Claudin-1 / EGFR bispecific antibodies

Abstract

The present disclosure relates to bispecific antibodies that bind Claudin 1 and EGFR and their use in treating diseases, for example, cancer.

Description

CLAUDIN-l / EGFR BISPECIFIC ANTIBODIESCROSS REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims the benefit of U.S. Provisional Patent Application No. 63 / 694,100 filed September 12, 2024, which is incorporated herein by reference in its entirety.INCORPORATION-BY-REFERENCE OF SEQUENCE LISTING

[0002] The Sequence Listing XML associated with this application is provided electronically in XML file format and is hereby incorporated by reference into the specification. The name of the XML file containing the Sequence Listing XML is “ALNT- 014_001WO_SeqList”. The XML file is 40,422 bytes, created on September 8, 2025, and is being submitted electronically.FIELD

[0003] According to various aspects of this disclosure, the present disclosure relates to anti-Claudin 1 / anti-EGFR bispecific antibodies and methods of their use in treating disease.BACKGROUND

[0004] Epidermal growth factor receptor (EGFR) is a 170 kilodalton (kDa) transmembrane protein expressed on the surface of epithelial cells. EGFR is a member of the growth factor receptor family of protein tyrosine kinases, a class of cell cycle regulatory molecules. (W.J. Gullick et al., 1986, Cancer Res., 46: 285-292). EGFR is activated when its ligands (either EGF or TGF-a) bind to the extracellular domain, resulting in autophosphorylation of the receptor’s intracellular tyrosine kinase domain (S. Cohen et al., 1980, J. Biol. Chem., 255: 4834-4842; A.B. Schreiber et al., 1983, J. Biol. Chem., 258: 846- 853). EGFR is the protein product of a growth promoting oncogene, ErbB or ErbBl, that is one member of a family, i.e., the ERBB family of protooncogenes. EGFR is expressed in a wide range of human tissues and its over-expression is associated with a number of malignant tumors of epithelia cell origin, such as colorectal cancer, non-small cell lung cancer, head and neck cancer, bladder, lung, and stomach cancer as well as glioblastomas.

[0005] Through binding to its ligand EGF, EGFR undergoes a transition from an inactive monomeric form to an active homodimer or heterodimer, and then induces tyrosinephosphorylation and downstream signal leading to tumor cells uncontrolled proliferation. EGFR also binds other ligands, including AREG, EREG, EPGN, albeit with lower affinity compared to EGF, all of which induce EGFR phosphorylation. Two EGFR-targeted antibodies, cetuximab (Erbitux) and panitumumab (Vectibix), have been approved by the US Food and Drug Administration for the treatment of colon cancers and head and neck cancers.

[0006] According to clinical statistics, varying anti-tumor efficacy was observed with existing EGFR-specific therapeutic antibodies, and refractory or relapsed EGFR-expressing tumors proved disappointing clinical outcomes. Especially patients with EGFR mutation, KRAS, BRAF mutations, which comprise approximately 40-50% of colorectal cancers, do not respond to anti-EGFR therapeutic antibodies.

[0007] Claudins function as major constituents of the tight junction complexes that regulate the permeability of epithelia. While some claudin family members play essential roles in the formation of impermeable barriers, others mediate the permeability to ions and small molecules. Often, several claudin family members are co-expressed and interact with each other, and this determines the overall permeability. CLDN1 is an integral membrane protein with two major roles: (1) together with other proteins it contributes to the barrier function by tight junctions; (2) it is expressed outside the tight junctions in the basolateral membrane of epithelial cells, where CLDN1 has been shown to mediate pro-carcinogenic signaling, epithelial-mesenchymal transition (EMT) and cell fate. Moreover, it has been shown that CLDN1 is also expressed by nonepithelial cells such as myofibroblasts of liver, lung, and kidney.

[0008] Claudin-1 (CLDN1) is required to prevent the paracellular diffusion of small molecules through tight junctions in the epidermis and is required for the normal barrier function of the skin. The term “human Claudin-1” or “CLDN1” refers to a protein having the sequence shown in NCBI Accession Number NP_066924.1, or any naturally occurring variants commonly found in HCV permissive human populations.

[0009] Claudin (CLDN) 1 is the most studied claudin in cancers and to date, its role as either a tumor promoter or suppressor (or both) is not established. In some cancers, lower expression of CLDN1 is shown to be associated with cancer progression and invasion, while in others, loss of CLDN1 improves the patient’s survival. Another topic of discussion regarding the significance of CLDN1 is its localization (nuclear or cytoplasmic vs peri- junctional) in diseased states. The association of CLDN1 with patient survival or recurrencein many cancers suggests its importance as a prognostic marker and as a potential therapeutic target.

[0010] Therefore, there exists a need for development of bispecific antibodies that target CLDN 1 and EGFR as an alternative immunotherapy strategy for the patients with the tumor resistant or refractory to EGFR monoclonal antibodies, such as cetuximab (Erbitux) and panitumumab (Vectibix) treatment.SUMMARY

[0011] In one aspect, provided herein is a bispecific antibody comprising (a) a Claudin 1 (CLDN1) binding moiety comprising a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 1, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 2, a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 3, a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 4, a light chain CDR2 comprising the amino acid sequence GAS, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 5, and (b) an Epidermal Growth Factor Receptor (EGFR) binding moiety comprising: a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 8, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 9, a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 10, a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 11, a light chain CDR2 comprising the amino acid sequence of YAS, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 13.

[0012] In some embodiments, the antibody induces improved antibody dependent cell cytotoxicity of cells co-expressing EGFR and CLDN1 in comparison to antibodies targeting CLDN1 and EGFR individually. In some embodiments, the antibody induces improved antibody dependent cell phagocytosis of cells co-expressing EGFR and CLDN1 in comparison to antibodies targeting CLDN1 and EGFR individually.

[0013] In some embodiments, (a) the CLDN1 binding moiety comprises: (i) a heavy chain variable domain (VH1) sequence at least 85% identical to SEQ ID NO: 6, and (ii) a light chain variable domain (VL1) sequence at least 85% identical to SEQ ID NO: 7; and (b) the anti-EGFR antibody comprises: (i) a heavy chain variable domain (VH2) sequence at least 85% identical to SEQ ID NO: 14, and (ii) a light chain variable domain (VL2) sequence at least 85% identical to SEQ ID NO: 15.

[0014] In some embodiments, (a) the CLDN1 binding moiety comprises: (i) a heavy chain variable domain (VH1) sequence at least 90% identical to SEQ ID NO: 6, and (ii) alight chain variable domain (VL1) sequence at least 90% identical to SEQ ID NO: 7; and (b) the anti-EGFR antibody comprises: (i) a heavy chain variable domain (VH2) sequence at least 90% identical to SEQ ID NO: 14, and (ii) a light chain variable domain (VL2) sequence at least 90% identical to SEQ ID NO: 15.

[0015] In some embodiments, (a) the CLDN1 binding moiety comprises: (i) a heavy chain variable domain (VH1) sequence at least 95% identical to SEQ ID NO: 6, and (ii) a light chain variable domain (VL1) sequence at least 95% identical to SEQ ID NO: 7; and (b) the anti-EGFR antibody comprises: (i) a heavy chain variable domain (VH2) sequence at least 95% identical to SEQ ID NO: 14, and (ii) a light chain variable domain (VL2) sequence at least 95% identical to SEQ ID NO: 15.

[0016] In some embodiments, (a) the CLDN1 binding moiety comprises: (i) a heavy chain variable domain (VH1) comprising the sequence of SEQ ID NO: 6, and (ii) a light chain variable domain (VL1) comprising the sequence of SEQ ID NO: 7; and (b) the anti- EGFR antibody comprises: (i) a heavy chain variable domain (VH2) comprising the sequence of SEQ ID NO: 14, and (ii) a light chain variable domain (VL2) comprising the sequence of SEQ ID NO: 15.

[0017] In some embodiments, the bispecific antibody comprises four polypeptide chains: (i) a first heavy chain comprising the sequence of any one of SEQ ID NOs: 16-19; (ii) a first light chain represented comprising the sequence of SEQ ID NO: 24; (iii) a second heavy chain comprising the sequence of any one of SEQ ID NOs: 20-23, and (iv) a second light chain comprising the sequence of any one of SEQ ID NOs: 25-28. In some embodiments, the first heavy chain comprises the sequence of SEQ ID NO: 16, the first light chain comprises the sequence of SEQ ID NO: 24, the second heavy chain comprises the sequence of SEQ ID NO: 21, and the second light chain comprises the sequence of SEQ ID NO: 25.

[0018] In some embodiments, the first heavy chain comprises the sequence of SEQ ID NO: 16, the first light chain comprises the sequence of SEQ ID NO: 24, the second heavy chain comprises the sequence of SEQ ID NO: 21, and the second light chain comprises the sequence of SEQ ID NO: 26.

[0019] In some embodiments, the first heavy chain comprises the sequence of SEQ ID NO: 16, the first light chain comprises the sequence of SEQ ID NO: 24, the second heavy chain comprises the sequence of SEQ ID NO: 20, and the second light chain comprises the sequence of SEQ ID NO: 27.

[0020] In some embodiments, the first heavy chain comprises the sequence of SEQ ID NO: 16, the first light chain comprises the sequence of SEQ ID NO: 24, the second heavy chain comprises the sequence of SEQ ID NO: 20, and the second light chain comprises the sequence of SEQ ID NO: 28.

[0021] In some embodiments, the first heavy chain comprises the sequence of SEQ ID NO: 17, the first light chain comprises the sequence of SEQ ID NO: 24, the second heavy chain comprises the sequence of SEQ ID NO: 21, and the second light chain comprises the sequence of SEQ ID NO: 25.

[0022] In some embodiments, the first heavy chain comprises the sequence of SEQ ID NO: 17, the first light chain comprises the sequence of SEQ ID NO: 24, the second heavy chain comprises the sequence of SEQ ID NO: 21, and the second light chain comprises the sequence of SEQ ID NO: 26.

[0023] In some embodiments, the first heavy chain of SEQ ID NO: 17, a first light chain of SEQ ID NO: 24, a second heavy chain of SEQ ID NO: 21, and a second light chain of SEQ ID NO: 27.

[0024] In some embodiments, the first heavy chain comprises the sequence of SEQ ID NO: 17, the first light chain comprises the sequence of SEQ ID NO: 24, the second heavy chain comprises the sequence of SEQ ID NO: 21, and the second light chain comprises the sequence of SEQ ID NO: 28.

[0025] In some embodiments, the first heavy chain comprises the sequence of SEQ ID NO: 18, the first light chain comprises the sequence of SEQ ID NO: 24, the second heavy chain comprises the sequence of SEQ ID NO: 22, and the second light chain comprises the sequence of SEQ ID NO: 26.

[0026] In some embodiments, the first heavy chain comprises the sequence of SEQ ID NO: 18, the first light chain comprises the sequence of SEQ ID NO: 24, the second heavy chain comprises the sequence of SEQ ID NO: 22, and the second light chain comprises the sequence of SEQ ID NO: 28.

