211> 1335 <212> DNA <213> Mus musculus <400> 188 gacgtgcagc ttcaggagtc aggacctagc ctcgtgaaac cttctcagac tctgtccctc 60 acctgttctg tcactggcga ctccatcacc agtgattact ggagctggat ccggaaattc 120 ccagggaata gacttgagta catggggtac gtaagctaca gtggtagcac ttactacaat 180 ccatctctca aaagtcgaat ctccatcacc cgagacacat ccaagaacca gtactacctg 240 gatttgaatt ctgtgactac tgaggacaca gccacatatt actgtgcaaa ctgggacggt 300 gattactggg gccaagggac tctggtcact gtctcttcag cagctaaaac aacagcccca 360 tcggtctatc cactggcccc tgtgtgtgga gatacaactg gctcctcggt gactctagga 420 tgcctggtca agggttattt ccctgagcca gtgaccttga cctggaactc tggatccctg 480 tccagtggtg tgcacacctt cccagctgtc ctgcagtctg acctctacac cctcagcagc 540 tcagtgactg taacctcgag cacctggccc agccagtcca tcacctgcaa tgtggcccac 600 ccggcaagca gcaccaaggt ggacaagaaa attgagccca gagggcccac aatcaagccc 660 tgtcctccat gcaaatgccc agcacctaac ctcttgggtg gaccatccgt cttcatctttc 720 cctccaaaga tcaaggatgt actcatgatc tcctgagcc catagtcac atgtgtggtg 780 gtggatgtga gcgaggatga cccagatgtc cagatcagct ggtttgtgaa caacgtggaa 840 gtacacacag ctcagacaca aacccataga gaggattaca acagtactct ccgggtggtc 900 agtgccctcc ccatccagca ccaggactgg atgagtggca aggagttcaa atgcaaggtc 960 aacaacaaag acctcccagc gcccatcgag agaaccatct caaaacccaa agggtcagta 1020 agagctccac aggtatatgt cttgcctcca ccagaagaag agatgactaa gaaacaggtc 1080 actctgacct gcatggtcac agacttcatg cctgaagaca tttacgtgga gtggaccaac 1140 aacgggaaaa cagagctaaa ctacaagaac actgaaccag tcctggactc tgatggttct 1200 tacttcatgt acagcaagct gagagtggaa aagaagaact gggtggaaag aatagctac 1260 tcctgttcag tggtccacga gggtctgcac aatcaccaca cgactaagag cttctcccgg 1320 actccgggta aatga 1335 <210> 189 <211> 8806 <212> DNA <213> Artificial sequence <220> <223> Synthetic peptide <400> 189 aatgtagtct tatgcaatac tcttgtagtc ttgcaacatg gtaacgatga gttagcaaca 60 tgccttacaa ggagagaaaa agcaccgtgc atgccgattg gtggaagtaa ggtggtacga 120 tcgtgcctta ttaggaaggc aacagacggg tctgacatgg attggacgaa ccactgaatt 180 gccgcattgc agagatattg tatttaagtg cctagctcga tacataaacg ggtctctctg 240 gttagaccag atctgagcct gggagctctc tggctaacta gggaacccac tgcttaagcc 300 tcaataaagc ttgccttgag tgcttcaagt agtgtgtgcc cgtctgttgt gtgactctgg 360 taactagaga tccctcagac ccttttagtc agtgtggaaa atctctagca gtggcgcccg 420 aacagggact tgaaagcgaa agggaaacca gaggagctct ctcgacgcag gactcggctt 480 gctgaagcgc gcacggcaag aggcgagggg cggcgactgg tgagtacgcc aaaaattttg 540 actagcggag gctagaagga gagagatggg tgcgagagcg tcagtattaa gcgggggaga 600 attagatcgc gatgggaaaa aattcggtta aggccagggg gaaagaaaaa atataaatta 660 aaacatatag tatgggcaag cagggagcta gaacgattcg cagttaatcc tggcctgtta 720 gaaacatcag aaggctgtag aaaatactg ggacagctac aaccatccct tcagacagga 780 tcagagaac ttagatcatt atataca gtagcaaccc tctattgtgt gcatcaagg 840 frequently frequently frequently aaaaaagt 900 agaccaccg cacagcaagc ggccgctgat cttcagacct ggaggagg atatgaggga 960 attgaatt taggtagc 1020 acccaccaag gcaagagaa gagtggtgca gagaaaaa agagcagtgg gataggagc 1080 ttgttcctt gggttcttgg gagcagcagg aagcactatg ggcgcagcgt caatgacgct 1140 gacggtacag gccagacaat tattgtctgg tatagtgcag cagcagaaca atttgctgag 1200 ggctattgag gcgcacagc atctgttgca accacagtc tggggcatca agcagctcca 1260 ggcaagaatc ctggctgtgg aaagatacct aaaggatcaa cagctcctgg ggatttgggg 