Claudin 6 bispecific antibody

Abstract

The present disclosure provides bispecific antigen-binding proteins that bind to claudin 6 (CLDN6) and a second antigen. In various aspects, the antigen-binding proteins bind to extracellular loop 2 (EL2) of the extracellular domain of CLDN6. Related polypeptides, nucleic acids, vectors, host cells, and complexes are further provided herein. Kits and pharmaceutical compositions comprising such entities are also provided. Also provided are methods of making the antigen-binding proteins and methods of treating a subject with cancer.

Description

Technical Field

[0001] Cross-reference to Related Applications This application claims the benefit of U.S. Provisional Application No. 62 / 821,399, filed Mar. 20, 2019, the entire contents of which are hereby incorporated by reference in their entirety.

[0002] Incorporation by Reference of Electronically Submitted Materials Incorporated by reference in its entirety is a computer-readable nucleotide / amino acid sequence listing identified as a 344,064 ASCII (text) file named “54086P2_Seqlisting.txt” created on Mar. 20, 2019, which was filed concurrently with the application.

Background Art

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

[0004] More than 12 monoclonal antibodies are currently approved by the U.S. Food and Drug Administration for treating cancer. Among these agents are alemtuzumab (Campath®), which is indicated for chronic lymphocytic leukemia (CLL), and trastuzumab (Herceptin®), which is used to treat breast cancer. Some antibodies are labeled with chemotherapeutic agents, such as brentuximab vedotin (Adcetris®) and trastuzumab emtansine (Kadcyla®). Other antibody products, such as blinatumomab (Blincyto), are designed to recognize and bind two different antigens. Despite the availability of such antibody products, current cancer incidence and cancer deaths remain high. Cancer incidence is reported to be more than 450 per 100,000 person-years for both men and women, and cancer mortality is reported to be more than 170 per 100,000 person-years for both men and women.

SUMMARY OF THE INVENTION

[0005] This specification provides an antigen-binding protein that binds to Claudin 6 (CLDN6) and bispecific forms thereof. In various embodiments, the antigen-binding proteins of the present disclosure bind to human CLDN6 and optionally to mouse CLDN6. In various embodiments, the antigen-binding protein binds to the extracellular domain (ECD) of CLDN6. In various cases, the antigen-binding protein binds to extracellular loop 2 (EL2) of the ECD of CLDN6. In various embodiments, the antigen-binding protein binds to EL2 and does not bind to extracellular loop 1 (EL1) of the ECD of CLDN6. In various cases, the antigen-binding protein binds to additional members of the human claudin family, such as Claudin 3 (CLDN3), Claudin 4 (CLDN4), and Claudin 9 (CLDN9), etc. In various cases, the antigen-binding protein binds to CLDN6 and at least one of CLDN4 and CLDN9. In various cases, the antigen-binding protein binds to CLDN6 and does not bind to any other member of the claudin family. In various embodiments, the antigen-binding protein binds to CLDN6 that is endogenously expressed by human ovarian cancer cells, such as 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, such as a human, without including any other moiety bound to this antigen-binding protein.

[0006] Also provided herein are antigen-binding proteins conjugated to a heterologous moiety (e.g., conjugated to any chemotherapeutic agent, drug, or toxic moiety) that inhibit tumor growth in a subject, e.g., a human. In various cases, the conjugated antigen-binding protein is a monoclonal antibody. In various cases, the antibody is conjugated to an agent that alters microtubule dynamics, e.g., MMAE. In various cases, the conjugate comprises a cleavable linker, e.g., MC-VC-PAB. In various embodiments, the conjugate is a homogeneous conjugate or a heterogeneous conjugate. In various embodiments, the heterologous moiety is conjugated at a specific site of the antigen-binding protein. In various cases, the conjugated antigen-binding protein is a bispecific antigen-binding protein.

[0007] In various embodiments, the bispecific antigen-binding protein binds to CLDN6 expressed by human cancer cells. In various embodiments, the antigen-binding protein inhibits the binding interaction between human CLDN6 and an anti-CLDN6 reference antibody. Without being bound by a particular theory, due to the inhibitory effect of the antigen-binding proteins provided herein, such entities are useful in methods for treating a subject with a tumor or cancer by suppressing tumor growth. As further discussed herein, in various embodiments, the antigen-binding protein is an antibody, an antigen-binding antibody fragment thereof, or an antibody protein product.

[0008] The disclosure also provides bispecific antigen-binding proteins comprising at least 3, 4, 5, or all of a particular group of amino acid sequences. In various embodiments, the antigen-binding protein comprises at least 3, 4, 5, or 6 complementarity determining region (CDR) amino acid sequences of a CLDN6 antibody disclosed herein.

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

[0010] Also provided is a method for producing a bispecific antigen-binding protein. In various embodiments, the method comprises culturing a host cell comprising a nucleic acid encoding such a bispecific antigen-binding protein or polypeptide so as to express the bispecific antigen-binding protein or polypeptide as described herein.

[0011] Further provided herein is a method for 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 cancer in the subject.

[0012] Also provided is a method for treating a subject having CLDN6-expressing cancer, which comprises administering to the subject a pharmaceutical composition described herein. Further contemplated is a method for inhibiting tumor growth in a subject, which comprises administering to the subject a pharmaceutical composition described herein.

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

[0014] Also provided herein is a method for treating cancer in a subject diagnosed as a CLDN6 low overexpressing individual, which comprises administering to the subject a pharmaceutical composition described herein.

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

[0016]

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BEST MODE FOR CARRYING OUT THE INVENTION

[0017] Claudin Family

[0018] Tight junctions, also known as occluding junctions or zonulae occludentes, are vertebrate structures located between two adjacent cells that control paracellular permeability and maintain cell polarity in epithelial and endothelial cell sheets. Genes of the claudin (CLDN) family encode membrane proteins that are important components of tight junctions.

[0019] 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 in adjacent cells interact with each other to closely adhere the cell sheet and control paracellular transport between the lumen and the lateral bottom gap.

[0020] CLDN proteins are involved in various human diseases and pathologies. For example, mutations in the CLDN1 gene have been shown to cause progressive scaling of the skin along with bile duct obstruction. Mutants of the CLDN16 gene cause magnesium wasting disorders. Mutations in CLDN19 result in eye diseases such as macular colobomata and myopia, and mutations in CLDN14 can result in asymptomatic recessive hearing loss. CLDN3 and CLDN4 are known to be surface receptors for Clostridium perfringens enterotoxin in the intestinal tract, and CLDN1, CLDN6, and CLDN9 are co-receptors involved in the entry of hepatitis C virus (HCV). Some CLDN proteins have been shown to be abnormally expressed in cancer. For example, CLDN1 is downregulated in breast cancer and colon cancer, while CLDN3 and CLDN4 are highly upregulated in multiple cancers.

[0021] Claudin 6 (CLDN6) is a member of the claudin family. The gene encoding the human CLDN6 protein is located at 16p13.3 on the short arm of human chromosome 16 and is conserved in chimpanzee, rhesus monkey, dog, cow, mouse, rat, zebrafish, and frog. CLDN6 is generally expressed as a precursor protein of 220 amino acids in humans, and the first 21 amino acids among them constitute a signal peptide. The amino acid sequence of the CLDN6 precursor protein is publicly available as NCBI Reference Sequence NP_067018.2 on the website of the National Center for Biotechnology Information (NCBI) 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 having Val at position 143 is provided herein as SEQ ID NO: 178.

[0022] Antigen-Binding Protein

[0023] Provided herein is an antigen-binding protein that binds to claudin 6 (CLDN6). The antigen-binding protein of the present disclosure can take any one of several forms of antigen-binding proteins known in the art. In various embodiments, the antigen-binding protein of the present disclosure takes the form of an antibody, or an antigen-binding antibody fragment, or an antibody protein product.

[0024] In various embodiments of the present disclosure, the antigen-binding protein comprises, consists essentially of, or consists of an antibody. As used herein, the term “antibody” refers to a protein having the normal immunoglobulin format, including heavy and light chains, and including variable and constant regions. For example, an antibody can be an IgG in which two identical pairs of polypeptide chains have a “Y-shaped” structure, with each pair having one “light” chain (typically having a molecular weight of about 25 kDa) and one “heavy” chain (typically having a molecular weight of about 50-70 kDa). Antibodies have variable and constant regions. In the IgG format, the variable region generally has more than about 100-110 amino acids, contains three complementarity-determining regions (CDRs), is primarily involved in antigen recognition, and is substantially different from other antibodies that bind different antigens. The constant region enables the antibody to recruit cells and molecules of the immune system. The variable region is formed at the N-terminal region of each of the light and heavy chains, and the constant region is formed at the C-terminal portion of each of the heavy and light chains. (Janeway et al., “Structure of the Antibody Molecule and the Immunoglobulin Genes”, Immunobiology: The Immune System in Health and Disease, 4 th ed. Elsevier Science Ltd. / Garland Publishing, (1999)).

[0025] The general structure and characteristics of antibody CDRs are reported in the art. Briefly, in the antibody scaffold, the CDRs are embedded within the frameworks of the variable regions of the heavy and light chains and constitute regions that are largely involved in antigen binding and recognition. The variable regions typically contain at least three CDRs of the heavy or light chain (see also Kabat et al., 1991, Sequences of Proteins of Immunological Interest, Public Health Service N.I.H., Bethesda, Md., and Chothia and Lesk, 1987, J. Mol. Biol. 196:901-917, Chothia et al., 1989, Nature 342:877-883) within the framework regions (FR1, FR2, FR3, and FR4 of the designated framework regions 1-4 according to Kabat et al., 1991; see also Chothia and Lesk, 1987 supra).

[0026] The antibody can include any constant region known in the art. Human light chains are classified into kappa and lambda light chains. Heavy chains are classified into mu, delta, gamma, alpha, or epsilon, which define the antibody isotypes as IgM, IgD, IgG, IgA, and IgE, respectively. IgG has several subclasses, including but not limited to IgG1, IgG2, IgG3, and IgG4. IgM has subclasses, including but not limited to IgM1 and IgM2. Embodiments of the present disclosure include all such classes or isotypes of antibodies. The light chain constant region can be, for example, a kappa or lambda light chain constant region, such as a human kappa or lambda light chain constant region. The heavy chain constant region can be, for example, an alpha, delta, epsilon, gamma, or mu heavy chain constant region, such as a human alpha, delta, epsilon, gamma, or mu heavy chain constant region. Thus, in various embodiments, the antibody is an antibody of isotype IgA, IgD, IgE, IgG, or IgM, which includes any one of IgG1, IgG2, IgG3, or IgG4. In various aspects, the antibody includes a constant region that contains one or more amino acid modifications compared to the naturally occurring amino acids to improve the half-life / stability or to make the antibody more suitable with respect to expression / producibility. In various cases, the antibody includes a constant region in which the C-terminal Lys residue present in the naturally occurring constant region has been removed or clipped.

[0027] The antibody can be a monoclonal antibody. In some embodiments, the antibody comprises a sequence substantially similar to that of a naturally occurring antibody produced by a mammal, such as a mouse, rabbit, goat, horse, chicken, hamster, human, etc. In this regard, the antibody can be regarded as a mammalian antibody, such as a mouse antibody, rabbit antibody, goat antibody, horse antibody, chicken antibody, hamster antibody, human antibody, etc. In certain embodiments, the antigen-binding protein is an antibody such as a human antibody. In certain embodiments, the antigen-binding protein is a chimeric antibody or a humanized antibody. The term "chimeric antibody" refers to an antibody containing domains from two or more different antibodies. A chimeric antibody can, for example, contain a constant domain from one species and a variable domain from a second species, or more generally, can contain intervals of amino acid sequences from at least two species. A chimeric antibody can also contain domains from two or more different antibodies within the same species. The term "humanized" as used in connection with an antibody refers to an antibody having at least the CDR regions from a non-human material that has been engineered to have a structure and immunological function more similar to that of a true human antibody than the original source antibody. For example, humanizing can involve transplanting the CDRs from a non-human antibody such as a mouse antibody into a human antibody. Humanizing can also involve selecting amino acid substitutions to create non-human sequences more similar to human sequences. Information such as sequence information for the constant regions of the heavy and light chains of a human antibody is publicly available from the Uniprot database and other databases well known to those of skill in the art of antibody engineering and production. For example, the IgG2 constant region is available from the Uniprot database as Uniprot number P01859 and is incorporated herein by reference.

[0028] The antibody can be cleaved into fragments by enzymes such as papain and pepsin. Papain cleaves the antibody to produce two Fab fragments and a single Fc fragment. Pepsin cleaves the antibody to produce F(ab’) 2Generate fragments and pFc’ fragments. In various aspects of the present disclosure, the antigen-binding protein of the present disclosure is an antigen-binding fragment of an antibody (also known as an antigen-binding antibody fragment, antigen-binding fragment, or antigen-binding portion). In various cases, the antigen-binding antibody fragment is a Fab fragment or F(ab’) 2 fragment.

[0029] The structure of antibodies has been utilized to create a wide range of alternative antibody formats that are expanding, with a molecular weight range of at least about 12 to 150 kDa and a valence (n) range starting from monomer (n = 1) to dimer (n = 2), trimer (n = 3), tetramer (n = 4), and potentially even higher. Such alternative antibody formats are referred to herein as "antibody protein products". Antibody protein products include those based on the complete antibody structure, as well as antibody fragments that retain full antigen-binding ability, such as those mimicking scFv, Fab, and VHH / VH (discussed below). Among the antigen-binding fragments, the smallest antigen-binding fragment that retains its complete antigen-binding site is the Fv fragment, which consists of only the variable (V) regions. To stabilize the molecule, a soluble and flexible amino acid peptide linker is used to connect the V regions into an scFv (single-chain variable region) fragment, or a constant (C) domain is added to the V region to generate a Fab fragment [fragment, antigen-binding]. Both scFv and Fab fragments can be easily produced in host cells, such as prokaryotic host cells. Other antibody protein products include various formats such as diabodies, triabodies, and tetrabodies composed of scFv linked to an oligomerization domain, or dimeric and multimeric antibody formats such as minibodies (miniAb), including ds-scFv (disulfide bond-stabilized scFv), scFab (single-chain Fab), etc. The smallest fragments are VHH / VH of camelid heavy-chain Abs and single-domain Abs (sdAb). The most frequently used component for creating new antibody formats is the single-chain variable (V) domain antibody fragment (scFv), which contains the V domains (VH domain and VL domain) from the heavy and light chains linked by a peptide linker of about 15 amino acid residues. Peptibodies or peptide-Fc fusions are yet another type of antibody protein product. The structure of peptibodies consists of bioactive peptides transplanted into the Fc domain. Peptibodies are well-described in the art. See, for example, Shimamoto et al., mAbs 4(5):586-591 (2012).

[0030] Other antibody protein products include single-chain antibodies (SCA), diabodies, triabodies, tetra-bodies, bispecific or trispecific antibodies, etc. Bispecific antibodies are divided into five major classes, namely, BsIgG, IgG with additions, bispecific antibody (BsAb) fragments, bispecific fusion proteins, and BsAb complexes. See, for example, Spiess et al., Molecular Immunology 67(2) Part A:97-106(2015).

[0031] In various embodiments, the antigen-binding protein of the present disclosure comprises, consists essentially of, or consists of any one of these antibody protein products. In various embodiments, the antigen-binding protein of the present disclosure comprises, consists essentially of, or consists of any one of scFv, Fab VHH / VH, Fv fragment, ds-scFv, scFab, dimeric antibody, multimeric antibody (e.g., diabody, triabody, tetrabody), miniAb, peptibody VHH / VH of camelid heavy-chain antibody, sdAb, diabody; triabody; tetrabody; bispecific or trispecific antibody, BsIgG, IgG with additions, BsAb fragment, bispecific fusion protein, and BsAb complex.

[0032] In various cases, the antigen-binding protein of the present disclosure is an antibody protein product in monomeric form, or in polymeric, oligomeric, or multimeric form. In certain embodiments where the antibody comprises fragments of two or more distinct antigen-binding regions, the antibody is considered bispecific, trispecific, or multispecific, or bivalent, trivalent, or multivalent, depending on the number of distinct epitopes that the antibody recognizes and binds to.

[0033] In various embodiments, the anti-CLDN6 antibody or its variant antibody is selected from the group consisting of human antibodies, humanized antibodies, chimeric antibodies, monoclonal antibodies, recombinant antibodies, antigen-binding antibody fragments, single-chain antibodies, monomeric antibodies, diabodies, triabodies, tetra-bodies, Fab fragments, IgG1 antibodies, IgG2 antibodies, IgG3 antibodies, and IgG4 antibodies.

[0034] In various aspects, the antigen-binding protein of the present disclosure is conjugated to a therapeutic agent. As described below, the therapeutic agent can be any known in the art and includes, but is not limited to, chemotherapeutic agents, cytokines and growth factors, cytotoxic drugs, etc. See the "complex" below.

[0035] Bispecific Format

[0036] In an exemplary aspect, the antigen-binding protein is bispecific and thus can bind to two different distinct antigens. In an exemplary embodiment, the antigen-binding protein is bispecific and binds to CLDN6 and a second antigen.

[0037] In an exemplary case, the second antigen is a cell surface protein expressed by T cells. In an exemplary embodiment, the cell surface protein is a component of the T cell receptor (TCR), such as CD3. In an exemplary case, the second antigen is a co-stimulatory molecule that aids in T cell activation, such as CD40 or 4-1BB (CD137). In an exemplary embodiment, the second antigen is an Fc receptor. In various embodiments, the Fc receptor is an Fc gamma receptor, an Fc-alpha receptor, or an Fc-epsilon receptor. In an exemplary embodiment, the Fc receptor is CD64 (Fc-gamma RI), CD32 (Fc-gamma RIIA), CD16A (Fc-gamma RIIIA), CD16b (Fc-gamma RIIIb), FcεRI, CD23 (Fc-epsilon RII), CD89 (Fc-epsilon RI), Fcα / μR, or FcRn. In an exemplary embodiment, the Fc receptor is CD16A. In an exemplary case, the second antigen is an immune checkpoint molecule, such as a protein involved in the immune checkpoint pathway. The immune checkpoint pathway and the molecules or proteins that function within the pathway are known in the art. See, for example, Pardoll, Nat Rev Genet 12(4):252-264(2012). In an exemplary case, the immune checkpoint molecule is A2AR, B7-H3, B7-H4, BTLA, CTLA4, IDO, KIR, LAG3, NOX2, PD-1, TIM3, VISTA, or SIGLEC7. Optionally, the immune checkpoint molecule is PD-1, LAG3, TIM3, or CTLA4.

[0038] Over 50 formats of bispecific antigen-binding proteins are known in the art, some of which are described in Kontermann and Brinkmann, Drug Discovery Today 20(7):838 - 847(2015), Zhang et al., Exp Hematol Oncol 6:12(2017), Spiess et al., Mol Immunol.;67(2 Pt A):95 - 106(2015). In an exemplary embodiment, the bispecific antigen-binding protein of the present disclosure is produced by chemical engineering, genetic engineering, or quadroma technology.

[0039] In an exemplary embodiment, the bispecific antigen-binding protein is constructed using some or all of the constant domains of an antibody. In an exemplary embodiment, the bispecific antigen-binding protein of the present disclosure includes an Fc polypeptide and retains effector function by Fc. In various cases, the bispecific antigen-binding protein is, for example, a bispecific monoclonal antibody formed by chemical cross-linking of two monoclonal antibodies (mabs) or by the knob and hold technology. In an exemplary embodiment, the bispecific antigen-binding protein is produced by the "knobs-into-holes" technology, in which case different mutations are introduced into two CH3 domains to generate an asymmetric antibody, thereby forcing H-chain heterodimerization. A "knob" mutation is made in one HC and a "hole" mutation is made in the other HC to promote heterodimerization. In an exemplary embodiment, the bispecific antigen-binding protein is a bispecific antibody produced by quadroma technology based on somatic cell fusion of two different hybridoma cells that generate monoclonal antibodies with the desired specificities. Zhang et al., 2017, supra. In an exemplary embodiment, the bispecific antigen-binding protein is crossMab, ortho-Fab IgG, DVD-Ig, two in one IgG, IgG-scFv and scFv 2-Fc (Kontermann and Brinkmann, 2015, supra). In various embodiments, the bispecific antigen-binding protein is an Ig-scFv fusion that adds a new antigen-binding moiety to a full-length IgG, resulting in a tetravalent fusion protein for two distinct antigens, e.g., an IgG C-terminal scFv fusion and an IgG N-terminal scFv fusion. In an exemplary case, the bispecific antigen-binding protein is a dual variable domain IgG (DVD-IgG), where the variable regions of the LC and HC of an IgG specific for one antigen are fused via a linker to the N-terminus of the variable regions of the LC and HC of an IgG specific for a second antigen, forming the DVD-IgG. In an exemplary embodiment, the bispecific antigen-binding protein is a diabody-Fc fusion involving replacement of the Fab fragment of an IgG with a bispecific diabody.

[0040] In alternative examples, the bispecific antigen-binding proteins of the disclosure do not contain an Fc polypeptide. In an exemplary embodiment, the bispecific antigen-binding protein contains the variable domains of each parental monoclonal antibody, and the linkers are cloned and ligated to form a single-chain bispecific antibody. In an exemplary embodiment, the bispecific antigen-binding protein is a tandem scFv, diabody format, single-chain diabody, tandem diabody (TandAb), dual affinity retargeting molecule (DART), dock-and-lock (DNL), and nanobody (Fan et al., J Hematol Oncol. 2015;8:130). In various embodiments, the bispecific antigen-binding protein is a bispecific F(mab 1 ) 2, scFv, bispecific diabody (BsDb), single-chain bispecific diabody (scBsDb), single-chain bispecific tandem variable domain (scBsTaFv), dock-and-lock trivalent Fab (DNL-(Fab)3), single-domain antibody (sdAb), or bispecific single-domain antibody (BssdAb). In an exemplary embodiment, the bispecific antigen-binding protein is a tandem scFv comprising two scFv fragments linked by an additional peptide linker such as a glycine-serine repeat motif. Optionally, the tandem scFv is VL A - linker 1 - VH A - linker 2 - VH B - linker 3 - VL B and has the structure (where VL and VH are derived from single-chain antibody fragments, and A and B represent parental monoclonal antibody A and parental monoclonal antibody B). In an exemplary embodiment, the bispecific antigen-binding protein is a TandAb and contains two pairs of VL domains and VH domains that are connected into a single polypeptide chain (Reusch et al., MAbs. 2015;7(3):584 - 604). The two polypeptide products dimerize in a head-to-tail manner and form a homodimer with a large molecular weight (about 105 kDa) upon expression. In an exemplary embodiment, the bispecific antigen-binding protein is prepared using the crossMab technology described in PNAS 108(27):11187 - 92(2011). CrossMab is prepared by a method that has no chemical linker or connector and enforces correct light-chain association in bispecific heterodimeric IgG antibodies. In an exemplary embodiment, CrossMab is a bivalent (1 + 1), trivalent (2 + 1), and tetravalent (2 + 2) bispecific crossMab, or a crossMab using a non-Fc tandem antigen-binding fragment (Fab). In an exemplary case, CrossMab is CrossMab Fab , CrossMab VH-VL , or CrossMab CH1-CL .

[0041] In an exemplary embodiment, the bispecific antigen-binding protein comprises a single-domain antibody or nanobody comprising a single monomeric variable antibody domain. Optionally, the variable domain is based on a heavy-chain variable domain. In an alternative embodiment, the variable domain is based on a light-chain variable domain.

[0042] In an exemplary embodiment, the bispecific antigen-binding protein is a bispecific T cell Engager or BiTE®. BiTE is a bivalent small molecule comprising only the variable regions of antibodies, in which the scFvs are connected by a flexible peptide linker. In an exemplary embodiment, the bispecific antigen-binding protein comprises an scFV comprising the LC variable region and the HC variable region of the CLDN6 antibody disclosed herein, and the LC variable region and the HC variable region of a second antibody specific for a second antigen. In some embodiments, the BiTE comprises the LC variable region and the HC variable region of a second antibody specific for CD3. In some embodiments, CD3 is CD3E.

[0043] In an exemplary case, the bispecific antigen-binding protein is a dual-affinity retargeting (DART), which, unlike BiTEs®, has a covalent bond between the two chains of the DART that restricts the freedom of the antigen-binding site. Thus, DART is structurally small and can form a stable contact between the target and the effector cell. DART comprises two engineered Fv fragments in which the VH of one is exchanged with the VH of the other. The mutually exchanged Fv domains advantageously release the mutant fragments from the conformational constraints by a short linker peptide.

[0044] In an exemplary embodiment, the bispecific antigen-binding protein is an HSABody comprising two scFvs fused to a modified HSA. HSABody is described in McDonagh et al., Mol Cancer Ther. 2012;11(3):582-93.

[0045] Thus, in an exemplary embodiment, the bispecific antigen-binding protein comprises an antigen-binding fragment of any of the CLDN6 antibodies disclosed herein. In an exemplary embodiment, the antigen-binding fragment is a Fab. In an exemplary embodiment, the bispecific antigen-binding protein comprises an scFv comprising the variable regions of the light chain and heavy chain of any of the CLDN6 antibodies disclosed herein. In an exemplary embodiment, the scFv comprises the amino acid sequence of SEQ ID NO: 514 or 515. In various embodiments, the antigen-binding fragment is based on the heavy chain variable region, and in other embodiments, the antigen-binding fragment is based on the light chain variable region. In an exemplary embodiment, the antigen-binding fragment comprises at least a portion of both the HC variable region and the LC variable region. In an exemplary embodiment, the bispecific antigen-binding protein comprises at least one of the LC variable region or the HC variable region of any of the CLDN6 antibodies disclosed herein, and at least one of the LC variable region and the HC variable region of a second antibody specific for a second antigen. In an exemplary case, the bispecific antigen-binding protein comprises an scFV comprising the LC variable region and the HC variable region of a CLDN6 antibody disclosed herein, and the LC variable region and the HC variable region of a second antibody specific for a second antigen. 2 ’. In an exemplary embodiment, the bispecific antigen-binding protein comprises an scFv comprising the variable regions of the light chain and heavy chain of any of the CLDN6 antibodies disclosed herein. In an exemplary embodiment, the scFv comprises the amino acid sequence of SEQ ID NO: 514 or 515. In various embodiments, the antigen-binding fragment is based on the heavy chain variable region, and in other embodiments, the antigen-binding fragment is based on the light chain variable region. In an exemplary embodiment, the antigen-binding fragment comprises at least a portion of both the HC variable region and the LC variable region. In an exemplary embodiment, the bispecific antigen-binding protein comprises at least one of the LC variable region or the HC variable region of any of the CLDN6 antibodies disclosed herein, and at least one of the LC variable region and the HC variable region of a second antibody specific for a second antigen. In an exemplary case, the bispecific antigen-binding protein comprises an scFV comprising the LC variable region and the HC variable region of a CLDN6 antibody disclosed herein, and the LC variable region and the HC variable region of a second antibody specific for a second antigen.

[0046] CLDN6 and Epitope

[0047] The antigen-binding protein of the present disclosure binds to CLDN6. In various embodiments, CLDN6 is a human CLDN6 having the following amino acid sequence. TIFF0007682797000001.tif40160Here, X is Ile or Val (SEQ ID NO: 202).

[0048] In various embodiments, human CLDN6 comprises the amino acid sequence of any one of SEQ ID NOs: 1, 178, and 200-202.

[0049] In various aspects, the antigen-binding protein of the present disclosure binds to an epitope within the amino acid sequence of CLDN6. In various aspects, CLDN6 is human CLDN6, and the antigen-binding protein of the present disclosure binds to an epitope within the amino acid sequence of human CLDN6, for example, within SEQ ID NOs: 1, 178, and 200-202. An "epitope" means a region of CLDN6 or a region within CLDN6 to which the antigen-binding protein binds. In some embodiments, the epitope is a linear epitope. A "linear epitope" refers to a region of CLDN6 or a region within CLDN6 to which the antigen-binding protein binds, and that region is composed of contiguous amino acids of the amino acid sequence of CLDN6. The amino acids of the linear epitope are adjacent to each other in the primary structure of CLDN6. Thus, a linear epitope is a fragment or portion of the antigen, i.e., the amino acid sequence of CLDN6. In various other embodiments, the epitope is a conformational epitope or a structural epitope. A "conformational epitope" or "structural epitope" means an epitope composed of amino acids that are located close to each other only when CLDN6 is in its properly folded state. Unlike a linear epitope, the amino acids of a conformational epitope or a structural epitope are not adjacent to each other in the primary structure (i.e., the amino acid sequence) of CLDN6. A conformational epitope or a structural epitope is not made of contiguous amino acids of the amino acid sequence of the antigen (CLDN6).

[0050] In various aspects, the epitope is located within the extracellular domain (ECD) of CLDN6, such as 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 protein of the present disclosure binds to the N-terminal portion of SEQ ID NO: 2, such as TAHAIIRDFYNPL (SEQ ID NO: 3). In various aspects, the antigen-binding protein of the present disclosure binds to the C-terminal portion of SEQ ID NO: 2, such as LVAEAQKREL (SEQ ID NO: 4). In various cases, the antigen-binding protein of the present disclosure binds to EL2 but does not bind to extracellular loop 1 (EL1) of CLDN6. In various aspects, the epitope(s) to which the antigen-binding protein of the present disclosure binds is different from the epitope to which 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 binds. In various aspects, the epitope(s) to which the antigen-binding protein of the present disclosure binds is different from the epitope to which 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 binds.