[0027] In some embodiments, the first heavy chain comprises the sequence of SEQ ID NO: 19, the first light chain comprises the sequence of SEQ ID NO: 24, the second heavy chain comprises the sequence of SEQ ID NO: 23, and the second light chain comprises the sequence of SEQ ID NO: 28.

[0028] In some embodiments, the first heavy chain comprises the sequence of SEQ ID NO: 32, the first light chain comprises the sequence of SEQ ID NO: 24, the second heavychain comprises the sequence of SEQ ID NO: 33, and the second light chain comprises the sequence of SEQ ID NO: 26. In some embodiments, the first heavy chain comprises the sequence of SEQ ID NO: 34, the first light chain comprises the sequence of SEQ ID NO: 36, the second heavy chain comprises the sequence of SEQ ID NO: 35, and the second light chain comprises the sequence of SEQ ID NO: 37.

[0029] In some embodiments, the CLDN1 binding moiety and the EGFR binding moiety are connected by a linker.

[0030] In some embodiments, the antibody comprises a human Fc region. In some embodiments, the human Fc region is a human IgG4 or IgGl Fc region.

[0031] In some embodiments, the antibody comprises one or more mutations in the Fc region, wherein the one or more mutation is selected from L234F, L235E, and P331S, wherein the residues are defined by the EU numbering system. In some embodiments, the antibody comprises one or more mutations in the Fc region, wherein the one or more mutation is selected from S354C, T366W, T366S and L368A, wherein the residues are defined by the EU numbering system.

[0032] In some embodiments, the bispecific antibody is a humanized antibody.

[0033] In another aspect, provided herein is an isolated nucleic acid molecule, comprising a nucleic acid sequence encoding a bispecific antibody described herein.

[0034] In another aspect, provided herein is a vector comprising a nucleic acid molecule described herein.

[0035] In another aspect, provided herein is a host cell comprising a nucleic acid molecule or a vector provided herein.

[0036] In another aspect, provided herein is a pharmaceutical composition comprising a bispecific antibody described herein and a pharmaceutically acceptable carrier.

[0037] In another aspect, provided herein is a method for producing a bispecific described herein, comprising the steps of: (a) expressing a bispecific antibody disclosed herein in the host cell; and (b) isolating the bispecific antibody from the host cell.

[0038] In another aspect, provided herein is a method for modulating an immune response in a subject, comprising administering to the subject a bispecific antibody or a pharmaceutical composition of described herein.

[0039] In another aspect, provided herein is a method for inhibiting growth of a tumor cell in a subject, comprising administering an effective amount of a bispecific antibody or a pharmaceutical composition disclosed herein. In some embodiments, the tumor cell is from acancer selected from the group consisting of Head and Neck Squamous Cell Carcinoma, Esophageal Cancer, and Hepatocellular Carcinoma.

[0040] In another aspect, provided herein is a method for treating cancer, comprising administering an effective amount of a bispecific antibody or a pharmaceutical composition disclosed herein to the subject. In some embodiments, the tumor cell is from a cancer selected from the group consisting of Head and Neck Squamous Cell Carcinoma, Esophageal Cancer, and Hepatocellular Carcinoma.

[0041] In another aspect, provided herein is the use of a bispecific antibody disclosed herein in the manufacture of a medicament for modulating an immune response or inhibiting growth of a tumor cell in a subject. In some embodiments, the tumor cell is from a cancer selected from the group consisting of Head and Neck Squamous Cell Carcinoma, Esophageal Cancer, and Hepatocellular Carcinoma.

[0042] In another aspect, provided herein is the use of a bispecific antibody disclosed herein in the manufacture of a medicament for treating cancer in a subject. In some embodiments, the tumor cell is from a cancer selected from the group consisting of Head and Neck Squamous Cell Carcinoma, Esophageal Cancer, and Hepatocellular Carcinoma.

[0043] In another aspect, provided herein is a kit, comprising a container comprising a bispecific antibody disclosed herein.DESCRIPTION OF FIGURES

[0044] FIGs. 1 A-1C show co-expression of CLDN1 and EGFR in head and neck cancer cells (FIG. 1 A), squamous cell lung cancer cells (FIG. IB), and esophageal Cancer Cells (FIG. 1C) based on TCGA data.

[0045] FIGs. 2A-2B show efficient binding of anti-EGFR (FIG. 2A) and anti-CLDNl (FIG. 2B) antibodies to their targets in Huh7 cells.

[0046] FIG. 3 shows an illustrative anti-CLDNl / EGFR bispecific antibody in Crossmab KZ. format.

[0047] FIG. 4A shows CLDN1 and EGFR expression level by western blot and FIGs. 4B-4D show binding of ALE-C04 and an Fc competent anti-CLNDl / EGFR bispecific antibody in crossmab format (BsAbl) as well their isotype and monovalent controls to Huh7 wildtype, CLND1 KO and EGFR KO cells, respectively.

[0048] FIGs. 5A-5C show in vitro ADCC activity of BsAbl, ALE.C04, Cetuximab, and an IgGl control. The activity was measured by a Jurkat NFAT CD 16a luciferase reporter assay in a Cal27 Head & Neck Squamous Cell Cancer cell line (FIG. 5A), a KYSE30Esophageal Cancer cell line (FIG. 5B), and a Huh-7 Hepatocellular Carcinoma cell line (FIG. 5C).

[0049] FIGs. 6A-6C show in vitro ADCP activity of BsAbl, ALE.C04 alone, Cetuximab alone, and an IgGl isotype control. The activity was measured by a Jurkat NFAT CD32 luciferase reporter assay in a KYSE30 Esophageal Cancer cell line (FIG. 6A), a Cal27 Head & Neck Squamous Cell Cancer cell line (FIG. 6B), and a Detroit 562 Head & Neck Squamous Cell Cancer cell line (FIG. 6C).

[0050] FIG. 7 shows binding of BsAbl, cetuximab, ALE.C04 and isotype control to CLNDl-transudced NCI-H1975 cells as determined by FACS.

[0051] FIG. 8 shows binding of BsAbl, ALE.F02, cetuximab and isotype control to CLND1 on SW480 cells expressing low levels of CLND1.

[0052] FIGs. 9A and 9B show binding of an Fc non-competent anti-CLNDl / EGFR bispecific antibody in crossmab format (BsAb2), ALE.F02, Cetuximab and isotype control to SW480 cells with low and high CLND1 expression, respectively.

[0053] FIGs. 10A and 10B show internalization of BsAbl, ALE.F02, cetuximab and isotype control into NCI-H1975 cells. FIG. 10A shows illustrative FACS scatterplots at the 4- hour time point and FIG. 10B shows kinetics of internalization as measured by FACS over time.

[0054] FIGs. 11 A and 1 IB show internalization of BsAbl, ALE.F02, cetuximab and isotype control into SW480 expressing low levels of CLND1 cells. FIG. 11 A shows illustrative FACS scatterplots at the 2-hour time point and FIG. 1 IB shows kinetics of internalization as measured by FACS over time.

[0055] FIG. 12 shows internalization of BsAbl, ALE.F02, cetuximab and isotype control into Huh7 cells.DETAILED DESCRIPTION

[0056] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. In case of conflict, the present application including the definitions will control. Unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. All publications, patents and other references mentioned herein are incorporated by reference in their entirety for all purposes as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference.

[0057] Although methods and materials similar or equivalent to those described herein can be used in practice or testing of the present disclosure, suitable methods and materials are described below. The materials, methods and examples are illustrative only and are not intended to be limiting. Other features and advantages of the disclosure will be apparent from the detailed description and from the claims.

[0058] In order to further define this disclosure, the following terms and definitions are provided.

[0059] As used herein, the term “about” is used to mean approximately, roughly, around, or in the regions of. When the term “about” is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values set forth. In general, the term “about” is used herein to modify a numerical value above and below the stated value by a variance of 10 percent, up or down (higher or lower).

[0060] Throughout this disclosure, various aspects of this invention are presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub-ranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range. Numeric ranges recited are inclusive of the numbers defining the range and include each integer within the defined range.

[0061] Units, prefixes, and symbols are denoted in their Systeme International de Unites (SI) accepted form. Numeric ranges are inclusive of the numbers defining the range. Where a range of values is recited, it is to be understood that each intervening integer value, and each fraction thereof, between the recited upper and lower limits of that range is also specifically disclosed, along with each subrange between such values. The upper and lower limits of any range can independently be included in or excluded from the range, and each range where either, neither or both limits are included is also encompassed within the disclosure. Thus, ranges recited herein are understood to be shorthand for all of the values within the range, inclusive of the recited endpoints. For example, a range of 1 to 10 isunderstood to include any number, combination of numbers, or sub-range from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10.

[0062] Where a value is explicitly recited, it is to be understood that values which are about the same quantity or amount as the recited value are also within the scope of the disclosure. Where a combination is disclosed, each subcombination of the elements of that combination is also specifically disclosed and is within the scope of the disclosure.Conversely, where different elements or groups of elements are individually disclosed, combinations thereof are also disclosed. Where any element of a disclosure is disclosed as having a plurality of alternatives, examples of that disclosure in which each alternative is excluded singly or in any combination with the other alternatives are also hereby disclosed; more than one element of a disclosure can have such exclusions, and all combinations of elements having such exclusions are hereby disclosed.

[0063] As used herein, the term “and / or” is to be taken as specific disclosure of each of the two specified features or components with or without the other. Thus, the term “and / or” as used in a phrase such as “A and / or B” herein is intended to include “A and B,” “A or B,” “A” (alone), and “B” (alone). Likewise, the term “and / or” as used in a phrase such as “A, B, and / or C” is intended to encompass each of the following aspects: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).Bispecific Antibodies

[0064] In one aspect, provided herein are bispecific antibodies that bind to both Claudin 1 and epidermal growth factor receptor (anti-CLDNl / anti-EGFR bispecific antibodies).

[0065] A native intact antibody (or immunoglobulin) generally is a Y-shaped tetrameric protein comprising two heavy (H) and two light (L) polypeptide chains held together by covalent disulfide bonds and non-covalent interactions. Light chains of an antibody may be classified into K and X light chain. Heavy chains may be classified into p, 5, y, a and a, which define isotypes of an antibody as IgM, IgD, IgG, IgA and IgE, respectively. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). A heavy chain constant region consists of 3 domains (CHI, CH2 and CH3). Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). VH and VL region can further be divided into hypervariable regions (called complementary determining regions (CDR)), which are interspaced by relatively conservativeregions (called framework region (FR)). Each VH and VL consists of 3 CDRs and 4 FRs in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 from N-terminal to C- terminal. The variable region (VH and VL) of each heavy / light chain pair forms antigen binding sites, respectively. Distribution of amino acids in various regions or domains follows the definition in Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991) ) , or Chothia & Lesk (1987) J. Mol. Biol. 196: 901-917; Chothia et al., (1989) Nature 342: 878-883. Antibodies may be of different antibody isotypes, for example, IgG (e.g., IgGl, IgG2, IgG3 or IgG4 subtype), IgAl, IgA2, IgD, IgE or IgM antibody.