1320 ttgctctgga aaactcattt gcaccactgc tgtgccttgg atgctagtt ggagtaataa 1380 atctctggaa cagatttgga atcacacgac ctggatggag tgggacag aattacaa 1440 ttacacaagc ttatacact ccttaattga agaatcgcaaaccagcaag aaagaatga 1500 acaagaatta ttggattag aataatgggc aagtttgtgg aattggtttta acatacaaa 1560 ttggctgtgg tatataaat tattcataat gatagtagga ggcttggtag gtttaagaat 1620 agttttgct gtactttcta tagtgaatag agttaggcag ggattattcac cattatcgtt 1680 1740 1740 1740 tggagagaga gagagaca gatccattcg attagtgaac ggatctcgac ggtatcggtt 1800 aacttttaaa agaaaagggg ggattggggg gtacagtgca ggggaagaa tagtagacat 1860 atagcaaca gaatacaa ctaagaatt aaaaaaaaaaaaa ttcaaattt 1920 tccgataagc ttgggagttc cgcgttacat aacttacggt aaatggcccg cctggctgac 1980 cgcccaacga cccccgccca tgacgtca taatgacgta tgttcccata gtaacgccaa 2040 tagggacttt ccattgacgt caatgggtgg agtatttacg gtaaactgcc cacttggcag 2100 tacatcaagt gtatcatatg ccaagtacgc cccctattga cgtcaatgac ggtaaatggc 2160 ccgcctggca ttatgcccag tacatgacct tatgggactt tcctacttgg cagtacatct 2220 acgtattagt catcgctatt accatggtga tgcggttttg gcagtacatc aatgggcgtg 2280 gatagcggtt tgactcacgg ggatttccaa gtctccaccc cattgacgtc aatgggagtt 2340 tgttttggca ccaaaatcaa cgggactttc caaaatgtcg taacaactcc gccccattga 2400 cgcaaatggg cggtaggcgt gtacggtggg aggtctatat aagcagagct cgtttagtga 2460 accgtcagat cgcctggaga cgccatccac gctgttttga cctccataga agacaccgac 2520 tctagaacta gtggatcccc cgggctgcag gaattcgtcg actggatccg gtaccgagga 2580 gatctgccgc cgcgatcgcc ggcgcgccag atctcaagct taactagtta gcggaccgac 2640 gcgtacgcgg ccgctcgaga tgagcggggg cgaggagctg ttcgccggca tcgtgcccgt 2700 gctgatcgag ctggacggcg acgtgcacgg ccacaagttc agcgtgcgcg gcgagggcga 2760 gggcgacgcc gactacggca agctggagat caagttcatc tgcaccaccg gcaagctgcc 2820 cgtgccctgg cccaccctgg tgaccaccct ctgctacggc atccagtgct tcgcccgcta 2880 ccccgagcac atgaagatga acgacttctt caagagcgcc atgcccgagg gctacatcca 2940 ggagcgcacc atccagttcc aggacgacgg caagtacaag acccgcggcg aggtgaagtt 3000 cgagggcgac accctggtga accgcatcga gctgaagggc aaggacttca aggaggacgg 3060 caacatcctg ggccacaagc tggagtacag cttcaacagc cacaacgtgt acatccgccc 3120 cgacaaggcc aacaacggcc tggaggctaa cttcaagacc cgccacaaca tcgagggcgg 3180 cggcgtgcag ctggccgacc actaccagac caacgtgccc ctgggcgacg gccccgtgct 3240 gatccccatc aaccactacc tgagcactca gaccaagatc agcaaggacc gcaacgaggc 3300 ccgcgaccac atggtgctcc tggagtcctt cagcgcctgc tgccacaccc acggcatgga 3360 cgagctgtac aggtccggac tcagataagt ttaaacccga tatcatcatc tagggcggcc 3420 aattccgccc ctctcccccc ccctaacgtt actggccgaa gccgcttgga ataaggccgg 3480 tgtgcgtttg tctatatgtt attttccacc atattgccgt cttttggcaa tgtgagggcc 3540 cggaaacctg gccctgtctt cttgacgagc attcctaggg gtctttcccc tctcgccaaa ggaatgcaag gtctgttgaa tgtcgtgaag gaagcagttc ctctggaagc ttcttgaaga 3720. caaacaacgt ctgtagcgac cctttgcagg cagcggaacc ccccacctgg cgacaggtgc ctctgcggcc aaaagccacg tgtataagat acacctgcaa aggcggcaca accccagtgc cacgttgtga gttggatagt tgtggaaaga gtcaaatggc tctcctcaag cgtattcaac aaggggctga aggatgccca gaaggtaccc cattgtatgg gatctgatct ggggcctcgg 3960. tgcacatgct ttacatgtgt ttagtcgagg ttaaaaaaac gtctaggccc cccgaaccac ggggacgtgg ttttcctttg aaaaacacga tgataagctt gccacaaccc acaaggagac 4020 gaccttccat gaccgagtac aagcccacgg tgcgcctcgc cacccgcgac gacgtccccc 4080 