[0051] In various aspects, the antigen-binding protein binds to human CLDN6 and non-human CLDN6. In various cases, the non-human CLDN6 is CLDN6 of chimpanzee, rhesus monkey, dog, cow, mouse, rat, zebrafish, or frog. In various cases, the antigen-binding protein binds to human CLDN6 and mouse CLDN6.

[0052] Affinity and Binding Strength

[0053] 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 represented by its affinity, which is 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 bind to a large amount of CLDN6 in a shorter period than low-affinity antigen-binding proteins. In various aspects, the antigen-binding protein has an equilibrium association constant K A 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 or at least 10 10 mol -1 or at least 10 A mol

[0054] In various embodiments, the binding strength of the antigen-binding protein to CLDN6 can be represented by its sensitivity. K D is the equilibrium dissociation constant that is the ratio of k off / k on between the antigen-binding protein and CLDN6. K D and K A are inversely correlated. The K D value is related to the concentration of the antigen-binding protein (the amount of antigen-binding protein required for a specific experiment), so the lower the K D value (lower concentration), the higher the affinity of the antigen-binding protein. In various aspects, the binding strength of the antigen-binding protein to CLDN6 is K DIt can be represented by. In various embodiments, the K of the antigen-binding protein provided herein D is about 10 -1 about 10 -2 about 10 -3 about 10 -4 about 10 -5 about 10 -6 or less. In various embodiments, the K of the antigen-binding protein provided herein D is micromolar, nanomolar, picomolar or femtomolar. In various embodiments, the K of the antigen-binding protein provided herein D is about 10 -4 to 10 -6 or 10 -7 to 10 -9 or 10 -10 to 10 -12 or 10 -13 to 10 -15 within the range of. In various embodiments, the K of the antigen-binding protein provided herein D is in the range of about 1.0×10 -12 M to about 1.0×10 -8 M. In various embodiments, the K of the antigen-binding protein D is in the range of about 1.0×10 -11 M to about 1.0×10 -9 M.

[0055] In various aspects, the affinity of an antigen-binding protein is measured or ranked using an assay based on flow cytometry - or fluorescence-activated cell sorting (FACS). 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 an antigen-binding protein is measured or ranked using Trikha et al., Int J Cancer 110:326-335(2004) and Tam et al., Circulation 98(11):1085-1091(1998), as well as the competitive assays described below. See the section entitled "Competitive Assays" below. In Trikh et al., cells expressing the antigen were used in a radioassay. 125 The binding of an I-labeled antigen-binding protein (e.g., antibody) to a cell surface antigen is measured using cells in suspension. In various aspects, the relative affinity of a CLDN6 antibody is determined by an FACS-based assay, in which various concentrations of the CLDN6 antibody conjugated to a fluorophore are incubated with CLDN6-expressing cells and the fluorescence emitted (a direct measure of antibody-antigen binding) is determined. A curve is created by plotting each dose or concentration. The maximum value is the lowest concentration at which the fluorescence plateaus or reaches a maximum, i.e., when binding saturation is occurring. Half of the maximum value is considered the EC50 or IC50, and the antibody with the lowest EC50 / IC50 is considered to have the highest affinity compared to other antibodies tested in the same manner. Such an assay is described in Example 5 herein.

[0056] In various aspects, the IC 50 value determined in a competitive binding inhibition assay is the K Dis approximated. In various cases, as discussed below, the competitive assay is a FACS-based assay performed using a reference antibody, a fluorophore-conjugated secondary antibody, and cells expressing CLDN6. In various embodiments, the cells are genetically engineered to overexpress CLDN6. In some embodiments, the cells are HEK293T cells transduced with a viral vector to express CLDN6. In alternative embodiments, the cells endogenously express CLDN6. Prior to performing the FACS-based assay, in some embodiments, cells that endogenously express CLDN6 are pre-determined to be CLDN6 low-expressing cells or CLDN6 high-expressing cells. In some embodiments, the cells are cancer or tumor cells. In various embodiments, the cells are cells from a cell line, e.g., an ovarian cell line, an endometrial cell line, a bladder cell line, a lung cell line, an upper gastrointestinal (GI) cell line, a hepatocyte cell line, a lung cell line, etc. In various embodiments, cells that endogenously express CLDN6 are selected from the group consisting of OVCA429 ovarian cells, ARK2 endometrial cells, OAW28 ovarian cells, UMUC-4 bladder cells, PEO14 ovarian cells, OV177 ovarian cells, H1693 lung cells, MKN7 upper gastrointestinal cells, OV-90 ovarian cells, HUH-7 hepatocytes, JHOS-4 ovarian cells, H1435 lung cells, and NUGC3 upper gastrointestinal cells. In various embodiments, the antigen-binding protein inhibits the binding interaction between human CLDN6 expressed by the cells and the reference antibody, where the reference antibody is known to bind to CLDN6 but is not the antigen-binding protein of the present disclosure. In various cases, the antigen-binding protein of the present disclosure competes with the reference antibody for binding to human CLDN6, thereby reducing the amount of human CLDN6 bound to the reference antibody as determined in an in vitro competitive binding assay. In various embodiments, the antigen-binding protein of the present disclosure inhibits the binding interaction between human CLDN6 and the reference antibody, and the inhibition is characterized by an IC 50 In various embodiments, the antigen-binding protein inhibits the binding interaction between human CLDN6 and the reference antibody with an IC 50is shown. In various embodiments, the antigen-binding protein has 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 100 nM 50 is shown. In various embodiments, the antigen-binding protein has 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 10 nM 50 is shown. In various cases, the antigen-binding proteins of the present disclosure compete for binding to CLDN6 with a reference antibody known to bind to CLDN6 (the reference antibody is different from any of the antigen-binding proteins of the present disclosure). See below "Competition assay" for details.

[0057] From the binding strength, a measure of the overall strength of the antibody-antigen complex is obtained. This varies depending on three main parameters, namely, 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 more antigen (CLDN6) it can bind. In various embodiments, the antigen-binding protein has a strong binding force to CLDN6. In various embodiments, the antigen-binding protein is multivalent. In various embodiments, the antigen-binding protein is bivalent. In various cases, the antigen-binding protein is monovalent.

[0058] Cross-Reactivity

[0059] In various embodiments, the antigen-binding protein of the present disclosure binds to CLDN6 and does not bind to any other member of the CLDN family, e.g., does not cross-react with any other member of the CLDN family. In various cases, the antigen-binding protein of the present disclosure is CLDN6-specific. In various embodiments, the antigen-binding protein of the present disclosure has selectivity for CLDN6 and is at least 10-fold, 5-fold, 4-fold, 3-fold, 2-fold greater than the selectivity of the antigen-binding protein for CLDN3, CLDN4, CLDN9, or a combination thereof. In various embodiments, the antigen-binding protein of the present disclosure has selectivity for CLDN6 and is at least 10-fold, 5-fold, 4-fold, 3-fold, 2-fold greater than the selectivity of the antigen-binding protein for each of CLDN3, CLDN4, and CLDN9. Selectivity may be based on K D and K D can be determined by techniques known in the art, e.g., surface plasmon resonance, FACS-based affinity assays.

[0060] In various aspects, the antigen-binding protein of the present disclosure binds to CLDN6 and does not bind to any of claudin 3 (CLDN3), claudin 4 (CLDN4), and claudin 9 (CLDN9). In various aspects, the antigen-binding protein does not bind to any of CLDN3, CLDN4, and CLDN9 and has an IC 50is shown. In various embodiments, the antigen-binding protein does not bind to any of CLDN3, CLDN4, and CLDN9, and the concentration at which 50% of binding saturation is achieved in OVCA429 cells that endogenously express CLDN6 is less than about 1200 nM (e.g., less than about 1000 nM, less than about 750 nM, less than about 500 nM, less than about 250 nM). In various embodiments, the antigen-binding protein exhibits at least 5-fold greater selectivity for CLDN6 than for CLDN3, CLDN4, and CLDN9, and the concentration at which 50% of binding saturation is achieved in OVCA429 cells that endogenously express CLDN6 is less than about 1200 nM (e.g., less than about 1000 nM, less than about 750 nM, less than about 500 nM, less than about 250 nM). In various embodiments, the antigen-binding protein exhibits 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 of more than about 5-fold that differentiates the IC50 of CLDN6 from that of CLDN3, CLDN4, and / or CLDN9. 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 exhibits an IC50 that is at least 5-fold greater than that IC50 for any one of CLDN3, CLDN4, and CLDN9.

[0061] In various embodiments, the antigen-binding proteins of the disclosure bind to CLDN6 and cross-react (e.g., bind) with at least one other member of the CLDN family. In various embodiments, the antigen-binding proteins of the disclosure bind to CLDN6 and one or more of CLDN3, CLDN4, and CLDN9. In various embodiments, the antigen-binding proteins of the disclosure bind to CLDN6 and CLDN4 or CLDN9, but not to CLDN3. In various cases, the antigen-binding proteins of the disclosure bind to CLDN6 and CLDN4, but not to CLDN3 or CLDN9. In various cases, the antigen-binding proteins of the disclosure bind to CLDN6 and CLDN9, but not to either CLDN3 or CLDN4.

[0062] Competitive Assay

[0063] In various embodiments, the antigen-binding protein inhibits the binding interaction between human CLDN6 and a reference antibody, where the reference antibody is known to bind to CLDN6 but is not the antigen-binding protein of the present disclosure. In various cases, the antigen-binding protein of the present disclosure competes with the reference antibody for binding to human CLDN6, thereby reducing the amount of human CLDN6 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 the light chain variable sequence of SEQ ID NO: 181 and the 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 characterized by an IC 50 characterized thereby. In various aspects, the antigen-binding protein exhibits an IC 50 of less than about 2500 nM when inhibiting the binding interaction between human CLDN6 and the reference antibody. In various aspects, the antigen-binding protein exhibits an IC 50 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 100 nM when inhibiting the binding interaction between human CLDN6 and the reference antibody. In various aspects, the antigen-binding protein exhibits an IC 50 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 10 nM when inhibiting the binding interaction between human CLDN6 and the reference antibody.

[0064] In various cases, the antigen-binding protein of the present disclosure competes with a reference antibody for binding to human CLDN6, thereby reducing the amount of human CLDN6 bound to the reference antibody as determined by an in vitro competitive binding assay. In various embodiments, the in vitro competitive binding assay is a FACS-based assay that measures the fluorescence of a fluorophore-conjugated secondary antibody that binds to the Fc of the reference antibody in the absence or presence of a specific amount of the antigen-binding protein of the present disclosure. Such FACS-based assays are described in the Examples herein. In various embodiments, the FACS-based assay is performed using a reference antibody, a fluorophore-conjugated secondary antibody, and cells that express CLDN6. In various embodiments, the cells are genetically engineered to overexpress CLDN6. In some embodiments, the cells are HEK293T cells transduced with a viral vector to express CLDN6. In alternative embodiments, the cells endogenously express CLDN6. Prior to performing the FACS-based assay, in some embodiments, cells that endogenously express CLDN6 are pre-determined to be CLDN6 low-expressing cells or CLDN6 high-expressing cells. In some embodiments, the cells are cancer or tumor cells. In various embodiments, 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, an upper gastrointestinal (GI) cell line, a hepatocyte cell line, a lung cell line, etc. In various embodiments, cells that endogenously express CLDN6 are selected from the group consisting of OVCA429 ovarian cells, ARK2 endometrial cells, OAW28 ovarian cells, UMUC-4 bladder cells, PEO14 ovarian cells, OV177 ovarian cells, H1693 lung cells, MKN7 upper gastrointestinal cells, OV-90 ovarian cells, HUH-7 hepatocytes, JHOS-4 ovarian cells, H1435 lung cells, and NUGC3 upper gastrointestinal cells. In various cases, the antigen-binding protein of the present disclosure binds with high affinity to CLDN6 endogenously expressed by one or more of ARK2 cells, OVCA429 cells, LS513 cells, or MCF7 cells. In various embodiments, the antigen-binding protein is determined by an IC in a FACS-based competitive binding inhibition assay using one or more of ARK2 cells, OVCA429 cells, LS513 cells, or MCF7 cells 50shows less than about 3000 nM. In various embodiments, the antigen-binding protein has an IC determined by a FACS-based competitive binding inhibition assay using one or more of ARK2 cells, OVCA429 cells, LS513 cells, or MCF7 cells 50 shows 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. In various embodiments, the antigen-binding protein has an IC determined by a FACS-based competitive binding inhibition assay using one or more of ARK2 cells, OVCA429 cells, LS513 cells, or MCF7 cells 50 shows 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.

[0065] Other binding assays for testing the ability of an antibody to compete with a second antibody for binding to an antigen or its epitope, such as competitive binding assays or competition assays, 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). Also, 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 constants of an antibody and a second antibody and to compare those two binding constants.

[0066] Antibody Preparation Method and Related Methods

[0067] Suitable methods for producing 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, e.g., in Harlow and Lane (eds.), Antibodies: A Laboratory Manual, CSH Press (1988), and C.A. Janeway et al. (eds.), Immunobiology, 5 th Ed., Garland Publishing, New York, NY (2001)). Various methods for preparing the CLDN6 monoclonal antibodies of the present disclosure are provided in the examples herein.

[0068] Depending on the host species, various adjuvants can be used to enhance the immune response that results in high levels of antibody production by the host. Such adjuvants include, but are not limited to, Freund's adjuvant, mineral gels such as aluminum hydroxide, and surfactants such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, and dinitrophenol. BCG (bacilli Calmette-Guerin) and Corynebacterium parvum may be useful human adjuvants.

[0069] Other antibody production methods are summarized in Table 1.

Table 1

[0070] Regardless of how an antibody is produced, methods for testing the ability of an antibody to bind to an epitope of CLDN6 are known in the art and include any antibody-antigen binding assay, such as radioimmunoassay (RIA), ELISA, Western blot, immunoprecipitation, SPR, and competitive inhibition assays, etc. (see, for example, Janeway et al., below, and U.S. Patent Application Publication No. 2002 / 0197266, and the above sections related to competitive assays).

[0071] Sequence / Structure

[0072] In this specification, (a) the amino acid sequence of the heavy chain (HC) complementarity-determining region (CDR) 1 described in Table A, or a sequence selected from the group consisting of SEQ ID NOs: 11, 17, 23, 29, 35, 41, 47, 53, 59, 65, 71, 77, 83, 89, 95, 101, 107, 113, 119, 125, and 131, or a variant sequence thereof, wherein only one or two amino acids are different, or having about or at least 70% (e.g., at least about 80%, at least about 85%, at least about 90%, at least about 95%) sequence identity; (b) the amino acid sequence of the HC CDR2 described in Table A, or a sequence selected from the group consisting of SEQ ID NOs: 12, 18, 24, 30, 36, 42, 48, 54, 60, 66, 72, 78, 84, 90, 86, 102, 108, 114, 120, 126, and 132, or a variant sequence thereof, wherein only one or two amino acids are different, or having about or at least 70% (e.g., at least about 80%, at least about 85%, at least about 90%, at least about 95%) sequence identity; (c) the amino acid sequence of the HC CDR3 described in Table A, or a sequence selected from the group consisting of SEQ ID NOs: 13, 19, 25, 31, 37, 43, 49, 55, 61, 67, 73, 79, 85, 91, 97, 103, 109, 115, 121, 127, and 133, or a variant sequence thereof, wherein only one or two amino acids are different, or having about or at least 70% (e.g., at least about 80%, at least about 85%, at least about 90%, at least about 95%) sequence identity; (d) the amino acid sequence of the light chain (LC) CDR1 described in Table A, or a sequence selected from the group consisting of SEQ ID NOs: 8, 14, 20, 32, 38, 44, 50, 56, 62, 68, 74, 80, 86, 92, 98, 104, 110, 116, 122, and 128, or a variant sequence thereof, wherein only one or two amino acids are different, or having about or at least 70% (e.g., at least about 80%, at least about 85%, at least about 90%, at least about 95%) sequence identity; (e) the LC described in Table AThe amino acid sequence of CDR2, or a sequence selected from the group consisting of SEQ ID NOs: 9, 15, 21, 27, 33, 39, 45, 51, 57, 63, 69, 75, 81, 87, 93, 99, 105, 111, 117, 123, and 129, or a variant sequence thereof, wherein only one or two amino acids are different, or having about or at least 70% (e.g., at least about 80%, at least about 85%, at least about 90%, at least about 95%) sequence identity; (f) the amino acid sequence of LC CDR3 described in Table A, or a sequence selected from the group consisting of SEQ ID NOs: 10, 16, 22, 28, 34, 40, 46, 52, 58, 64, 70, 76, 82, 88, 94, 100, 106, 112, 118, 124, and 130, or a variant sequence thereof, wherein only one or two amino acids are different, or having about or at least 70% (e.g., at least about 80%, at least about 85%, at least about 90%, at least about 95%) sequence identity; or (g) an antigen-binding protein comprising any combination of two or more of (a)-(f) is provided. [Table 2]

[0073] In various embodiments, 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 described in Table A and at least one or two of the HC CDR amino acid sequences described in Table A. In various embodiments, 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 described in Table A and at least one or two of the LC CDR amino acid sequences described in Table A.

[0074] In various embodiments, the antigen-binding protein comprises at least 3, 4, or 5 of the amino acid sequences designated by the SEQ ID NOs in a single row of Table A. In various embodiments, the antigen-binding protein comprises each of the LC CDR amino acid sequences designated by the SEQ ID NOs in a single row of Table A and at least 1 or 2 of the HC CDR amino acid sequences designated by the SEQ ID NOs within the single row of Table A. In various embodiments, the antigen-binding protein comprises each of the HC CDR amino acid sequences designated by the SEQ ID NOs in a single row of Table A and at least 1 or 2 of the LC CDR amino acid sequences designated by the SEQ ID NOs in a single row of Table A. In various embodiments, the antigen-binding protein comprises all 6 of the CDR amino acid sequences designated by the SEQ ID NOs in a single row of Table A. In various embodiments, the antigen-binding protein comprises 6 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: 116-121, (r) SEQ ID NOs: 8-13, (s) SEQ ID NOs: 68-73, (t) SEQ ID NOs: 14-19, and (u) SEQ ID NOs: 20-25.

[0075] In various cases, the amino acid sequences in Table A are separated by at least one or more (e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) intervening amino acid(s). In various cases, there are about 10 to about 20 amino acids between the sequences of LC CDR1 and LC CDR2, and about 25 to about 40 amino acids between the sequences of LC CDR2 and LC CDR3. In various cases, there are about 14 to about 16 amino acids between the sequences of LC CDR1 and LC CDR2, and about 30 to about 35 amino acids between the sequences of LC CDR2 and LC CDR3. In various cases, there are about 10 to about 20 amino acids between the sequences of HC CDR1 and HC CDR2, and about 25 to about 40 amino acids between the sequences of HC CDR2 and HC CDR3. In various cases, there are about 14 to about 16 amino acids between the sequences of HC CDR1 and HC CDR2, and about 30 to about 35 amino acids between the sequences of HC CDR2 and HC CDR3.

[0076] In various embodiments, the antigen-binding protein comprises (a) a heavy-chain variable region amino acid sequence set forth in Table B, or a sequence selected from the group consisting of SEQ ID NOs: 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, and 175, or a variant sequence thereof, wherein only one or two amino acids are different, or having about or at least 70% (e.g., at least about 80%, at least about 85%, at least about 90%, at least about 95%) sequence identity, or (b) a light-chain variable region amino acid sequence set forth in Table B, or a sequence selected from the group consisting of SEQ ID NOs: 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, and 176, or a variant sequence thereof, wherein only one or two amino acids are different, or having about or at least 70% (e.g., at least about 80%, at least about 85%, at least about 90%, at least about 95%) sequence identity, or (c) both (a) and (b).

Table 3

[0077] 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.

[0078] In various embodiments, the antigen-binding protein does not include a pair of amino acid sequences encoded by the sequences of SEQ ID NOs: 179 and 180. In various embodiments, the antigen-binding protein does not include a pair of amino acid sequences of SEQ ID NOs: 181 and 182. In various embodiments, the antigen-binding protein does not include a pair of amino acid sequences encoded by the sequences of SEQ ID NOs: 183 and 184. In various embodiments, the antigen-binding protein does not include a pair of amino acid sequences of SEQ ID NOs: 185 and 186.

[0079] In various embodiments, the antigen-binding protein includes amino acid sequences that are similar to the aforementioned amino acid sequences, but the antigen-binding protein still substantially retains its biological functions, e.g., the ability to bind to human CLDN6, reduce tumor growth, and treat cancer.

[0080] In various embodiments, the antigen-binding protein includes an amino acid sequence that differs from the aforementioned amino acid sequence(s) by only 1, 2, 3, 4, 5, 6, or more amino acids. In various embodiments, the antigen-binding protein includes a variant sequence of the recited sequence, and the variant sequence differs from the recited sequence by only 1 or 2 amino acids. In various embodiments, the antigen-binding protein includes one or more amino acid substitutions that occur outside of the CDRs, e.g., one or more amino acid substitutions that occur within the framework region(s) of the heavy or light chain. In various embodiments, the antigen-binding protein includes one or more amino acid substitutions, but the antigen-binding protein still retains the amino acid sequences of the 6 CDRs. In various embodiments, the antigen-binding protein includes an amino acid sequence having only 1, 2, 3, 4, 5, 6, or more conservative amino acid substitutions compared to the aforementioned amino acid sequence(s). As used herein, the term "conservative amino acid substitution" refers to the substitution of one amino acid with another amino acid having similar properties, e.g., size, charge, hydrophobicity, hydrophilicity, and / or aromaticity, and this includes exchanges within one of the following 5 groups. TIFF0007682797000006.tif70161

[0081] In various embodiments, the conservative amino acid substitutions are exchanges within one of the following groups of amino acids. TIFF0007682797000007.tif70161

[0082] In various embodiments, the antigen-binding protein comprises an amino acid sequence having at least about 30% or higher, about 50% or higher, or about 70% or higher sequence identity to the foregoing amino acid sequence. In various embodiments, the antigen-binding protein comprises an amino acid sequence having at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, or higher than 90% sequence identity to the foregoing amino acid sequence. In various embodiments, the antigen-binding protein comprises an amino acid sequence having at least 70%, at least 80%, at least 85%, at least 90%, or higher than 90% sequence identity along the entire length of the foregoing amino acid sequence. In various embodiments, the antigen-binding protein comprises an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity along the entire length of the foregoing amino acid sequence.

[0083] In various embodiments, the antigen-binding protein comprises a variant sequence of the recited sequence, and the variant sequence has about or at least 70% sequence identity to the foregoing sequence. In various embodiments, the antigen-binding protein comprises a variant sequence of the recited sequence, and the variant sequence has about or at least 80% sequence identity to the foregoing sequence. In various embodiments, the antigen-binding protein comprises a variant sequence of the recited sequence, and the variant sequence has about or at least 90% sequence identity to the foregoing sequence. In various embodiments, the antigen-binding protein comprises a variant sequence of the recited sequence, and the variant sequence has about or at least 95% sequence identity to the foregoing sequence.

[0084] In various embodiments, the antigen-binding protein comprises one, two, three, four, or five of the sequences within a single row of Table A having SEQ ID NOs, and at least one variant sequence having about or at least 70% (e.g., at least about 80%, at least about 85%, at least about 90%, at least about 95%) sequence identity to any of SEQ ID NOs: 8-133. In various embodiments, the antigen-binding protein comprises one, two, three, four, or five of the sequences selected from the sequence sets 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: 116-121, (r) SEQ ID NOs: 8-13, (s) SEQ ID NOs: 68-73, (t) SEQ ID NOs: 14-19, and (u) SEQ ID NOs: 20-25, and such antigen-binding protein further comprises at least one variant sequence having about or at least 70% (e.g., at least about 80%, at least about 85%, at least about 90%, at least about 95%) sequence identity to at least one of the sequences of the set. For example, in various aspects, the antigen-binding protein comprises four of the sequences of SEQ ID NOs: 74-79, namely SEQ ID NOs: 74-77, where the antigen-binding protein comprises two variant sequences, one having about or at least 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 having about or at least 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.

[0085] In various embodiments, the antigen-binding protein comprises a pair of variant sequences having about or at least 70% (e.g., at least about 80%, at least about 85%, at least about 90%, at least about 95%) sequence identity to any of SEQ ID NOs: 134-175. In various cases, the antigen-binding protein comprises a pair of variant sequences having about or at least 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: 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 antigen-binding protein comprises a pair of sequences, one being a sequence in Table B and the other being a variant sequence having about or at least 70% (e.g., at least about 80%, at least about 85%, at least about 90%, at least about 95%) sequence identity to any of SEQ ID NOs: 134-175.In various embodiments, the antigen-binding protein comprises a pair of sequences, one of which is a sequence selected from (a) SEQ ID NO: 156 and 157, (b) SEQ ID NO: 148 and 149, (c) SEQ ID NO: 172 and 173, (d) SEQ ID NO: 140 and 141, (e) SEQ ID NO: 174 and 175, (f) SEQ ID NO: 144 and 145, (g) SEQ ID NO: 152 and 153, (h) SEQ ID NO: 158 and 159, (i) SEQ ID NO: 146 and 147, (j) SEQ ID NO: 160 and 161, (k) SEQ ID NO: 166 and 167, (l) SEQ ID NO: 150 and 151, (m) SEQ ID NO: 142 and 143, (n) SEQ ID NO: 168 and 169, (o) SEQ ID NO: 164 and 165, (p) SEQ ID NO: 162 and 163, (q) SEQ ID NO: 170 and 171, (r) SEQ ID NO: 134 and 135, (s) SEQ ID NO: 154 and 155, (t) SEQ ID NO: 136 and 137, and (u) SEQ ID NO: 138 and 139, and the other sequence is a variant sequence having about or at least 70% (e.g., at least about 80%, at least about 85%, at least about 90%, at least about 95%) sequence identity to the sequences of (a)-(u). For example, in various aspects, the antigen-binding protein comprises the sequence of SEQ ID NO: 134, and such antigen-binding protein further comprises a variant sequence having about or at least 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.

[0086] In various cases, the antigen-binding protein comprises the amino acid sequence of the aforementioned amino acid sequence and has one or more amino acid substitutions to reduce or remove reactive amino acids so as to reduce or prevent undesirable side chain reactions. For example, the antigen-binding protein comprises the amino acid sequence of the aforementioned amino acid sequence and has one or more of the following substitutions: (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 embodiments, the antigen-binding protein comprises the amino acid sequence of the aforementioned amino acid sequence and has amino acid substitutions that are predicted to have higher binding affinity, higher stability, or other beneficial properties based on SHM events or statistical analysis of a large number of other similar antibody sequences. In some embodiments, the antigen-binding protein is (a) the HC CDR1 amino acid sequence set forth in Table A1, or a sequence selected from the group consisting of SEQ ID NOs: 452, 455, 461, 465, 71, and 472, or a variant sequence thereof, wherein only one or two amino acids are different, or having about or at least 70% (e.g., at least about 80%, at least about 85%, at least about 90%, at least about 95%) sequence identity; (b) the HC CDR2 amino acid sequence set forth in Table A1, or a sequence selected from the group consisting of SEQ ID NOs: 475, 456, 462, 466, 468, and 473, or a variant sequence thereof, wherein only one or two amino acids are different, or having about or at least 70% (e.g., at least about 80%, at least about 85%, at least about 90%, at least about 95%) sequence identity; (c) the HC set forth in Table A1The CDR3 amino acid sequence, or a sequence selected from the group consisting of SEQ ID NOs: 453, 457, 463, 467, 469, and 474, or a variant sequence thereof, wherein only one or two amino acids are different, or having about or at least 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 sequence set forth in Table A1, or a sequence selected from the group consisting of SEQ ID NOs: 449, 476, 458, 464, 68, and 470, or a variant sequence thereof, wherein only one or two amino acids are different, or having about or at least 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 sequence set forth in Table A1, or a sequence selected from the group consisting of SEQ ID NOs: 450, 477, 459, 57, 69, and 471, or a variant sequence thereof, wherein only one or two amino acids are different, or having about or at least 70% (e.g., at least about 80%, at least about 85%, at least about 90%, at least about 95%) sequence identity; (f) the LC CDR3 amino acid sequence set forth in Table A1, or a sequence selected from the group consisting of SEQ ID NOs: 451, 454, 460, 58, 70, and 112, or a variant sequence thereof, wherein only one or two amino acids are different, or having about or at least 70% (e.g., at least about 80%, at least about 85%, at least about 90%, at least about 95%) sequence identity; or (g) any combination of two or more of (a) to (f).

Table 4

[0087] In some embodiments, HC CDR1 contains Gly immediately adjacent to the N-terminus of SEQ ID NO: 452, and optionally, in some embodiments, HC CDR1 contains MX immediately adjacent to the C-terminus of SEQ ID NO: 452, where X is H, N, or S. In various embodiments, HC CDR3 contains Ala immediately adjacent to the N-terminus of SEQ ID NO: 453. In various embodiments, LC CDR1 further contains TAS immediately adjacent to the N-terminus of SEQ ID NO: 449, and optionally contains XH immediately adjacent to the C-terminus of SEQ ID NO: 449, where X is H, S, Y, or Q. In some embodiments, as described below, the first amino acid of SEQ ID NO: 449 is S or Q. In some embodiments, as described below, the first amino acid of SEQ ID NO: 451 is S or Q.