[0066] The term “antigen-binding portion” or “antigen-binding fragment” of an antibody, which can be interchangeably used in the context of the application, refers to polypeptides comprising fragments of a full-length antibody, which retain the ability of specifically binding to an antigen that the full-length antibody specifically binds to, and / or compete with the full-length antibody for binding to the same antigen. Generally, see Fundamental Immunology, Ch. 7 (Paul, W., ed., the second edition, Raven Press, N.Y. (1989), which is incorporated herein by reference for all purposes. Antigen-binding fragments of an antibody may be derived, e.g., from full antibody molecules using any suitable standard techniques such as proteolytic digestion or recombinant genetic engineering techniques involving the manipulation and expression of DNA encoding antibody variable and optionally constant domains. Such DNA is known and / or is readily available from, e.g., commercial sources, DNA libraries (including, e.g., phage-antibody libraries), or can be synthesized. The DNA may be sequenced and manipulated chemically or by using molecular biology techniques, for example, to arrange one or more variable and / or constant domains into a suitable configuration, or to introduce codons, create cysteine residues, modify, add or delete amino acids, etc.

[0067] Non-limiting examples of antigen-binding fragments include: (i) Fab fragments; (ii) F (ab’)2 fragments; (iii) Fd fragments; (iv) Fv fragments; (v) single-chain Fv (scFv) molecules; (vi) dAb fragments; and (vii) minimal recognition units consisting of the amino acid residues that mimic the hypervariable region of an antibody (e.g., an isolated complementarity determining region (CDR) such as a CDR3 peptide), or a constrained FR3- CDR3-FR4 peptide. Other engineered molecules, such as domain-specific antibodies, single domain antibodies, domain-deleted antibodies, chimeric antibodies, CDR-grafted antibodies, diabodies, triabodies, tetrabodies, minibodies, nanobodies (e.g. monovalent nanobodies,bivalent nanobodies, etc.), small modular immunopharmaceuticals (SMIPs), and shark variable IgNAR domains, are also encompassed within the expression “antigen-binding fragment, “ as used herein. In certain aspects, an antigen-binding fragment of an antibody may contain at least one variable domain covalently linked to at least one constant domain. The variable and constant domains may be either directly linked to one another or may be linked by a full or partial hinge or linker region. A hinge region may consist of at least 2 (e.g., 5, 10, 15, 20, 40, 60 or more) amino acids which result in a flexible or semi-flexible linkage between adjacent variable and / or constant domains in a single polypeptide molecule.

[0068] An antibody variable region or a fragment thereof generally comprises one or more CDRs. Although a variable domain may comprise an intact variable region (such as HCVR or LCVR), it is also possible to comprise less than an intact variable region yet still retain the capability of binding to an antigen or forming an antigen-binding site.

[0069] An antigen-binding moiety is capable of binding to the same antigen to which the parent antibody binds. In certain aspects, an antigen-binding moiety may comprise one or more CDRs from a particular human antibody grafted to a framework region from one or more different human antibodies. For more and detailed formats of antigen-binding moiety are described in Spiess et al., Mol. Immun. 67.2:95-106 (2015), and Brinkman et al., mAbs 9(2): 182-212 (2017), which are incorporated herein by their entirety.

[0070] In some aspects, the antibody is bivalent. A bivalent antibody or an antigenbinding fragment generally has two antigen-binding sites, while a monovalent antibody or an antigen-binding fragment generally only has one single antigen-binding site. A multivalent antibody or an antigen-binding fragment generally has multiple antigen-binding sites.

[0071] The Fc region of an antibody is the portion of the antibody comprising the second (CH2) and third (CH3) constant regions of a first heavy chain bound to the second and third constant regions of a second heavy chain via disulfide bonding. The CH2 domain includes the portion of a heavy chain molecule that extends, e.g., from about amino acid 244 to amino acid 360 of an IgG antibody using conventional numbering schemes (amino acids 244 to 360, Kabat numbering system; and amino acids 231-340, EU numbering system; see Kabat, E., et al., U.S. Department of Health and Human Services, (1983)). The CH3 domain extends from the CH2 domain to the C-terminus of the IgG molecule and comprises approximately 108 amino acids. Certain immunoglobulin classes, e.g., IgM, further include a CH4 region. The Fc portion of the antibody is responsible for various effector functions such as ADCC, and CDC, but does not function in antigen binding.

[0072] The antibodies described herein may be Fc competent or Fc non-competent. An antibody may be rendered Fc non-competent by introducing mutations that reduce or eliminate the binding of the Fc region to Fc receptors or that reduce or eliminate the antibody’s ability to induce Fc-mediated effector functions. Examples of such mutations include mutations at residues 234, 235 and / or 331 as defined by the EU numbering system. In some embodiments, an antibody descried herein comprises L234F / L235E / P331S substitutions (also referred to as an “LFLEPS”).

[0073] The hinge region of an antibody includes the portion of a heavy chain molecule that joins the CHI domain to the CH2 domain. This hinge region comprises approximately 25 amino acid residues and is flexible, thus allowing the two N-terminus antigen binding regions to move independently.

[0074] A bispecific antibody generally refers to an artificial antibody which has fragments derived from two different monoclonal antibodies and is capable of binding to two different epitopes. The two epitopes may be present on the same antigen, or they may be present on two different antigens. An epitope is generally understood as the specific group of atoms or amino acids on an antigen to which an antibody binds. Epitopes can be formed both from contiguous amino acids (also called linear or sequential epitope) or noncontiguous amino acids juxtaposed by tertiary folding of a protein (also called configurational or conformational epitope). Epitopes formed from contiguous amino acids are typically arranged linearly along the primary amino acid residues on the protein and the small segments of the contiguous amino acids can be digested from an antigen binding with major histocompatibility complex (MHC) molecules or retained on exposure to denaturing solvents whereas epitopes formed by tertiary folding are typically lost on treatment with denaturing solvents. An epitope typically includes at least 3, and more usually, at least 5, about 7, or about 8-10 amino acids in a unique spatial conformation. Two antibodies may bind the same or a closely related epitope within an antigen if they exhibit competitive binding for the antigen. For example, if an antibody or antigen-binding moiety blocks binding of a reference antibody to the antigen by at least 85%, or at least 90%, or at least 95%, then the antibody or antigen-binding moiety may be considered to bind the same / closely related epitope as the reference antibody.

[0075] Each antigen-binding domain within the bispecific antibody comprises at least one CDR that alone, or in combination with one or more additional CDRs and / or FRs, specifically binds to a particular antigen. In the context of the present disclosure, the firstantigen-binding site specifically binds to a first antigen (e.g., CLDN1), and the second antigen-binding site specifically binds to a second, distinct antigen (e.g., EGFR).

[0076] These anti-CLNDl / anti -EGFR bispecific antibodies comprise a CLDN1- targeting moiety and an EGFR-targeting moiety. The anti -CLNDl / anti -EGFR bispecific antibodies may be in any suitable format.

[0077] In some embodiments, an anti -CLNDl / anti -EGFR bispecific antibody provided herein a monoclonal antibody.

[0078] In some embodiments, an anti -CLNDl / anti -EGFR bispecific antibody provided herein is a human antibody. A human antibody is an antibody having variable regions in which both the framework and CDR regions are derived from human germline immunoglobulin sequences. Furthermore, if the antibody contains a constant region, the constant region also is derived from human germline immunoglobulin sequences. The human antibodies of the disclosure can include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo).

[0079] In some embodiments, an anti -CLNDl / anti -EGFR bispecific antibody provided herein is a humanized antibody. A humanized antibody is an antibody in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences. Additional framework region modifications may be made within the human framework sequences.

[0080] In some embodiments, an anti -CLNDl / anti -EGFR bispecific antibody provided herein is a chimeric antibody. A chimeric antibody is an antibody in which the variable region sequences are derived from one species and the constant region sequences are derived from another species, such as an antibody in which the variable region sequences are derived from a mouse antibody and the constant region sequences are derived from a human antibody.

[0081] An anti-CLNDl / anti-EGFR bispecific antibody provided herein may be isolated. An isolated antibody is generally understood to be an antibody that is substantially free of other antibodies having different antigenic specificities (e.g., an isolated antibody that specifically binds a CLDN1ZEGFR protein is substantially free of antibodies that specifically bind antigens other proteins than CLDN1ZEGFR). An isolated antibody that specifically binds a human CLDN1ZEGFR protein may, however, have cross-reactivity to other antigens, suchas CLDN1ZEGFR proteins from other species. Moreover, an isolated antibody can be substantially free of other cellular material and / or chemicals.CLDN1 -binding domains

[0082] The anti-CLNDl / anti-EGFR bispecific antibodies provided may comprise an anti-CLDNl antibody or antigen-binding fragment thereof, as a CLDN1 targeting moiety.

[0083] Antibodies directed against human Claudin-1 have been previously described to treat hepatitis c virus infection, hepatocellular carcinoma, and certain fibrotic diseases, such as lung fibrosis (see WO 2010 / 034812, WO 2016 / 146809, and WO 2021 / 094469). Anti- Claudin-1 antibodies that can be used in the practice of the present disclosure include any antibody raised against Claudin-1. Examples are disclosed in WO 2010 / 034812 and WO 2017 / 162678, each of which is incorporated herein by reference in its entirety for examples of antibodies that may be used in CLDN1 targeting moi eties herein. Other examples of suitable anti-Claudin-1 antibodies include those disclosed in European Patent No. EP 1,167,389, in U.S. Patent No. 6,627,439, in international patent application published under No. WO 2014 / 132307, in international patent applications published under No. WO 2015 / 014659 and No. WO 2015 / 014357, and in Yamashita et al., J. Pharmacol. Exp. Ther. 353(1): 112-118 (2015), each of which is incorporated herein by reference in its entirety for examples of antibodies that may be used in CLDN1 targeting moi eties herein.

[0084] In some aspects, the six complementarity determining regions (CDRs) of the anti-Claudin-1 antibody are the same as those in the anti-Claudin-1 monoclonal antibody secreted by a hybridoma cell line deposited at the DSMZ on July 29, 2008 under an Accession Number DSM ACC2938.

[0085] In some aspects, the CLDNl-binding domain is one previously described in WO 2017 / 162678, which is incorporated herein by reference in its entirety. Illustrative sequences for the anti-CLDNl antibody or antigen binding fragment provided herein are described in Table 1.Table 1: CLDNl-binding domain sequences

[0086] In one aspect, the anti-CLDNl binding moiety comprises: a heavy chain CDR (complementarity determining region)-Hl (CDR-H1) comprising an amino acid sequence of SEQ ID NO: 1; CDR-H2 comprising an amino acid sequence of SEQ ID NO: 2; a CDR-H3 comprising an amino acid sequence of SEQ ID NO: 3; a light chain CDR-L1 comprising an amino acid sequence of SEQ ID NO: 4; a light chain CDR-L2 comprising the amino acidsequence GAS; and a light chain CDR-L3 comprising an amino acid sequence of SEQ ID NO: 5.