gggccgtacg caccctcgcc gccgcgttcg ccgactaccc cgccacgcg cacaccgtcg 4140 acccggaccg ccacatcgag cgggtcaccg agctgcaaga actcttcctc acgcgcgtcg 4200. ggctcgacat cggcaaggtg tgggtcgcgg acgacggcgc cgcggtggcg gtctggacca 4260 cgccggagag cgtcgaagcg ggggcggtgt tcgccgagat cggcccgcgc atggccgagt 4320 tgagcggttc ccggctggcc gcgcagcaac agatggaagg cctcctggcg ccgcaccggc 4380 ccaaggagcc cgcgtggttc ctggccaccg tcggcgtctc gcccgaccac cagggcaagg 4440 gtctgggcag cgccgtcgtg ctccccggag tggaggcggc cgagcgcgcc ggggtgcccg 4500 ccttcctgga gacctccgcg ccccgcaacc tccccttcta cgagcggctc ggcttcaccg 4560 tcaccgccga cgtcgaggtg cccgaaggac cgcgcacctg gtgcatgacc cgcaagcccg 4620 gtgcctgaaa ttagatcgat accgtcgaca atcaacctct ggattacaaa atttgtgaaa 4680 gattgactgg tattcttaac tatgttgctc cttttacgct atgtggatac gctgctttaa 4740 tgcctttgta tcatgctatt gcttcccgta tggctttcat tttctcctcc ttgtataaat 4800 cctggttgct gtctctttat gaggagttgt ggcccgttgt caggcaacgt ggcgtggtgt 4860 gcactgtgtt tgctgacgca accccccactg gttggggcat tgccaccacc tgtcagctcc 4920 tttccgggac tttcgctttc cccctcccta ttgccacggc ggaactcatc gccgcctgcc 4980 ttgcccgctg ctggacaggg gctcggctgt tgggcactga caattccgtg gtgttgtcgg 5040 ggaagctgac gtcctttcca tggctgctcg cctgtgttgc cacctggatt ctgcgcggga 5100 cgtccttctg ctacgtccct tcggccctca atccagcgga ccttccttcc cgcggcctgc 5160 tgccggctct gcggcctctt ccgcgtcttc gccttcgccc tcagacgagt cggatctccc 5220 tttgggccgc ctccccgcct gaatacgagc tcggtacctt taagaccaat gacttacaag 5280 gcagctgtag atcttagcca ctttttaaaa gaaaaggggg gactggaagg gctaattcac 5340 tcccaacgaa gacaagatct gctttttgct tgtactgggt ctctctggtt agaccagatc 5400 tgagcctggg agctctctgg ctaactaggg aacccactgc ttaagcctca ataaagcttg 5460 ccttgagtgc ttcaagtagt gtgtgcccgt ctgttgtgtg actctggtaa ctagagatcc 5520 ctcagaccct tttagtcagt gtggaaaatc tctagcagta gtagttcatg tcatcttatt 5580 attcagtatt tataacttgc aaagaaatga atatcagaga gtgagaggaa cttgtttatt 5640 gcagcttata atggttacaa ataaagcaat agcatcacaa atttcacaaa taaagcattt 5700 ttttcactgc attctagttg tggtttgtcc aaactcatca atgtatctta tcatgtctgg 5760 ctctagctat cccgccccta actccgccca tcccgcccct aactccgccc agttccgccc 5820 attctccgcc ccatggctga ctaatttttt ttatttatgc agaggccgag gccgcctcgg 5880 cctctgagct attccagaag tagtgaggag gcttttttgg aggcctaggg acgtacccaa 5940 ttcgccctat agtgagtcgt attacgcgcg ctcactggcc gtcgttttac aacgtcgtga 6000 ctgggaaaac cctggcgtta cccaacttaa tcgccttgca gcacatcccc ctttcgccag 6060 ctggcgtaat agcgaagagg cccgcaccga tcgcccttcc caacagttgc gcagcctgaa 6120 tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg 6180 cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc 6240 ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg 6300 gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc 6360 acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt 6420 ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc 6480 ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta 6540 acaaaaattt aacgcgaatt ttaacaaaat attaacgttt acaatttccc aggtggcact 6600 tttcgggggaa atgtgcgcgg aacccctatt tgttttttt tctaaataca ttcaaatatg 6660 tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa aaaggaagt 6720 atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct 6780 gtttttgctc acccagaaac gctggtgaaa gtaaa...