[0088] In various embodiments, HC CDR1 contains Gly immediately adjacent to the N-terminus of SEQ ID NO: 455, and optionally, in various embodiments, HC CDR1 contains MX immediately adjacent to the C-terminus of SEQ ID NO: 455, where X is N, S, or H. In some embodiments, HC CDR2 contains Gln immediately adjacent to the N-terminus of SEQ ID NO: 456, and optionally contains H immediately adjacent to the C-terminus of SEQ ID NO: 456. In various embodiments, LC CDR1 contains RIS immediately adjacent to the N-terminus of SEQ ID NO: 476, and optionally contains LA immediately adjacent to the C-terminus of SEQ ID NO: 476. In various embodiments, LC CDR2 contains XLVE immediately adjacent to the C-terminus of SEQ ID NO: 477, where X is I or S.

[0089] In various embodiments, HC CDR1 contains MH immediately adjacent to the C-terminus of SEQ ID NO: 461. In various embodiments, HC CDR2 contains Tyr immediately adjacent to the N-terminus of SEQ ID NO: 462, and optionally contains TH immediately adjacent to the C-terminus of SEQ ID NO: 462. In an exemplary embodiment, HC CDR3 does not contain the first two amino acids of SEQ ID NO: 463. In various embodiments, LC CDR1 contains RSS immediately adjacent to the N-terminus of SEQ ID NO: 458, and optionally contains LN immediately adjacent to the C-terminus of SEQ ID NO: 458. In various embodiments, LC CDR2 contains XRFS immediately adjacent to the C-terminus of SEQ ID NO: 459, where X is Q, S, A, or D.

[0090] In various embodiments, HC CDR1 includes MH immediately adjacent to the C-terminus of SEQ ID NO: 465. In various embodiments, HC CDR2 includes YI immediately adjacent to the N-terminus of SEQ ID NO: 466 and, optionally, Xaa immediately adjacent to the C-terminus of SEQ ID NO: 466, where Xaa is N, S, Q, or A. In various embodiments, LC CDR1 includes LAS 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 embodiments, LC CDR2 includes SLAD immediately adjacent to the C-terminus of SEQ ID NO: 57.

[0091] In various embodiments, HC CDR1 includes MH immediately adjacent to the C-terminus of SEQ ID NO: 71. In various embodiments, HC CDR2 includes Tyr immediately adjacent to the N-terminus of SEQ ID NO: 468 and, optionally, IY immediately adjacent to the C-terminus of SEQ ID NO: 468. In various embodiments, LC CDR1 includes RAS immediately adjacent to the N-terminus of SEQ ID NO: 68 and, optionally, SYIH immediately adjacent to the C-terminus of SEQ ID NO: 68. In various embodiments, LC CDR2 includes XLES immediately adjacent to the C-terminus of SEQ ID NO: 69, where X is N, Q, S, A, or D.

[0092] In various embodiments, LC CDR1 includes KSS immediately adjacent to the N-terminus of SEQ ID NO: 470 and, optionally, YLA immediately adjacent to the C-terminus of SEQ ID NO: 470. In various embodiments, LC CDR2 includes TRES immediately adjacent to the C-terminus of SEQ ID NO: 471. In various embodiments, HC CDR1 includes MN immediately adjacent to the C-terminus of SEQ ID NO: 472. In various embodiments, HC CDR2 includes Xaa immediately adjacent to the N-terminus of SEQ ID NO: 473, where Xaa is N, Q, S, or A, and, optionally, Thr immediately adjacent to the C-terminus of SEQ ID NO: 473.

[0093] In various embodiments, the antigen-binding protein comprises the LC CDR1 amino acid sequence, the LC CDR2 amino acid sequence, and the LC CDR3 amino acid sequence set forth in Table A1, and at least one or two of the HC CDR amino acid sequences set forth in Table A1. In various embodiments, the antigen-binding protein comprises the HC CDR1 amino acid sequence, the HC CDR2 amino acid sequence, and the HC CDR3 amino acid sequence set forth in Table A1, and at least one or two of the LC CDR amino acid sequences set forth in Table A1.

[0094] In various embodiments, the antigen-binding protein comprises at least 3, 4, or 5 of the amino acid sequences designated by the SEQ ID NOs in a row across Table A1. In various embodiments, the antigen-binding protein comprises each of the LC CDR amino acid sequences designated by the SEQ ID NOs in a row across Table A1 and at least one or two of the HC CDR amino acid sequences designated by the SEQ ID NOs within the same row across Table A1. In various embodiments, the antigen-binding protein comprises each of the HC CDR amino acid sequences designated by the SEQ ID NOs in a row across Table A1 and at least one or two of the LC CDR amino acid sequences designated by the SEQ ID NOs in the same row across Table A1. In various embodiments, the antigen-binding protein comprises all 6 of the CDR amino acid sequences designated by the SEQ ID NOs in a row across Table A1. In various embodiments, the antigen-binding protein comprises 6 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 NO: 112, and 470-474.

[0095] In various cases, the amino acid sequences in Table A1 are separated by at least one or more (e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) intervening amino acid(s). In various cases, there are about 10 to about 20 amino acids between the sequences of LC CDR1 and LC CDR2, and about 25 to about 40 amino acids between the sequences of LC CDR2 and LC CDR3. In various cases, there are about 14 to about 16 amino acids between the sequences of LC CDR1 and LC CDR2, and about 30 to about 35 amino acids between the sequences of LC CDR2 and LC CDR3. In various cases, there are about 10 to about 20 amino acids between the sequences of HC CDR1 and HC CDR2, and about 25 to about 40 amino acids between the sequences of HC CDR2 and HC CDR3. In various cases, there are about 14 to about 16 amino acids between the sequences of HC CDR1 and HC CDR2, and about 30 to about 35 amino acids between the sequences of HC CDR2 and HC CDR3.

[0096] In various embodiments, the antigen-binding protein comprises (a) a heavy chain variable region amino acid sequence as set forth in Table B1, or a sequence selected from the group consisting of SEQ ID NOs: 478, 480, 482, 484, 486, and 488, or a variant sequence thereof that differs by only one or two amino acids or has about or at least 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 as set forth in Table B1, or a sequence selected from the group consisting of SEQ ID NOs: 479, 481, 483, 485, 487, and 489, or a variant sequence thereof that differs by only one or two amino acids or has about or at least 70% (e.g., at least about 80%, at least about 85%, at least about 90%, at least about 95%) sequence identity, or (c) both (a) and (b).

Table 5

[0097] In various embodiments, the antigen-binding protein comprises a pair of amino acid sequences selected from the group consisting of (a) SEQ ID NO: 478 and 479, (b) SEQ ID NO: 480 and 481, (c) SEQ ID NO: 482 and 483, (d) SEQ ID NO: 484 and 485, (e) SEQ ID NO: 486 and 487, and (f) SEQ ID NO: 488 and 489. In various aspects, the antigen-binding protein comprises a variant sequence of a sequence having a SEQ ID NO: described in Table B1, which differs by only one or two amino acids or has about or at least 70% (e.g., at least about 80%, at least about 85%, at least about 90%, at least about 95%) sequence identity, wherein the differing amino acid(s) are present at the positions described in "humanized antibody" below.

[0098] Humanized Antibody

[0099] In various aspects, the antigen-binding protein is a humanized version of the antigen-binding protein described in Table A, Table A1, Table B, or Table B1.

[0100] Humanized AB1

[0101] In various embodiments, the antigen-binding protein is a humanized version of AB1 as set forth in Table B or B1 and 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, or 35) amino acid substitutions at one or more of the following positions within the heavy chain variable region: 5, 8, 11, 12, 13, 20, 31, 33, 35, 38, 40, 48, 50, 55, 57, 59, 61, 65, 66, 67, 68, 70, 72, 74, 76, 79, 80, 82, 87, 90, 91, 98, 101, and 116. In various cases, the antigen-binding protein comprises the amino acid sequence of SEQ ID NO: 428. In various embodiments, the antigen-binding protein is a humanized version of AB1 as set forth in Table B or B1 and has one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12) amino acid substitutions at one or more of the following positions within 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 embodiments, the amino acids at the positions listed above are selected from the amino acids according to the following table.

Table 6

[0102] In various embodiments, the antigen-binding protein is a humanized version of AB1 as set forth in Table B or B1 and has one or more amino acid substitutions at one or more of the following positions within the light chain variable region: 1, 3, 4, 9, 10, 11, 15, 17, 21, 24, 27, 29, 32, 34, 35, 43, 44, 48, 51, 52, 53, 54, 55, 56, 61, 67, 71, 72, 73, 79, 80, 81, 84, 90, 92, 93, 94, 95, 96, 101, 107, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, or 41). In various cases, the antigen-binding protein comprises the amino acid sequence of SEQ ID NO: 430. In various embodiments, the antigen-binding protein is a humanized version of AB1 as set forth in Table B or B1 and has one or more amino acid substitutions at one or more of the following positions within the light chain variable region: 4, 21, 32, 34, 48, 51, 53, 61, 67, 79, 84, 91, and 93, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13). In various cases, the antigen-binding protein comprises the amino acid sequence of SEQ ID NO: 431. In various embodiments, the amino acids at the positions listed above are selected from the amino acids according to the following table.

Table 7

[0103] Humanized AB3

[0104] In various embodiments, the antigen-binding protein is a humanized version of AB3 as set forth in Table B or B1 and has one or more amino acid substitutions at one or more of the following positions within the heavy chain variable region: 3, 5, 18, 19, 23, 31, 33, 35, 40, 42, 49, 50, 52, 53, 54, 55, 56, 57, 58, 59, 61, 64, 76, 79, 80, 81, 87, 94, 95, 99, 106, 112, 114 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33). In various cases, the antigen-binding protein comprises the amino acid sequence of SEQ ID NO: 432. In various embodiments, the antigen-binding protein is a humanized version of AB3 as set forth in Table B or B1 and has one or more amino acid substitutions at one or more of the following positions within the heavy chain variable region: 31, 35, 50, 55, 79, 99, 106 (e.g., one, two, three, four, five, six, or seven). In various cases, the antigen-binding protein comprises the amino acid sequence of SEQ ID NO: 433. In various embodiments, the amino acids at the positions listed above are selected from the amino acids according to the following table.

[0105]

Table 8

[0106] In various embodiments, the antigen-binding protein is a humanized version of AB3 as set forth in Table B or B1 and has one or more amino acid substitutions at one or more of the following positions within the light chain variable region: 9, 17, 18, 25, 27, 28, 30, 34, 40, 43, 45, 48, 50, 52, 53, 55, 56, 70, 72, 74, 76, 84, 85, 90, 91, 93, 94, 97, and 100 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29). In various cases, the antigen-binding protein comprises the amino acid sequence of SEQ ID NO: 434. In various embodiments, the antigen-binding protein is a humanized version of AB3 as set forth in Table B or B1 and has one or more amino acid substitutions at one or more of the following positions within the light chain variable region: 25, 34, 48, 53, 55, 84, 85, 90, and 93 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, or 9). In various cases, the antigen-binding protein comprises the amino acid sequence of SEQ ID NO: 435. In various embodiments, the amino acids at the positions listed above are selected from the amino acids according to the following table.

Table 9

[0107] Humanized AB4

[0108] In various embodiments, the antigen-binding protein is a humanized version of AB4 as set forth in Table B or B1 and has one or more amino acid substitutions at one or more of the following positions within the heavy chain variable region: 5, 11, 12, 13, 20, 29, 31, 33, 37, 38, 40, 45, 48, 50, 55, 56, 57, 59, 61, 62, 65, 66, 67, 68, 70, 72, 74, 76, 79, 82, 84, 87, 91, 97, 101, 117 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36). In various cases, the antigen-binding protein comprises the amino acid sequence of SEQ ID NO: 436. In various embodiments, the antigen-binding protein is a humanized version of AB4 as set forth in Table B or B1 and has one or more amino acid substitutions at one or more of the following positions within the heavy chain variable region: 20, 29, 31, 37, 45, 48, 56, 59, 61, 62, 65, 66, 68, 70, 74, 79, 84, 97, and 101 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19). In various cases, the antigen-binding protein comprises the amino acid sequence of SEQ ID NO: 437. In various embodiments, the amino acids at the positions listed above are selected from the amino acids according to the following table.

Table 10

[0109] In various embodiments, the antigen-binding protein is a humanized version of AB4 as set forth in Table B or B1 and has one or more amino acid substitutions at one or more of the following positions within the light chain variable region: 7, 14, 17, 18, 31, 33, 39, 41, 42, 44, 50, 51, 55, 57, 60, 81, 88, 92, 94, 95, 96, 99, 100, 105 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24). In various cases, the antigen-binding protein comprises the amino acid sequence of SEQ ID NO: 438. In various embodiments, the antigen-binding protein is a humanized version of AB4 as set forth in Table B or B1 and has one or more amino acid substitutions at one or more of the following positions within the light chain variable region: 33, 39, 55, 57, 81, 95, and 96 (e.g., 1, 2, 3, 4, 5, 6, or 7). In various cases, the antigen-binding protein comprises the amino acid sequence of SEQ ID NO: 439. In various embodiments, the amino acids at the positions listed above are selected from the amino acids according to the following table.

Table 11

[0110] Humanized AB18

[0111] In various embodiments, the antigen-binding protein is a humanized version of AB18 as set forth in Table B or B1 and has 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 within the heavy-chain variable region: 5, 9, 11, 12, 20, 38, 40, 41, 43, 44, 48, 61, 65, 67, 68, 70, 72, 74, 76, 79, 82, 84, 87, 91, and 116, and optionally has one or more amino acid substitutions at one or more (e.g., 1, 2, 3, 4, or 5) of the following positions: 20, 48, 68, 70, 79. In various cases, the antigen-binding protein comprises the amino acid sequence of SEQ ID NO: 440 or 441. In various embodiments, the amino acids at the positions listed above are selected from the amino acids according to the following table. [Table 12]

[0112] In various embodiments, the antigen-binding protein is a humanized version of AB18 as set forth in Table B or B1 and has 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 within the light-chain variable region: 1, 3, 9, 15, 18, 19, 21, 22, 49, 51, 69, 93, 84, 78, 105, and 111, and optionally has one or more amino acid substitutions at 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 embodiments, the amino acids at the positions listed above are selected from the amino acids according to the following table.

[0113] [Table 13]

[0114] Humanized AB9

[0115] In various embodiments, the antigen-binding protein is a humanized version of AB9 as described in Table B or B1 and has one or more amino acid substitutions at one or more of the following positions within the heavy-chain variable region: 1, 5, 9, 11, 12, 20, 38, 40, 41, 43, 44, 48, 61, 63, 65, 67, 69, 70, 72, 73, 74, 76, 79, 84, 87, 91, 93, 112, and 113 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29). In various cases, the antigen-binding protein comprises the amino acid sequence of SEQ ID NO: 444. In various embodiments, the amino acids at the positions listed above are selected from the amino acids according to the following table. [Table 14]

[0116] In various embodiments, the antigen-binding protein is a humanized version of AB9 as described in Table B or B1 and has one or more amino acid substitutions at one or more of the following positions within the light-chain variable region: 9, 11, 15, 17, 18, 43, 45, 70, 72, 73, 74, 80, 84, 85, and 100 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15). In various cases, the antigen-binding protein comprises the amino acid sequence of SEQ ID NO: 445. In various embodiments, the amino acids at the positions listed above are selected from the amino acids according to the following table. [Table 15]

[0117] Humanized AB11

[0118] In various embodiments, the antigen-binding protein is a humanized version of AB11 as set forth in Table B or B1, and has one or more amino acid substitutions at one or more of the following positions within the heavy chain variable region: 1, 15, 18, 19, 42, 49, 63, 75, 76, 78, 80, 84, 88, and 93 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15). In various cases, the antigen-binding protein comprises the amino acid sequence of SEQ ID NO: 446. In various embodiments, the amino acids at the positions listed above are selected from the amino acids according to the following table.

Table 16

[0119] In various embodiments, the antigen-binding protein is a humanized version of AB11 as set forth in Table B or B1, and has one or more amino acid substitutions at one or more of the following positions within the light chain variable region: 4, 9, 17, 22, 64, 78, 80, 81, 82, 83, 84, 87, 89, 104, and 110 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15), optionally, at one or more of the following positions: 4, 82, 110. In various cases, the antigen-binding protein comprises the amino acid sequence of SEQ ID NO: 447 or 448. In various embodiments, the amino acids at the positions listed above are selected from the amino acids according to the following table.

Table 17

[0120] In various embodiments, the antigen-binding protein comprises (a) the heavy-chain variable region amino acid sequence set forth 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 variant sequence thereof, wherein only one or two amino acids are different, or having a sequence identity of about or at least 70%, or about 80%, or about 85%, or about 90%, or about 95%, or (b) the light-chain variable region amino acid sequence set forth 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 a variant sequence thereof, wherein only one or two amino acids are different, or having a sequence identity of about or at least 70%, or about 80%, or about 85%, or about 90%, or about 95%, or (c) both (a) and (b).

[0121]

Table 18

[0122] In various embodiments, the humanized antigen-binding protein comprises a pair of amino acid sequences shown in Table D.

Table 19

[0123] In various embodiments, the antigen-binding protein comprises a pair of variant sequences, each having a sequence identity of about or at least 70% (e.g., at least about 80%, at least about 85%, at least about 90%, at least about 95%) to the sequence numbers listed in Table C. In various embodiments, the antigen-binding protein comprises a pair of sequences, one being a sequence selected from the sequence numbers listed in Table C, and the other sequence being a sequence having a sequence number listed in Table D, or a variant sequence having a sequence identity of about or at least 70% (e.g., at least about 80%, at least about 85%, at least about 90%, at least about 95%) to the sequence having a sequence number listed in Table C.

[0124] In various embodiments, the antigen-binding protein comprises a pair of sequences, one being a sequence selected from the sequence numbers listed in Table D, and the other sequence being a variant sequence having a sequence identity of about or at least 70% (e.g., at least about 80%, at least about 85%, at least about 90%, at least about 95%) to the sequence having a sequence number listed in Table D. For example, in various aspects, the antigen-binding protein comprises the sequence of SEQ ID NO: 419, and such antigen-binding protein further comprises a variant sequence having a sequence identity of about or at least 70% (e.g., at least about 80%, at least about 85%, at least about 90%, at least about 95%) to SEQ ID NO: 421.

[0125] In various cases, the antigen-binding protein is a humanized antigen-binding protein having one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35) amino acid substitutions within the heavy chain (HC) variable region or the light chain (LC) variable region, or both, as described in Table D. In an exemplary embodiment, the antigen-binding protein is a humanized antigen-binding protein, AB1-11, having one or more amino acid substitutions in the HC variable region, the LC variable region, or both. In an exemplary embodiment, the antigen-binding protein comprises the HC of SEQ ID NO: 379 and has one, two, three, four, or five amino acid substitutions. In an exemplary embodiment, the antigen-binding protein comprises the HC CDR1 of SEQ ID NO: 504, the HC CDR2 of SEQ ID NO: 505, the HC CDR3 of SEQ ID NO: 506, or a combination thereof. In an exemplary case, the antigen-binding protein comprises the HC of SEQ ID NO: 503. In some embodiments, the antigen-binding protein comprises any one of the HCs of SEQ ID NOs: 496 - 501. In some embodiments, the antigen-binding protein comprises the HC sequence shown as S7 - S12 in Figure 22. In various cases, the light chain variable region comprises the LC CDR1 of SEQ ID NO: 449, the LC CDR2 of SEQ ID NO: 450, the LC CDR3 of SEQ ID NO: 451, or a combination thereof. In some embodiments, the antigen-binding protein comprises any one of the LCs of SEQ ID NOs: 380 - 383 and 479. In an exemplary case, the antigen-binding protein comprises the LC of SEQ ID NO: 383. In some embodiments, the antigen-binding protein comprises the LC sequence shown as S7 - S12 in Figure 22. In an exemplary embodiment, the antigen-binding protein is a humanized antigen-binding protein, AB3-7, having one or more amino acid substitutions in the HC variable region, the LC variable region, or both. In an exemplary embodiment, the antigen-binding protein comprises the HC of SEQ ID NO: 387 and has one, two, three, four, five, or six amino acid substitutions. In an exemplary embodiment, the antigen-binding protein comprises the HC CDR1 of SEQ ID NO: 507, the HC CDR2 of SEQ ID NO: 508, the HC CDR3 of SEQ ID NO: 509, or a combination thereof.In an exemplary case, the antigen-binding protein comprises the HC of SEQ ID NO: 502. In some embodiments, the antigen-binding protein comprises any one of the HCs of SEQ ID NOs: 490-495. In some embodiments, the antigen-binding protein comprises the HC sequences designated as S1-S6 in FIG. 22. In various cases, the light chain variable region comprises the LC CDR1 of SEQ ID NO: 476, the LC CDR2 of SEQ ID NO: 477, the LC CDR3 of SEQ ID NO: 454, or a combination thereof. In some embodiments, the antigen-binding protein comprises any one of the LCs of SEQ ID NOs: 388-390 and 481. In an exemplary case, the antigen-binding protein comprises the LC of SEQ ID NO: 389. In some embodiments, the antigen-binding protein comprises the LC sequences designated as S1-S6 in FIG. 22. In an exemplary embodiment, the antigen-binding protein is a humanized antigen-binding protein of AB3 and has one or more amino acid substitutions in the HC variable region, the LC variable region, or both. In an exemplary embodiment, the antigen-binding protein comprises the HC of SEQ ID NO: 139 and has one, two, three, four, or five (or more) amino acid substitutions. In some embodiments, the antigen-binding protein comprises any one of the HCs of SEQ ID NO: 510. In some embodiments, the antigen-binding protein comprises the HC sequence of SEQ ID NO: 510 and has one, two, three, four, or five (or more) amino acid substitutions as shown in FIG. 23. In an exemplary embodiment, the antigen-binding protein comprises the HC of SEQ ID NO: 138 and has one, two, three, four, or five (or more) amino acid substitutions. In some embodiments, the antigen-binding protein comprises any one of the HCs of SEQ ID NO: 511. In some embodiments, the antigen-binding protein comprises the HC sequence of SEQ ID NO: 511 and has one, two, three, four, or five (or more) amino acid substitutions as shown in FIG. 24. In an exemplary embodiment, the antigen-binding protein is a humanized antigen-binding protein of AB1 and has one or more amino acid substitutions in the HC variable region, the LC variable region, or both. In an exemplary embodiment, the antigen-binding protein comprises the HC of SEQ ID NO: 135 and has one, two, three, four, or five (or more) amino acid substitutions. In some embodiments, the antigen-binding protein comprises any one of the HCs of SEQ ID NO: 513.In some embodiments, the antigen-binding protein comprises the HC sequence of SEQ ID NO: 513 and has one, two, three, four, or five (or more) amino acid substitutions as shown in FIG. 25. In an exemplary embodiment, the antigen-binding protein comprises the HC of SEQ ID NO: 134 and has one, two, three, four, or five (or more) amino acid substitutions. In some embodiments, the antigen-binding protein comprises any one of the HCs of SEQ ID NO: 512. In some embodiments, the antigen-binding protein comprises the HC sequence of SEQ ID NO: 512 and has one, two, three, four, or five (or more) amino acid substitutions as shown in FIG. 26.

[0126] Defucosylated Antibody

[0127] Many secreted proteins undergo post-translational glycosylation, a process in which sugar moieties (e.g., glycans, sugars) are covalently attached to specific amino acids of the protein. In eukaryotic cells, two types of glycosylation reactions occur: (1) N-linked glycosylation, in which a glycan is attached to asparagine in the recognition sequence Asn-X-Thr / Ser where "X" is any amino acid other than proline, and (2) O-linked glycosylation, in which a glycan is attached to serine or threonine. Regardless of the type of glycosylation (N-linked [glycosylation] or O-linked [glycosylation]), there is microheterogeneity of the protein's glycoforms due to the large range of glycan structures that associate with each site (O or N).

[0128] All N-glycans have a common core sugar sequence: Manα1-6(Manα1-3)Manβ1-4GlcNAcβ1-4GlcNAcβ1-Asn-X-Ser / Thr(Man 3 GlcNAc 2It has Asn and is classified into one of three types, namely, (A) a high-mannose (HM) or oligomannose (OM) type consisting of two N-acetylglucosamine (GalNAc) moieties and a large number (e.g., 5, 6, 7, 8, or 9) of mannose (Man) residues, (B) a complex type containing more than two GlcNAc moieties and any number of other types of sugars, or (C) a hybrid type containing Man residues in one branch and GlcNAc at the base of the complex branch. Figure 1A (obtained from Stanley et al., Chapter 8: N-Glycans, Essentials of Glycobiology, 2 nd nd ed., Cold Spring Harbor Laboratory Press; 2009) shows the three types of N-glycans.

[0129] N-linked glycans typically contain one or more monosaccharides such as galactose (Gal), N-acetylgalactosamine (GalNAc), galactosamine (GalN), glucose (GLc), N-acetylglucosamine (ClcNAc), glucosamine (GlcN), mannose (Man), N-acetylmannosamine (ManNAc), mannosamine (ManN), xylose (Xyl), N-acetylneuraminic acid (Neu5Ac), N-glycolylneuraminic acid (Neu5Gc), 2-keto-3-doxynononic acid (Kdn), fucose (Fuc), glucuronic acid (GLcA), iduronic acid (IdoA), galacturonic acid (Gal A), mannuronic acid (Man A). The symbols commonly used for such sugars are shown in Figure 29A.

[0130] N-linked glycosylation begins in the endoplasmic reticulum (ER) and through a complex series of reactions results in the attachment of a core glycan structure consisting essentially of two GlcNAc residues and three Man residues. The glycan complex formed in the ER is modified by the action of enzymes within the Golgi apparatus. If the sugar is relatively inaccessible to the enzyme, the sugar typically remains in its original HM form. If the enzyme can access the sugar, many of the Man residues are cleaved and the sugar is further modified to yield a complex-type N-glycan structure. For example, mannosidase-1 located in the cis Golgi can cleave or hydrolyze the HM glycan, and fucosyltransferase FUT-8 located in the medial Golgi fucosylates the glycan (Hanrue Imai-Nishiya (2007), BMC Biotechnology, 7:84).

[0131] Therefore, the sugar composition and structural configuration of the glycan structure vary depending, among other factors, particularly on the glycosylation machinery in the ER and the Golgi apparatus, the accessibility of the enzymes of the machinery to the glycan structure, the order of action of each enzyme, and the stage at which the protein is released from the glycosylation machinery.

[0132] In exemplary embodiments of the present disclosure, the antigen-binding protein comprises an Fc polypeptide. As used herein, the term "Fc polypeptide" includes polypeptides in native and mutant protein forms derived from the Fc region of an antibody. In an exemplary aspect, the Fc polypeptide of the antigen-binding protein disclosed herein contains a glycan. In various cases, the glycan lacks fucose or is defucosylated. In an exemplary aspect, the antigen-binding protein contains a defucosylated glycan. As used herein, the term "defucosylated glycan" or "afuco glycan" or "defucosylated glycoform" or "Afuc" refers to a glycoform lacking a core fucose, e.g., an α1,6-linked fucose on a GlcNAc residue involved in an amide bond with Asn of an N-glycosylation site. Defucosylated glycoforms include, but are not limited to, A1G0, A2G0, A2G1a, A2G1b, A2G2, and A1G1M5. Additional defucosylated glycans include, for example, A1G1a, G0[H3N4], G0[H4N4], G0[H5N4], FO-N[H3N3]. See, for example, Reusch and Tejada, Glycobiology 25(12):1325-1334(2015).

[0133] The present disclosure also provides a composition comprising an antigen-binding protein comprising an Fc polypeptide comprising a defucosylated glycan, e.g., a pharmaceutical composition. In an exemplary embodiment, about or at least 25% of the antigen-binding proteins present in the composition are antigen-binding proteins comprising an Fc polypeptide comprising a defucosylated glycan. In an exemplary embodiment, about or at least 25% of the antigen-binding proteins present in the composition are defucosylated. Optionally, at least 30%, 40%, 50%, 60%, 70%, 80%, or 90% or more of the antigen-binding proteins present in the composition are defucosylated. Methods for producing compositions comprising antigen-binding proteins with a specific glycoprofile are known in the art. In an exemplary embodiment, the antigen-binding protein is recombinant and is produced in cells genetically engineered to alter the activity of an enzyme in the de novo or salvage pathway. These two fucose metabolic pathways are shown in Figure 29B. In an exemplary embodiment, the cells are genetically engineered to alter the activity of any one or more of fucosyltransferase (FUT, e.g., FUT1, FUT2, FUT3, FUT4, FUT5, FUT6, FUT7, FUT8, FUT9), fucose kinase, GDP-fucose pyrophosphorylase, GDP-D-mannose-4,6-dehydratase (GMD), and GDP-keto-6-deoxymannose-3,5-epimerase, 4-reductase (FX). In an exemplary embodiment, the cells are genetically engineered to knockout the gene encoding FX. See, e.g., International Patent Publication No. WO2017 / 079165A1, Kanda et al., J Biotechnol 130, 2007, 300-310, Yamane-Ohunuki et al., Biotechnol Bioeng 87, 2004, 614-622, Malphettes et al., Biotechnol Bioeng 106, 2010, 774-783.