[0087] In another aspect, the anti-CLDNl binding moiety comprises a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 6, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 7.

[0088] In some aspects, the anti-CLDNl binding moiety comprises a VH comprising an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 6. In some aspects, the anti-CLDNl a binding moiety comprises a VL comprising an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 7.

[0089] In some aspects, the anti-CLDNl binding moiety comprises a VH comprising an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 6, wherein the VH comprises a CDR-H1 comprising an amino acid sequence of SEQ ID NO: 1; CDR-H2 comprising an amino acid sequence of SEQ ID NO: 2; a CDR-H3 comprising an amino acid sequence of SEQ ID NO: 3.

[0090] In some aspects, the anti-CLDNl binding moiety comprises a VL comprising an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 7, wherein the VL comprises a light chain CDR-L1 comprising an amino acid sequence of SEQ ID NO: 4; a light chain CDR-L2 comprising the amino acid sequence GAS; and a light chain CDR-L3 comprising an amino acid sequence of SEQ ID NO: 5.

[0091] In some aspects, the anti- binding moiety comprises (a) a VH comprising an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 6, wherein the VH comprises a CDR-H1 comprising an amino acid sequence of SEQ ID NO: 1; CDR-H2 comprising an amino acid sequence of SEQ ID NO: 2; a CDR-H3 comprising an amino acid sequence of SEQ ID NO: 3; and (b) a VL comprising an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or atleast 99% sequence identity to SEQ ID NO: 7, wherein the VL comprises a light chain CDR- L1 comprising an amino acid sequence of SEQ ID NO: 4; a light chain CDR-L2 comprising the amino acid sequence GAS; and a light chain CDR-L3 comprising an amino acid sequence of SEQ ID NO: 5.

[0092] In another aspect, the anti-CLDNl binding moiety comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 30, and a light chain comprising the amino acid sequence of SEQ ID NO: 29. In another aspect, the anti-CLDNl binding moiety comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 31, and a light chain comprising the amino acid sequence of SEQ ID NO: 29.

[0093] In some aspects, the anti-CLDNl binding moiety comprises a heavy chain comprising an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 30. In some aspects, the anti-CLDNl binding moiety comprises a heavy chain comprising an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 31. In some aspects, the anti-CLDNl a binding moiety comprises a light chain comprising an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 29.

[0094] In some aspects, the anti-CLDNl binding moiety comprises a heavy chain comprising an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 30, wherein the heavy chain comprises a CDR- H1 comprising an amino acid sequence of SEQ ID NO: 1; CDR-H2 comprising an amino acid sequence of SEQ ID NO: 2; a CDR-H3 comprising an amino acid sequence of SEQ ID NO: 3.

[0095] In some aspects, the anti-CLDNl binding moiety comprises a heavy chain comprising an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 31, wherein the heavy chain comprises a CDR- H1 comprising an amino acid sequence of SEQ ID NO: 1; CDR-H2 comprising an aminoacid sequence of SEQ ID NO: 2; a CDR-H3 comprising an amino acid sequence of SEQ ID NO: 3.

[0096] In some aspects, the anti-CLDNl binding moiety comprises a light chain comprising an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 29, wherein the light chain comprises a light chain CDR-L1 comprising an amino acid sequence of SEQ ID NO: 4; a light chain CDR-L2 comprising the amino acid sequence GAS; and a light chain CDR-L3 comprising an amino acid sequence of SEQ ID NO: 5.

[0097] In some aspects, the anti- binding moiety comprises (a) a heavy chain comprising an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 30, wherein the heavy chain comprises a CDR- H1 comprising an amino acid sequence of SEQ ID NO: 1; CDR-H2 comprising an amino acid sequence of SEQ ID NO: 2; a CDR-H3 comprising an amino acid sequence of SEQ ID NO: 3; and (b) a light chain comprising an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 29, wherein the VL comprises a light chain CDR-L1 comprising an amino acid sequence of SEQ ID NO: 4; a light chain CDR-L2 comprising the amino acid sequence GAS; and a light chain CDR-L3 comprising an amino acid sequence of SEQ ID NO: 5.

[0098] In some aspects, the anti- binding moiety comprises (a) a heavy chain comprising an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 31, wherein the heavy chain comprises a CDR- H1 comprising an amino acid sequence of SEQ ID NO: 1; CDR-H2 comprising an amino acid sequence of SEQ ID NO: 2; a CDR-H3 comprising an amino acid sequence of SEQ ID NO: 3; and (b) a light chain comprising an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 29, wherein the VL comprises a light chain CDR-L1 comprising an amino acid sequence of SEQ ID NO: 4; a light chain CDR-L2 comprising the amino acid sequence GAS; and a light chain CDR-L3 comprising an amino acid sequence of SEQ ID NO: 5.EGFR-binding domains