Claims

1. An antigen binding protein that binds to tight junction protein 6, comprising: (a) HC CDR1 sequence consisting of GFTFSNYW; (b) HC CDR2 sequence consisting of IRLKSDNYAT; (c) a HC CDR3 sequence consisting of NDGPPSGX, wherein X consists of any one of S, A, C or Y; (d) LC CDR1 sequence consisting of ENIYSY; (e) a LC CDR2 sequence consisting of NAK; and (f) LC CDR3 sequence consisting of QHHYTVPWT.

2. The antigen binding protein of claim 1, wherein X is S and the HC CDR3 sequence consists of NDGPPSGS.

3. The antigen binding protein of claim 1, wherein X is A and the HC CDR3 sequence consists of NDGPPSGA.

4. The antigen binding protein of claim 1, wherein X is C and the HC CDR3 sequence consists of NDGPPSGC.

5. The antigen binding protein of claim 1, wherein X is Y and the HC CDR3 sequence consists of NDGPPSGY. The antigen binding protein of claim 1 , wherein the antigen binding protein is an antibody or an antigen binding fragment of an antibody.

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

8. The antigen binding protein of claim 1, wherein the antigen binding protein is a monoclonal antibody.

9. The antigen binding protein of claim 1, wherein the antigen binding protein is a human antibody, a chimeric antibody or a humanized antibody.

10. The antigen-binding protein of claim 1, wherein the antigen-binding protein comprises the heavy chain variable region amino acid sequence listed in SEQ ID NO: 387 and the light chain variable region amino acid sequence listed in SEQ ID NO:

389. The antigen binding protein of claim 10 , wherein the antigen binding protein is an antibody or an antigen binding fragment of an antibody.

12. The antigen-binding protein of claim 11, wherein the antigen-binding fragment of the antibody is selected from the group consisting of scFv, F(ab')2, Fab and Fv.