[0134] Nucleic Acid

[0135] The present disclosure further provides a nucleic acid comprising a nucleotide sequence encoding an antigen-binding protein of the present disclosure. As used herein, "nucleic acid" includes "polynucleotide", "oligonucleotide", and "nucleic acid molecule", and generally means a polymer of DNA or RNA, or a modified form thereof, which can be single-stranded or double-stranded, synthetic, or obtained from naturally occurring substances (e.g., isolated and / or purified), and can contain natural, non-natural, or modified nucleotides, and can also contain natural, non-natural, or modified internucleotide linkages, such as phosphoramidate linkages or phosphorothioate linkages, instead of the phosphodiester found between nucleotides of unmodified oligonucleotides. The nucleic acid can include any nucleotide sequence encoding any of the antigen-binding proteins of the present disclosure. In various embodiments, the nucleic acid is (a) the amino acid sequence of heavy chain (HC) complementarity determining region (CDR) 1 set forth in Table A or A1, or a sequence selected from the group consisting of SEQ ID NOs: 11, 17, 23, 29, 35, 41, 47, 53, 59, 65, 71, 77, 83, 89, 95, 101, 107, 113, 119, 125, 131, 452, 455, 461, 465, and 472, or a variant sequence thereof, wherein only one or two amino acids are different, or having about or at least 70% (e.g., about or at least 80%, about or at least 85%, about or at least 90%, about or at least 95%) sequence identity, (b) the HC CDR2 amino acid sequence set forth in Table A or A1, or a sequence selected from the group consisting of SEQ ID NOs: 12, 18, 24, 30, 36, 42, 48, 54, 60, 66, 72, 78, 84, 90, 86, 102, 108, 114, 120, 126, 132, 475, 456, 462, 466, 468, and 473, or a variant sequence thereof, wherein only one or two amino acids are different, or having about or at least 70% (e.g., about or at least 80%, about or at least 85%, about or at least 90%, about or at least 95%) sequence identity, (c) the HC CDR3 amino acid sequence set forth in Table A or A1, or a sequence selected from the group consisting of SEQ ID NOs: 13, 19, 25, 31, 37, 43, 49, 55, 61, 67,An array selected from the group consisting of 73, 79, 85, 91, 97, 103, 109, 115, 121, 127, 133, 453, 457, 463, 467, 469, and 474, or a variant array thereof, wherein only one or two amino acids are different, or having about or at least 70% (e.g., about or at least 80%, about or at least 85%, about or at least 90%, about or at least 95%) sequence identity, (d) the light chain (LC) CDR1 amino acid sequence described in Table A or A1, or a sequence selected from the group consisting of SEQ ID NOs: 8, 14, 20, 32, 38, 44, 50, 56, 62, 68, 74, 80, 86, 92, 98, 104, 110, 116, 122, 128, 449, 476, 458, 464, and 470, or a variant array thereof, wherein only one or two amino acids are different, or having about or at least 70% (e.g., about or at least 80%, about or at least 85%, about or at least 90%, about or at least 95%) sequence identity, (e) the LC CDR2 amino acid sequence described in Table A or A1, or a sequence selected from the group consisting of SEQ ID NOs: 9, 15, 21, 27, 33, 39, 45, 51, 57, 63, 69, 75, 81, 87, 93, 99, 105, 111, 117, 123, 129, 450, 477, 459, and 471, or a variant array thereof, wherein only one or two amino acids are different, or having about or at least 70% (e.g., about or at least 80%, about or at least 85%, about or at least 90%, about or at least 95%) sequence identity, (f) the amino acid sequence of LC CDR3 described in Table A, or a sequence selected from the group consisting of SEQ ID NOs: 10, 16, 22, 28, 34, 40, 46, 52, 58, 64, 70, 76, 82, 88, 94, 100, 106, 112, 118, 124, 130, 451, 454, and 460, or a variant array thereof, wherein only one or two amino acids are different, or having about or at least 70% (e.g., about or at least 80%, about or at least 85%, about or at least 90%, about or at least 95%) sequence identityAlternatively, it includes a nucleotide sequence encoding an antigen-binding protein comprising any combination of two or more of (g)(a) to (f). In various embodiments, the nucleic acid encodes an antigen-binding protein comprising the LC CDR1 amino acid sequence, LC CDR2 amino acid sequence, and LC CDR3 amino acid sequence described in Table A or A1, and at least one or two of the HC CDR amino acid sequences described in Table A or A1. In various embodiments, the nucleic acid encodes an antigen-binding protein comprising the HC CDR1 amino acid sequence, HC CDR2 amino acid sequence, and HC CDR3 amino acid sequence described in Table A or A1, and at least one or two of the LC CDR amino acid sequences described in Table A or A1. In various embodiments, the nucleic acid comprises (a) at least 3, 4, or 5 of the amino acid sequences specified by the SEQ ID NOs in a row in Table A or A1, (b) each of the LC CDR amino acid sequences specified by the SEQ ID NOs in a row in Table A or A1 and at least one or two of the HC CDR amino acid sequences specified by the SEQ ID NOs within the same row in Table A or A1, (c) each of the HC CDR amino acid sequences specified by the SEQ ID NOs in a row in Table A or A1 and at least one or two of the LC CDR amino acid sequences specified by the SEQ ID NOs in a row in Table A or A1, (d) all 6 sequences of the CDR amino acid sequences specified by the SEQ ID NOs in a row in Table A, and / or (e) (a) SEQ ID NOs 74 - 79, (b) SEQ ID NOs 50 - 55, (c) SEQ ID NOs 122 - 127, (d) SEQ ID NOs 26 - 31, (e) SEQ ID NOs 128 - 133, (f) SEQ ID NOs 38 - 43, (g) SEQ ID NOs 62 - 67, (h) SEQ ID NOs 80 - 85, (i) SEQ ID NOs 44 - 49, (j) SEQ ID NOs 86 - 91, (k) SEQ ID NOs 104 - 109, (l) SEQ ID NOs 56 - 61, (m) SEQ ID NOs 32 - 37, (n) SEQ ID NOs 110 - 115, (o) SEQ ID NOs 98 - 103, (p) SEQ ID NOs 92 - 97, (q) SEQ ID NOs 116 - 121, (r) SEQ ID NOs 8 - 13, (s) SEQ ID NOs 68 - 73, (t) SEQ ID NOs 14 - 19, (u) SEQ ID NOs 20 - 25, (v) SEQ ID NOs 449 - 453 and 475, (w) SEQ ID NOs 476 - 477, 454 - 457, (x) SEQ ID NOs 458 - 463,It comprises a nucleotide sequence encoding an antigen-binding protein comprising 6 CDR amino acid sequences selected from the group consisting of (y) SEQ ID NOs: 57, 58, 464-467, (z) SEQ ID NOs: 68-71 and 468-469, and (aa) SEQ ID NOs: 112, and 470-474. In various embodiments, the nucleic acid is (a) the heavy chain variable region amino acid sequence set forth in Table B or B1, 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 variant sequence thereof, wherein only 1 or 2 amino acids are different, or having about or at least 70% (e.g., about or at least 80%, about or at least 85%, about or at least 90%, about or at least 95%) sequence identity, or (b) the light chain variable region amino acid sequence set forth in Table B or B1, 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 a variant sequence thereof, wherein only 1 or 2 amino acids are different, or having about or at least 70% (e.g., about or at least 80%, about or at least 85%, about or at least 90%, about or at least 95%) sequence identity, or (c) both (a) and (b), and comprises a nucleotide sequence encoding an antigen-binding protein. In various embodiments, the nucleic acid is (a) SEQ ID NOs: 156 and 157, (b) SEQ ID NOs: 148 and 149, (c) SEQ ID NOs: 172 and 173, (d) SEQ ID NOs: 140 and 141, (e) SEQ ID NOs: 174 and 175, (f) SEQ ID NOs: 144 and 145, (g) SEQ ID NOs: 152 and 153, (h) SEQ ID NOs: 158 and 159, (i) SEQ ID NOs: 146 and 147, (j) SEQ ID NOs: 160 and 161, (k) SEQ ID NOs: 166 and 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) A pair of amino acid sequences selected from the group consisting of 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, encoding an antigen-binding protein. In various embodiments, the nucleic acid comprises a nucleotide sequence encoding an antigen-binding protein comprising a pair of amino acid sequences selected from the group consisting of pairs described in Table D. In various aspects, the nucleic acid comprises a nucleotide sequence comprising a sequence of any one or more of SEQ ID NOs: 208 to 375. In some embodiments, the nucleic acid does not include any insertions, deletions, inversions, and / or substitutions. In other embodiments, the nucleic acid includes one or more insertions, deletions, inversions, and / or substitutions.,

[0136] In various embodiments, the nucleic acid comprises a nucleotide sequence encoding an antigen-binding protein that is a humanized antigen-binding protein having one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35) amino acid substitutions within the heavy chain (HC) variable region and / or the light chain (LC) variable region as set forth in Table D. In an exemplary embodiment, the nucleic acid comprises a nucleotide sequence encoding an antigen-binding protein that is a humanized antigen-binding protein having one or more amino acid substitutions in the HC variable region, the LC variable region, or both, such as AB1-11. In an exemplary embodiment, the nucleic acid comprises a nucleotide sequence encoding an antigen-binding protein comprising the HC of SEQ ID NO: 379 having one, two, three, four, or five amino acid substitutions. In an exemplary embodiment, the nucleic acid comprises a nucleotide sequence encoding an antigen-binding protein comprising the HC CDR1 of SEQ ID NO: 504, the HC CDR2 of SEQ ID NO: 505, the HC CDR3 of SEQ ID NO: 506, or a combination thereof. In an exemplary case, the nucleic acid comprises a nucleotide sequence encoding an antigen-binding protein comprising the HC of SEQ ID NO: 503. In some embodiments, the nucleic acid comprises a nucleotide sequence encoding an antigen-binding protein comprising any one of the HCs of SEQ ID NOs: 496-501. In some embodiments, the nucleic acid comprises a nucleotide sequence encoding an antigen-binding protein comprising the HC sequence shown as S7-S12 in Figure 22. In various cases, the nucleic acid comprises a nucleotide sequence encoding a light chain variable region comprising the LC CDR1 of SEQ ID NO: 449, the LC CDR2 of SEQ ID NO: 450, the LC CDR3 of SEQ ID NO: 451, or a combination thereof. In some embodiments, the nucleic acid comprises a nucleotide sequence encoding an antigen-binding protein comprising any one of the LCs of SEQ ID NOs: 380-383 and 479. In an exemplary case, the nucleic acid comprises a nucleotide sequence encoding an antigen-binding protein comprising the LC of SEQ ID NO: 383. In some embodiments, the nucleic acid comprises a nucleotide sequence encoding an antigen-binding protein comprising the LC sequence shown as S7-S12 in Figure 22.In an exemplary embodiment, the nucleic acid comprises a nucleotide sequence encoding an antigen-binding protein that is a humanized antigen-binding protein, AB3-7, having one or more amino acid substitutions in the HC variable region, the LC variable region, or both. In an exemplary embodiment, the nucleic acid comprises a nucleotide sequence encoding an antigen-binding protein having 1, 2, 3, 4, 5, or 6 amino acid substitutions, comprising the HC of SEQ ID NO: 387. In an exemplary embodiment, the nucleic acid comprises a nucleotide sequence encoding an antigen-binding protein comprising the HC CDR1 of SEQ ID NO: 507, the HC CDR2 of SEQ ID NO: 508, the HC CDR3 of SEQ ID NO: 509, or a combination thereof. In an exemplary case, the nucleic acid comprises a nucleotide sequence encoding an antigen-binding protein comprising the HC of SEQ ID NO: 502. In some embodiments, the nucleic acid comprises a nucleotide sequence encoding an antigen-binding protein comprising any one of the HCs of SEQ ID NOs: 490-495. In some embodiments, the nucleic acid comprises a nucleotide sequence encoding an antigen-binding protein comprising the HC sequences designated as S1-S6 in FIG. 22. In various cases, the nucleic acid comprises a nucleotide sequence encoding a light chain variable region comprising the LC CDR1 of SEQ ID NO: 476, the LC CDR2 of SEQ ID NO: 477, the LC CDR3 of SEQ ID NO: 454, or a combination thereof. In some embodiments, the nucleic acid comprises a nucleotide sequence encoding an antigen-binding protein comprising any one of the LCs of SEQ ID NOs: 388-390, and 481. In an exemplary case, the nucleic acid comprises a nucleotide sequence encoding an antigen-binding protein comprising the LC of SEQ ID NO: 389. In some embodiments, the nucleic acid comprises a nucleotide sequence encoding an antigen-binding protein comprising the LC sequences designated as S1-S6 in FIG. 22.

[0137] In an exemplary embodiment, the nucleic acid comprises a nucleotide sequence encoding an antigen-binding protein that is a humanized antigen-binding protein of AB3 having one or more amino acid substitutions in the HC variable region, the LC variable region, or both. In an exemplary embodiment, the nucleic acid comprises a nucleotide sequence encoding an antigen-binding protein having one, two, three, four, or five (or more) amino acid substitutions and comprising the HC of SEQ ID NO: 139. In some embodiments, the nucleic acid comprises a nucleotide sequence encoding an antigen-binding protein comprising any one of the HCs of SEQ ID NO: 510. In some embodiments, the nucleic acid comprises a nucleotide sequence encoding an antigen-binding protein having one, two, three, four, or five (or more) amino acid substitutions as shown in FIG. 23 and comprising the HC sequence of SEQ ID NO: 510. In an exemplary embodiment, the nucleic acid comprises a nucleotide sequence encoding an antigen-binding protein having one, two, three, four, or five (or more) amino acid substitutions and comprising the HC of SEQ ID NO: 138. In some embodiments, the nucleic acid comprises a nucleotide sequence encoding an antigen-binding protein comprising any one of the HCs of SEQ ID NO: 511. In some embodiments, the nucleic acid comprises a nucleotide sequence encoding an antigen-binding protein having one, two, three, four, or five (or more) amino acid substitutions as shown in FIG. 24 and comprising the HC sequence of SEQ ID NO: 511. In an exemplary embodiment, the nucleic acid comprises a nucleotide sequence encoding an antigen-binding protein that is a humanized antigen-binding protein of AB1 having one or more amino acid substitutions in the HC variable region, the LC variable region, or both. In an exemplary embodiment, the nucleic acid comprises a nucleotide sequence encoding an antigen-binding protein having one, two, three, four, or five (or more) amino acid substitutions and comprising the HC of SEQ ID NO: 135. In some embodiments, the nucleic acid comprises a nucleotide sequence encoding an antigen-binding protein comprising any one of the HCs of SEQ ID NO: 513. In some embodiments, the nucleic acid comprises a nucleotide sequence encoding an antigen-binding protein having one, two, three, four, or five (or more) amino acid substitutions as shown in FIG. 25 and comprising the HC sequence of SEQ ID NO: 513.In an exemplary aspect, the nucleic acid comprises a nucleotide sequence encoding an antigen-binding protein having one, two, three, four, or five (or more) amino acid substitutions, including the HC of SEQ ID NO: 134. In some aspects, the nucleic acid comprises a nucleotide sequence encoding an antigen-binding protein comprising any one of the HCs of SEQ ID NO: 512. In some aspects, the nucleic acid comprises a nucleotide sequence encoding an antigen-binding protein having one, two, three, four, or five (or more) amino acid substitutions as shown in FIG. 26, including the HC sequence of SEQ ID NO: 512. In some embodiments, the nucleic acid does not include any insertions, deletions, inversions, and / or substitutions. In other embodiments, the nucleic acid includes one or more insertions, deletions, inversions, and / or substitutions.

[0138] In some aspects, the nucleic acids of the present disclosure are recombinant. As used herein, the term "recombinant" refers to (i) a molecule constructed by joining, outside a living cell, a natural or synthetic nucleic acid segment to a nucleic acid molecule capable of replicating within a living cell, or (ii) a molecule obtained from the replication of the molecule described in (i) above. For purposes herein, replication can be either in vitro or in vivo replication.

[0139] In some embodiments, the nucleic acid is constructed based on chemical synthesis and / or enzymatic ligation reactions using procedures known in the art. See, for example, Sambrook et al. (supra), and Ausubel et al. (supra). For example, the nucleic acid can be chemically synthesized using naturally occurring nucleotides or various modified nucleotides (e.g., phosphorothioate derivatives and acridine substituted nucleotides) designed to enhance the biological stability of the molecule or to enhance the physical stability of the duplex formed upon hybridization. Examples of modified nucleotides that can be used to generate nucleic acids include 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxymethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N 6 -isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N-substituted adenine, 7-methylguanine, 5-methylammomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N 6- Isopentenyladenine, uracil-5-oxyacetic acid (v), vibtoxin, pseudouracil, queuosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, methyl ester of uracil-5-oxyacetic acid, 3-(3-amino-3-N-2-carboxypropyl)uracil, and 2,6-diaminopurine are included, but not limited thereto. Alternatively, one or more of the nucleic acids of the present disclosure can be purchased from companies such as Macromolecular Resources (Fort Collins, CO) and Synthegen (Houston, TX).

[0140] Vector

[0141] The nucleic acids of the present disclosure in some embodiments are incorporated into vectors. In this regard, the present disclosure provides vectors comprising any of the nucleic acids disclosed herein. In various embodiments, the vector is a recombinant expression vector. For the purposes herein, the term "recombinant expression vector" means an oligonucleotide or polynucleotide construct in which a gene has been recombined to enable the expression of mRNA, protein, polypeptide, or peptide by a host cell, where the construct contains a nucleotide sequence encoding such mRNA, protein, polypeptide, or peptide, and the vector contacts the cell under conditions sufficient to cause the cell to express the mRNA, protein, polypeptide, or peptide intracellularly. The vectors of the present disclosure are generally not naturally occurring. However, portions of the vector may be naturally occurring. The vectors disclosed herein can contain any type of nucleotide, including but not limited to DNA and RNA, which can be single-stranded or double-stranded, synthetic, or partially obtained from naturally occurring substances, and can contain natural, non-natural, or modified nucleotides. The vector can contain natural nucleotide linkages, non-natural nucleotide linkages, or both types of linkages. In some embodiments, the modified nucleotides or non-natural nucleotide linkages do not interfere with the transcription or replication of the vector.

[0142] The vectors of the present disclosure can be any suitable vectors and can be used for transducing, transforming or transfecting any suitable host. Suitable vectors include those designed for propagation and / or expansion and / or expression, such as plasmids and viruses. The vector can be an expression vector using a plasmid. In various embodiments, the vector 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). Bacteriophage vectors, such as λGTIO, λGTl1, λZapII (Stratagene), λEMBL4, and λNMl149, etc. can also be used. Examples of plant expression vectors include pBIOl, pBI101.2, pBI101.3, pBI121 and pBIN19 (Clontech). Examples of animal expression vectors include pEUK-Cl, pMAM and pMAMneo (Clontech). In some embodiments, the vector is a viral vector, such as a retroviral vector. In various embodiments, 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 embodiments, the vector is a baculovirus vector that infects arthropods, such as insects. In various embodiments, the baculovirus vector is Autographacalifornica multiple nuclear virus (AcMNPV) or Bombyxmorinuclear polyhedrosis (BmNPV).See, for example, 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).

[0143] The vectors of the present disclosure can be prepared using standard recombinant DNA techniques as described, for example, in Sambrook et al. (supra) and Ausubel et al. (supra). The construct of the expression vector, which can be circular or linear, can be prepared to contain a replication system that functions in a host prokaryotic cell or a host eukaryotic cell. The replication system can be derived from, for example, CoIEl, 2μ plasmid, λ, SV40, bovine papillomavirus, etc.

[0144] In some embodiments, the vector includes control sequences such as start codons and stop codons for transcription and translation that are specific to the type of host (e.g., bacteria, fungi, plants, or animals) into which the vector is to be introduced, and the DNA or RNA nature of the vector is considered as necessary.

[0145] The vector can include one or more marker genes that enable the selection of transformed or transfected hosts. Marker genes include biocide resistance, e.g., resistance to antibiotics, heavy metals, etc., and complementarity in auxotrophic hosts to provide prototrophy. Suitable marker genes for the expression vectors disclosed in the present application include, for example, neomycin / G418 resistance gene, hygromycin resistance gene, histidinol resistance gene, tetracycline resistance gene, and ampicillin resistance gene.

[0146] A vector can comprise a natural or standard promoter that is functionally linked to a nucleotide sequence encoding a polypeptide (including its functional parts and functional variants), or a nucleotide sequence that is complementary to or hybridizes to a nucleotide sequence encoding a polypeptide. The selection of a promoter, for example, strong, weak, inducible, tissue-specific, development-specific, etc., is within the scope of the ordinary skill of those in the art. Similarly, combining a nucleotide sequence with a promoter is also within the scope of the skill of those in the art. The promoter can be a non-viral promoter or a viral promoter, such as the cytomegalovirus (CMV) promoter, SV40 promoter, RSV promoter, and the promoter found in the long terminal repeat of murine stem cell virus.

[0147] Host Cell

[0148] Provided herein are host cells comprising a nucleic acid or vector of the present disclosure. As used herein, the term "host cell" refers to any type of cell that can contain the vectors disclosed in the present application and can produce an expression product encoded by a nucleic acid (e.g., mRNA, protein). In some embodiments, the host cell is an adherent cell or a floating cell, i.e., a cell that grows in a floating state. In various embodiments, 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 of any cell type, can be derived from any type of tissue, and can be at any developmental stage.

[0149] In various embodiments, the antigen-binding protein is a glycosylated protein and the host cell is a cell having glycosylation ability. In various embodiments, the cell having glycosylation ability is a eukaryotic cell, including but not limited to yeast cells, filamentous fungal cells, protozoan cells, algal cells, insect cells, or mammalian cells. Such host cells have been reported in the art. See, for example, Frenzel, et al., Front Immunol 4:217(2013). In various embodiments, the eukaryotic cell is a mammalian cell. In various embodiments, the mammalian cell is a non-human mammalian cell. In some embodiments, the cell is a Chinese hamster ovary (CHO) cell and cells derived therefrom (e.g., CHO-K1, CHO pro-3), mouse myeloma cells (e.g., NS0, GS-NS0, Sp2 / 0), cells engineered to lack dihydrofolate reductase (DHFR) activity (e.g., DUKX-X11, DG44), human embryonic kidney 293 (HEK293) cells or cells derived therefrom (e.g., HEK293T, HEK293-EBNA), African green monkey kidney cells (e.g., COS cells, VERO cells), human cervical cancer cells (e.g., HeLa), human osteosarcoma epithelial cells U2-OS, human alveolar basal epithelial adenocarcinoma cells A549, human fibrosarcoma cells HT1080, mouse brain tumor cells CAD, embryonal carcinoma cells P19, mouse embryonic fibroblast NIH 3T3, mouse fibroblast L929, mouse neuroblastoma cells N2a, human breast cancer cells MCF-7, retinoblastoma cells Y79, human retinoblastoma cells SO-Rb50, human hepatocellular carcinoma cells Hep G2, mouse myeloma B cells J558L, or baby hamster kidney (BHK) cells (Gaillet et al.2007; Khan, Adv Pharm Bull 3(2):257-263(2013)).

[0150] For the purpose of amplifying or replicating the vector, in some embodiments the host cell is a prokaryotic cell, e.g., a bacterial cell.

[0151] Also provided in the present disclosure is a cell population comprising at least one host cell described herein. In some embodiments, the cell population is a heterogeneous population that includes host cells containing the described vectors, in addition to at least one other cell that does not contain any of the vectors. Alternatively, in some embodiments, the cell population is a substantially homogeneous population, and the population primarily comprises (e.g., consists essentially of) host cells containing the vector. In some embodiments, the population is a clonal population of cells, and all of the cells in the population are clones of a single host cell containing the vector, and all of the cells in the population contain the vector. In various embodiments of the present disclosure, the cell population is a clonal population that includes host cells containing the vectors described herein.

[0152] Manufacturing Method

[0153] Also provided herein is a method for producing an antigen-binding protein that binds to CLDN6. In various embodiments, the method includes culturing a host cell comprising a nucleic acid comprising a nucleotide sequence encoding the antigen-binding protein in a cell culture medium as described herein, and recovering the antigen-binding protein from the cell culture medium. The host cell can be any of the host cells described herein. In various aspects, the host cell is selected from the group consisting of CHO cells, NS0 cells, COS cells, VERO cells, and BHK cells. In various aspects, the step of culturing the host cell includes culturing the host cell in a growth medium to support the growth and proliferation of the host cell. In various aspects, the growth medium increases the cell density, viability, and productivity during culture at an appropriate time. In various aspects, the growth medium includes 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 fully synthetic medium consisting 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 the pH and osmotic pressure. Some growth media are commercially available and reported in the art. See, for example, Arora, “Cell Culture Media: A Review” MATER METHODS 3:175 (2013).

[0154] In various aspects, the method includes culturing the host cell in a fed-batch medium. In various aspects, the method includes culturing in a fed-batch medium by a fed-batch culture method. Methods for producing recombinant proteins are known in the art. See, for example, Li et al., “Cell culture processes for monoclonal antibody production” MAbs 2(5):466-477 (2010).

[0155] The method for producing an antigen-binding protein can include purifying the protein from a cell culture or its supernatant, and preferably one or more steps for recovering the purified protein. In various embodiments, the method includes one or more chromatography steps, such as affinity chromatography (e.g., protein A affinity chromatography), ion exchange chromatography, hydrophobic interaction chromatography. In various embodiments, the method includes purifying the protein using a protein A affinity chromatography resin.

[0156] In various embodiments, the method further includes steps for formulating the purified protein and the like, thereby obtaining a formulation containing the purified protein. Such steps are described in Formulation and Process Development Strategies for Manufacturing, eds. Jameel and Hershenson, John Wiley & Sons, Inc. (Hoboken, NJ), 2010.

[0157] In various embodiments, the antigen-binding protein and the antigen-binding protein linked to the polypeptide are part of a fusion protein. Accordingly, the present disclosure further provides a method for producing a fusion protein comprising an antigen-binding protein that binds to CLDN6. In various embodiments, the method includes culturing a host cell containing a nucleic acid comprising a nucleotide sequence encoding the fusion protein in a cell culture medium as described herein, and recovering the fusion protein from the cell culture medium.

[0158] Complex

[0159] The present disclosure also provides an antigen-binding protein that is attached, linked, or conjugated to a second moiety (e.g., a heterologous moiety, a complex moiety). Thus, the present disclosure provides a complex comprising an antigen-binding protein and a heterologous moiety. As used herein, the term "heterologous moiety" is synonymous with "complex moiety" and refers to any molecule (chemical or biochemical molecule, naturally occurring molecule, or non-coded molecule) that is different from the antigen-binding protein of the present disclosure. Various heterologous moieties include, but are not limited to, polymers, carbohydrates, lipids, nucleic acids, oligonucleotides, DNA or RNA, amino acids, peptides, polypeptides, proteins, therapeutic agents (e.g., cytotoxic drugs, cytokines), or diagnostic agents.

[0160] In some embodiments, the heterologous moiety is a polymer. The polymer may be branched or unbranched. The polymer can have any molecular weight. The polymer in some embodiments has an average molecular weight of about 2 kDa to about 100 kDa (the term "about" indicates that in a preparation of a water-soluble polymer, there are molecules that are more than the described molecular weight and there are also molecules that are less). The average molecular weight of the polymer is, in some aspects, about 5 kDa to about 50 kDa, about 12 kDa to about 40 kDa, or about 20 kDa to about 35 kDa.

[0161] 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. The polymer in some embodiments is water-soluble so that the protein to which it binds does not precipitate in an aqueous environment such as a physiological environment. In some embodiments, for example, when the composition is used therapeutically, the polymer is pharmaceutically acceptable. Further, in some aspects, the polymer is a mixture of polymers, such as a copolymer, a block copolymer.

[0162] 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 acid esters and methacrylic acid esters (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)), polyvinyl polymers (including polyvinyl alcohol, polyvinyl ether, polyvinyl ester, halogenated polyvinyl, poly(vinyl acetate), and polyvinyl pyrrolidone), polyglycolide, polysiloxane, polyurethanes and their copolymers, cellulose (including alkyl cellulose, hydroxyalkyl cellulose, cellulose ether, cellulose ester, nitrocellulose, methyl cellulose, ethyl cellulose, hydroxypropyl cellulose, hydroxy - propyl methyl cellulose, hydroxybutyl methyl cellulose, cellulose acetate, cellulose propionate, cellulose acetate butyrate, cellulose acetate phthalate, carboxyethyl cellulose, triacetate cellulose, and sodium salt of cellulose sulfate), polypropylene, polyethylene (including poly(ethylene glycol), poly(ethylene oxide), and poly(ethylene terephthalate)), and polystyrene.

[0163] 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 with a wide range of molecular weights and is soluble in water and most organic solvents.

[0164] 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, carrageenan, laminarin, xylan, mannan, fucoidan, galactomannan).

[0165] 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., mono-substituted, di-substituted, tri-substituted glycerol), a glycerophospholipid (e.g., phosphatidylcholine, phosphatidylinositol, phosphatidylethanolamine, phosphatidylserine), a sphingolipid (e.g., sphingosine, ceramide), a sterol lipid (e.g., steroid, cholesterol), a prenol lipid, a glycolipid, or a polyketide, an oil, a wax, cholesterol, a sterol, a fat-soluble vitamin, a monoglyceride, a diglyceride, a triglyceride, a phospholipid.