[0099] The anti-CLDNl / anti-EGFR bispecific antibody may comprise an anti- EGFR antibody or an antigen-binding fragment thereof as an EGFR binding moiety.[000100] In one aspect, the anti-EGFR antibody is cetuximab. The antigen binding fragment of the anti-EGFR antibody can be a scFv, (scFv)2, Fab, Fab’ or F(ab’)2 of an anti- EGFR antibody, for example cetuximab.[000101] The anti-EGFR antibody or an antigen-binding fragment thereof may be an anti-EGFR antibody or an antigen-binding fragment thereof comprising six CDRs of cetuximab.[000102] The amino acid sequences of the CDRs of the anti-EGFR antibody or an antigen-binding fragment are illustrated in Table 2.Table 2: EGFR-binding domain sequences[000103] In one aspect, the EGFR binding moiety thereof may comprise: a CDR-H1 comprising an amino acid sequence of SEQ ID NO: 8; a CDR-H2 comprising an amino acid sequence of SEQ ID NO: 9; a CDR-H3 comprising an amino acid sequence of SEQ ID NO: 10; a CDR-L1 comprising an amino acid sequence of SEQ ID NO: 11; a CDR-L2 comprising an amino acid sequence of YAS; and a CDR-L3 comprising an amino acid sequence of SEQ ID NO: 13.[000104] In another aspect, the EGFR binding moiety comprises the VH and VL of cetuximab. In one aspect, the cetuximab VH and VL comprise amino acid substitutions. In one aspect, the VH comprises an amino acid substitution of NDT— >DDT to reduce glycosylation. In another aspect, the EGFR binding moiety comprises a heavy chain variableregion comprising the amino acid sequence of SEQ ID NO: 14, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 15.[000105] In some aspects, the EGFR binding moiety comprises a VH comprising an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 14. In some aspects, the anti-EGFR antibody comprises a VL comprising an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 15.[000106] In some aspects, the EGFR binding moiety comprises a VH comprising an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 14, wherein the VH comprises CDR-H1 comprising an amino acid sequence of SEQ ID NO: 8; a CDR-H2 comprising an amino acid sequence of SEQ ID NO: 9; a CDR-H3 comprising an amino acid sequence of SEQ ID NO: 10.[000107] In some aspects, the EGFR binding moiety comprises a VL comprising an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 15, wherein the VL comprises ; a CDR-L1 comprising an amino acid sequence of SEQ ID NO: 11; a CDR-L2 comprising an amino acid sequence of YAS; and a CDR-L3 comprising an amino acid sequence of SEQ ID NO: 13.[000108] In some aspects, the EGFR binding moiety comprises (a) a VH comprising an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 14, wherein the VH comprises a CDR-H1 comprising an amino acid sequence of SEQ ID NO: 8; a CDR-H2 comprising an amino acid sequence of SEQ ID NO: 9; a CDR-H3 comprising an amino acid sequence of SEQ ID NO: 10; and (b) a VL comprising an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 15, wherein the VL comprises ; a CDR-L1 comprising an amino acid sequence of SEQ ID NO: 11; a CDR-L2 comprising an amino acid sequence of YAS; and a CDR-L3 comprising an amino acid sequence of SEQ ID NO: 13.Bispecific antibody[000109] In one embodiment, the present disclosure provides an anti -CLDN1 / anti - EGFR bispecific antibody comprising: (1) an anti-CLDNl antibody or an antigen-binding fragment thereof, as an CLDN1 binding moiety, which is capable of specifically recognizing and / or binding to CLDN1 protein, and (2) an anti -EGFR antibody or an antigen-binding fragment thereof, as an EGFR binding moiety, which is capable of specifically recognizing and / or binding to EGFR protein.[000110] In some embodiments, an anti-CLDNl / anti-EGFR bispecific antibody comprises one or more mutations that enhance its antibody dependent cell cytotoxicity (ADCC) and / or its antibody dependent cell phagocytosis (ADCP) activity. Examples of such mutations include S239D, I332E and A330L (referring to the EU numbering system).[000111] Illustrative sequences of anti-CLDNl / anti-EGFR bispecific antibodies are set forth in Table 3.Table 3: Illustrative Bispecific Antibody Domain Sequences[000112] Accordingly, in one aspect, the anti-CLDNl / anti-EGFR bispecific antibody comprises: (a) a CLDN1 binding moiety comprising a heavy chain variable region comprising a CDR-H1 of SEQ ID NO: 1, a CDR-H2 of SEQ ID NO: 2, and a CDR-H3 of SEQ ID NO: 3, a first light chain variable region comprising a CDR-L1 of SEQ ID NO: 4, a CDR-L2 with the amino acid sequence GAS, and a CDR-L3 of SEQ ID NO: 5; and (b) an EGFR binding moiety comprising a heavy chain variable region of comprising (i) a CDR-H1 of SEQ ID NO: 8, a CDR-H2 of SEQ ID NO: 9, and a CDR-H3 of SEQ ID NO: 10, and (ii) a second light chain variable region comprising a CDR-L1 of SEQ ID NO: 11, a CDR-L2 of YAS, and a CDR-L3 of SEQ ID NO: 13.[000113] A person of skill in the art will appreciate that the sequences of the scFvs and Fabs set forth in Table 3 can be freely combined to construct bispecific antibodies targeting CLDN1 and EGFR.[000114] In one aspect, the anti-CLDNl / anti-EGFR bispecific antibody comprises: (a) a CLDN1 binding moiety comprising the heavy chain of SEQ ID NO: 16 and the light chain of SEQ ID NO: 24; and (b) an EGFR binding moiety comprising the heavy chain of SEQ ID NO: 20 and the light chain of SEQ ID NO: 25.[000115] In one aspect, the anti-CLDNl / anti-EGFR bispecific antibody comprises: (a) a CLDN1 binding moiety comprising the heavy chain of SEQ ID NO: 16 and the light chain of SEQ ID NO: 24; and (b) an EGFR binding moiety comprising the heavy chain of SEQ ID NO: 20 and the light chain of SEQ ID NO: 26.[000116] In one aspect, the anti-CLDNl / anti-EGFR bispecific antibody comprises: (a) a CLDN1 binding moiety comprising the heavy chain of SEQ ID NO: 16 and the light chain ofSEQ ID NO: 24; and (b) an EGFR binding moiety comprising the heavy chain of SEQ ID NO: 20 and the light chain of SEQ ID NO: 27.[000117] In one aspect, the anti-CLDNl / anti-EGFRbispecific antibody comprises: (a) a CLDN1 binding moiety comprising the heavy chain of SEQ ID NO: 16 and the light chain of SEQ ID NO: 24; and (b) an EGFR binding moiety comprising the heavy chain of SEQ ID NO: 20 and the light chain of SEQ ID NO: 28.[000118] In one aspect, the anti-CLDNl / anti-EGFRbispecific antibody comprises: (a) a CLDN1 binding moiety comprising the heavy chain of SEQ ID NO: 17 and the light chain of SEQ ID NO: 24; and (b) an EGFR binding moiety comprising the heavy chain of SEQ ID NO: 21 and the light chain of SEQ ID NO: 25.[000119] In one aspect, the anti-CLDNl / anti-EGFRbispecific antibody comprises: (a) a CLDN1 binding moiety comprising the heavy chain of SEQ ID NO: 17 and the light chain of SEQ ID NO: 24; and (b) an EGFR binding moiety comprising the heavy chain of SEQ ID NO: 21 and the light chain of SEQ ID NO: 26.[000120] In one aspect, the anti-CLDNl / anti-EGFRbispecific antibody comprises: (a) a CLDN1 binding moiety comprising the heavy chain of SEQ ID NO: 17 and the light chain of SEQ ID NO: 24; and (b) an EGFR binding moiety comprising the heavy chain of SEQ ID NO: 21 and the light chain of SEQ ID NO: 27.[000121] In one aspect, the anti-CLDNl / anti-EGFRbispecific antibody comprises: (a) a CLDN1 binding moiety comprising the heavy chain of SEQ ID NO: 17 and the light chain of SEQ ID NO: 24; and (b) an EGFR binding moiety comprising the heavy chain of SEQ ID NO: 21 and the light chain of SEQ ID NO: 28.[000122] In one aspect, the anti-CLDNl / anti-EGFRbispecific antibody comprises: (a) a CLDN1 binding moiety comprising the heavy chain of SEQ ID NO: 18 and the light chain of SEQ ID NO: 24; and (b) an EGFR binding moiety comprising the heavy chain of SEQ ID NO: 22 and the light chain of SEQ ID NO: 26.[000123] In one aspect, the anti-CLDNl / anti-EGFRbispecific antibody comprises: (a) a CLDN1 binding moiety comprising the heavy chain of SEQ ID NO: 18 and the light chain of SEQ ID NO: 24; and (b) an EGFR binding moiety comprising the heavy chain of SEQ ID NO: 22 and the light chain of SEQ ID NO: 28.[000124] In one aspect, the anti-CLDNl / anti-EGFRbispecific antibody comprises: (a) a CLDN1 binding moiety comprising the heavy chain of SEQ ID NO: 19 and the light chain ofSEQ ID NO: 24; and (b) an EGFR binding moiety comprising the heavy chain of SEQ ID NO: 23 and the light chain of SEQ ID NO: 28.[000125] In one aspect, the anti-CLDNl / anti-EGFRbispecific antibody comprises: (a) a CLDN1 binding moiety comprising the heavy chain of SEQ ID NO: 32 and the light chain of SEQ ID NO: 24; and (b) an EGFR binding moiety comprising the heavy chain of SEQ ID NO: 33 and the light chain of SEQ ID NO: 26.[000126] In one aspect, the anti-CLDNl / anti-EGFRbispecific antibody comprises: (a) a CLDN1 binding moiety comprising the heavy chain of SEQ ID NO: 34 and the light chain of SEQ ID NO: 36; and (b) an EGFR binding moiety comprising the heavy chain of SEQ ID NO: 35 and the light chain of SEQ ID NO: 37.[000127] Table 4 further illustrates the heavy and light chain combinations that may be used in the anti-CLDNl / anti-EGFR bispecific antibodies disclosed herein.Table 4: Exemplary bispecific antibodies in Crossmab format[000128] Without wishing to be bound by theory, it is hypothesized that the bispecific antibodies described herein induce antibody dependent cell cytotoxicity (ADCC) and antibody dependent cell phagocytosis (ADCP) in their target cells to a greater degree than an antibody targeting CLND-1 or EGFR individually.[000129] Thus, in some embodiments, a bispecific antibody described herein ADCC in a target cell by about 25% to about 50% compared to an antibody targeting EGFR or CLDN- 1 individually. In some embodiments, a bispecific antibody described herein ADCC in atarget cell by about 50% to about 75% compared to an antibody targeting EGFR or CLDN-1 individually. In some embodiments, a bispecific antibody described herein ADCC in a target cell about 2-fold compared to an antibody targeting EGFR or CLDN-1 individually. In some embodiments, a bispecific antibody described herein increases ADCC in a target cell about 2- fold to about 3 -fold compared to an antibody targeting EGFR or CLDN-1 individually. In some embodiments, a bispecific antibody increases ADCC in a target cell about 3-fold to about 4-fold compared to an antibody targeting EGFR or CLDN-1 individually. In some embodiments, a bispecific antibody described increases ADCC in a target cell about 4-fold to about 5-fold compared to an antibody targeting EGFR or CLDN-1 individually.[000130] In some embodiments, a bispecific antibody described herein increases ADCP in a target cell by about 25% to about 50% compared to an antibody targeting EGFR or CLDN-1 individually. In some embodiments, a bispecific antibody described herein increases ADCP in a target cell by about 50% to about 75% compared to an antibody targeting EGFR or CLDN-1 individually. In some embodiments, a bispecific antibody described herein increases ADCP in a target cell about 1-fold to about 2-fold compared to an antibody targeting EGFR or CLDN-1 individually. In some embodiments, a bispecific antibody described herein increases ADCP in a target cell about 2-fold to about 3-fold compared to an antibody targeting EGFR or CLDN-1 individually. In some embodiments, a bispecific antibody described herein increase ADCP in a target cell about 3 -fold to about 4-fold compared to an antibody targeting EGFR or CLDN-1 individually. In some embodiments, a bispecific antibody described herein increases ADCP in a target cell about 4-fold to about 5- fold compared to an antibody targeting EGFR or CLDN-1 individually. In some embodiments, the target cell co-expresses EGFR and CLND1.[000131] ADCP and ADCC may be assessed using any suitable assay known in the art or described herein, including, for example the Jurkat NF AT CD16a Luc reporter assay, or the CD32 Luc reporter assay.[000132] In addition, the bispecific antibodies described herein show improved target binding compared to monospecific antibodies targeting EGFR (e.g., cetuximab) and monospecific antibodies targeting CLND1 (e.g., ALE.C04 or ALE.F02). The present disclosure also shows that the bispecific antibodies disclosed herein are internalized into target cells to a greater degree than monospecific antibodies targeting EGFR (e.g., cetuximab) and monospecific antibodies targeting CLND1 (e.g., ALE.C04 or ALE.F02). TheADCP and ADCC effects of the bispecific antibodies disclosed herein are believed to be due to the improved binding of the bispecific antibodies.Methods of Production[000133] The anti-CLDNl / anti-EGFR bispecific antibodies disclosed herein may be produced using any suitable method known in the art. For example, an antibody may be prepared, expressed, created or isolated by recombinant means, such as antibodies isolated from an animal that is transgenic for another species’ immunoglobulin genes, antibodies expressed using a recombinant expression vector transfected into a host cell, antibodies isolated from a recombinant, combinatorial antibody library, or antibodies prepared, expressed, created or isolated by any other means that involves splicing of immunoglobulin gene sequences to other DNA sequences.