13. The antigen binding protein of claim 11, wherein the antibody is a monoclonal antibody.

14. The antigen binding protein of claim 11, wherein the antibody is a human antibody, a chimeric antibody or a humanized antibody.

15. A conjugate comprising the antigen binding protein of claim 1, and a cytotoxic agent.

16. The conjugate of claim 15, wherein the cytotoxic agent is a chemotherapeutic agent.

17. The conjugate of claim 16, wherein the chemotherapeutic agent is an antimitotic alkaloid.

18. The conjugate of claim 16, wherein the chemotherapeutic agent is monomethylauristatin E (MMAE). A fusion protein comprising the antigen-binding protein according to claim 1 .

20. A fusion protein comprising the antigen binding protein according to claim 10.

21. A nucleic acid comprising a nucleotide sequence encoding the antigen-binding protein of claim 1 or a fusion protein comprising the antigen-binding protein of claim 1.

22. A nucleic acid comprising a nucleotide sequence encoding the antigen-binding protein of claim 10 or a fusion protein comprising the antigen-binding protein of claim 10.

23. A vector comprising the nucleic acid of claim 21.

24. A vector comprising the nucleic acid of claim 22.

25. A host cell comprising the nucleic acid of claim 21.

26. A host cell comprising the nucleic acid of claim 22.

27. A method for producing an antigen binding protein that binds to a claudin 6 (CLDN6) protein, comprising (i) culturing the host cell of claim 25 or 26 in a cell culture medium, and (ii) harvesting the antigen binding protein from the cell culture medium.

28. A method for producing a fusion protein comprising an antigen binding protein that binds to a Claudin 6 (CLDN6) protein, comprising (i) culturing the host cell of claim 25 or 26 in a cell culture medium, and (ii) harvesting the fusion protein from the cell culture medium.

29. A method of producing a pharmaceutical composition comprising combining an antigen binding protein as described in any one of claims 1 to 14, a conjugate as described in any one of claims 15 to 18, a fusion protein as described in claim 19 or 20, a nucleic acid as described in claim 21 or 22, a vector as described in claim 23 or 24, a host cell as described in claim 25 or 26, or a combination thereof, and a pharmaceutically acceptable carrier, diluent or excipient.

30. A pharmaceutical composition comprising an antigen binding protein as described in any one of claims 1 to 14, a conjugate as described in any one of claims 15 to 18, a fusion protein as described in claim 19 or 20, a nucleic acid as described in claim 21 or 22, a vector as described in claim 23 or 24, a host cell as described in claim 25 or 26, or a combination thereof, and a pharmaceutically acceptable carrier, diluent or excipient.

31. Use of the pharmaceutical composition of claim 30 in the manufacture of a medicament for treating a cancer expressing CLDN6, wherein the cancer is selected from the group consisting of lung cancer, endometrial cancer, melanoma cancer, bladder cancer and ovarian cancer.

32. The use according to claim 31, wherein the cancer expressing CLDN6 is a low overexpresser of CLDN6.

33. The use of claim 31, wherein the pharmaceutical composition induces apoptosis of tumor cells, wherein the tumor is selected from the group consisting of lung tumors, endometrial tumors, melanoma tumors, bladder tumors and ovarian tumors.

34. The use according to claim 31, wherein the pharmaceutical composition induces apoptosis of cells expressing CLDN6.

35. An in vitro or ex vivo method for inhibiting tumor growth, comprising contacting cancer cells with the pharmaceutical composition of claim 30 in an amount effective to inhibit tumor growth, wherein the tumor is selected from the group consisting of lung tumors, endometrial tumors, melanoma tumors, bladder tumors, and ovarian tumors.

36. A method for detecting tight junction protein 6 (CLDN6) in a sample for non-diagnostic purposes, comprising contacting the sample with an antigen binding protein according to any one of claims 1 to 14, a conjugate according to any one of claims 15 to 18, or a fusion protein according to claim 19 or 20, and measuring an immune complex comprising the antigen binding protein, the conjugate or the fusion protein bound to CLDN6.

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