[0166] In some embodiments, the heterologous moiety is a therapeutic agent. The therapeutic agent can be any of the therapeutic agents known in the art. Examples of therapeutic agents contemplated herein include natural enzymes, proteins derived from naturally occurring substances, recombinant proteins, natural peptides, synthetic peptides, cyclic peptides, antibodies, receptor agonists, cytotoxic drugs, immunoglobulins, beta - adrenergic blockers, calcium channel blockers, coronary vasodilators, cardiac glycosides, antiarrhythmic drugs, cardiac sympathetic nerve agents, angiotensin - converting enzyme (ACE) inhibitors, diuretics, cardiotonics, cholesterol and triglyceride - lowering agents, bile acid sequestrants, fibrates, 3 - hydroxy - 3 - methylglutaryl (HMG) - CoA reductase inhibitors, niacin derivatives, anti - adrenergic agents, alpha - adrenergic blockers, central anti - adrenergic agents, vasodilators, potassium - retaining agents, thiazide - related agents, angiotensin II receptor antagonists, peripheral vasodilators, anti - androgenic agents, estrogens, antibiotics, retinoids, insulin analogs, alpha - glucosidase inhibitors, biguanide agents, meglitinides, sulfonylureas, thiazolidinediones, androgens, progesterones, bone metabolism regulators, anterior pituitary hormones, hypothalamic hormones, posterior pituitary hormones, gonadotropins, gonadotropin - releasing hormone antagonists, ovulation stimulants, selective estrogen receptor modulators, anti - thyroid drugs, thyroid hormones, bulking agents, laxatives, motility inhibitors, bacterial flora modifiers, intestinal adsorbents, intestinal anti - infectives, antianorexics, anticachexics, antibulimics, anorectic agents, anti - obesity drugs, antacids, upper gastrointestinal agents, anticholinergics, aminosalicylate derivatives, biologic response modifiers, corticosteroids, antispasmodics, 5 - HT 4 partial agonists, antihistamines, cannabinoids, dopamine antagonists, serotonin antagonists, cytoprotective agents, histamine H2 - receptor antagonists, mucosal protectants, proton pump inhibitors, H. pylori eradication therapy, erythropoiesis - stimulating agents, hematopoietic agents, anemia drugs, heparin, antifibrinolytics, hemostatic agents, blood coagulation factors, adenosine diphosphate inhibitors, glycoprotein receptor inhibitors, fibrinogen - platelet binding inhibitors, thromboxane - A 2Inhibitors, plasminogen activators, antithrombotic drugs, glucocorticoids, mineralocorticoids, corticosteroids, selective immunosuppressants, antifungal drugs, drugs associated with prophylactic treatment, AIDS-related infectious diseases, cytomegalovirus, non-nucleoside reverse transcriptase inhibitors, nucleoside analog reverse transcriptase inhibitors, protease inhibitors, anemia, Kaposi's sarcoma (idiopathic multiple hemorrhagic sarcoma), aminoglycoside drugs, carbapenem drugs, cephalosporin drugs, glycopeptides, lincosamide drugs, macrolide drugs, oxazolidinone drugs, penicillin drugs, streptogramin drugs, sulfonamides, trimethoprim and derivatives, tetracycline drugs, anthelmintics, antiamebic drugs, biguanide drugs, quinine alkaloids, folic acid antagonists, quinoline derivatives, Pneumocystis carinii treatment drugs, hydrazides, imidazoles, triazoles, nitroimidazoles, cyclic amines, neuraminidase inhibitors, nucleosides, phosphorus adsorbents, anticholinesterase drugs, adjuvant therapy drugs, barbituric acid and derivatives, benzodiazepines, gamma-aminobutyric acid derivatives, hydantoin derivatives, iminostilbene derivatives, succinimide derivatives, anticonvulsants, ergot alkaloids, antiheadache drug preparations, biological response modifiers, carbamic acid eater (carbamic acideater), tricyclic derivatives, depolarizing agents, non-depolarizing agents, neuromuscular paralytic agents, central nervous system stimulants, dopamine agonists, monoamine oxidase inhibitors, COMT inhibitors, alkyl sulfonates, ethyleneimines, imidazotetrazines, nitrogen mustard analogs, nitrosoureas, platinum-containing compounds, antimetabolites, purine analogs, pyrimidine analogs, urea derivatives, anthracycline agents, actinomycin d, camptothecin derivatives, epipodophyllotoxins, taxanes, vinca alkaloid analogs, antiandrogen agents, antiestrogen agents, non-steroidal aromatase inhibitors, protein kinase inhibitors, anti-cancer drugs, azaspirodecanedione derivatives, anti-anxiety drugs, stimulants, monoamine reuptake inhibitors, selective serotonin reuptake inhibitors, antidepressants, benzisoxazole derivatives, butyrophenone derivatives, dibenzodiazepine derivatives, dibenzothiazepine derivatives, diphenylbutylpiperazine derivatives, phenothiazines, thienobenzodiazepine derivatives, thioxanthene derivatives, allergen extracts, non-steroidal drugs, leukotriene receptor antagonists, xanthines, endothelin receptor antagonists, prostaglandins, lung surfactant, mucolytics, anti-mitotic drugs, uricosuric agents, xanthine oxidase inhibitors, phosphodiesterase inhibitors, methenamine salts, nitrofurans derivatives, quinolone agents, smooth muscle relaxants, parasympathomimetics, halogenated hydrocarbons, esters of aminobenzoic acid, amides (e.g., lidocaine, articaine hydrochloride, bupivacaine hydrochloride), antipyretics, hypnotics and sedatives, cyclopyrrolones, pyrazolopyrimidines, non-steroidal anti-inflammatory drugs, opioids, para-aminophenol derivatives, alcohol dehydrogenase inhibitors, heparin antagonists, adsorbents, emetics, opioid antagonists, cholinesterase reactivators, nicotine replacement therapy drugs, vitamin A analogs and antagonists, vitamin B analogs and antagonists, vitamin C analogs and antagonists, vitamin D analogs and antagonists, vitamin E analogs and antagonists, vitamin K analogs and antagonists, but are not limited thereto.

[0167] The antigen-binding protein of the present disclosure can bind to one or more cytokines and growth factors that are effective in inhibiting tumor metastasis, where the cytokine or growth factor has been shown to have an antiproliferative 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 in this specification include angiogenin, bone morphogenetic protein-1, bone morphogenetic protein-2, bone morphogenetic protein-3, bone morphogenetic protein-4, bone morphogenetic protein-5, bone morphogenetic protein-6, bone morphogenetic protein-7, bone morphogenetic protein-8, bone morphogenetic protein-9, bone morphogenetic protein-10, bone morphogenetic protein-11, bone morphogenetic protein-12, bone morphogenetic protein-13, bone morphogenetic protein-14, bone morphogenetic protein-15, bone morphogenetic protein receptor IA, bone morphogenetic protein receptor IB, brain-derived neurotrophic factor, ciliary neurotrophic factor, ciliary neurotrophic factor receptor α, cytokine-induced neutrophil chemoattractant 1, cytokine-induced neutrophil chemoattractant 2α, cytokine-induced neutrophil chemoattractant 2β, β-endothelial cell growth factor, endothelin 1, epithelial-derived neutrophil-activating peptide, glial cell line-derived neurotrophic factor receptor α1, glial cell line-derived neurotrophic factor receptor α2, growth-associated protein, growth-associated protein α, growth-associated protein β, growth-associated protein γ, heparin-binding epidermal growth factor, hepatocyte growth factor, hepatocyte growth factor receptor, insulin-like growth factor I, insulin-like growth factor receptor, insulin-like growth factor II, insulin-like growth factor binding protein, keratinocyte growth factor, leukemia inhibitory factor, leukemia inhibitory factor receptor α, 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 α, transforming growth factor β, transforming growth factor β1, transforming growth factor β1.2, transforming growth factor β2, transforming growth factor β3, transforming growth factor β5, latent transforming growth factor β1, transforming growth factor β-binding protein I, transforming growth factor β-binding protein II, transforming growth factor β-binding protein III, tumor necrosis factor receptor type I, tumor necrosis factor receptor type II, urokinase-type plasminogen activator receptor, and chimeric proteins and their biological or immunological active fragments are included.

[0168] In some embodiments, the conjugate comprises a compound described herein and a cytotoxic agent. A cytotoxic agent is any molecule (chemical or biochemical) that is toxic to cells. In some aspects, when a cytotoxic agent is conjugated to a compound of the invention, the resulting effect is synergistic. In other words, the efficacy of the combination therapy of the compound and the cytotoxic agent is synergistic, i.e., the efficacy is greater than the efficacy predicted from the individual additive effects of each. Thus, the dosage of the cytotoxic agent can be reduced, and thus the risks of toxicity problems and other side effects are simultaneously reduced. In some embodiments, the cytotoxic agent is a chemotherapeutic agent. Chemotherapeutic agents are known in the art and include, but are not limited to, platinum coordination compounds, topoisomerase inhibitors, antibiotics, anti-mitotic alkaloids, and difluoronucleosides as described in U.S. Patent No. 6,630,124.

[0169] 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, the platinum coordination compound is cis-diamminediaquoplatinum(II)-ion; chloro(diethylenetriamine)-platinum(II) chloride; dichloro(ethylenediamine)-platinum(II), diammin(1,1-cyclobutanedicarboxylato)platinum(II) (carboplatin); spiroplatin; iplatin; diammin(2-ethylmalonato)-platinum(II); ethylenediaminemalonatoplatinum(II); aqua(1,2-diaminocyclohexane)-sulfatoplatinum(II); (1,2-diaminocyclohexane)malonatoplatinum(II); (4-carboxyphthalato)(1,2-diaminocyclohexane)platinum(II); (1,2-diaminocyclohexane)-(isocitrato)platinum(II); (1,2-diaminocyclohexane)cis(pyruvato)platinum(II); (1,2-diaminocyclohexane)oxalatoplatinum(II); ormaplatin; and tetraplatin.

[0170] In some embodiments, cisplatin is the platinum coordination compound used in the compositions and methods of the present invention. Cisplatin is commercially available from Bristol Myers-Squibb Corporation under the generic name PLATINOL™ and is available as a powder for constitution with water, sterile saline or other suitable vehicles. Other platinum coordination compounds suitable for use in the present invention are known and can be utilized commercially and / or prepared by conventional techniques. Cisplatin, or cis-dichlorodiammineplatinum II, has been used for many years as a chemotherapeutic agent in the treatment of various human solid malignancies with good results. More recently, other diamino-platinum complexes have also been shown to be effective as chemotherapeutic agents in the treatment of various human solid malignancies. Such diamino-platinum complexes include, but are not limited to, spiroplatinum and carboplatin. Cisplatin and other diamino-platinum complexes are widely used as chemotherapeutic agents in humans, but high-dose delivery is required, which can cause toxicity problems such as kidney damage.

[0171] In some embodiments, the chemotherapeutic agent is a topoisomerase inhibitor. Topoisomerases are enzymes that can change the DNA topology of eukaryotic cells. They are important for cell function and cell proliferation. Generally, in eukaryotic cells, there are two classes of topoisomerases, type I and type II. Topoisomerase I is a monomeric enzyme with a molecular weight of about 100,000. This enzyme binds to DNA, introduces a transient single-strand break, allows the double helix to unwind (or enables unwinding), and then religates the break before dissociating from the DNA strand. Various topoisomerase inhibitors have recently shown clinical efficacy in the treatment of humans suffering from ovarian, cancer, esophageal or non-small cell lung cancer.

[0172] In some embodiments, the topoisomerase inhibitor is camptothecin or a camptothecin analog. Camptothecin is a water-insoluble cytotoxic alkaloid produced by the tree Camptotheca accuminata native to China and the tree Nothapodytes foetida native to India. Camptothecin exhibits tumor cell growth inhibitory activity against some tumor cells. Camptothecin analog class compounds are typically specific inhibitors of DNA topoisomerase I. The term "topoisomerase inhibitor" means any tumor cell growth inhibitory compound structurally related to camptothecin. Examples of camptothecin analog class compounds include, but are not limited to, topotecan, irinotecan, and 9-amino-camptothecin.

[0173] In additional embodiments, the cytotoxic agent is any tumor cell growth inhibitory camptothecin analog that is claimed or described in U.S. Patent No. 5,004,758, issued April 2, 1991, and European Patent Application Publication No. EP0 321 122, published June 21, 1989, as European Patent Application No. 88311366.4; U.S. Patent No. 4,604,463, issued August 5, 1986, and European Patent Application Publication No. EP0 137 145, published April 17, 1985; U.S. Patent No. 4,473,692, issued September 25, 1984, and European Patent Application Publication No. EP0 074 256, published March 16, 1983; U.S. Patent No. 4,545,880, issued October 8, 1985, and European Patent Application Publication No. EP0 074 256, published March 16, 1983; European Patent Application Publication No. EP0 088 642, published September 14, 1983; Wani et al., J. Med. Chem., 29, 2358-2363 (1986), Nitta et al., Proc. 14th International Congr. Chemotherapy, Kyoto, 1985, Tokyo Press, Anticancer Section 1, p. 28-30 (particularly the compound called CPT-11). CPT-11 is a camptothecin analog having a 4-(piperidino)-piperidine side chain linked via a carbamate bond at C-10 of 10-hydroxy-7-ethylcamptothecin. CPT-11 is currently being tested in humans and is also called irinotecan.Wani et al, J.Med.Chem., 23, 554 (1980), Wani et.al., J.Med.Chem., 30, 1774 (1987), U.S. Patent No. 4,342,776 issued on August 3, 1982; U.S. Patent Application No. 581,916 filed on September 13, 1990 and European Patent Application Publication No. EP418 099 published on March 20, 1991; U.S. Patent No. 4,513,138 issued on April 23, 1985 and European Patent Application Publication No. EP0 074 770 published on March 23, 1983; U.S. Patent No. 4,399,276 issued on August 16, 1983 and European Patent Application Publication No. 0 056 692 published on July 28, 1982; the entire disclosure of each of them is incorporated herein by reference. Any of the camptothecin analog class compounds listed above can be commercially available and / or prepared by conventional techniques including those described in the references listed above. The topoisomerase inhibitor can be selected from the group consisting of topotecan, irinotecan and 9-aminocamptothecin.

[0174] In some embodiments, the camptothecin analog is an active metabolite of irinotecan (CPT-11). In some such embodiments, the camptothecin analog is 7-ethyl-10-hydroxycamptothecin (SN-38). As a metabolite, SN-38 is formed by hydrolysis of irinotecan by carboxylesterase. In some embodiments, SN-38 has one of the following structures.

Chemical formula

[0175] In some embodiments, the camptothecin analog is exatecan mesylate. Exatecan mesylate exhibits more potent topoisomerase I inhibitory activity and antitumor activity than water-soluble camptothecin (CPT) and other CPT analogs. Furthermore, exatecan is effective against p-glycoprotein (P-gp)-mediated multidrug-resistant cells.

[0176] In some embodiments, the camptothecin analog is deruxtecan (Dxd), a potent derivative of exatecan, which has a topoisomerase I inhibitory ability 10 times higher than that of SN-38. In some embodiments, Dxd has the following structure.

Chemical formula

[0177] Dxd is described in US Patent Application No. 6,407,115, US Patent Application No. 10,195,288, US Patent Application No. 9,808,537, and US Patent Application No. 6,407,115.

[0178] The preparation of preparations of a number of camptothecin analog class compounds (including pharmaceutically acceptable salts, their hydrates and solvates) and oral and parenteral pharmaceutical compositions containing such camptothecin analog class compounds and an inert pharmaceutically acceptable carrier or diluent are described in detail in US Patent No. 5,004,758, issued April 2, 1991, and European Patent Application No. 88311366.4, published as EP0 321 122 on June 21, 1989, the teachings of which are incorporated herein by reference.

[0179] In still further other embodiments of the present invention, the chemotherapeutic agent is a combination antibiotic. Suitable antibiotics include, but are not limited to, doxorubicin, mitomycin, bleomycin, daunorubicin, and streptozocin.

[0180] In some embodiments, the chemotherapeutic agent is an anti-mitotic alkaloid. Generally, anti-mitotic alkaloids can be extracted from Cantharanthus roseus and have been shown to be effective as anti-cancer chemotherapeutic agents. A number of semi-synthetic derivatives have been studied both chemically and pharmacologically (see O. Van Tellingen et al, Anticancer Research, 12, 1699-1716 (1992)). Examples of anti-mitotic alkaloids of the present invention include, but are not limited to, vinblastine, vincristine, vindesine, taxol, and vinorelbine. The latter two anti-mitotic alkaloids are commercially available from Eli Lilly and Company, and Pierre Fabre Laboratories, respectively (see U.S. Patent No. 5,620,985). In one embodiment, the anti-mitotic alkaloid is vinorelbine.

[0181] In other embodiments of the present invention, the chemotherapeutic agent is a difluoronucleoside. 2'-Deoxy-2',2'-difluoronucleoside is known in the art to have anti-viral activity. Such compounds are disclosed and taught in U.S. Patent Nos. 4,526,988 and 4,808,614. European Patent Application Publication No. 184,365 discloses that these same difluoronucleosides have tumor-lysing activity. In certain embodiments, the 2'-deoxy-2',2'-difluoronucleoside used in the compositions and methods of the present invention is 2'-deoxy-2',2'-difluorocytidine hydrochloride, also known as gemcitabine hydrochloride. Gemcitabine can be obtained commercially or synthesized in a multi-step process as disclosed and taught in U.S. Patent Nos. 4,526,988, 4,808,614, and 5,223,608, the teachings of which are incorporated herein by reference.

[0182] In various aspects, the chemotherapeutic agent is an anti-mitotic agent that inhibits cell division by blocking tubulin polymerization, destabilizing microtubules, or altering microtubule dynamics, and includes, for example, maytansinoids or derivatives thereof (e.g., DM1 or DM4), auristatins or derivatives thereof. In various cases, the chemotherapeutic agent is an auristatin. For example, in some aspects, the auristatin is dolastatin, monomethyl auristatin E (MMAE), monomethyl auristatin E (MMAE), or PF-06380101. Auristatins are reported in the art. See, for example, Maderna, A.; et al., Mol Pharmaceutics 12(6):1798-1812 (2015). In various aspects, the conjugate includes the antibody of the present disclosure in combination with MMAE. Optionally, the conjugate includes a linker. In some aspects, the linker includes a cleavable linking moiety. In various cases, the conjugate includes an antibody of the present disclosure linked to a linking group linked to a cathepsin-cleavable linker and linked to a spacer linked to MMAE. In an aspect, the linking group is linked to the antibody via a Cys residue in the Fc region of the antibody. In an exemplary aspect, the linking group includes a structure of Formula I.

Chemical formula

[0183] In an exemplary aspect, the cathepsin-cleavable linker includes a structure of Formula II.

Chemical formula

[0184] In an exemplary aspect, the spacer includes a structure of Formula III.

Chemical formula

[0185] In some embodiments, MMAE has the following structure. [Chemical]

[0186] The present disclosure also provides a complex comprising an antigen-binding protein of the present disclosure linked to a polypeptide, such that the complex is a fusion protein. Thus, the present disclosure provides a fusion protein comprising an antigen-binding protein of the present disclosure linked to a polypeptide. In various embodiments, the polypeptide is a diagnostic label, such as a fluorescent protein like green fluorescent protein, or another tag, 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.

[0187] Linker

[0188] In some embodiments, the complex is directly linked to the heterologous moiety. In alternative embodiments, the complex comprises a linker that links a compound of the present disclosure to the heterologous moiety. In some aspects, the linker comprises a chain of 1 to about 60 atoms, or 1 to 30 atoms or more, 2 to 5 atoms, 2 to 10 atoms, 5 to 10 atoms, or 10 to 20 atoms in length. In some embodiments, all of the chain atoms are carbon atoms. In some embodiments, the chain atoms within the linker backbone are selected from the group consisting of C, O, N, and S. The chain atoms and the linker can be selected according to their predicted solubility (hydrophilicity) such that a more soluble complex is provided. In some embodiments, the linker provides a functional group that is subject to cleavage by an enzyme or other catalyst, or by hydrolytic conditions found in the target tissue or organ or cell. In some embodiments, the length of the linker is sufficient to reduce the likelihood of steric hindrance. In some embodiments, the linker is an amino acid or a peptidyl linker. Such peptidyl linkers can be of any length. Various linkers are about 1 to 50 amino acids in length, 5 to 50, 3 to 5, 5 to 10, 5 to 15, or 10 to 30 amino acids in length.

[0189] A variety of suitable linkers are known in the art. The linker can be cleavable, for example, under physiological conditions, such as intracellular conditions (cleavable linker), such that cleavage of the linker releases the drug into the intracellular environment. Alternatively, the linker can be cleavable under extracellular conditions, such as outside or in the vicinity of tumor cells, such that cleavage of the linker releases the drug that selectively penetrates into the tumor cells. In other embodiments, the linker is not cleavable (non-cleavable linker), and the drug is released, for example, by degradation of the antibody.

[0190] The linker can be attached to a chemically reactive group on the antibody moiety, such as a free amino group, imino group, hydroxyl group, thiol group, or carboxyl group (e.g., at the N-terminus or C-terminus, the epsilon amino group of one or more lysine residues, the free carboxylic acid group of one or more glutamic acid residues or aspartic acid residues, the sulfhydryl group of one or more cysteinyl residues, or the hydroxyl group of one or more serine residues or threonine residues). The site to which the linker is attached can be a native residue within the amino acid sequence of the antibody moiety, and can also be introduced into the antibody moiety, for example, by DNA recombination techniques (e.g., by introducing a cysteine or protease cleavage site into the amino acid sequence) or by protein biochemistry (e.g., reduction, pH adjustment, or proteolysis). The site to which the linker is attached can also be a non-natural amino acid. The site to which the linker is attached can also be a glycan on the antibody.

[0191] Typically, the linker is substantially inert under conditions where the two groups it connects are linked. The terms "bifunctional crosslinker", "bifunctional linker" or "crosslinker" refer to a modifier having two reactive groups at each end of the linker, such that one reactive group can first be reacted with a cytotoxic compound to provide a linker moiety-bearing compound, and then the second reactive group can be reacted with an antibody. Alternatively, one end of the bifunctional crosslinker can first be reacted with an antibody to provide an antibody bearing the linker moiety and the second reactive group, which can then react with a cytotoxic compound. The linking moiety can contain a chemical bond that allows for the release of the cytotoxic moiety at a specific site. Suitable chemical bonds are well known in the art and include disulfide bonds, thioether bonds, acid-labile bonds, photo-labile bonds, protease / peptidase-labile bonds, and esterase-labile bonds. See, for example, U.S. Pat. Nos. 5,208,020, 5,475,092, 6,441,163, 6,716,821, 6,913,748, 7,276,497, 7,276,499, 7,368,565, 7,388,026, and 7,414,073. In some embodiments, the bond is a disulfide bond, thioether, and / or protease / peptidase-labile bond. Other linkers that can be used in the present invention include cleavable linkers such as those detailed in US20050169933, which are hereby expressly incorporated by reference herein, and charged or hydrophilic linkers such as those described in US2009 / 0274713, US2010 / 0129314, and WO2009 / 134976.

[0192] In some embodiments, the linker is a hydrophilic linker that imparts hydrophilicity to the complex. In some embodiments, the hydrophilic linker comprises polyethylene glycol (PEG). In some embodiments, the hydrophilic linker is CLA2. In some embodiments, the CLA2 linker has the following structure.

Chemical formula

[0193] In some embodiments, the hydrophilic linker is CL2E. In some embodiments, CL2E has the following structure.

Chemical formula

[0194] In some embodiments, the linker is cleavable by a cleavage substance present in the intracellular environment (e.g., inside a lysosome or an endosome or a caveola). The linker can be, for example, a peptide linker that is cleaved by an intracellular or extracellular peptidase or protease enzyme, including but not limited to those containing lysosomal or endosomal proteases. In some embodiments, the peptide linker comprises at least 2 amino acids in length, at least 3 amino acids in length, at least 4 amino acids in length, or at least 5 amino acids in length.

[0195] In some embodiments, the peptide linker is MC-VC-PAB that contains a valine residue and a citrulline residue. In some embodiments, the MC-VC-PAB linker has the following structure.

Chemical formula

[0196] In some embodiments, the peptide linker is maleimidocaproyl glycine-glycine-phenylalanine-glycine (MC-GGFG). In some embodiments, the MC-GGFG linker has the following structure. [Chemical formula] MC-GGFG is described in U.S. Patent Nos. 9,808,537 and 10,195,288.

[0197] In other embodiments, the cleavable linker is pH-sensitive, i.e., sensitive to hydrolysis at a specific pH value. In some embodiments, the pH-sensitive linker is hydrolyzable under acidic conditions. For example, acid-labile linkers that are hydrolyzable within lysosomes (e.g., hydrazones, semicarbazones, thiosemicarbazones, cis-aconitamides, orthoesters, acetals, ketals, etc.) can be used (see, e.g., U.S. Patent Nos. 5,122,368, 5,824,805, 5,622,929, Dubowchik and Walker, 1999, Pharm. Therapeutics 83:67-123, Neville et al, 1989, Biol. Chem. 264:14653-14661). Such linkers are relatively stable under neutral pH conditions such as in blood, but are unstable at pH values below 5.5 or 5.0, which is the approximate pH of lysosomes. In certain embodiments, the hydrolyzable linker is a thioether linker (e.g., a thioether bonded to a therapeutic agent via an acylhydrazone linkage (see, e.g., U.S. Patent No. 5,622,929).

[0198] In other embodiments, the linker is cleavable under reducing conditions (e.g., disulfide linker). Bifunctional crosslinkers that allow the antibody to be linked to a cytotoxic compound via a disulfide bond include, but are not limited to, N-succinimidyl-4-(4-nitropyridyl-2-dithio)butanoate, N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), N-succinimidyl-4-(2-pyridyldithio)pentanoate (SPP), N-succinimidyl-4-(2-pyridyldithio)butanoate (SPDB), N-succinimidyl-4-(2-pyridyldithio)-2-sulfobutanoate (sulfo-SPDB). Sulfo-SPDB is described, for example, in U.S. Patent No. 8,236,319, which is incorporated herein by reference. Alternatively, crosslinkers that introduce thiol groups, such as 2-iminothiolane, homocysteine ​​thiolactone, or S-acetylsuccinic anhydride, can be used. In other embodiments, the linker may contain a combination of one or more of the aforementioned peptide linkers, pH-sensitive linkers, or disulfide linkers.

[0199] A "heterobifunctional crosslinker" is a bifunctional crosslinker that has two different reactive groups. Heterobifunctional crosslinkers that contain both an amine-reactive N-hydroxysuccinimide group (NHS group) and a carbonyl-reactive hydrazine group can also be used to link antibodies to cytotoxic compounds. Examples of such commercially available heterobifunctional crosslinkers include succinimidyl 6-hydrazinonicotinamide acetone hydrazone (SANH), succinimidyl 4-hydrazidoterephthalate hydrochloride (SHTH) and succinimidyl hydrazinium nicotinate hydrochloride (SHNH). Conjugates with acid-labile bonds can also be prepared using the hydrazine-bearing benzodiazepine derivatives of the invention. Examples of bifunctional crosslinkers that can be used include succinimidyl-p-formylbenzoate (SFB) and succinimidyl-p-formylphenoxyacetate (SFPA).

[0200] The linkers described herein may be used in any combination with the heterologous moieties described herein. Any of the linkers and heterologous moieties described above can be commercially available and / or prepared by conventional techniques including those described in the references cited above.

[0201] Complexation

[0202] The ratio of heterologous moiety to antigen-binding protein (HAR) represents the number of linked heterologous moieties per antigen-binding molecule. In some embodiments, the HAR ranges from 1 to 15, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 to 2. In some embodiments, the HAR ranges from 2 to 10, 2 to 9, 2 to 8, 2 to 7, 2 to 6, 2 to 5, 2 to 4, or 2 to 3. In other embodiments, the HAR is about 2, about 2.5, about 3, about 4, about 5, or about 6. In some embodiments, the HAR ranges from about 2 to about 4. The HAR can be characterized by conventional means such as mass spectrometry, UV / Vis spectroscopy, ELISA assays, and / or HPLC.

[0203] In some embodiments, the complex is a heterogeneous complex (also referred to as a "conventional type"), and the antigen-binding protein is bound to different numbers of heterologous moieties. In some embodiments, the heterogeneous complex follows a Gaussian or quasi-Gaussian distribution of the complex, in which case the center of the distribution comes to the average value of the heterologous moiety loading, where some antigen-binding proteins bind at higher values than the average and some antigen-binding proteins bind at lower values than the average.

[0204] In some embodiments, the complex is a homogeneous complex, in which case a significant proportion of the antigen-binding proteins are bound to a predetermined number of heterologous moieties. In some embodiments, the homogeneous complex comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 HARs. In some embodiments, the homogeneous complex comprises 2, 4, 6, or 8 HARs. In a preferred embodiment, the homogeneous complex comprises 4 HARs. In another preferred embodiment, the homogeneous complex comprises 2 HARs. In some embodiments, the homogeneous complex comprises 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 percent or more of complexes having a defined HAR. In some embodiments, the homogeneous complex comprises about 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 percent of complexes having a defined HAR. In some embodiments, the homogeneous complex comprises at least 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 percent of complexes having a defined HAR. In some embodiments, the homogeneous complex comprises a HAR distribution that is neither Gaussian nor quasi-Gaussian. In some embodiments, the homogeneity of the homogeneous complex is determined by chromatography, e.g., HPLC or any suitable chromatography. In some embodiments, the chromatography is HIC chromatography. The homogeneous complex can be generated by site-specific complexation.