[000134] Also provided herein are vectors comprising nucleic acid sequences encoding the anti-CLDNl / anti-EGFR bispecific antibodies disclosed herein. When the vector allows for the expression of the protein encoded by the polynucleotide inserted therein, the vector is called an expression vector. The vector can have the carried genetic material elements expressed in a host cell by transformation, transduction, or transfection into the host cell. Vectors are well known by a person skilled in the art, including, but not limited to plasmids, phages, cosmids, artificial chromosome such as yeast artificial chromosome (YAC), bacterial artificial chromosome (BAC) or Pl -derived artificial chromosome (PAC); phage such as X phage or Ml 3 phage and animal virus. The animal viruses that can be used as vectors, include, but are not limited to, retrovirus (including lentivirus), adenovirus, adeno-associated virus, herpes virus (such as herpes simplex virus), pox virus, baculovirus, papillomavirus, papova virus (such as SV40). A vector may comprise multiple elements for controlling expression, including, but not limited to, a promoter sequence, a transcription initiation sequence, an enhancer sequence, a selection element and a reporter gene. In addition, a vector may comprise origin of replication.[000135] The elements present in a vector may be operably linked, with or without a spacer or linker. If two or more biological sequences of interest are operably linked, they are generally understood to be arranged in such a way that they are in a relationship permitting them to function in an intended manner. For one instance, when a polynucleotide encoding a polypeptide is operably linked to a regulatory sequence (e.g., promoter, enhancer, silencer sequence, etc.), it is intended to mean that the polynucleotide sequences are linked in such a way that permits regulated expression of the polypeptide from the polynucleotide.[000136] Any suitable host cell known in the art may be used to produce the anti- CLDNl / anti-EGFR bispecific antibodies disclosed herein. Host cells include, without limitation, cultured cells, e.g., mammalian cultured cells derived from rodents (rats, mice, guinea pigs, or hamsters) such as CHO, BHK, NSO, SP2 / 0, YB2 / 0; or human tissues or hybridoma cells; cells from prokaryotes or eukaryotic microbes, such as bacteria cells (e.g., eubacteria, such as Gram-negative or Gram-positive organisms, for example, Enterob acteriaceae such as Escherichia, e.g., E. coli), and fungal cells (e.g., yeast cells, filamentous fungi cells, etc.); plant cells or animal cells such as insect cells, and cells comprised within a transgenic animal or cultured tissue. A host cell may refer to not only the particular subject cell but also the progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not be identical to the parent cell, but may still included within the scope of the term “host cell.”[000137] Protocols and techniques for transfection include but not limited to lipid transfection and chemical and physical methods such as electroporation. A number of transfection techniques are well known in the art and are disclosed herein. See, e.g., Graham et al., 1973, Virology 52: 456; Sambrook et al., 2001, Molecular Cloning: A Laboratory Manual; Davis et al., 1986, Basic Methods in Molecular Biology, Elsevier; Chu et al, 1981, Gene 13: 197.[000138] In some embodiments, the bispecific CLDN1 / EGFR antibodies of the present disclosure comprise different antigen binding moieties, fused to one or the other of the two subunits of the Fc domain, thus the two subunits of the Fc domain are typically comprised in two non-identical polypeptide chains. Recombinant co-expression of these polypeptides and subsequent dimerization leads to several possible combinations of the two polypeptides. To improve the yield and purity of the bispecific antibodies of the present disclosure in recombinant production, it can be advantageous to promote heterodimerization by a variety of means.[000139] In one aspect, heterodimerization can be directed using CrossMab technology. Schaefer et al. (Roche Diagnostics GmbH), describe a method to express two heavy and two light chains, derived from two existing antibodies, as human bivalent bispecific IgG antibodies without use of artificial linkers (PNAS (2011) 108(27): 11187-11192 and US 2009 / 0232811). The method involves exchanging one or more heavy chain and light chain domains within the antigen-binding fragment (Fab) of one half of the bi-specific antibody(CrossMab). Correct association of the light chains and their cognate heavy chains is achieved by exchange of heavy-chain and light-chain domains within the antigen binding fragment (Fab) of one half of the bispecific antibody. This “crossover” retains the antigenbinding affinity but makes the two arms so different that light-chain mispairing can no longer occur. See W02009 / 080251, W02009 / 080252, W02009 / 080253, W02009 / 080254, WO 2010 / 115589, WO 2010 / 136172, WO 2010 / 145792, and WO 2010 / 145793, each incorporated herein by reference in its entirety. Despite these recent advantages e.g. due to development of methodologies like “knobs into holes (KiH) or the “CrossMab” technology the expression of multispecific antibodies may still lead to undesired formation of productspecific impurities specifically associated with their production. These product-specific impurities for example may include Yi antibodies (comprising a single heavy-chain / light- chain pair),3 / 4 antibodies (comprising a complete antibody lacking a single light chain) or a 5 / 4 antibody by-product (comprising an additional heavy or light chain variable domain). [000140] Bispecific antibodies may also be assembled using knob-in-hole (KIH) technology. In this method, mutations are introduced in the constant region of a heavy chain. Examples of such mutations include S354C, T366W, T366S and L368A, where the residues are defined by the EU numbering system. Examples of VH domains comprising KIH mutations are set forth in SEQ ID NO: 34 (ALE.F02) and SEQ ID NO: 35 (Cetuximab). An example of a bispecific antibody assembled using KIH technology is BsAbl2 (comprising ALEF02-VHl-FcGlm3-Knob- LFLEPS).[000141] The bispecific antibodies disclosed herein may comprise additional mutations to make them easier to purify, for example, a H435R mutation (as numbered by the EU system).Pharmaceutical Compositions[000142] Also provided herein are pharmaceutical compositions comprising an anti- CLDNl / anti-EGFR bispecific antibody described herein and at least one pharmaceutically acceptable carrier or excipient. Any vehicle, diluent, excipient and / or salts thereof that chemically and / or physically is compatible with other ingredients in the formulation, and physiologically compatible with the recipient may be used as the carrier. Examples of pharmaceutically acceptable carriers and excipients are well known in the art (see, e.g., Remington’s Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), and include, but are not limited to pH adjusters, surfactants, adjuvants and ionic strength enhancers.[000143] The optimal pharmaceutical formulation can be varied depending upon the route of administration and desired dosage. Such formulations may influence the physical state, stability, rate of in vivo release, and rate of in vivo clearance of the administered active ingredient.[000144] The pharmaceutical compositions of the present disclosure may be formulated in dosage unit form for ease of administration and uniformity of dosage. It will be understood, however, that the total daily dosage of the compositions will be decided by the attending physician within the scope of sound medical judgement.Methods of Use[000145] The present disclosure includes methods of treating a disease or disorder in a subject in need thereof comprising administering to the subject an anti -CLDN1 / anti -EGFR bispecific antibody described herein or a pharmaceutical composition comprising the same. [000146] The terms “subject” and “patient” are used interchangeably herein to refer to a human or non-human animal, including a mammal or a primate, in need of diagnosis, prognosis, amelioration, prevention, and / or treatment of a disease or condition. Mammalian subjects include humans, domestic animals, farm animals, and zoo, sports, or pet animals such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, swine, cows, bears, and so on.[000147] In some embodiments, the subject exhibits symptoms or indicia of cancer (e.g., a subject expressing a tumor or suffering from any of the cancers mentioned herein below), or who otherwise would benefit from an inhibition or reduction in EGFR activity or a regression of EGFR+ tumors.[000148] Any disease or disorder in which stimulation, activation and / or targeting of an immune response would be beneficial may be treated using the methods disclosed herein. In particular, the anti-CLDNl / anti-EGFR bispecific antibodies of the disclosure may be used for the treatment of any disease or disorder associated with or mediated by EGFR or CLDN1 expression or activity.[000149] In one aspect, the disease or disorder is cancer. Examples of cancers that may be treated in accordance with the methods disclosed herein include lymphomas, lymphocytic leukemias, lung cancer, non-small cell lung (NSCL) cancer, bronchioloalveolar cell lung cancer, bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck (e.g. Head and Neck Squamous Cell Carcinoma), cutaneous or intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, gastric cancer, colon cancer, breast cancer, uterine cancer, carcinoma of the fallopian tubes, carcinoma of theendometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, Hodgkin’s Disease, cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the urethra, cancer of the penis, prostate cancer, cancer of the bladder, cancer of the kidney or ureter, renal cell carcinoma, carcinoma of the renal pelvis, mesothelioma, hepatocellular cancer, biliary cancer, neoplasms of the central nervous system (CNS), spinal axis tumors, brain stem glioma, glioblastoma multiforme, astrocytomas, schwanomas, ependymonas, medulloblastomas, meningiomas, squamous cell carcinomas, pituitary adenoma and Ewings sarcoma, including refractory versions of any of the above cancers, or a combination of one or more of the above cancers. The term “treatment,” “treating” or “treated,” as used herein in the context of treating a condition, pertains generally to treatment and therapy, whether of a human or an animal, in which some desired therapeutic effect is achieved, for example, the inhibition of the progress of the condition, and includes a reduction in the rate of progress, a halt in the rate of progress, regression of the condition, amelioration of the condition, and cure of the condition. Treatment as a prophylactic measure (i.e., prophylaxis, prevention) is also included. For cancer, “treating” may refer to dampen or slow the tumor or malignant cell growth, proliferation, or metastasis, or some combination thereof. For tumors, “treatment” includes removal of all or part of the tumor, inhibiting or slowing tumor growth and metastasis, preventing or delaying the development of a tumor, or some combination thereof.[000150] A method of treatment described herein may achieve one or more desired therapeutic effects, for example, the inhibition of the progress of the condition, a reduction in the rate of progress, a halt in the rate of progress, regression of the condition, or amelioration of the condition. With respect to cancer, “treating” may refer to dampen or slow the tumor or malignant cell growth, proliferation, or metastasis, or some combination thereof. Treatment may result in removal of all or part of the tumor, inhibiting or slowing tumor growth and metastasis, preventing or delaying the development of a tumor, or some combination thereof. [000151] A method of treatment may also result in a reduction of tumor burden or tumor regression. Reduction of tumor burden or tumor regression includes the partial or complete disappearance of a tumor or tumors in a subject. It is understood that tumor regression represents a trend toward a lower tumor burden or less severe state of disease. As such, regression is a progressive decline elimination of measurable malignancies in the body, including decline in tumor size and / or decline in number of tumors. Reduction of tumordevelopment includes a partial or complete inhibition or suppression of further or new tumor growth. The anti-CLDNl / anti-EGFRbispecific antibodies of the present disclosure are administered in an amount sufficient to reduce tumor burden, produce tumor regression, inhibit tumor growth or reduce tumor development in the subject.[000152] The present disclosure also includes methods for treating residual cancer in a subject. As used herein, the term “residual cancer” means the existence or persistence of one or more cancerous cells in a subject following treatment with an anti-cancer therapy. Thus, in some embodiments, a subject treated in accordance with a method disclosed herein has previously received cancer treatment (e.g., chemotherapy). In some embodiments, a subject treated in accordance with the methods disclosed herein has a cancer that is resistant to a cancer treatment (e.g., chemotherapy).[000153] The anti-CLDN 1 / anti-EGFR bispecific antibody described herein or a pharmaceutical composition comprising the same can be administered to a subject in need thereof by any suitable route. Various delivery systems are known and can be used to administer antibodies, including tablets, capsules, injectable solutions, encapsulation in liposomes, microparticles, microcapsules, etc. Methods of administration include, but are not limited to, dermal, intradermal, intramuscular, intraperitoneal, intralesional, intravenous, subcutaneous, intranasal, pulmonary, epidural, and oral routes. An anti-CLDN 1 / anti-EGFR bispecific antibody, or a pharmaceutical composition thereof, may be administered by any convenient or other appropriate route, for example, by infusion or bolus injection, by absorption through epithelial or mucosa linings (e.g., oral mucosa, bronchial mucosa, rectal and intestinal mucosa, etc.). Administration can be systemic or local.[000154] In some aspects, the anti-CLDNl / anti-EGFR bispecific antibody described herein or a pharmaceutical composition comprising the same is administered intratumorally, intravenously, intraperitoneally, intramuscularly, intrathecally or subcutaneously.[000155] An anti-CLDNl / anti-EGFR bispecific antibody described herein or a pharmaceutical composition comprising the same may be administered in a dosage such that the amount delivered is effective for the intended purpose. The route of administration, formulation and dosage administered will depend on the therapeutic effect desired, the severity of the condition to be treated if already present, the presence of any infection, the age, sex, weight, and general health condition of the patient as well as upon the potency, bioavailability, and in vivo half-life of the antibody or composition used, the use (or not) of concomitant therapies, and other clinical factors. These factors are readily determinable bythe attending physician in the course of the therapy. Alternatively or additionally, the dosage to be administered can be determined from studies using animal models (e.g., non-human primates or rodents). Adjusting the dose to achieve maximal efficacy based on these or other methods are well known in the art and are within the capabilities of trained physicians. As studies are conducted using anti-CLDNl / anti-EGFRbispecific antibody, further information will emerge regarding the appropriate dosage levels and duration of treatment.Kits[000156] In another aspect, the present disclosure provides a pharmaceutical pack or kit comprising one or more containers (e.g., vials, ampoules, test tubes, flasks or bottles) containing one or more ingredients of an inventive pharmaceutical composition, allowing administration of an anti-CLDNl / anti-EGFR bispecific antibody.[000157] Different ingredients of a pharmaceutical pack or kit may be supplied in a solid (e.g., lyophilized) or liquid form. Each ingredient will generally be suitable as aliquoted in its respective container or provided in a concentrated form. Pharmaceutical packs or kits may include media for the reconstitution of lyophilized ingredients. Individual containers of the kits will preferably be maintained in close confinement for commercial sale.[000158] Optionally associated with the container(s) can be a notice or package insert in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceutical or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration. The notice of package insert may contain instructions for use of a pharmaceutical composition according to methods of treatment disclosed herein.[000159] An identifier, e.g., a bar code, radio frequency, ID tags, etc., may be present in or on the kit. The identifier can be used, for example, to uniquely identify the kit for purposes of quality control, inventory control, tracking movement between workstations, etc.EXAMPLES[000160] The following examples are illustrative and do not limit the scope of the claimed aspects.Example 1. Bispecific anti-CLDNl / anti-EGFR antibodies[000161] CLDN1 and EGFR1 are co-expressed in head and neck cancer (FIG. 1A), squamous cell lung cancer (FIG. IB), and esophageal cancer (FIG. 1C).[000162] In order to take advantage of this co-expression, the bispecific anti- CLDNl / anti-EGFR antibodies with Crossmab KZ. format were designed according to the Crossmab methodology (FIG. 3). First, binding of monospecific, bivalent versions of anti- EGFR antibodies (FIG. 2A; Table 5) and anti-CLDNl antibodies (FIG. 2B; Table 6) to their respective targets in Huh7 cells was confirmed.Table 5: Monospecific anti-EGFR Antibody Target binding in Huh7 CellsTable 6: Monospecific anti-CLDN 1 Antibody Target binding in Huh7 CellsGeneration of Bispecific Anti-CLDNl / anti-EGFR Antibodies[000163] Next, DNA sequences encoding two different heavy and two different light chains were generated (Table 3). The anti-CLDNl arm heavy chains contain the Variable Heavy (VH) domains fused to the human Kappa constant (cK) domain and human IgGl CH2 and CH3 domains. The anti-CLDNl arm light chain was designed to contain the Variable Light (VL) domain fused to the human CHI domain. The anti-EGFR heavy chains arm corresponds to the canonical definition with VH domain fused to human IgGl CHI, CH2 and CH3 domains. The anti-EGFR Light chain was designed to fuse the VL domain to the human Lambda (cL) constant domain. The DNAs encoding these two different heavy chains and two different light chains were then synthesized and cloned into separate plasmids.[000164] The four plasmids encoding these heavy and light chains were co-transfected in mammalian cells according to table 4 and using lipofection procedures. Thanks to the Crossmab methodology, the supernatant of transfected cells contains three different species with a mixture of two monoclonal antibodies and one bispecific antibody. The first monoclonal antibody corresponds to two anti-CLDNl binding arms with only Kappa constant domains. The second monoclonal antibody corresponds to two anti-EGFR binding arms with only Lambda constant domains. The bispecific antibody contains one anti-CLDNl and one anti-EGFR binding arms with both Kappa and Lambda constant domains.[000165] To isolate the desired bispecific antibody, a three-affinity step purification process was designed. The first step consists in the recovery of the three different antibodies using affinity purification reagents targeting IgG Fc-region. Non-limiting examples of IgG Fc-binding resins include protein A resins like MabSelect PrismA, Byzen Pro, UNOsphere SUPrA, CaptureSelect IgG-Fc and CaptureSelect Fc-XL affinity matrices. The second and third affinity steps are using resin agents binding specifically to the human cK or human cL domains. Examples of human cK or human cL binding resins include CaptureSelect KappaXL and CaptureSelect LC Lambda affinity resins, respectively. The second and third affinity steps are sequentially applied to separate the bispecific antibody from monoclonal antibody contaminants. The monoclonal antibodies only bind to either the CaptureSelect KappaXL or CaptureSelect LC Lambda resins but not to the combined resins in contrast to the bispecific antibody having both Kappa and Lambda constant domains. The purified bispecific antibody with Crossmab KZ. format can be then formulated and used for further applications.[000166] The ability of an Fc competent bispecific anti-CLDNl / anti-EGFR antibody (BsAbl) to bind to its targets was confirmed through a binding assay towards Huh-7 cells, Huh-7 cells transfected with Cas9 alone (FIG. 4B), Huh-7 cells with CLDN1 knocked out via CRISPR-Cas9 (FIG. 4C), and Huh-7 cells with EGFR knocked out via CRISPR-Cas9 (FIG. 4D). Binding affinities are summarized in Table 7.Table 7: Binding of anti-CLNDl, anti-EGFR, and bispecific anti-CLND 1 / anti-EGFR Antibodies to Huh7 Cells[000167] A Jurkat NF AT CD16a Luc reporter assay was used to determine ADCC induction by BsAbl, cetuximab and ALE.C04 in the Cal27 Head and Neck Squamous Cell Carcinoma cell line (effectortarget ratio 2: 1), a KYSE30 Esophageal Cancer cell line (effectortarget ratio 4: 1) and a Huh7 Hepatocellular Carcinoma cell line (effectortarget ratio 2: 1). The bispecific anti-CLDNl / anti-EGFR BsAbl antibody BsAbl enhanced ADCC response (IC50 value) relative to ALE.C04 in all three cell lines and relative to cetuximab inCal27 and Huh7 cells, as shown in FIGs. 5A-5C and Table 8. The bispecific anti-CLDN1 / anti -EGFR BsAbl antibody BsAbl enhanced the maximum ADCC effect relative toALE.C04 and cetuximab in all three cell lines FIGs. 5A-5C.Table 8: ADCC Induction by BsAbl and ControlsNA: Not applicable since the ADCC was too low to calculate an IC50 value[000168] This data shows that the bispecific anti -CLDN1 / anti -EGFR antibody BsAbl is better at driving ADCC than both Cetuximab alone or ALE.C04 alone.[000169] A Jurkat NF AT CD32 Luc reporter assay was used to measure ADCP induction by BsAbl, cetuximab and ALE.C04 in an Esophageal Cancer cell line (KYSE30, effectortarget ratio 4:1), and two Head and Neck Squamous Cell Carcinoma cell lines (Cal27, effectortarget ratio 4: 1 and Detroit 562; effectortarget ratio 4: 1). The bispecific anti- CLDN1 / anti -EGFR BsAbl antibody enhanced ADCP in all three cell lines as shown in FIGs. 6A-6C and Table 9.Table 9: ADCP Induction by BsAbl and ControlsND: Not detectable[000170] This data shows that the bispecific anti -CLDN1 / anti -EGFR antibody BsAbl is better at driving ADCP than both Cetuximab alone or ALE.C04 alone.Example 2: Target Binding of Bispecific AntibodiesMethods[000171] CLDN1 -transduced NCI-H1975 cells were harvested after cell expansion in aT75 culture flask. In a flat bottom 96-well plate, 5xl04cells were seeded in 100 mL of cell culture medium for 24h. Cell medium was then removed, and cells washed with 100 mL ofPBS. Cells were incubated in 100 mL of cell dissociation buffer until they detach. Detached cells were collected and transferred into a V-bottom plate, and the plate was spun for 3min at 350 g. The supernatants were discarded and cells incubated in 100 mL of primary antibodies or their isotype control hlgGl for Ih at 4°C (antibodies were used in a dose-range). Cells were washed twice in cold PBS and incubated in 100 mL of secondary antibody solution (Alexa Fluor® 647 AffiniPure Goat Anti -Human IgG (H+L) diluted 1 :400) containing Zombie Violet Live / Dead cell marker (1 :500 dilution) for 30 min at 4°C. Cells were washed twice with 100 mL of FACS buffer, and resuspended in 45 mL of FACS buffer, before flow cytometry analysis on a CytoFLEX benchtop flow cytometer (Beckman Coulter) for Zombie Violet negative and Alexa Fluor® 647 positive cells.Results[000172] BsAbl showed an improved binding compared to Cetuximab or ALE.C04 in H1975 cells expressing CLDN1 (FIG. 7), with a top MFI of 3.7xl05for BsAbl compared to 2.9xl05for ALE.C04 and 1.6xl05for cetuximab (Table 10).Table 10: Binding of Bs Ab 1 to H1975 Cells[000173] BsAbl also showed an improved binding compared to Cetuximab or ALE.F02 in SW480 cells expressing low levels of CLND1 (FIG. 8) with binding MFIs of 3.7xl05for BsAbl compared to 1.7xl05for ALE.F02 and 1.3xl05for cetuximab (Table 11).Table 11: Binding of BsAbl to SW480 Cells[000174] Similar to the Fc-competent BsAbl, an Fc non-competent bispecific anti- CLDNl / anti-EGFR antibody (BsAb2) showed an improved binding compared to Cetuximab or ALE.F02 in SW480 cells. This effect was observed in both SW480 cells with low CLDN1 expression (FIG. 9A) and in SW480 cells with high CLND1 expression (FIG. 9B). Binding affinities and top MFI are summarized in Table 12.Table 12: Binding ofBsAb2 to SW480 CellsExample 3: Internalization of Bispecific AntibodiesMethodsAntibody labeling with Zenon pHrodo reagent[000175] Zenon pHrodo IgG labeling reagent was used to label anti-CLDNl antibodies, Cetuximab and their isotype controls. A 4x working solution of Zenon™ pHrodo™ iFL Red Human IgG Labeling Reagent was prepared at 480 mM and mixed with a 40 mg / mL antibody solution for 5 min at room temperature to allow labelling complexes to form between the Fab fragments from the Zenon pHrodo reagent and the Fc portion of IgG antibodies.Internalization assay by FACS[000176] NCLH1975 or SW480 cells were seeded at 35xl03(CLDN1 -transduced cells) in a flat bottom 96-well plate in 100 mL of cell culture medium and incubated for 18h. Then the medium was removed and replaced by a mix of 50 mL of fresh media and 50 mL of the Zenon pHrodo labeled complex (Antibody or isotype), and the cells were incubated from 1 h to 6 h under standard cell culture conditions to allow antibodies to internalize (pHrodo dye is pH sensitive, little to no fluorescent signal at neutral pH and fluoresces brightly in acidic environments like lysosomal vesicles). When incubation was completed, the medium was removed, and the cells were washed with PBS. 100 mL of Accutase was added in each well to allow cells to detach. The detached cells were transferred in a V-bottom plate and washed twice with cold PBS and incubated in 50 mL of Zombie Violet Live / Dead cell marker (1 :500 dilution) for 15 min at 4°C. After a final washing step with PBS, cells were resuspended in 40 mL of FACS buffer and analyzed by flow cytometry on a CytoFLEX benchtop flow cytometer (Beckman Coulter) for Zombie Violet negative and pHrodo-positive cells (excitation at 560 nm, emission at 585 nm).[000177] FACS scatter plot graphs were generated for the 4h internalization timepoint (NCI-H1975-CLDN1) or 2h internalization timepoint (SW480-CLDN1). GFP channel corresponds to CLDN1 expression and APC channel to pHrodo detection (internalization) (pHrodo dye is pH sensitive, little to no fluorescent signal at neutral pH and fluoresces brightly in acidic environments like lysosomal vesicles).Results[000178] The improved target binding of BsAbl demonstrated in Example 2 translated into a better internalization for bispecific antibody BsAbl compared to ALE.F02, cetuximab and isotype control in NCI-H1975 cells (FIGs. 10A and 10B). Similar effects were observed in SW480 cells expressing low levels of CLND1 (FIG. 11 A and 1 IB) and Huh7 cells (FIG. 12).