[0205] In some embodiments, the heterologous moiety is site-specifically conjugated to an antigen-binding protein (e.g., an antibody). Various methods of site-specific conjugation are known in the art, for example, thiomab or TDC or conjugation with mismatched cysteine residues (Junutula et al. (2008) Nat. Biotechnol. 26:925-932, Dimasi et al. (2017) Mol. Pharm. 14:1501-1516, Shen et al. (2012) Nat. Biotechnol. 30:184-9), thiol crosslinker (Behrens et al. (2015) Mol. Pharm. 12:3986-98), conjugation with glutamine using transglutaminase (Dennler et al. (2013) Methods Mol. Bio. 1045:205-15, Dennler et al. (2014) Bioconjug Chem. 25:569-78), conjugation with engineered unnatural amino acid residues (Axup et al. (2012) Proc Natl Acad Sci U.S.A. 104-16101-6, Tian et al. (2014) Proc Natl Acad Sci U.S.A. 111:1766-71, VanBrunt et al. (2015) Bioconjug Chem 26:2249-60, Zimmerman et al. (2014) Bioconjug Chem 25:351-61), selenocysteine conjugation (Li et al. (2017) Cell Chem Biol 24:433-442), glycan-mediated conjugation (Okeley et al. (2013) Bioconjug Chem 24:1650-5), conjugation with galactose or GalNAc analogs (Ramakrishnan and Qasba (2002) J Biol Chem 277:20833-9, van Geel et al. (2015) Bioconjug Chem 26:2233-42), by glycan engineering Zhou et al. (2014) Bioconjug Chem 25:510-20, Tang et al.((2017) Nat Protoc 12:1702-1721), those using the manipulation of glutamine tags or short-chain peptide tags such as sortase A-mediated peptide transfer (Strop et al. (2013) Chem Biol 20:161-7, Beerli et al. (2015) PLoS One 10:e0131177), and those using aldehyde tags (Wu et al. (2009) Proc Natl Acad Sci U.S.A. 106:3000-5).

[0206] Predictive Uncertainty of Complex (e.g., ADC)

[0207] Based solely on the antibody profile or the drug payload profile, it is impossible to predict in advance which antibody-drug conjugates are sufficiently safe and effective for clinical applications. For example, a particular drug payload may function perfectly when conjugated to an antibody directed against one target, but may hardly function, or may not function at all, when conjugated to an antibody directed against a different target, or when conjugated to a different antibody directed against the same target. The reasons why different antibody-drug conjugates exhibit different antitumor activities in vivo have not been fully elucidated, and thus accurate prediction is not possible in the design of new antibody-drug conjugates. It is presumed that the unpredictable interactions of many factors are involved. These factors may include, for example, the binding affinity of the antibody-drug conjugate for the target antigen, the solid tumor penetration ability of the conjugate, and the circulating blood half-life for appropriate exposure to the tumor without causing toxicity.

[0208] Complexity and unpredictability are not sufficiently indicated by antibody affinity alone. High-affinity antibodies or antibody-drug conjugates leave a trail of better uptake into cells and release of higher levels of cytotoxic payload inside the cells. High affinity is also known to enhance antibody-dependent cell cytotoxicity (ADCC). All of these attributes are favorable for the cell-killing properties of antibody-drug conjugates. However, it is also known that high affinity of an antibody or antibody-drug conjugate can impede efficient tumor penetration by the "antigen barrier effect", suggesting that for achieving strong anti-tumor activity in vivo, the affinity of the antibody-drug conjugate should be neither too high nor too low, but just right. To date, there is no known way to predict which level of affinity is the most efficient or effective for a given antibody-drug conjugate.

[0209] Furthermore, anti-tumor activity in vivo cannot be predicted from the mechanism of the linker and payload alone. For example, O. Ab et al, Mol. Cancer Ther. 14(&):1605 - 1613(2015) demonstrated that when the same antibody was conjugated to the same anti-tubulin toxin with different linkers and tested in preclinical cancer models, dramatically different anti-tumor activities were shown. This example is particularly surprising because the chemical structures of the two linkers are very similar. Furthermore, the linker present in the superior conjugate contained a hydrophilic moiety. Hydrophilic metabolites generally have low membrane permeability and slower efflux from lysosomes (the site where the conjugate is degraded), resulting in a delay in the anti-tubulin activity of the released payload. This finding argues for an "ideal" kinetics of payload delivery, but currently there is no insight into what constitutes such kinetics. What further complicates this is the unresolved question of whether the ideal kinetics of payload delivery, while defined for a particular cell type, applies to all cell types. Therefore, it is impossible to predict the most effective in vivo anti-tumor activity simply from the chemical composition of the linker or payload.

[0210] Composition, Pharmaceutical Composition and Formulation

[0211] Compositions are provided herein that include an antigen-binding protein, nucleic acid, vector, host cell, or complex as disclosed in the present application. In some embodiments, the composition includes the antigen-binding protein in an isolated and / or purified form. In some embodiments, the composition includes a single type (e.g., structure) of the antigen-binding protein of the present disclosure, or a combination of two or more antigen-binding proteins of the present disclosure, such combination including two or more antigen-binding proteins of different types (e.g., structures).

[0212] In some embodiments, the composition includes an agent that enhances the chemical and physical characteristics of the antigen-binding protein, e.g., stabilizing the antigen-binding protein at a specific temperature, e.g., room temperature, extending the shelf life, reducing degradation, e.g., degradation by oxidative proteases, extending the half-life of the antigen-binding protein, etc. In some embodiments, the composition includes any of the agents disclosed herein as a heterologous or complex moiety, optionally formulated as a mixture with the antigen-binding protein of the present disclosure or included by binding to such antigen-binding protein.

[0213] In various embodiments of the present disclosure, the composition further includes a pharmaceutically acceptable carrier, diluent, or additive. In some embodiments, an antigen-binding protein, nucleic acid, vector, host cell, or complex as disclosed in the present application (hereinafter referred to as "active substance") is formulated into a pharmaceutical composition including the active substance together with a pharmaceutically acceptable carrier, diluent, or additive. In this regard, the present disclosure further provides a pharmaceutical composition including an active substance intended for administration to a subject, e.g., a mammal.

[0214] In some embodiments, the active substance is present in the pharmaceutical composition at a purity level suitable for administration to a patient. In some embodiments, the active substance 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 additive. In some embodiments, the composition contains the active substance at a concentration of about 0.001 to about 30.0 mg / ml.

[0215] In various aspects, the pharmaceutical composition includes a pharmaceutically acceptable carrier. As used herein, the term "pharmaceutically acceptable carrier" includes any standard pharmaceutical carrier, such as phosphate buffered saline, water, emulsions such as oil / water or water / oil emulsions, and various wetting agents. The term also encompasses any agent approved by the regulatory authorities of the federal government of the United States, or any agent listed in the United States Pharmacopeia for use in animals including humans.

[0216] The pharmaceutical composition can contain any pharmaceutically acceptable ingredients, including, for example, acidifying agents, additives, adsorbents, aerosol propellants, air displacement agents, alkalizing agents, anti-caking agents, anticoagulants, antibacterial preservatives, antioxidants, disinfectants, bases, binders, buffers, chelating agents, coating agents, coloring agents, desiccants, detergents, diluents, germicides, disintegrants, dispersants, solubilizing agents, pigments, emollients, emulsifiers, emulsion stabilizers, excipients, film-forming agents, flavoring agents, fragrance agents, flow promoters, gelling agents, granulating agents, humectants, lubricants, mucoadhesives, ointment bases, ointments, oily vehicles, organic bases, suppository bases, pigments, plasticizers, gloss agents, preservatives, blocking agents, skin penetration enhancers, solubilizers, solvents, stabilizers, suppository bases, surfactants, surface-active agents, suspending agents, sweetening agents, therapeutic agents, thickening agents, isotonic agents, toxic agents, thickening agents, water-absorbing agents, water-miscible co-solvents, water softeners, or wetting agents. See, for example, Handbook of Pharmaceutical Excipients, Third Edition, A.H. Kibbe (Pharmaceutical Press, London, UK, 2000), which is incorporated herein by reference in its entirety. Remington’s Pharmaceutical Sciences, Sixteenth Edition, E.W. Martin (Mack Publishing Co., Easton, Pa., 1980), which is incorporated herein by reference in its entirety.

[0217] In various embodiments, the pharmaceutical composition contains formulation materials that are non-toxic to the recipient at the dosages and concentrations used. In specific embodiments, a pharmaceutical composition containing an active substance, and one or more pharmaceutically acceptable salts, polyols, surfactants, osmotic balance maintainers, isotonic agents, antioxidants, antibiotics, antifungal agents, bulking agents, lyoprotectants, defoaming agents, chelating agents, preservatives, coloring agents, analgesics, or additional pharmaceutical agents. In various embodiments, the pharmaceutical composition contains one or more polyols and / or one or more surfactants, and optionally, one or more additional additives, including, but not limited to, pharmaceutically acceptable salts, osmotic balance maintainers (isotonic agents), antioxidants, antibiotics, antifungal agents, bulking agents, lyoprotectants, defoaming agents, chelating agents, preservatives, coloring agents, and analgesics.

[0218] In certain embodiments, the pharmaceutical composition can contain formulation materials for altering, maintaining, or preserving, for example, the pH, osmotic pressure, viscosity, clarity, color, isotonicity, odor, sterility, stability, rate of dissolution or release, adsorption, or permeability of the composition. In such embodiments, suitable formulation materials include amino acids (such as glycine, glutamine, asparagine, arginine, or lysine); antibacterial agents; antioxidants (such as ascorbic acid, sodium sulfite, or sodium bisulfite); buffers (such as borate, bicarbonate, Tris-HCl, citrate, phosphate, or other organic acids); bulking agents (such as mannitol or glycine); chelating agents (such as ethylenediaminetetraacetic acid (EDTA)); complexing agents (such as caffeine, polyvinylpyrrolidone, beta-cyclodextrin, or hydroxypropyl-beta-cyclodextrin); excipients; monosaccharides; disaccharides; and other carbohydrates (such as glucose, mannose, or dextrin); proteins (such as serum albumin, gelatin, or immunoglobulins); coloring agents, flavoring agents, and diluents; emulsifying agents; 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, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid, or hydrogen peroxide); solvents (such as glycerin, 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, triton, tromethamine, lecithin, cholesterol, tyloxapal); stability enhancing substances (such as sucrose or sorbitol); isotonicity enhancing agents (such as alkali metal halides, preferably sodium chloride or potassium chloride, mannitol, sorbitol); delivery vehicles; diluents; additives; and / or pharmaceutical adjuvants, but are not limited thereto. See REMINGTON‘S PHARMACEUTICAL SCIENCES, 18″ Edition, (A.R. Genrmo, ed.), 1990, Mack Publishing Company.

[0219] 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 to about 8.0, or between about 4.5 to about 7.5, or between about 5.0 to about 7.5. In various embodiments, the pH of the pharmaceutical composition is 5.5 to 7.5.

[0220] The present disclosure provides a method for producing a pharmaceutical composition. In various aspects, the method includes combining an antigen-binding protein, complex, fusion protein, nucleic acid, vector, host cell, or combination thereof with a pharmaceutically acceptable carrier, diluent, or additive.

[0221] Route of Administration

[0222] With respect to the present disclosure, the active substance, or the pharmaceutical composition containing the same, can be administered to a subject via any suitable route of administration. For example, the active substance can be administered to the subject by parenteral administration, nasal administration, oral administration, pulmonary administration, topical administration, intravaginal administration, or rectal administration. The following considerations regarding the route of administration are provided only to illustrate various embodiments and should not be construed as limiting in any way.

[0223] Formulations suitable for parenteral administration can contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, aqueous and non-aqueous isotonic sterile injectable solutions, and aqueous and non-aqueous sterile suspensions that can contain suspending agents, solubilizing agents, thickening agents, stabilizing agents, and preservatives. The term "parenteral" means via some other route than through the digestive tract, such as subcutaneously, intramuscularly, intraspinally, or intravenously. The active substances of the present disclosure can be administered using a pharmaceutical carrier containing a physiologically acceptable diluent such as a sterile liquid or liquid mixture, which includes water, physiological saline, aqueous dextrose and related sugar solutions, alcohols such as ethanol or hexadecyl alcohol, glycols such as propylene glycol or polyethylene glycol, dimethyl sulfoxide, glycerol, ketals such as 2,2-dimethyl-l,3-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), pectins, carbomers, methylcellulose, hydroxypropylmethylcellulose, or carboxymethylcellulose, or suspending agents, or emulsifying agents, and other pharmaceutical adjuvants.

[0224] Oils that can be used in parenteral formulations include petroleum, animal, vegetable, or synthetic oils. Specific examples of oils include peanut, soybean, sesame, cottonseed, corn, olive, petrolatum, and mineral. Suitable fatty acids for use in parenteral formulations include oleic acid, stearic acid, and isostearic acid. Ethyl oleate and isopropyl myristate are examples of suitable fatty acid esters.

[0225] Suitable soaps for parenteral use include salts of aliphatic alkali metals, ammonium, and triethanolamine. Suitable detergents include: (a) cationic detergents such as dimethyldialkylammonium halides and alkylpyridinium halides; (b) anionic detergents such as alkyl sulfonates, aryl sulfonates, olefin sulfonates, alkyl sulfates, olefin sulfates, ether sulfates, monoglyceride sulfates, and sulfosuccinates; (c) nonionic detergents such as aliphatic amine oxides, fatty acid alkanolamides, and copolymers of polyoxyethylene and polypropylene; (d) amphoteric detergents such as alkyl-β-aminopropionates and 2-alkyl-imidazoline quaternary ammonium salts; and (e) mixtures thereof.

[0226] The parenteral formulations in some embodiments contain from about 0.5 wt% to about 25 wt% of the active substance solution of the present disclosure. Preservatives and buffers can be used. To minimize or eliminate irritation at the injection site, such compositions can contain one or more nonionic surfactants having a hydrophilic-lipophilic balance (HLB) of from about 12 to about 17. The amount of surfactant in such formulations typically ranges from about 5 wt% to about 15 wt%. Suitable surfactants include polyethylene glycol sorbitan fatty acid esters such as sorbitan monooleate, and high molecular weight adducts of ethylene oxide having a hydrophobic base formed by the condensation of propylene oxide and propylene glycol. The parenteral formulations in some aspects are in single-dose or multi-dose sealed containers such as ampoules and vials and can be stored in a freeze-dried state that requires only the addition of a sterile liquid additive for injection, such as water, immediately before use. The ready-to-use injection solutions and suspensions in some aspects are prepared from sterile powders, granules, and tablets of the types described above.

[0227] Injectable formulations are in accordance with the present disclosure. Requirements for an effective pharmaceutical carrier for injectable compositions are well known to those skilled in the art (see, for example, Pharmaceutics and Pharmacy Practice, J.B. Lippincott Company, Philadelphia, PA, Banker and Chalmers, eds., pages 238-250 (1982), and ASHP Handbook on Injectable Drugs, Toissel, 4th ed., pages 622-630 (1986)).

[0228] Dosage

[0229] The disclosed active substances are believed to be useful in other methods as detailed herein, including methods of inhibiting tumor growth and methods of treating or preventing cancer. For purposes of the disclosure, the amount or dosage of the active substance administered should be sufficient for an effect, such as a therapeutic or prophylactic response, in the subject or animal over a reasonable time frame. For example, the dosage of the active substances of the present disclosure should be sufficient to treat the cancers described herein for a period of about 1 to 4 minutes, 1 to 4 hours, or 1 to 4 weeks, or more, for example, 5 to 20 weeks, or more weeks, from the time of administration. In certain embodiments, the time period may be longer. The dosage is determined by the effectiveness of the particular active substance and the condition of the animal (e.g., human), as well as the weight of the animal (e.g., human) to be treated.

[0230] Many assays for determining dosage are known in the art. For the purposes herein, an assay is contemplated that can be used to determine the starting dosage to be administered to a mammal, including comparing the degree to which cancer is treated upon administration of a given dosage of the active substance of the present disclosure among groups of mammals to which different dosages of the active substance are administered per group. The degree to which cancer is treated upon administration of a specific dosage can be represented, for example, by the degree of tumor regression achieved by the active substance in a mouse xenograft model. Methods for evaluating tumor regression are known in the art and are described in the Examples herein.

[0231] The dosage of the active substance of the present disclosure is also determined by the presence, nature, and extent of any adverse side effects that may be associated with the administration of the specific active substance of the present disclosure. Typically, the attending physician determines the dosage of the active substance of the present disclosure for treating each individual patient, taking into account various factors such as age, body weight, general health, diet, sex, the active substance of the present disclosure to be administered, the route of administration, and the severity of the condition being treated. Without intending to limit the present disclosure, by way of example, the dosage of the active substance of the present disclosure can be from about 0.0001 to about 1 g / kg body weight (body weight of the subject being treated) / day, from about 0.0001 to about 0.001 g / kg body weight / day, or from about 0.01 mg to about 1 g / kg body weight / day.

[0232] Controlled Release Formulation

[0233] In some embodiments, the active substances described herein can be modified into depot forms such that the manner in which the active substances of the present disclosure are released into the body into which they are administered is controlled with respect to time and location within the body (see, e.g., U.S. Patent No. 4,450,150). The depot form of the active substance of the present disclosure can be, for example, an implantable composition comprising the active substance and a porous or non-porous material such as a polymer, where the active substance is encapsulated by such material or diffused throughout the material and / or diffused through the degradation of the non-porous material. The depot is then implanted at a desired location within the body of the subject, and the active substance is released from the implant tablet at a predetermined rate.

[0234] In certain embodiments, a pharmaceutical composition comprising an active agent is modified to have any type of in vivo release profile. In some embodiments, the pharmaceutical composition is a formulation of immediate release, controlled release, sustained release, extended release, delayed release, or biphasic release. Methods of formulating release controlling peptides are known in the art. See, for example, Qian et al., J Pharm 374:46-52 (2009) and International Patent Application Publication Nos. WO2008 / 130158, WO2004 / 033036, WO2000 / 032218, and WO1999 / 040942.

[0235] The composition can further include, for example, micelles or liposomes, or some other form of encapsulation, and can be administered in a sustained release form to provide long term storage and / or delivery effects.

[0236] Use

[0237] The antigen-binding proteins of the disclosure are useful for inhibiting tumor growth. Without being bound by a particular theory, the inhibitory action of the antigen-binding proteins provided herein renders such entities useful in methods of treating cancer.

[0238] Accordingly, provided herein are methods of inhibiting tumor growth in a subject and methods of reducing tumor size in a subject. In various embodiments, the methods include administering to the subject a pharmaceutical composition of the disclosure in an amount effective to inhibit tumor growth in the subject or reduce tumor size. In various embodiments, the growth of ovarian tumors, melanoma tumors, bladder tumors, or endometrial tumors is inhibited. In various embodiments, the size of ovarian tumors, melanoma tumors, bladder tumors, or endometrial tumors is reduced.

[0239] As used herein, the terms “inhibit” or “reduce” and words stemming therefrom may not be 100% or complete inhibition or reduction. Rather, the degree of inhibition or reduction that would be recognized by one of ordinary skill in the art as having potential benefit or therapeutic effect varies. 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 about or at least 10% inhibition (e.g., about or at least 20% inhibition, about or at least 30% inhibition, about or at least 40% inhibition, about or at least 50% inhibition, about or at least 60% inhibition, about or at least 70% inhibition, about or at least 80% inhibition, about or at least 90% inhibition, about or at least 95% inhibition, about or at least 98% inhibition). In various embodiments, the reduction provided by the methods of the present disclosure is about or at least 10% reduction (e.g., about or at least 20% reduction, about or at least 30% reduction, about or at least 40% reduction, about or at least 50% reduction, about or at least 60% reduction, about or at least 70% reduction, about or at least 80% reduction, about or at least 90% reduction, about or at least 95% reduction, about or at least 98% reduction).

[0240] The present specification further provides a method for treating a subject having cancer, such as CLDN6-expressing cancer. In various embodiments, the method comprises administering to the subject an effective amount of the pharmaceutical composition of the present disclosure to treat the cancer in the subject.

[0241] For the purposes herein, cancer in the methods disclosed herein can be any cancer, such as any malignant growth or tumor resulting from abnormal and uncontrolled cell division that can spread to other parts of the body, for example, via the lymphatic system or bloodstream. Cancer in some embodiments can be acute lymphoblastic cancer, acute myeloid leukemia, alveolar rhabdomyosarcoma, bone cancer, brain tumor, breast cancer, anal, anal canal, or anorectal cancer, eye cancer, intrahepatic bile duct cancer, joint cancer, cervical, gallbladder, or pleural cancer, nasal, nasal cavity, or middle ear cancer, oral cancer, vulvar cancer, chronic lymphocytic leukemia, chronic myeloid cancer, colon cancer, esophageal cancer, cervical cancer, gastrointestinal carcinoid tumor, Hodgkin lymphoma, hypopharyngeal cancer, kidney cancer, laryngeal cancer, liver cancer, lung cancer, malignant mesothelioma, melanoma, multiple myeloma, nasopharyngeal cancer, non-Hodgkin lymphoma, ovarian cancer, pancreatic cancer, peritoneal, omentum, and mesentery cancer, pharyngeal cancer, prostate cancer, rectal cancer, renal cancer (e.g., renal cell carcinoma (RCC)), small intestine cancer, soft tissue cancer, stomach cancer, testicular cancer, thyroid cancer, ureteral cancer, and bladder cancer, which is selected from the group consisting of. In certain embodiments, 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, stomach cancer, breast cancer, endometrial cancer, and colorectal cancer, hepatocellular carcinoma, glioblastoma, bladder cancer, lung cancer, for example, non-small cell lung cancer (NSCLC), bronchioloalveolar carcinoma. In various embodiments, the cancer is ovarian cancer, melanoma, bladder cancer, lung cancer, liver cancer, endometrial cancer. In various embodiments, the cancer is any cancer characterized by medium to high expression of CLDN6. For example, refer to FIGS. 1-3. In various embodiments, the cancer is acute myeloid leukemia, large cell type B cell lymphoma, stomach 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 oncocytoma, low grade glioma, adrenocortical carcinoma, glioblastoma, papillary renal cell carcinoma, lung squamous cell carcinoma, thyroid cancer, lung adenocarcinoma, pancreatic cancer, ovarian endometrioid carcinoma, uterine carcinosarcoma, or ovarian cancer. In various embodiments, the cancer is selected from ovarian cancer, endometrioid carcinoma, uterine cancer, lung cancer, stomach cancer, breast cancer head and neck squamous cell carcinoma (HNSCC), cervical cancer, and bladder cancer.

[0242] As used herein, the term "treat" and related terms do not necessarily mean 100% or complete treatment. Rather, the degree of treatment recognized by those of ordinary skill in the art as having potential benefit or therapeutic effect varies. In this regard, in the methods of treating cancer of the present disclosure, any amount or level of treatment can be provided. Further, the treatment provided by the methods of the present disclosure can include treatment of one or more conditions or symptoms or signs of the cancer being treated. Also, the treatment provided by the methods of the present disclosure can include slowing the progression of cancer. For example, the methods can treat cancer in terms of enhancing T cell activity or the immune response against cancer, suppressing tumor or cancer growth, reducing metastasis of tumor cells, increasing cell death of tumor or cancer cells, etc. In various embodiments, the 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, 2 months, 3 months, 4 months, 6 months, 1 year, 2 years, 3 years, 4 years, or more. In various embodiments, the methods treat by extending the survival of the subject.

[0243] The antigen-binding proteins of the present disclosure can also be used to detect CLDN6 in a sample or to diagnose CLDN6-positive cancer. Accordingly, the present disclosure provides a method for detecting claudin 6 (CLDN6) in a sample. In various embodiments, the method includes contacting the sample with an antigen-binding protein, complex, or fusion protein as described herein and evaluating an immune complex comprising the antigen-binding protein, complex, or fusion protein bound to CLDN6. The present disclosure also provides a method for diagnosing claudin 6 (CLDN6)-positive cancer in a subject. In various embodiments, the method includes contacting a biological sample comprising cells or tissue obtained from the subject with an antigen-binding protein, complex, or fusion protein as described herein and evaluating an immune complex comprising the antigen-binding protein, complex, or fusion protein bound to CLDN6.

[0244] Subject

[0245] In some embodiments of the present disclosure, the subject is a mammal, including but not limited to rodents such as mice and hamsters, lagomorphs such as rabbits, mammals from the order Carnivora including Felidae (cats) and Canidae (dogs), mammals from the order Artiodactyla including Bovidae (female cows) and Suidae (pigs), or mammals from the order Perissodactyla including Equidae (horses). In some aspects, the mammal is a primate mammal, a superfamily of Old World monkeys, or a Simoid (monkey) or euprimate mammal (humans and apes). In some aspects, the mammal is a human.

[0246] Kit

[0247] In some embodiments, the antigen-binding proteins of the present disclosure are provided in a kit. In various aspects, the kit contains the antigen-binding protein(s) as a unit dose. For the purposes herein, "unit dose" refers to an individual amount dispersed in a suitable carrier. In various aspects, the unit dose is an amount sufficient to provide the desired effect to a subject, such as inhibition of tumor growth, reduction of tumor size, treatment of cancer. Accordingly, provided herein is a kit comprising the antigen-binding protein of the present disclosure, optionally provided in unit doses. In various aspects, the kit contains several unit doses, for example, a one-week or one-month supply of unit doses, optionally each individually packaged or otherwise distinguishable from other unit doses. In some embodiments, the kit / unit dose components are packaged with instructions for administration to a patient. In some embodiments, the kit contains one or more devices for administration to a patient, such as needles and syringes. In some aspects, the antigen-binding protein of the present disclosure, its pharmaceutically acceptable salts, complexes containing the antigen-binding protein, or multimers or dimers containing the antigen-binding protein are already packaged in a ready-to-use form, such as a syringe, an intravenous bag, etc. In some aspects, the kit further contains other therapeutic or diagnostic agents or pharmaceutically acceptable carriers (e.g., solvents, buffers, diluents, etc.), including those described herein. In certain aspects, the kit contains the antigen-binding protein of the present disclosure together with an agent used in chemotherapy or radiotherapy, such as a therapeutic agent.

[0248] Various Embodiments

[0249] In various embodiments of the present disclosure, the bispecific antigen-binding protein binds to the human Claudin 6 (CLDN6) protein (SEQ ID NO: 200) and a second antigen, and (a) the antigen-binding protein binds to extracellular loop 2 (EL2) of the extracellular domain (ECD) of CLDN6 and does not bind to extracellular loop 1 (EL1) of the ECD of CLDN6, or (b) does not bind to any of Claudin 3 (CLDN3), Claudin 4 (CLDN4), and Claudin 9 (CLDN9), and inhibits the binding of a reference antibody to endogenously expressed CLDN6 by OVCA429 cells at less than about 1200 nM, or (c) a combination thereof. In various cases, the bispecific antigen-binding protein binds to an epitope within the amino acid sequence of WTAHAIIRDFYNPLVAEAQKREL (SEQ ID NO: 2), or to the amino acid sequence of TAHAIIRDFYNPL (SEQ ID NO: 3) or LVAEAQKREL (SEQ ID NO: 4) of CLDN6. In various aspects, the bispecific antigen-binding protein does not bind to one or more of Claudin 3 (CLDN3), Claudin 4 (CLDN4), and Claudin 9 (CLDN9). In various cases, the bispecific antigen-binding protein does not bind to CLDN3. In various cases, the bispecific antigen-binding protein binds to CLDN6, CLDN4, and CLDN9, but does not bind to CLDN3. In various cases, the bispecific antigen-binding protein binds to CLDN6 and CLDN4, but does not bind to CLDN3 or CLDN9. In various aspects, the bispecific antigen-binding protein binds to CLDN6 and CLDN9, but does not bind to CLDN3 or CLDN4.

[0250] In various cases, the bispecific antigen-binding protein of the present disclosure inhibits the binding of a reference antibody to CLDN6 endogenously expressed by OVCA429 cells at less than about 1200 nM, and the reference antibody comprises the light chain variable sequence of SEQ ID NO: 181 and the heavy chain variable sequence of SEQ ID NO: 182 or the light chain variable sequence of SEQ ID NO: 185 and the heavy chain variable sequence of SEQ ID NO: 186. In various embodiments, the bispecific antigen-binding protein of the present disclosure inhibits the binding of a reference antibody to CLDN6 endogenously expressed by OVCA429 cells at less than 1000 nM or less than 750 nM (e.g., less than 500 nM, less than 250 nM, less than about 100 nM), and the reference antibody comprises the light chain variable sequence of SEQ ID NO: 181 and the heavy chain variable sequence of SEQ ID NO: 182 or the light chain variable sequence of SEQ ID NO: 185 and the heavy chain variable sequence of SEQ ID NO: 186.

[0251] In various embodiments, the bispecific antigen-binding protein comprises: (a) the amino acid sequence of the heavy-chain CDR1 set forth in Table A or A1, or a sequence selected from the group consisting of SEQ ID NOs: 11, 17, 23, 29, 35, 41, 47, 53, 59, 65, 71, 77, 83, 89, 95, 101, 107, 113, 119, 125, 131, 452, 455, 461, 465, and 472, or a variant sequence thereof that differs by only one or two amino acids or has about or at least 70% (e.g., about or at least 85%, about or at least 90%) sequence identity; (b) the amino acid sequence of the heavy-chain CDR2 set forth in Table A or A1, or a sequence selected from the group consisting of SEQ ID NOs: 12, 18, 24, 30, 36, 42, 48, 54, 60, 66, 72, 78, 84, 90, 86, 102, 108, 114, 120, 126, 132, 475, 456, 462, 466, 468, and 473, or a variant sequence thereof that differs by only one or two amino acids or has about or at least 70% (e.g., about or at least 85%, about or at least 90%) sequence identity; (c) the amino acid sequence of the heavy-chain CDR3 set forth in Table A or A1, or a sequence selected from the group consisting of SEQ ID NOs: 13, 19, 25, 31, 37, 43, 49, 55, 61, 67, 73, 79, 85, 91, 97, 103, 109, 115, 121, 127, 133, 453, 457, 463, 467, 469, and 474, or a variant sequence thereof that differs by only one or two amino acids or has about or at least 70% (e.g., about or at least 85%, about or at least 90%) sequence identity; (d) the amino acid sequence of the light-chain CDR1 set forth in Table A or A1, or a sequence selected from the group consisting of SEQ ID NOs: 8, 14, 20, 32, 38, 44, 50, 56, 62, 68, 74, 80, 86, 92, 98, 104, 110, 116, 122, 128, 449, 476, 458, 464, and 470, or a variant sequence thereof that differs by only one or two amino acids or has about or at least 70% (e.g., about or at least 85%, about or at least 90%) sequence identity;(e) The amino acid sequence of the light chain CDR2 described in Table A or A1, or a sequence selected from the group consisting of SEQ ID NOs: 9, 15, 21, 27, 33, 39, 45, 51, 57, 63, 69, 75, 81, 87, 93, 99, 105, 111, 117, 123, 129, 450, 477, 459, and 471, or a variant sequence thereof, wherein only one or two amino acids are different, or having about or at least 70% (e.g., about or at least 85%, about or at least 90%) sequence identity; (f) The amino acid sequence of the light chain CDR3 described in Table A or A1, or a sequence selected from the group consisting of SEQ ID NOs: 10, 16, 22, 28, 34, 40, 46, 52, 58, 64, 70, 76, 82, 88, 94, 100, 106, 112, 118, 124, 130, 451, 454, and 460, or a variant sequence thereof, wherein only one or two amino acids are different, or having about or at least 70% (e.g., about or at least 85%, about or at least 90%) sequence identity; (g) A combination of any two or more of (a) to (f).

[0252] In various embodiments, the bispecific antigen-binding protein comprises the light chain CDR1 amino acid sequence, the light chain CDR2 amino acid sequence, and the light chain CDR3 amino acid sequence set forth in Table A or A1, and one or two of the heavy chain CDR amino acid sequences set forth in Table A or A1. In some cases, the bispecific antigen-binding protein comprises the heavy chain CDR1 amino acid sequence, the heavy chain CDR2 amino acid sequence, and the heavy chain CDR3 amino acid sequence set forth in Table A or A1, and one or two of the light chain CDR amino acid sequences set forth in Table A or A1. In various embodiments, the bispecific 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: 116-121, (r) SEQ ID NOs: 8-13, (t) SEQ ID NOs: 68-73, (u) SEQ ID NOs: 14-19, (v) SEQ ID NOs: 20-25, (v) SEQ ID NOs: 449-453 and 475, (w) SEQ ID NOs: 476-477, 454-457, (x) SEQ ID NOs: 458-463, (y) SEQ ID NOs: 57, 58, 464-467, (z) SEQ ID NOs: 68-71 and 468-469, and (aa) SEQ ID NO: 112, and 470-474.In various aspects, the bispecific antigen-binding protein comprises (a) a heavy chain variable region amino acid sequence as set forth in Table B, or a sequence selected from the group consisting of SEQ ID NOs: 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, and 175, or a variant sequence thereof, wherein only one or two amino acids are different, or which has about or at least 70% (e.g., about or at least 85%, about or at least 90%) sequence identity, or (b) a light chain variable region amino acid sequence as set forth in Table B, or a sequence selected from the group consisting of SEQ ID NOs: 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, and 176, or a variant sequence thereof, wherein only one or two amino acids are different, or which has about or at least 70% (e.g., about or at least 85%, about or at least 90%) sequence identity, or both (a) and (b). In various aspects, the bispecific 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.

[0253] In various embodiments, the bispecific antigen-binding protein comprises (a) a heavy chain variable region amino acid sequence as set forth in Table B1 or 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 a variant sequence thereof that differs by only one or two amino acids or has about or at least 70%, or about 80%, or about 90%, or about 95% sequence identity, or (b) a light chain variable region amino acid sequence as set forth in Table B1 or C, or a sequence selected from the group consisting of SEQ ID NOs: 380-383, 388-390, 397-402, 409-411, 414, 415, 420, 421, and 479, 481, 483, 485, 487, and 489, or a variant sequence thereof that differs by only one or two amino acids or has about or at least 70%, or about 80%, or about 90%, or about 95% sequence identity, or (c) both (a) and (b). In various aspects, the bispecific antigen-binding protein comprises a pair of amino acid sequences as set forth in Table D.

[0254] The present disclosure provides a bispecific antigen-binding protein comprising: (A) an HC CDR1 comprising the amino acid sequence YTFTXYT, wherein X is T, V, D, or S (SEQ ID NO: 452), and optionally comprising the amino acid sequence YTFTTYT (SEQ ID NO: 11); (B) an HC CDR2 comprising the amino acid sequence IXPSSGYT, wherein X is Q, S, A, or N (SEQ ID NO: 475), and optionally comprising the amino acid sequence INPSSGYT (SEQ ID NO: 12); (C) an HC CDR3 comprising the amino acid sequence AXGDYYVAY, wherein X is N, Q, H, or D (SEQ ID NO: 453), and optionally comprising the amino acid sequence ANGDYYVAY (SEQ ID NO: 13); (D) an LC CDR1 comprising the amino acid sequence SSVSSXY, wherein X is T, V, F, or D (SEQ ID NO: 449), and optionally comprising the amino acid sequence SSVSSTY (SEQ ID NO: 8); (E) an LC CDR2 comprising the amino acid sequence XTX, wherein X at position 1 is S, T, Q, or A, and X at position 3 is S, T, D, or Q (SEQ ID NO: 450), and optionally comprising the amino acid sequence STS (SEQ ID NO: 9); and (F) an LC CDR3 comprising the amino acid sequence HXYXRSPLT, wherein X at position 2 is Q, H, or S, and X at position 4 is H, Y, Q, or S (SEQ ID NO: 451), and optionally comprising the amino acid sequence HQYHRSPLT (SEQ ID NO: 10).

[0255] A bispecific antigen-binding protein is provided, which comprises: (A) an HC CDR1 having an amino acid sequence of FTFSXYX, wherein X at position 5 is N, S, R, Q, or A, and X at position 7 is W, H, Y, F (SEQ ID NO: 455), and optionally comprises an amino acid sequence of FTFSNYW (SEQ ID NO: 23); (B) an HC CDR2 having an amino acid sequence of IRLKXDXYAT, wherein X at position 5 is S, N, A, or T, and X at position 7 is Q, S, A, N (SEQ ID NO: 456), and optionally comprises an amino acid sequence of IRLKSDNYAT (SEQ ID NO: 24); (C) an HC CDR3 having an amino acid sequence of XDGPPSGX, wherein X at position 1 is N, D, or T, and X at position 8 is S, T, A, C, or Y (SEQ ID NO: 457), and optionally comprises an amino acid sequence of NDGPPSGC (SEQ ID NO: 25); (D) an LC CDR1 having an amino acid sequence of EXIYSY, wherein X is Q, S, A, D, or N (SEQ ID NO: 476), and optionally comprises an amino acid sequence of ENIYSY (SEQ ID NO: 20); (E) an LC CDR2 having an amino acid sequence of XAK, wherein X at position 1 is Q, S, A, D, or N (SEQ ID NO: 477), and optionally comprises an amino acid sequence of NAK (SEQ ID NO: 21); and (F) an LC CDR3 having an amino acid sequence of QXHYXVPWT, wherein X at position 2 is H, Q, S, or T, and X at position 5 is T, S, N, or G (SEQ ID NO: 454), and optionally comprises an amino acid sequence of QHHYTVPWT (SEQ ID NO: 22).

[0256] A bispecific antigen-binding protein is provided, which comprises: (A) an HC CDR1 comprising the amino acid sequence YTX TXYT, wherein X at position 3 is F, Y, S, or T, and X at position 5 is S, T, Y, or D (SEQ ID NO: 461), and optionally comprising the amino acid sequence YTFTSYT (SEQ ID NO: 29); (B) an HC CDR2 comprising the amino acid sequence IXPSSXYT, wherein X at position 2 is Q, S, A, or N, and X at position 6 is T, S, V, D, or G (SEQ ID NO: 462), and optionally comprising the amino acid sequence INPSSTYT (SEQ ID NO: 30); (C) an HC CDR3 comprising the amino acid sequence XRGEXGGFAY, wherein X at position 1 is S, A, T, or V, and X at position 5 is L, V, or F (SEQ ID NO: 463), and optionally comprising the amino acid sequence SRGELGGFAY (SEQ ID NO: 31); (D) an LC CDR1 comprising the amino acid sequence QSLVHSXGXTY, wherein X at position 7 is D, N, E, Q, S, or A, and X at position 9 is Q, S, A, D, or N (SEQ ID NO: 458), and optionally comprising the amino acid sequence QSLVHSDGNTY (SEQ ID NO: 26); (E) an LC CDR2 comprising the amino acid sequence XVX, wherein X at position 1 is K, Q, or R, and X at position 3 is S, T, or V (SEQ ID NO: 459), and optionally comprising the amino acid sequence KVS (SEQ ID NO: 27); and (F) an LC CDR3 comprising the amino acid sequence SXXTHVPYT, wherein X at position 2 is Q, H, or T, and X at position 3 is S, G, T, or D (SEQ ID NO: 460), and optionally comprising the amino acid sequence SQSTHVPYT (SEQ ID NO: 28).

[0257] In various embodiments, the bispecific antigen-binding protein comprises the following. (a) The heavy chain CDR1 amino acid sequence of SEQ ID NO: 504 or SEQ ID NO: 507, or a variant sequence thereof, wherein only one or two amino acids are different, or having about or at least 70% sequence identity. (b) The heavy chain CDR2 amino acid sequence of SEQ ID NO: 505 or SEQ ID NO: 508, or a variant sequence thereof, wherein only one or two amino acids are different, or which has about or at least 70% sequence identity, (c) The heavy chain CDR3 amino acid sequence of SEQ ID NO: 506 or SEQ ID NO: 509, or a variant sequence thereof, wherein only one or two amino acids are different, or which has about or at least 70% sequence identity, (d) The light chain CDR1 amino acid sequence of SEQ ID NO: 449 or SEQ ID NO: 476, or a variant sequence thereof, wherein only one or two amino acids are different, or which has about or at least 70% sequence identity, (e) The light chain CDR2 amino acid sequence of SEQ ID NO: 450 or SEQ ID NO: 477, or a variant sequence thereof, wherein only one or two amino acids are different, or which has about or at least 70% sequence identity, (f) The light chain CDR3 amino acid sequence of SEQ ID NO: 451 or SEQ ID NO: 454, or a variant sequence thereof, wherein only one or two amino acids are different, or which has about or at least 70% sequence identity, (g) A combination of any two or more of (a) to (f).

[0258] Optionally, the variant sequence has at least about 80%, about or at least 85%, about or at least 90% or about or at least 95% sequence identity.

[0259] In an exemplary embodiment, the bispecific antigen-binding protein comprises the light chain CDR1 amino acid sequence of SEQ ID NO: 449, the light chain CDR2 amino acid sequence or SEQ ID NO: 450, and the light chain CDR3 amino acid sequence or SEQ ID NO: 451, and one or two of the heavy chain CDR1 amino acid sequence of SEQ ID NO: 504, the heavy chain CDR2 amino acid sequence or SEQ ID NO: 505, and the heavy chain CDR3 amino acid sequence or SEQ ID NO: 506. In various cases, the antigen-binding protein comprises the light chain CDR1 amino acid sequence of SEQ ID NO: 476, the light chain CDR2 amino acid sequence or SEQ ID NO: 477, and the light chain CDR3 amino acid sequence or SEQ ID NO: 454, and one or two of the heavy chain CDR1 amino acid sequence of SEQ ID NO: 507, the heavy chain CDR2 amino acid sequence or SEQ ID NO: 508, and the heavy chain CDR3 amino acid sequence or SEQ ID NO: 509. Optionally, the antigen-binding protein comprises six CDR amino acid sequences selected from the group consisting of SEQ ID NOs: 449-451 and 504-506, and SEQ ID NOs: 476, 477, 454 and 507-509.

[0260] In an exemplary embodiment, the bispecific antigen-binding protein comprises (a) any one of the heavy chain variable region amino acid sequences of SEQ ID NOs: 490-503, or the heavy chain variable region amino acid sequences shown as S1-S12 in FIG. 22, or a variant sequence thereof, wherein only one or two amino acids are different, or having about or at least 70% sequence identity, or (b) any one of the light chain variable region amino acid sequences of SEQ ID NOs: 380-383, 388-390, 479, and 481, or the light chain variable region amino acid sequences shown as S1-S12 in FIG. 22, or a variant sequence thereof, wherein only one or two amino acids are different, or having about or at least 70% sequence identity, or (c) both (a) and (b). In some embodiments, the variant sequence has at least about 80% or at least about 85% sequence identity, or the variant sequence has at least about 90% or at least about 95% sequence identity.

[0261] In an exemplary case, the bispecific antigen-binding protein comprises the following pair of amino acid sequences. Array numbers 389 and 490, Array numbers 389 and 491, Array numbers 389 and 492, Array numbers 389 and 493, Array numbers 389 and 494, Array numbers 389 and 495, Array numbers 383 and 496, Array numbers 383 and 497, Array numbers 383 and 498, Array numbers 383 and 499, Array numbers 383 and 500, Array numbers 383 and 501, Array numbers 383 and 503, Array numbers 389 and 502, The sequence of the heavy chain variable region displayed as S1 in Figure 22 and the sequence of the light chain variable region displayed as S1 in Figure 22, The sequence of the heavy chain variable region displayed as S2 in Figure 22 and the sequence of the light chain variable region displayed as S2 in Figure 22, The sequence of the heavy chain variable region displayed as S3 in Figure 22 and the sequence of the light chain variable region displayed as S3 in Figure 22, The sequence of the heavy chain variable region displayed as S4 in Figure 22 and the sequence of the light chain variable region displayed as S4 in Figure 22, The sequence of the heavy chain variable region displayed as S5 in Figure 22 and the sequence of the light chain variable region displayed as S5 in Figure 22, The sequence of the heavy chain variable region displayed as S6 in Figure 22 and the sequence of the light chain variable region displayed as S6 in Figure 22, The sequence of the heavy chain variable region displayed as S7 in Figure 22 and the sequence of the light chain variable region displayed as S7 in Figure 22, The sequence of the heavy chain variable region displayed as S8 in Figure 22 and the sequence of the light chain variable region displayed as S8 in Figure 22, The sequence of the heavy chain variable region displayed as S9 in Figure 22 and the sequence of the light chain variable region displayed as S9 in Figure 22, The sequence of the heavy chain variable region displayed as S10 in Figure 22 and the sequence of the light chain variable region displayed as S10 in Figure 22, The sequence of the heavy chain variable region shown as S11 in FIG. 22 and the sequence of the light chain variable region shown as S11 in FIG. 22, or the sequence of the heavy chain variable region shown as S12 in FIG. 22 and the sequence of the light chain variable region shown as S12 in FIG. 22.

[0262] In some embodiments, the bispecific antigen-binding protein is an antibody, e.g., a monoclonal antibody. In various embodiments, the antibody is an IgG. Optionally, the antigen-binding protein inhibits at least about 50% of colony growth in a soft agar 3D proliferation assay, inhibits tumor growth in xenograft mice injected with human cancer cells, inhibits tumor growth in xenograft mice injected with ovarian cancer cells, melanoma cancer cells, bladder cancer cells, or endometrial cancer cells, or inhibits at least 50% of tumor growth in xenograft mice injected with ovarian cancer cells, bladder cancer cells, or endometrial cancer cells.

[0263] Accordingly, in various embodiments, the present disclosure provides a bispecific antigen-binding protein comprising the following. (a) The heavy chain CDR1 amino acid sequence of SEQ ID NO: 504 or SEQ ID NO: 507, or a variant sequence thereof, wherein only one or two amino acids are different, or having about or at least 70% sequence identity, (b) The heavy chain CDR2 amino acid sequence of SEQ ID NO: 505 or SEQ ID NO: 508, or a variant sequence thereof, wherein only one or two amino acids are different, or having about or at least 70% sequence identity, (c) The heavy chain CDR3 amino acid sequence of SEQ ID NO: 506 or SEQ ID NO: 509, or a variant sequence thereof, wherein only one or two amino acids are different, or having about or at least 70% sequence identity, (d) The light chain CDR1 amino acid sequence of SEQ ID NO: 449 or SEQ ID NO: 476, or a variant sequence thereof, wherein only one or two amino acids are different, or having about or at least 70% sequence identity, (e) The light chain CDR2 amino acid sequence of SEQ ID NO: 450 or SEQ ID NO: 477, or a variant sequence thereof, wherein only 1 or 2 amino acids are different, or having about or at least 70% sequence identity, (f) The light chain CDR3 amino acid sequence of SEQ ID NO: 451 or SEQ ID NO: 454, or a variant sequence thereof, wherein only 1 or 2 amino acids are different, or having about or at least 70% sequence identity, or (g) A combination of any two or more of (a) to (f).

[0264] Also provided is a bispecific antigen-binding protein comprising 6 CDR amino acid sequences selected from the group consisting of SEQ ID NOs: 449 to 451 and 504 to 506, and SEQ ID NOs: 476, 477, 454 and 507 to 509.

[0265] The present disclosure provides a bispecific antigen-binding protein comprising the following. (a) Any one of the heavy chain variable region amino acid sequences of SEQ ID NOs: 490 to 503, or the heavy chain variable region amino acid sequences shown as S1 to S12 in FIG. 22, or a variant sequence thereof, wherein only 1 or 2 amino acids are different, or having about or at least 70% sequence identity, or (b) Any one of the light chain variable region amino acid sequences of SEQ ID NOs: 380 to 383, 388 to 390, 479, and 481, or the light chain variable region amino acid sequences shown as S1 to S12 in FIG. 22, or a variant sequence thereof, wherein only 1 or 2 amino acids are different, or having about or at least 70% sequence identity, or (c) Both (a) and (b).

[0266] In various embodiments, the variant sequence has at least about 85% sequence identity or about 90% or about 95% sequence identity.

[0267] The present disclosure also provides a bispecific antigen-binding protein comprising a pair of amino acid sequences selected from the group consisting of the following. SEQ ID NO: 389 and 490, SEQ ID NO: 389 and 491, SEQ ID NO: 389 and 492, SEQ ID NO: 389 and 493, SEQ ID NO: 389 and 494, SEQ ID NO: 389 and 495, SEQ ID NO: 383 and 496, SEQ ID NO: 383 and 497, SEQ ID NO: 383 and 498, SEQ ID NO: 383 and 499, SEQ ID NO: 383 and 500, SEQ ID NO: 383 and 501, SEQ ID NO: 383 and 503, SEQ ID NO: 389 and 502, The sequence of the heavy chain variable region shown as S1 in FIG. 22 and the sequence of the light chain variable region shown as S1 in FIG. 22, The sequence of the heavy chain variable region shown as S2 in FIG. 22 and the sequence of the light chain variable region shown as S2 in FIG. 22, The sequence of the heavy chain variable region shown as S3 in FIG. 22 and the sequence of the light chain variable region shown as S3 in FIG. 22, The sequence of the heavy chain variable region shown as S4 in FIG. 22 and the sequence of the light chain variable region shown as S4 in FIG. 22, The sequence of the heavy chain variable region shown as S5 in FIG. 22 and the sequence of the light chain variable region shown as S5 in FIG. 22, The sequence of the heavy chain variable region shown as S6 in FIG. 22 and the sequence of the light chain variable region shown as S6 in FIG. 22, The sequence of the heavy chain variable region shown as S7 in FIG. 22 and the sequence of the light chain variable region shown as S7 in FIG. 22, The sequence of the heavy chain variable region shown as S8 in FIG. 22 and the sequence of the light chain variable region shown as S8 in FIG. 22, The sequence of the heavy chain variable region shown as S9 in FIG. 22 and the sequence of the light chain variable region shown as S9 in FIG. 22, The sequence of the heavy chain variable region shown as S10 in FIG. 22 and the sequence of the light chain variable region shown as S10 in FIG. 22, the sequence of the heavy chain variable region shown as S11 in FIG. 22 and the sequence of the light chain variable region shown as S11 in FIG. 22, or the sequence of the heavy chain variable region shown as S12 in FIG. 22 and the sequence of the light chain variable region shown as S12 in FIG. 22.

[0268] This specification provides a bispecific antigen-binding protein comprising the following. (a) The heavy chain variable region amino acid sequence described as SEQ ID NO: 510 or 513, or shown in FIG. 23 or FIG. 25, or a variant sequence thereof, wherein only 1 or 2 amino acids are different, or having about or at least 70% sequence identity, or (b) The light chain variable region amino acid sequence described as SEQ ID NO: 511 or 512, or shown in FIG. 24 or FIG. 26, or a variant sequence thereof, wherein only 1 or 2 amino acids are different, or having about or at least 70% sequence identity, or (c) Both (a) and (b).

[0269] This disclosure also provides an antigen-binding protein comprising a pair of amino acid sequences, and such pair comprises the following. (a) The heavy chain variable region amino acid sequence described as SEQ ID NO: 510 and the light chain variable region amino acid sequence described as SEQ ID NO: 511, or a variant sequence thereof, wherein only 1 to 5 amino acids are different, or having about or at least 70% sequence identity, and optionally, the 1 to 5 different amino acids are those shown in FIG. 23 in the case of the heavy chain or FIG. 24 in the case of the light chain, or (b) The heavy-chain variable region amino acid sequence described as SEQ ID NO: 513 and the light-chain variable region amino acid sequence described as SEQ ID NO: 512, or a variant sequence thereof, in which only 1 to 5 amino acids are different, or which has about or at least 70% sequence identity, or optionally, the 1 to 5 different amino acids are those shown in Figure 25 in the case of the heavy chain or those shown in Figure 26 in the case of the light chain.

[0270] In various aspects, the antigen-binding proteins disclosed herein comprise an Fc polypeptide comprising a defucosylated glycan.

[0271] In various aspects, the bispecific antigen-binding proteins of the present disclosure are based on antibodies, such as monoclonal antibodies, such as IgG. In various aspects, the bispecific antigen-binding protein inhibits colony growth by at least about 50% in a soft agar 3D growth assay or inhibits tumor growth in xenograft mice injected with human cancer cells. In various aspects, the bispecific antigen-binding protein inhibits tumor growth in xenograft mice injected with ovarian cancer cells, melanoma cancer cells, bladder cancer cells, or endometrial cancer cells. In various cases, the bispecific antigen-binding protein inhibits tumor growth by at least 50% in xenograft mice injected with ovarian cancer cells, bladder cancer cells, or endometrial cancer cells.

[0272] The present disclosure provides a complex comprising a bispecific antigen-binding protein described herein and a heterologous moiety. In an exemplary aspect, the complex comprises a cytotoxic agent or chemotherapeutic agent, such as any of those described herein. Chemotherapeutic agents in various aspects are antimitotic agents that inhibit cell division by blocking tubulin polymerization. In some cases, the antimitotic agent is an auristatin and optionally MMAE.

[0273] The present disclosure also provides a fusion protein comprising a bispecific antigen-binding protein described herein. The present disclosure further provides a nucleic acid comprising a nucleotide sequence encoding the antigen-binding protein, complex, or fusion protein of the present disclosure. The present disclosure provides a vector comprising a nucleic acid comprising a nucleotide sequence encoding the antigen-binding protein, complex, or fusion protein of the present disclosure. The present disclosure additionally provides a host cell comprising the nucleic acid or vector of the present disclosure.

[0274] The present disclosure provides a method for producing a bispecific antigen-binding protein that binds to the Claudin 6 (CLDN6) protein, comprising (i) culturing the host cells of the present disclosure in a cell culture medium, wherein the host cells comprise a nucleic acid comprising a nucleotide sequence encoding the antigen-binding protein according to any one of the preceding claims, and (ii) collecting the antigen-binding protein from the cell culture medium. The present disclosure also provides a method for producing a fusion protein comprising a bispecific antigen-binding protein that binds to the Claudin 6 (CLDN6) protein, comprising (i) culturing the host cells of the present disclosure in a cell culture medium, wherein the host cells comprise a nucleic acid comprising a nucleotide sequence encoding the fusion protein of the present disclosure, and (ii) collecting the fusion protein from the cell culture medium.

[0275] The present disclosure further provides a method for producing a pharmaceutical composition comprising combining the bispecific antigen-binding protein, complex, fusion protein, nucleic acid, vector, host cell, or combination thereof of the present disclosure with a pharmaceutically acceptable carrier, diluent, or additive. The present disclosure also provides a pharmaceutical composition comprising the bispecific antigen-binding protein, complex, fusion protein, nucleic acid, vector, host cell, or combination thereof of the present disclosure and a pharmaceutically acceptable carrier, diluent, or additive.

[0276] Provided herein are methods of treating a subject having CLDN6-expressing cancer, including administering to the subject a pharmaceutical composition described herein in an amount effective to treat cancer. Also provided are methods of inhibiting tumor growth in a subject, including administering to the subject a pharmaceutical composition described herein in an amount effective to inhibit tumor growth. The present disclosure provides a method of reducing tumor size in a subject, including administering to the subject a pharmaceutical composition described herein in an amount effective to reduce tumor size. Further provided is a method of preventing cancer recurrence in a subject, including administering to the subject a pharmaceutical composition described herein in an amount effective to prevent cancer recurrence.

[0277] The present disclosure provides a method of detecting claudin 6 (CLDN6) in a sample, including contacting the sample with an antigen-binding protein, complex, or fusion protein of the present disclosure, and evaluating an immune complex comprising the antigen-binding protein, complex, or fusion protein bound to CLDN6. Also provided herein is a method of diagnosing a CLDN6-positive cancer in a subject, including contacting a biological sample comprising cells or tissue obtained from the subject with an antigen-binding protein, complex, or fusion protein of the present disclosure, and evaluating an immune complex comprising the antigen-binding protein, complex, or fusion protein bound to CLDN6.

[0278] The present disclosure also provides a method of treating cancer in a subject diagnosed as an individual with low overexpression of CLDN6. In various embodiments, the method includes administering to the subject a pharmaceutical composition disclosed herein in an amount effective to prevent recurrence of cancer. In some aspects, administration induces apoptosis in tumor cells and, optionally, administration induces apoptosis in CLDN6-expressing cells. In various aspects, the subject has a tumor, and the tumor is classified semi-quantitatively into one of four groups: high-expression individuals, moderate-expression individuals, low-expression individuals, and non-expression individuals. In various cases, high-expression individuals are defined as having CLDN6 RNA greater than 12 log Fragments Per Kilobase Million (FPKM), where the CLDN6 RNA is measured by RNASeq or the CLDN6 protein level is greater than 3+ when measured by immunohistochemistry (IHC). In various cases, moderate-expression individuals are defined as having CLDN6 RNA greater than 10 log FPKM, where the CLDN6 RNA is measured by RNASeq or the CLDN6 protein level is greater than 2+ when measured by IHC. In various cases, low-expression individuals are defined as having CLDN6 RNA greater than 6 log FPKM, where the CLDN6 RNA is measured by RNASeq or the CLDN6 protein level is greater than 1+ when measured by IHC. In various cases, non-expression individuals are defined as having CLDN6 RNA less than 6 log FPKM, where the CLDN6 RNA is measured by RNASeq or the CLDN6 protein level is below the IHC detection limit. In various aspects, the subject having the tumor is similarly described as a high-expression individual, moderate-expression individual, low-expression individual, or non-expression individual of CLDN6.

[0279] The following examples are provided solely to illustrate the present disclosure and are not intended to limit its scope in any way.

Examples

[0280] Example 1 This example shows the analysis of CLDN6 RNA levels in various cell and tissue sources.

[0281] To establish a baseline for CLDN6 expression in various source materials, the expression levels of CLDN6 in patient samples, normal tissues, and cell lines created by the Translational Oncology Research laboratory (TORL) were assayed.

[0282] The levels of CLDN6 RNA in patient samples were measured using information included in the The Cancer Genome Atlas (TCGA) database managed by the National Cancer Institute (NCI). The CLDN6 levels in normal tissues were measured using information from the Genotype-Tissue Expression (GTEx) database maintained by the Common Fund. Tissue analysis from the GTEx database showed that CLDN6 was detectable in various sites including, among others, the brain, pituitary gland, pancreas, kidney, lung, thyroid, and neck (Figure 1).

[0283] The CLDN6 expression levels were measured in TORL cancer cell lines using an Agilent 44K microarray (4x44K array chip, Agilent Technologies, Santa Clara, CA) and RNA sequencing (RNA-Seq) assays. RNASeq utilized the "RNASeq for quantification" service by BGI Americas (Cambridge, MA) and was performed by the company. As shown in Figures 2 and 3, cells from ovarian cancer, head and neck cancer, lung cancer, and bladder cancer expressed the highest levels of CLDN6, but CLDN6 expression levels were detectable in cells from breast cancer, kidney cancer, colon cancer, sarcoma, and liver cancer.

[0284] Example 2 This example shows the production of cells engineered to overexpress CLDN6.