Claims

CLAIMSWhat is claimed is:

1. A bispecific antibody, comprising(a) a Claudin 1 (CLDN1) binding moiety comprising a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 1, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 2, a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 3, a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 4, a light chain CDR2 comprising the amino acid sequence GAS, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 5, and(b) an Epidermal Growth Factor Receptor (EGFR) binding moiety comprising: a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 8, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 9, a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 10, a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 11, a light chain CDR2 comprising the amino acid sequence of YAS, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 13.

2. The bispecific antibody of claim 1, wherein the antibody increases antibody dependent cell cytotoxicity of cells co-expressing EGFR and CLDN1 by at least 50% in comparison to antibodies targeting CLDN1 and EGFR individually.

3. The bispecific antibody of claim 1, wherein the antibody increases antibody dependent cell phagocytosis of cells co-expressing EGFR and CLDN1 by at least 50% in comparison to antibodies targeting CLDN1 and EGFR individually.

4. The bispecific antibody of any one of claims 1-3, wherein (a) the CLDN1 binding moiety comprises: (i) a heavy chain variable domain (VH1) sequence at least 85% identical to SEQ ID NO: 6, and (ii) a light chain variable domain (VL1) sequence at least 85% identical to SEQ ID NO: 7; and (b) the anti-EGFR binding comprises: (i) a heavy chain variable domain (VH2) sequence at least 85% identical to SEQ ID NO: 14, and (ii) a light chain variable domain (VL2) sequence at least 85% identical to SEQ ID NO: 15.

5. The bispecific antibody of any one of claims 1-3, wherein (a) the CLDN1 binding moiety comprises: (i) a heavy chain variable domain (VH1) sequence at least 90% identical to SEQ ID NO: 6, and (ii) a light chain variable domain (VL1) sequence at least 90% identical to SEQ ID NO: 7; and (b) the anti-EGFR binding comprises: (i) a heavy chain variable domain (VH2) sequence at least 90% identical to SEQ ID NO: 14, and (ii) a light chain variable domain (VL2) sequence at least 90% identical to SEQ ID NO: 15.

6. The bispecific antibody of any one of claims 1-3, wherein (a) the CLDN1 binding moiety comprises: (i) a heavy chain variable domain (VH1) sequence at least 95% identical to SEQ ID NO: 6, and (ii) a light chain variable domain (VL1) sequence at least 95% identical to SEQ ID NO: 7; and (b) the anti-EGFR binding comprises: (i) a heavy chain variable domain (VH2) sequence at least 95% identical to SEQ ID NO: 14, and (ii) a light chain variable domain (VL2) sequence at least 95% identical to SEQ ID NO: 15.

7. The bispecific antibody of any one of claims 1-3, wherein (a) the CLDN1 binding moiety comprises: (i) a heavy chain variable domain (VH1) comprising the sequence of SEQ ID NO: 6, and (ii) a light chain variable domain (VL1) comprising the sequence of SEQ ID NO: 7; and (b) the anti-EGFR binding comprises: (i) a heavy chain variable domain (VH2) comprising the sequence of SEQ ID NO: 14, and (ii) a light chain variable domain (VL2) comprising the sequence of SEQ ID NO: 15.

8. The bispecific antibody of any one of claims 1-7, wherein the bispecific antibody comprises four polypeptide chains: (i) a first heavy chain comprising the sequence of any one of SEQ ID NOs: 16-19, 32 and 34; (ii) a first light chain comprising the sequence of SEQ ID NO: 24 or 36; (iii) a second heavy chain comprising the sequence of any one of SEQ ID NOs: 20-23, 33 and 35, and (iv) a second light chain comprising the sequence of any one of SEQ ID NOs: 25-28 and 37.

9. The bispecific antibody of claim 8, wherein the first heavy chain comprises the sequence of SEQ ID NO: 16, the first light chain comprises the sequence of SEQ ID NO: 24, the second heavy chain comprises the sequence of SEQ ID NO: 21, and the second light chain comprises the sequence of SEQ ID NO: 25.

10. The bispecific antibody of claim 8, wherein the first heavy chain comprises the sequence of SEQ ID NO: 16, the first light chain comprises the sequence of SEQ ID NO: 24, the second heavy chain comprises the sequence of SEQ ID NO: 21, and the second light chain comprises the sequence of SEQ ID NO: 26.

11. The bispecific antibody of claim 8, wherein the first heavy chain comprises the sequence of SEQ ID NO: 16, the first light chain comprises the sequence of SEQ ID NO: 24, the second heavy chain comprises the sequence of SEQ ID NO: 20, and the second light chain comprises the sequence of SEQ ID NO: 27.

12. The bispecific antibody of claim 8, wherein the first heavy chain comprises the sequence of SEQ ID NO: 16, the first light chain comprises the sequence of SEQ ID NO: 24, the second heavy chain comprises the sequence of SEQ ID NO: 20, and the second light chain comprises the sequence of SEQ ID NO: 28.

13. The bispecific antibody of claim 8, wherein the first heavy chain comprises the sequence of SEQ ID NO: 17, the first light chain comprises the sequence of SEQ ID NO: 24, the second heavy chain comprises the sequence of SEQ ID NO: 21, and the second light chain comprises the sequence of SEQ ID NO: 25.

14. The bispecific antibody of claim 8, wherein the first heavy chain comprises the sequence of SEQ ID NO: 17, the first light chain comprises the sequence of SEQ ID NO: 24, the second heavy chain comprises the sequence of SEQ ID NO: 21, and the second light chain comprises the sequence of SEQ ID NO: 26.

15. The bispecific antibody of claim 8, wherein the first heavy chain comprises the sequence of SEQ ID NO: 17, the first light chain comprises the sequence of SEQ ID NO: 24, the second heavy chain comprises the sequence of SEQ ID NO: 21, and the second light chain comprises the sequence of SEQ ID NO: 27.

16. The bispecific antibody of claim 8, wherein the first heavy chain comprises the sequence of SEQ ID NO: 17, the first light chain comprises the sequence of SEQ ID NO: 24,the second heavy chain comprises the sequence of SEQ ID NO: 21, and the second light chain comprises the sequence of SEQ ID NO: 28.

17. The bispecific antibody of claim 8, wherein the first heavy chain comprises the sequence of SEQ ID NO: 18, the first light chain comprises the sequence of SEQ ID NO: 24, the second heavy chain comprises the sequence of SEQ ID NO: 22, and the second light chain comprises the sequence of SEQ ID NO: 26.

18. The bispecific antibody of claim 8, wherein the first heavy chain comprises the sequence of SEQ ID NO: 18, the first light chain comprises the sequence of SEQ ID NO: 24, the second heavy chain comprises the sequence of SEQ ID NO: 22, and the second light chain comprises the sequence of SEQ ID NO: 28.

19. The bispecific antibody of claim 8, wherein the first heavy chain comprises the sequence of SEQ ID NO: 19, the first light chain comprises the sequence of SEQ ID NO: 24, the second heavy chain comprises the sequence of SEQ ID NO: 23, and the second light chain comprises the sequence of SEQ ID NO: 28.

20. The bispecific antibody of claim 8, wherein the first heavy chain comprises the sequence of SEQ ID NO: 29, the first light chain comprises the sequence of SEQ ID NO: 24, the second heavy chain comprises the sequence of SEQ ID NO: 30, and the second light chain comprises the sequence of SEQ ID NO: 26.

21. The bispecific antibody of claim 8, wherein the first heavy chain comprises the sequence of SEQ ID NO: 31, the first light chain comprises the sequence of SEQ ID NO: 33, the second heavy chain comprises the sequence of SEQ ID NO: 32, and the second light chain comprises the sequence of SEQ ID NO: 34.

22. The bispecific antibody of any one of claims 1-21, wherein the CLDN1 binding moiety and the EGFR binding moiety are connected by a linker.

23. The bispecific antibody of any one of claims 1-7, wherein the antibody comprises a human Fc region.

24. The bispecific antibody of claim 23, wherein the human Fc region is a human IgG4 or IgGl Fc region.

25. The bispecific antibody of any one of claim 1-7, wherein the antibody comprises one or more mutations in the Fc region, wherein the one or more mutation is selected from L234F, L235E, and P331S, wherein the residues are defined by the EU numbering system.

26. The bispecific antibody of any one of claim 1-7, wherein the antibody comprises one or more mutations in the Fc region, wherein the one or more mutation is selected from S354C, T366W, T366S and L368A, wherein the residues are defined by the EU numbering system.

27. The bispecific antibody of any one of claims 1-26, wherein the bispecific antibody is a humanized antibody.

28. An isolated nucleic acid molecule, comprising a nucleic acid sequence encoding the bispecific antibody of any one of claims 1-27.

29. A vector comprising the nucleic acid molecule of claim 28.

30. A host cell comprising the nucleic acid molecule of claim 28 or the vector of claim 29.

31. A pharmaceutical composition comprising the bispecific antibody of any one of claims 1-27 and a pharmaceutically acceptable carrier.

32. A method for producing the bispecific antibody of claims 1-27, comprising the steps of: (a) expressing the bispecific antibody as defined in any of claims 1-27 in the host cell of claim 30; and (b) isolating the bispecific antibody from the host cell.

33. A method for modulating an immune response in a subject, comprising administering an effective amount of the bispecific antibody of any of claims 1-27 or the pharmaceutical composition of claim 31 to the subject.

34. A method for inhibiting growth of a tumor cell in a subject, comprising administering an effective amount of the bispecific antibody of any of claims 1-27 or the pharmaceutical composition of claim 31 to the subject.

35. The method of claim 34, wherein the tumor cell is from a Head and Neck Squamous Cell Carcinoma, an Esophageal Cancer, or a Hepatocellular Carcinoma.

36. A method for treating a cancer in a subject in need thereof, comprising administering an effective amount of the bispecific antibody of any of claims 1-27 or the pharmaceutical composition of claim 31 to the subject.

37. The method of claim 36, wherein the cancer is a Head and Neck Squamous Cell Carcinoma, an Esophageal Cancer, or a Hepatocellular Carcinoma.

38. The bispecific antibody of any of claims 1-27 for use in treating or preventing CLDN1 -related and / or EGFR-related diseases in a subject39. Use of the bispecific antibody of any of claims 1-27 in the manufacture of a medicament for modulating an immune response or inhibiting growth of a tumor cell in a subject.

40. The use of claim 39, wherein the tumor cell is from a Head and Neck Squamous Cell Carcinoma, an Esophageal Cancer, or a Hepatocellular Carcinoma.

41. Use of the bi specific antibody or the antigen-binding portion thereof of any of claims 1-27 in the manufacture of a medicament for treating cancer in a subject.

42. The use of claim 41, wherein the cancer is a Head and Neck Squamous Cell Carcinoma, an Esophageal Cancer, and a Hepatocellular Carcinoma.

43. A kit, comprising a container comprising the bispecific antibody or the antigenbinding portion thereof of any one of claims 1-27.

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