[0285] A model engineered to overexpress CLDN6 was established. These models were used to determine the efficacy of the CLDN6 antibody described in Example 5. Briefly, the nucleotide sequence encoding CLDN6 was engineered into a bicistronic vector having a CMV promoter and an internal ribosome entry site (IRES) of a weakened encephalomyocarditis virus (EMCV) sequence. The IRES was positioned between the gene of interest (GOI) cDNA (CLDN6) and puromycin cDNA. The woodchuck post-transcriptional regulatory element (WPRE) was positioned downstream of the puromycin cDNA. The 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.

[0286] The expression vector was transduced into HEK293T cells (for screening) and NIH3T3 cells (for immunization) by virus. Positive transduced cells were selected based on survival in puromycin-containing medium (1 μg / ml). The positively selected cells were subcloned to obtain a clonal population of stable and homogeneous CLDN6-overexpressing cells.

[0287] The expression of the subcloned CLDN6 was confirmed by flow cytometry using a reference CLDN6 monoclonal antibody (mAb) on a BD Biosciences Accuri™ flow cytometer (San Jose, CA). Secondary antibody and conjugate: Goat anti-mouse IgG (minimal cross-reactivity) antibody Alexa Fluor® 647 (Biolegend, San Diego, CA; catalog number 405322) was used to detect the binding activity between the reference CLDN6 mAb and the CLDN6 expressed by the subclone.

[0288] The intracellular localization of CLDN6 was determined by fluorescence microscopy using a Cellavista (registered trademark) imaging system (Synentec (Mountain View, CA)) with cells expressing a CLDN6-green fluorescent protein (GFP) fusion protein. As shown in Figure 4, GFP fluorescence was detected in the cell membrane, demonstrating that CLDN6 is localized in the cell membrane.

[0289] Experimental Example 3 This example shows the production of reference antibodies and control antibodies.

[0290] Benchmark (reference) CLDN6-specific antibodies and control antibodies were prepared by cloning the heavy-chain variable region and light-chain variable region of the antibodies into the ExpiCHO (trademark) expression system (ThermoFisher Scientific, Waltham, MA) to produce recombinant mouse IgG2A chimeric antibodies. These antibodies were tested in parallel with the newly prepared CLDN6-specific antibodies described in Example 5.

[0291] Briefly, the ExpiCHO (trademark) expression system (catalog number: A29133, ThermoFisher Scientific, USA) was used according to the manufacturer's operating procedures to transfect a plasmid containing the sequence of the control antibody and the sequence of the benchmark antibody. The cells were cultured at 37 °C, 8% CO 2 on the first day, and then, after transfection, at 32 °C, 5% CO 2It was cultured at [condition]. The antibody was purified by centrifuging at 1,000 g for 10 minutes and then at 5,000 g for 30 minutes to clarify the ExpiCHO™ medium. Subsequently, the supernatant was filtered using a 0.45 μm filter and then a 0.22 μm filter. Subsequently, the supernatant was subjected to affinity purification using Protein A / G resin (Life Technologies, Carlsbad, CA; catalog number 20424) according to the manufacturer's procedure. Prior to ELISA purification, the antibody titer in the medium was roughly determined to ensure that the input medium volume occupied less than 80% of the resin binding capacity. After incubation, the resin was washed with PBS and eluted with elution buffer (Life Technologies, catalog number 21004). The eluted fraction was immediately adjusted to physiological pH by adding Tris buffer at pH 8.0. The purified antibody was then subjected to buffer exchange and protein concentration using an Amicon Ultra-15 centrifugal filter unit (Life Technologies, catalog number UFC900324) in PBS buffer. The antibody concentration was determined by BCA protein assay. SDS-PAGE and Coomassie staining were performed to test the antibody purity. The purified protein was aliquoted and stored at -80 °C for long-term storage or maintained at 4 °C for immediate use.

[0292] The integrity of the antibody was evaluated by comparing non-reducing and reducing conditions by SDS-PAGE followed by Coomassie staining. Under non-reducing conditions, one dominant band was observed around 150 kDa, and under reducing conditions, two bands were observed at 50 kDa and 25 kDa.

[0293] Antibodies specific for other CLDN family members with sequence similarity (Figure 5), namely CLDN3, CLDN4, and CLDN9, were prepared essentially in the same manner except that the antibody sequence contained in the plasmid was the antibody sequence specific for CLDN3, CLDN4, or CLDN9.

[0294] Example 4 This example shows the characterization of cell lines with high endogenous CLDN6 expression.

[0295] A panel of cancer cell lines was analyzed for their endogenous CLDN6 expression by FACS and Western blot. Briefly, the binding of antibodies to the target was evaluated by FACS using cells overexpressing CLDN6 (e.g., HEK293T cells overexpressing CLDN6 described in Example 2), and cell lines endogenously expressing CLDN6 at high or low levels determined in Example 1. CLDN6-expressing cells were incubated on ice for 30 minutes with a reference antibody or control antibody (described in Example 3), washed, and then incubated on ice for 30 minutes with an Alexa Fluor® 647-conjugated goat anti-mouse IgG (minimal cross-reactivity) antibody (Biolegend catalog number 405322). Fluorescence was read on a BD Biosciences Accuri™ flow cytometer (San Jose, CA).

[0296] Western blots were performed on nitrocellulose using reference and control antibodies. Briefly, samples from cell lysates were boiled to denature the protein contents. SDS-PAGE (SDS-polyacrylamide gel electrophoresis) was used to separate the denatured proteins by polypeptide length. The separated proteins were then transferred from the acrylamide gel to a nitrocellulose membrane. The membrane was blocked using a 2% bovine serum albumin (BSA) solution to minimize non-specific antibody binding. The membrane was incubated with a reference antibody or control antibody. The membrane was stained with a horseradish peroxidase (HRP)-conjugated secondary antibody that recognizes the reference or control antibody, and detection of the secondary antibody was performed by chemiluminescence.

[0297] Control and reference antibodies were evaluated using the overexpression strains, and at the time of evaluation, those control and reference antibodies were used to characterize the endogenous cell lines. Cells overexpressing CLDN6 were included in these assays as positive controls.

[0298] In the FACS assay, in addition to endometrial cancer cell lines, four ovarian cancer cell lines, a bladder cancer cell line, a lung cancer cell line, and an upper gastrointestinal cancer cell line were shown to express CLDN6 at high levels on the surface. High-level CLDN6 expression was also detected by Western blot. In two additional ovarian cancer cell lines, an additional liver cancer cell line, an additional lung cancer cell line, and an additional upper gastrointestinal cancer cell line, when detected by Western blot, CLDN6 was shown to be expressed at moderate levels on the surface. Endometrial tumor cells and bladder tumor cells also expressed high levels of CLDN6 as xenografts in vivo. The endogenous expression levels of CLDN6 by the cancer cell lines tested are summarized in Table 2.

Table 20

[0299] Example 5 This example shows the immunization of mice to produce CLDN6-specific antibodies.

[0300] Balb / c and CD1 mice were immunized to produce CLDN6-specific antibodies by using a mixture of three different peptide immunogens according to the method of the Fred Hutchinson Cancer Research Center. The three peptides covered the second loop (i.e., EL2) of the CLDN6 extracellular domain. The peptides included the full length of EL2, the peptide up to the first (N-terminal) half of EL2, and the peptide up to the remaining (C-terminal) half of EL2. The sequences of the three peptides are listed in Table 3.

Table 21

[0301] The mice were also immunized with 3T3 cells overexpressing full-length CLDN6 using a plasmid containing the human CLDN6-myc-DDK expression vector.

[0302] Spleen cells were collected from immunized mice and fused with a myeloma strain using BTX Electrofusion (BTX, Holliston, MA) to generate hybridomas. 7680 primary hybridoma cultures were generated and cultured in 384-well plates. The ability of the antibodies to bind to the peptides was evaluated by a bead array using beads expressing three different peptide targets. 1920 potential antibodies were re-aligned in 96-well plates and further screened by flow cytometry against an endogenous cell line model and an artificial cell line model.

[0303] Subsequently, the supernatants of the positive hybridomas were counter-screened by flow cytometry against an endogenous model and an artificial model of proteins having sequence similarity to the target region (e.g., other CLDN proteins). From the secondary screening and the counter-screening, approximately 20 CLDN6-specific antibodies were selected for further testing. These antibodies were subcloned and the variable heavy chain sequences and variable light chain sequences were determined. See Table B and the Sequence Listing.

[0304] The CLDN6 antibody was formatted as a full-length IgG antibody using ExpiCHO™ expression. The variable regions of the heavy and light chains of the antibody were cloned into an antibody expression vector engineered in the laboratory based on the pcDNA™ 3.4-TOPO® vector (Catalog No.: A14697, ThermoFisher Scientific, USA) and transfected into CHO cells (following the procedures provided in the kit (ExpiCHO™ Expression System, Catalog No.: A29133, ThermoFisher Scientific, USA)). The antibody was purified, and the cell surface binding of the antibody to CLDN6 and the IC50 of the antibody were determined by FACS. The CLDN6 antibody was directly conjugated to Alexa Fluor® 647 NHS ester (succinimidyl ester), Catalog No. A20106 (ThermoFisher Scientific) following the manufacturer's procedures. The CLDN6 antibody was tested at concentrations ranging from 0.32 nM to 1000 nM (serial dilution 1:5, 6 steps) in a volume of 50 μl using a system of 150,000 cells.

[0305] To determine the ability of the CLDN6 antibody to bind to cell surface CLDN6 and cross-react with other CLDN family members, CLDN6-expressing cells were used in a FACS assay. HEK293T 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 an artificial model of CLDN6 expression. Cells of ARK2, OVCA429, LS513, and MCF7 were used as an endogenous model of CLDN6 expression, as well as a model of CLDN3 / 4 expression.

[0306] For each of the cell types and each mAb tested, to protect cell surface proteins, cells were detached from the culture flask surface with EDTA (instead of trypsin). Subsequently, the detached cells were incubated for 30 minutes in the dark on ice at a preset concentration with CLDN6 mAb labeled with Alexa Fluor®. The CLDN6 mAb was directly labeled with Alexa Fluor® 647 NHS ester (succinimidyl ester). After washing, cells were read on a BD Accuri™ C6 flow cytometer to detect antibody-antigen protein binding in channel FL4H. Each antibody was tested at various concentrations and a dose-fluorescence curve was generated. Using the online-available Very Simple IC50 Tool kit, which can plot biological dose-response data and fit it to curve types to obtain EC50 / IC50, the EC50 / IC50 (antibody concentration at half the maximum value) of the antibody was calculated based on the FL4H values (gating on singlet live cells). The maximum value was taken as the lowest concentration of the antibody at which fluorescence reached its maximum. Antibodies were also screened for their ability to cross-react with other CLDN proteins such as CLDN9, CLDN3, and CLDN4. Using these values, the relative affinity of each antibody in the series of antibodies tested was determined. Cross-reactivity data were obtained using a similar method, but cells with different expression profiles for CLDN6, CLDN3, CLDN4, and CLDN9 were used.

[0307] The relative affinity data and cross-reactivity data determined in this way are presented in Tables 4 and 5.

Table 22

Table 23

[0308] Example 6 This example shows the characterization of IgG mAbs from chimeric mice.

[0309] To further clarify the characteristics of the mAbs described in Example 5, soft agar 3D growth assays and xenograft binding assays were performed. Briefly, for each well of a 48-well plate, 250 μL of an upper layer mixture containing 10,000 cells in 1× RPMI medium containing 0.6% SeaPlaque agarose was plated on top of 250 μL of a bottom layer of solidified 1× RPMI medium containing 0.6% SeaPlaque agarose. A 250 μL liquid feeder layer containing 1× RPMI medium was placed on top of the solidified upper layer. All three layers of the soft agar assay were prepared with or without trastuzumab, Cldn6 mAb, or mouse IgG2a control, starting at 150 ng / mL (1 μM) and ending at 1.5 ng / mL (diluted 1:10). Each test condition was performed in duplicate. Cells were allowed to form colonies for 3 weeks, then stained with 0.05% neutral red and imaged with an EVOS XL inverted optical microscope. Cell lines with a 20% or greater decrease in colony number compared to the untreated control were considered sensitive.

[0310] As shown in Table 6, many of the cell lines showed a decrease in colony number when treated with the indicated antibodies.

Table 24

[0311] An in vivo binding test was performed in xenograft mice injected with human cancer cell lines. Briefly, xenograft models of human cancer cell lines were established in 6-week-old CD-1 thymus-deficient nude mice (Charles River Laboratories). The following conditions were followed for subcutaneous injection of each cell line: ARK2 0.75×10 7 cells, UMUC4 1.0×10 7 cells, OV90 1.0×10 7 cells and M202 0.5×10 7Cells were all treated with 50% Matrigel (BD Biosciences). A sufficient number of mice were injected such that there were 8 mice per treatment group. When the tumors reached an average size of 150 - 300 mm 3 3 , the mice were randomly assigned to treatment groups. For treatment, each therapeutic antibody (AB3, AB2, reference Ab1, reference Ab2, reference Ab3 (trastuzumab), and non - target - directed IgG2 control) was diluted to a working concentration of 1 mg / ml in sterile saline for intravenous (IV) injection via the tail vein. In the M202 study, trametinib (DMSO solvate, MedChem Express) was orally administered at 1.0 mg / kg (10% Cremaphor, 10% PEG400) in the first week of the weekly cycle on a schedule of 5 days of dosing followed by 2 days off, and then the dose was reduced to 0.5 mg / kg for the remaining 2 weeks of dosing. Tumor xenografts were measured three times a week with calipers and the tumor volume was determined in mm 3 units by multiplying height × width × length. Mice were treated for 2 - 7 weeks. At the end of the study, the animals were euthanized and the tumor tissues were excised and divided for storage as snap - frozen tissues or formalin - fixed paraffin - embedded (FFPE) tissues for biomarker analysis. All work with animals was conducted based on procedures approved by the IACUC and the University of California at Los Angeles Animal Research Committee. Data were analyzed using StudyLog software (StudyDirector, San Francisco, CA). Results are presented as the average volume of each group. Error bars represent the standard error (SE) of the mean.

[0312] The results of the xenograft assays are shown in FIGS. 6-10. As shown in FIGS. 6A and 6B, in endometrial tumor-bearing mice, each of AB2 and AB3 resulted in a substantial average change in tumor volume relative to the control IgG2 antibody on day 14. As shown in FIGS. 7A and 7B, in bladder tumor-bearing mice, AB3 resulted in a substantial average change in tumor volume relative to the control IgG2 antibody on day 35. FIGS. 8A and 8B show that in ovarian tumor-bearing mice, each of AB2 and AB3 resulted in a substantial average change in tumor volume relative to the control IgG2 antibody on day 20. FIGS. 9A and 9B show that the models used in FIGS. 9A and 9B did not express any of CLDN6, CLDN3, CLDN4, and CLDN9 and were therefore used as negative controls, indicating that AB3 functions specifically for CLDN. The data in FIGS. 9A and 9B also suggest that AB3 has less off-target activity than reference Ab1 and reference Ab2. FIG. 10A summarizes the results of FIGS. 6-9. As shown in FIG. 10A, AB3 significantly inhibited tumor growth in mice bearing endometrial tumors, bladder tumors, and ovarian tumors that expressed CLDN6, respectively, but did not inhibit tumor growth in melanoma tumor-bearing mice that did not express CLDN6 (FIG. 10B). As shown in FIG. 11, there was no significant change in the average body weight of the mice during treatment, suggesting the safety of the treatment.

[0313] A second set of experiments was conducted in a xenograft model of the human ovarian cancer cell line OV90. One of the 10 mAbs described in Example 5, or a control antibody (mouse IgG2a antibody, reference CLDN6 ab), or a PBS vehicle control was injected into mice. The mice were in groups of 8, and each animal was intravenously injected with 10 mg / kg of antibody every 4 days. As shown in FIGS. 12A and 12B, some of the antibodies described in Example 5 reduced the tumor volume of ovarian tumor-bearing mice. Among them, AB3, AB4, AB7, and AB10 had the best results, but all the tested antibodies reduced the tumor volume compared to the vehicle control. As shown in FIG. 13, there were no significant changes in the body weights of the animals treated with AB3, AB4, AB7, or AB10, suggesting their safety.

[0314] Example 7 This example shows further characterization of the chimeric mouse IgG mAb.

[0315] An assay for quantifying internalization was performed. Briefly, the internalization of CLDN6 protein induced by the binding of reference Ab1, AB3, or AB4 was tested, and the transferrin receptor (TfR), a known cell surface receptor that internalizes after antibody binding and is expressed ubiquitously, was used as a positive control.

[0316] The TfR and CLDN6 antibodies were labeled with Texas Red™-X, succinimidyl ester, mixed isomers, catalog number T6134 (ThermoFisher Scientific). One day before antibody treatment, cells were seeded into μ-Slide 8-well chambers (catalog number 80826, ibidi Cells In Focus Inc.) and allowed to adhere and grow. The cells were incubated with the labeled antibodies in the dark on ice for 30 minutes. Subsequently, the chambers containing cells labeled with CLDN6 or TfR were read on an Echo lab fluorescence microscope, and images before internalization were collected. Then, the chambers were incubated at 37 °C for 40 minutes to allow internalization to proceed, and images were collected again on the Echo lab fluorescence microscope. For AB3 and AB4, the extent of CLDN6 internalization was greater compared to that brought about by the reference Ab1 (data not shown).

[0317] Example 8 This example shows further characterization of the chimeric mouse IgG mAb.

[0318] A two-dimensional (2D) proliferation assay was performed as follows using the selected antibodies described in Example 5: Cells were seeded in duplicate at 5,000 - 20,000 cells per well in a 24-well plate. The next day, the cells were treated with mAb at 6 dilutions from 1 - 5 (starting with either 100 nM trastuzumab, Cldn6 mAb, or mouse IgG2A control), a fixed concentration of 1 ng / μL monomethyl auristatin E (MMAE)-conjugated anti-mouse secondary antibody (Moradec, LLC) to generate a dose-response curve. To determine the range of cell proliferation, cells in untreated wells were quantified on day 1, the day of antibody treatment, and on day 6 thereafter. Wells treated with mAb were quantified on day 6, and proliferation under each treatment condition was determined as the normalized percent ratio to the proliferation of untreated cells. Quantification was performed using a Z1 Particle Counter (Beckman Coulter, Inc).

[0319] The results are shown in Figure 14. AB2, AB3, AB4, and AB5 showed the highest effectiveness in growth inhibition. The IC50 of each of these antibodies was 0.1 nM to 1 nM. Each of AB7, AB10, AB11, and AB15 also showed the ability to inhibit growth in this assay, although to a lesser extent than AB2, AB3, AB4, and AB5.

[0320] Example 9 This example shows the humanization of the antibodies of the present disclosure.

[0321] A subset of the antibodies listed in Table A was selected for humanization analysis. The heavy chain variable (VH) sequences and light chain variable (VL) sequences of the AB1 antibody, AB3 antibody, AB4 antibody, AB9 antibody, AB11 antibody, and AB18 antibody were compared with a library of known human germline sequences from the human VH gene and human VL kappa gene (IMGT® the international ImMunoGeneTics information system® www.imgt.org; founders and managers: Marie-Paule Lefranc, Montpellier, France) (the databases used were the IMGT human VH gene (F+ORF, 273 germline sequences) and the IMGT human VL kappa gene (F+ORF, 74 germline sequences)). An acceptor human germline was selected from among those with the most similar sequences to the parental antibody.

[0322] Table 7 provides information regarding the human germline sequence selected as the acceptor sequence and the selected human heavy chain joining region (J gene) for each of the VH and VL of each antibody. The joining region (J gene) was selected from the human joining region sequences stored in IMGT® the international ImMunoGeneTics information system® www.imgt.org (founders and managers: Marie-Paule Lefranc, Montpellier, France).

Table 25

[0323] The CDRs are defined according to the AbM definition (see the CDR definition comparison table on the website of Dr. Andrew C. R. Martin, www.bioinf.org.uk / abs / ).

[0324] Changes in the framework (i.e., non-CDR residues of VH and VL) positions of the human germline relative to the corresponding mouse parental sequences may be required to optimize the binding of the humanized antibody. The sequences of the humanized antibody versions are provided as SEQ ID NOs: 376 to 421.

[0325] In the case of AB1, each of Asn52 (consecutive numbering) of HC CDR2 and Asn54 of LC CDR2 was determined to have a low likelihood of deamidation based on sequence and conformation.

[0326] In the case of AB3, each of Asn31 (consecutive numbering) of HC CDR1, Asn57 of HC CDR2, Asn28 of LC CDR1, and Asn50 of LC CDR2 was determined to have a low likelihood of deamidation based on sequence and conformation. Trp33 of HC CDR1 was determined to have a potential for solvent exposure and thus a potential for oxidation, especially under stress conditions. In HC CDR3, there is a free Cys106 within the CDR that can be a problem during antibody production due to its potential for solvent exposure. Modification of this Cys residue to Tyr, Ser, or Ala was recommended. The binding maintenance of these modified antibodies was tested. Ile53 of LC CDR2 was determined to have a potential for solvent exposure and could cause non-specific binding. Modification of this Ile residue to Ser was proposed. The binding maintenance of this modified antibody was tested.

[0327] In the case of AB4, each of Asn52 (sequential numbering) in the CDR2 of the HC and Asn58 in the CDR2 of the LC was determined to have a low likelihood of deamidation based on sequence and conformation. The sequence DGNT within the CDR1 of the LC was determined to be a problem because it has a high likelihood of isoglutamate formation (sequence DG) and a potential for deamidation (sequence NT). This sequence was recommended for modification.

[0328] In the case of AB9, Asn33 (sequential numbering) within the CDR1 of the HC, and Asn52 and Asn59 within the CDR2 of the HC were judged to have a low likelihood of deamidation based on sequence and conformation. Asn54 was determined to have a moderate potential for deamidation based on sequence and conformation. The NGG sequence within the CDR2 of the HC was determined to have a high / moderate potential for deamidation following isoglutamate formation. Therefore, it was recommended that this amino acid sequence be modified. The free Cys106 within the CDR3 of the HC may pose a problem during antibody production because it has been determined to have a potential for solvent exposure. Modification of this Cys residue to Tyr, Ser, or Ala has been proposed. The binding maintenance of these modified antibodies is tested. Arg28 within the CDR1 of the HC is rarely found in human antibodies. This residue is modified to Thr and the binding maintenance is tested. In the case of AB9, Trp32 (sequential numbering) within the CDR1 of the LC has a potential for solvent exposure and can be oxidized, especially under stress conditions. Leu24 in the same CDR is rarely found in human antibodies. This residue is modified to Arg and the binding maintenance is tested.

[0329] In the case of AB11, Asp54 - Ser55 (sequential numbering) within the CDR2 of the HC was determined to have a low likelihood of isoglutamate formation. Asn57 within the CDR2 of the LC was determined to have a low likelihood of deamidation based on sequence and conformation.

[0330] In the case of AB18, Asn33 in CDR1 of HC and Asn50 in CDR-H2 (consecutive numbering) were determined to have a low likelihood of d...

Claims

1. A bispecific antigen-binding protein that binds to human Claudin 6 (CLDN6) protein (SEQ ID NO: 200) and a second antigen that is CD3 or CD16A, wherein the antigen-binding protein binds to human Claudin 6 (CLDN6) protein (SEQ ID NO: 200), a. HC CDR1 containing the amino acid sequence FTFSXYX (SEQ ID NO: 455), wherein X at position 5 is N and X at position 7 is W, b. HC CDR2 containing the amino acid sequence IRLKSDNYAT (SEQ ID NO: 24), c. HC CDR3 containing the amino acid sequence XDGPPSGX (SEQ ID NO: 457), wherein X at position 1 is N and X at position 8 is S; d. LC CDR1 containing the amino acid sequence ENIYSY (SEQ ID NO: 20), e. LC CDR2 containing the amino acid sequence NAK (SEQ ID NO: 21), and f. LC CDR3 containing the amino acid sequence QHHYTVPWT (SEQ ID NO: 22) is included in the bispecific antigen-binding protein.

2. The bispecific antigen-binding protein is an antibody that binds to CD3 or an antigen-binding antibody fragment thereof, i. HC CDR1 containing from glycine at position 270 to threonine at position 277 of SEQ ID NO: 518, corresponding to the amino acid sequence GYTFTRYT of SEQ ID NO: 518, ii. HC CDR2 containing from isoleucine at position 295 to threonine at position 302 of SEQ ID NO: 518, corresponding to the amino acid sequence INPSRGYT of SEQ ID NO: 518, iii. HC CDR3 containing from alanine at position 341 to tyrosine at position 352 of SEQ ID NO: 518, corresponding to the amino acid sequence ARYYDDHYCLDY of SEQ ID NO: 518, iv. LC CDR1 containing from serine at position 408 to tyrosine at position 412 of SEQ ID NO: 518, corresponding to the amino acid sequence SSVSY of SEQ ID NO: 518, v. LC CDR2 containing from aspartic acid at position 430 to serine at position 432 of SEQ ID NO: 518, corresponding to the amino acid sequence DTS of SEQ ID NO: 518, and vi. LC CDR3 containing from glutamine at position 469 to threonine at position 477 of SEQ ID NO: 518, corresponding to the amino acid sequence QQWSSNPLT of SEQ ID NO: 518, is included in the bispecific antigen-binding protein according to Claim 1.

3. The bispecific antigen-binding protein is an antibody that binds to CD16A or an antigen-binding antibody fragment thereof, i. HC CDR1 comprising glycine at position 26 to tyrosine at position 33 of SEQ ID NO: 522, corresponding to the amino acid sequence GYTFTSYY of SEQ ID NO: 522, ii. HC CDR2 comprising isoleucine at position 51 to threonine at position 58 of SEQ ID NO: 522, corresponding to the amino acid sequence INPSGGST of SEQ ID NO: 522, iii. HC CDR3 comprising alanine at position 97 to tyrosine at position 109 of SEQ ID NO: 522, corresponding to the amino acid sequence ARGSAYYYDFADY of SEQ ID NO: 522, iv. LC CDR1 comprising asparagine at position 397 to asparagine at position 402 of SEQ ID NO: 522, corresponding to the amino acid sequence NIGSKN of SEQ ID NO: 522, v. LC CDR2 comprising glutamine at position 420 to asparagine at position 422 of SEQ ID NO: 522, corresponding to the amino acid sequence QDN of SEQ ID NO: 522, and vi. LC CDR3 comprising glutamine at position 459 to leucine at position 467 of SEQ ID NO: 522, corresponding to the amino acid sequence QVWDNYSVL of SEQ ID NO: 522, The bispecific antigen-binding protein according to claim 1, comprising the same.

4. The bispecific antigen-binding protein according to claim 1, wherein the antigen-binding protein that binds to human CLDN6 protein is a humanized antibody comprising the heavy chain variable domain sequence set forth in SEQ ID NO: 387 and the light chain variable domain sequence set forth in SEQ ID NO:

389.

5. A pharmaceutical composition comprising the bispecific antigen-binding protein according to any one of claims 1 to 4 and a pharmaceutically acceptable carrier, diluent or additive.

6. The bispecific antigen-binding protein according to any one of claims 1 to 4, or the pharmaceutical composition according to claim 5, for use in the treatment of cancer, inhibition of tumor growth, reduction of tumor size, or prevention of cancer recurrence.

7. The bispecific antigen-binding protein according to any one of claims 1 to 6, wherein the bispecific antigen-binding protein is a bispecific antibody or a bispecific antigen-binding antibody fragment thereof.

8. The bispecific antigen-binding protein according to claim 7, wherein the bispecific antigen-binding protein is a bispecific monoclonal antibody.

9. The bispecific antigen-binding protein according to claim 7 or 8, wherein the bispecific antigen-binding protein is a chimeric or humanized bispecific antibody.

10. The bispecific antigen-binding protein according to any one of claims 1 to 9, wherein the bispecific antigen-binding protein is a bispecific T cell engager or a tandem diabody.

11. A pharmaceutically acceptable carrier, diluent or additive, and a. The bispecific antigen-binding protein according to claim 4, b. A complex comprising the bispecific antigen-binding protein according to claim 4, c. A bispecific antigen-binding protein fusion protein according to claim 4, d. A nucleic acid encoding the bispecific antigen-binding protein according to claim 4, e. A vector containing the nucleic acid of d, f. A host cell containing the nucleic acid of d or the vector of e, or g. The above combination A method for producing a pharmaceutical composition, comprising combining them.

12. i. The bispecific antigen-binding protein according to claim 4, or iii. A fusion protein comprising the bispecific antigen-binding protein according to claim 4 A nucleic acid comprising a nucleotide sequence encoding the same.

13. A method for producing a bispecific antigen-binding protein or a bispecific fusion protein thereof that binds to the Claudin 6 (CLDN6) protein and a second antigen, comprising: a. Culturing a host cell in a cell culture medium, wherein the host cell contains the nucleic acid according to claim 12, which contains a nucleotide sequence encoding a bispecific antigen-binding protein or a bispecific fusion protein thereof, and the culturing, and b. Collecting the bispecific antigen-binding protein or a bispecific fusion protein thereof from the cell culture medium. The method comprising the above.

14. Use of the bispecific antigen-binding protein according to any one of claims 1 to 4 and 7 to 10, or the pharmaceutical composition according to claim 5, in the manufacture of a medicament for the treatment of cancer, inhibition of tumor growth, reduction of tumor size, or prevention of cancer recurrence.

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